Nanomedicine Book

NANOMEDICINE
Editors:
Professor Alexander Seifalian
University College London, United Kingdom
Achala de Mel
Deepak M. Kalaskar
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Nanomedicine iii
Contents
Chapter 1 Long Circulating Nanoparticles for Tolerogenesis
Amy Tekrony, Vincent Wright, Anne Slaney, Usama Al-Atar, Amy Frederick, Teresa
Rodriguez, Jillian Buriak, David Cramb, Lori West………………………………………. 1
Chapter 2 In Vivo Applications of Quantum Dot Nanoparticles for Optical

Diagnostics and Therapy
Elnaz Yaghini, Alexander M. Seifalian, Alexander J. MacRobert……………………… 21
Chapter 3 Conjugation of Gold nanoparticles and liposomes for combined

vehicles of drug delivery in cancer
Sara Figueiredo1, Rita Cabral, Daniel Luís, Alexandra R. Fernandes and Pedro V.
Baptista…………………………………………………………………………………………… 48
Chapter 4 Multifunctional Core-Shell Nanostructures

M. Clara Gonçalves , M. Barbara Martins………………………………………………….83
Chapter 5 Biomaterial surface modification of titanium and titanium alloys for

medical applications
Mukta Kulkarni, Anca Mazare, Patrik Schmuki, Aleš Iglič……………………………. 111
Chapter 6 Experimental and Clinical Therapeutic Uses of Low-Molecular-

Weight Heparin/Protamine Micro/Nanoparticles
Masayuki Ishihara, Makoto Takikawa, Hidemi Hattori, Masanori Fujita, Miya
Ishihara and Shingo Nakamura………………………………………………………....... 137
Chapter 7 Nanophysical approach to diagnosis of epithelial tissues using Opto-

magnetic imaging spectroscopy
Lidija Matija, Branislava Jeftić, Gorana Nikolić, Aleksandra Dragičević, Ivana
Mileusnić, Jelena Munćan, Djuro Koruga ………………………………………………… 156
Chapter 8 Solid lipid nanoparticles – a promising drug delivery system

Hristo Svilenov, Christo Tzachev………………………………………………………...... 187
Chapter 9 Quantum Developments in Nanomedicine: Nanocurative Actions by

Soft Photons Sources and their Path Integrals
Francisco Bulnes, F H. Bulnes-Gonzalez………………………………………….......... 238
Nanomedicine iv
Chapter 10 Optimization of perfluorocarbon nanoemulsions for molecular

imaging by 19F MRI
Christoph Grapentin, Friederike Mayenfels, Sabine Barnert, Regine Süss, Rolf
Schubert…………………………………………………………………………………………. 268
Chapter 11 Designing polymeric nanoparticles for targeted drug delivery system

Ida Idayu Muhamad, Suguna Selvakumaran, Nurul Asmak Md Lazim……………. 287
Chapter 12 Surface modification of metallic implants with anodic oxide

nanotubular arrays via electrochemical anodization techniques
Lu-Ning Wang, Ming Jin……………………………………………………………………… 314
Chapter 13 Unsupervised medical image segmentation using stochastic

optimization methods
Ivan Cruz-Aceves1, Juan Gabriel Avina-Cervantes1, Juan Manuel Lopez-Hernandez,
Ma. de Guadalupe Garcia-Hernandez, Miguel Torres-Cisneros, Manuel Ornelas-
Rodriguez……………………………………………………………………………………….. 334
Chapter 14 Biological applications of Semiconductor Nanoparticles

Salam Massadeh, Manal Alaamery………………………………………………………. 357
Chapter 15 Biosensors and nanobiosensors: design and applications

Ahmed Touhami………………………………………………………………………………. 374
Chapter 16 Titanate Nanotubes as a Versatile Platform for Nanomedicine

Julien Boudon, Anne-Laure Papa, Jérémy Paris and Nadine Millot………………… 404
Chapter 17 Bismuth nanoparticles: antimicrobials of broad-spectrum, low cost

and safety
Cabral-Romero Claudio, Shankararaman Chellam……………………………………. 430
Chapter 18 Surface Modification of Porous Anodic Alumina for Medical and

Biological Applications
Morteza Aramesh and Jiri Cervenka………………………………………………………. 439
Chapter 19 Tissue visualization mediated by nanoparticles: from tissue staining

to mass spectrometry tissue profiling and imaging
Lenka Kolářová, Petr Vaňhara, Eladia María Peña-Méndez, Aleš Hampl, and Josef
Havel…………………………………………………………………………………………….. 468
Nanomedicine v
Chapter 20 Recent trends in tissue engineering applications of atom transfer

radical polymerization
Masoud Mozafari, Vahid Shabafrooz, Mostafa Yazdimamaghani, Daryoosh Vashaee
b, Lobat Tayebi………………………………………………………………………………… 490
Nanomedicine vi
Preface
Nanotechnology is influencing a great many disciplines that are essential to life and most significantly in
health and Medicine. Nanotechnology applied to medicine, addresses significant challenges in
advances in medical nanoscale-structured material, devices, biotechnology devices, molecular machine
systems, as well asnanorobotics.
Molecular-nano fabrications, surface modifications, quantum developments, nanoformulations are

some nanotechnology based methods by which systems are developed for biological and clinical
application of Nanomedicine. Such systems are applied in imaging/visualizingfor diagnostic and
therapy, monitoring, drug delivery, tissue engineering applications, experimental and clinical
therapeutics, targeting specific cells, for antibacterial effects, diagnostic methods in in-vitro models as
well as diagnosis and treatment of cancer.
I believe Nanotechnology in Medicine is still at a stage of infancy but has a huge potential for ultra-
rapid expansion and exciting developments over the next decade and beyond. Evidently,
Nanotechnology is already making a significant impactin the current era, and with my multidisciplinary
team, I implement this technology in research and development associated with Surgery, at myUCL
Centre for Nanotechnology and Regenerative Medicine, at Division of Surgery and Interventional
Sciences. We are applying nanotechnology in stem cell technology, cancer diagnostics, personalised
Medicine,development of ‘smart’ biomimetic materials and human organ development.
The authors of this book,who are experts in the field, and are currently active in the state of the art
developments of Nanotechnology and Nanomedicine. They are presenting these concepts, research
and development activities as well as their experience as a most exciting read whilst highlighting the
challenges and limitations in this field. This book I believe should entertain, educate as well as update
you as an expert in the field or even if you are a general readership. I hope this book will thoroughly
engage you in, Nanotechnology in Medicine, which is the most exciting development in this era in my
opinion, and inspire you with the power of NANO!
Professor Alexander Marcus Seifalian

University College London, UK
Dr Achala de Mel
Dr Deepak Kalaskar
Nanomedicine vii
Editors
Alexander Seifalian – Bio
Alexander M. Seifalian (AMS) who is Professor of

Nanotechnology and Regenerative Medicine and Head of
UCL Centre for Nanotechnology and Regenerative
Medicine. He is Fellow of the Institute of Nanotechnology
(FIoN) and Society of Biology (FSB), and has published over
410 peer-reviewed articles, 41 book chapters and 14
international patents. His current projects have led to the
development of cardiovascular implants using
nanomaterials and stem cell technology, development of
organs using tissue engineering and nanoparticles for the
detection and treatment of cancer. He was awarded the top prize in the field of nanomaterials for
cardiovascular implants in 2007 by Medical Future Innovation, and in 2009 received a Business
Innovation Award from UK Trade & Investment (UKTI) in the Life Sciences and Healthcare Category. He
received the most innovative new product award in 2012 for the ‘synthetic trachea’ by European Life
Science, Hamburg, Germany, and in 2013 was acknowledged by the international community for his
outstanding achievements in Nanoscience and Nanotechnology with the prestigious Nanosmat prize
award in Granada, Spain. AMS has a significant track record with regard to generating impact through
the translation of technology from the laboratory directly to the clinic, and has demonstrated that
nanocomposite polymers are core to the next generation of surgical implants with highly successful
outcomes in a number of pioneering projects.
This led to the development of the world’s
first synthetic trachea, nasal and ear
reconstruction, lacrimal duct conduit, and
small diameter bypass grafts for coronary
artery and vascular replacement, which has
enter clinical trials, all of which have been
used as first-in-man. His experience in
translational research has led to a number of
cutting edge projects at various stages of development. For example, we have developed abdominal
aortic aneurysm (AAA) stent-grafts, cardiac patches and transcatheter heart valves at the pre-clinical
stage of development and will be ready for clinical trials in early 2015. His research team is highly
experienced in reaching planned objectives and delivery of projects of this nature. During his career he
has led many large projects with multidisciplinary teams composed of scientists, engineers, and
clinicians in collaboration with UK industry, all with very successful outcomes in terms of
commercialisation and translation to benefit patient healthcare including:
i. commercialisation of bypass grafts for vascular access and haemodialysis;

ii. development of laser activated vascular sealants, which have been commercialised, and many
others medical devices currently in development.
Nanomedicine viii
Achala de Mel – Bio
Achala de Mel is a lecturer in Regenerative Medicine at UCLs Division of

Surgery and Interventional Sciences, and a member of the UCL’s centre for
Nanotechnology and Regenerative Medicine. Her primary interests are in
optimising cell-material interactions for Regenerative Medicine
applications. Together with Prof Alexander Seifalian she is currently leading
a research team funded by the EPSRC on 3D printing of thermoplastic
nanocomposites for surgical implants.
Deepak M. Kalaskar – Bio
Dr Deepak Kalaskar is Team Leader in development of artificial organs at

University College London (UCL). He is actively involved in research on
nanotechnology and tissue engineering solutions for artificial organs in Prof
Seifalian’s team. He had worked previously, at various universities in UK and
Belgium on number of developmental and applied research projects which
include high throughput culture for embryonic stem cells, Nanomaterials
coatings for orthopaedic implants, protein based nanoparticles for drug
delivery applications, on demand printing of cells using inkjet printer. His
primary interest includes development of smart materials for
nanomedicine, modification of biomaterial interfaces using Plasma
polymerisation and 3D printing of various artificial organs and their surface modification.
He also heads MSc course in Burns, Plastic and Reconstructive Surgery at Royal Free Hospital campus of
UCL, which is directed towards training scientists and clinicians in field of Regenerative Medicine and
Nanotechnology. He works as a consultant for UCL with various healthcare industries in UK and Europe.
Nanomedicine 1
1
Long Circulating Nanoparticles for
Tolerogenesis
1 2 2 2 1 1
Amy Tekrony ; Vincent Wright ; Anne Slaney ; Usama Al-Atar ; Amy Frederick ; Teresa Rodriguez ;
2 1,4 3
Jillian Buriak ; David Cramb ; Lori West
1. Department of Chemistry, University of Calgary

2. Department of Chemistry, University of Alberta
3. Departments of Pediatrics, Surgery, and Immunology, University of Alberta
4. Corresponding author.
Outline:
Introduction……………………………………………….………………………………………………………………………………..2
Blood Type Antigens and Immunology …………………………………………..……………………………………........ 2
Nanoparticles for Tolerogenesis …………………………………………………….…………………………………………… 6
Methods ………………………………………….…………………………………………………………………………………………. 8
Nanoparticle Design and Synthesis ………………………………………..…………………………………………………… 8
Nanoparticle Characterization in Solution ………………………………………………………………………………….. 10
CAM preparation ……………………………………….………………………………………………………………………………. 11
S2.4. Microinjection ……………………………………………………………………………………………………………………. 11
Chicken Embryo Chorioallantoic Membrane (CAM) Model for
Nanoparticle Characterization …………………………………………………………….……………………………………… 12
FCS Analysis ……………………………………………………………………………………………………………………………….. 12
S2.5. Zeta-Potential Measurement ………………………………………………………………..…………………………… 12
Results and Discussion ………………………………………………………………………..……………………………………… 12
Nanoparticle stability in aqueous solution and blood sera ………………………………………………………….. 12
Nanoparticles in the CAM …………………………………………………………………………………………………………… 14
Conclusion…………………………………………………………………………………………………………………………………… 17
Acknowledgements ……………………………………………………………………………………………………………………. 17
References……………………………………………………………………………………..…………………………………………… 17
Nanomedicine 2
Introduction
Blood Type Antigens and Immunology

Blood types are determined by blood type antigens, or sugars, that are expressed on membrane
proteins or glycolipids and can be found on red blood cells, epithelial cells, soluble glycans, and other
non-erythroid tissue [1, 2]. These sugars are oligosaccharide moieties that are formed on precursor
backbones by glycoprotein enzymes in the Golgi apparatus. In humans, there are three blood type
antigens: the A, B, and H antigens. The A and B antigens come from the same family, while the H
antigen is from a different family of sugars and is a precursor for the other two. Figure 1.1 shows the
structures of the A, B, and H antigens, which make up A, B, and O blood types, respectively [3]. The
subtle differences of the ABH antigens as seen in Figure 1.1 make up for major differences in
antigenicity. These differences can result in an immunological response mediated by natural
antibodies, which leads to the rejection of foreign blood and organs in transfusions and transplants [1].
As seen in Table 1.1, only certain blood types are compatible with others [4]. For example, if an A-
antigen is already expressed in the recipient, the recipient is able to accept A-type blood without
rejection since there will be very few or no anti-A antibodies. However, if the A-antigen is not present,
natural anti-A antibodies, present in humans with a healthy immune system, recognize the antigen as
foreign. As a result, this recognition and the binding of the anti-A antibody to the A-antigen will induce
an immunological response called hyperacute rejection [1, 2, 5]. In people with A-blood type, mostly
anti-B antibodies are present in the blood, for B-blood type, mostly anti-A antibodies are present, and
for O-blood type, anti-A and anti-B antibodies are present. Although it was previously thought that
natural antibodies that react with someone’s own antigens were not present in the immune system,
there is some evidence that serum contains these natural antibodies (i.e. anti-A antibodies in type A
blood type); however, these responses are not pathological when only low levels are present in the
blood [2]. As a result of these immune responses to foreign blood type antigens, O-blood type is the
universal donor and AB-blood is the universal acceptor (Table 1.1).
TABLE 1.1
Blood type compatibility
Variable Compatible Blood Groups Incompatible Blood Groups

Recipient’s Blood Type
O O A, B, AB
A A, O B, AB
B B, O A, AB
AB AB, B, A, O N/A
Donor’s Blood Type
O O, A, B, AB N/A
A A, AB O, B
B B, AB O, A
AB AB O, A, B
Nanomedicine 3
FIGURE 1.1
Structures of the O (H), A, and B blood type antigens for each blood type and resulting antibodies produced in
healthy immune system. R = glycoprotein or glycolipid.
The antibody responses in the immune system, as described above, play a large role in incompatible
blood type transplantation, which results in natural antibody-mediated hyperacute rejection by pre-
existing antibodies in the blood. Immunology is a very complex subject, and as a result, organ rejection
is also very complex, and factors other than natural antibody-mediated rejection, such as cellular-
mediated acute rejection of the human leukocyte antigen (HLA) and chronic rejection [4], also play
large roles in graft rejection. However, for the purposes of this chapter, only a general overview of
antibody-mediated hyperacute rejection will be given.
In antibody-mediated hyperacute rejection, the binding of an antibody to a foreign antigen can lead to
immediate graft destruction within minutes of the antibody recognizing the antigen. Most hyperacute
rejection responses occur within 24 hours of transplantation [5]. Graft function is lost as a result of a
biochemical cascade that helps the antibodies to clear pathogens, which begins when anti-donor
antibodies bind to blood vessels to activate the complement system in the newly transplanted organ.
Endothelial cell activation then promotes coagulation by the deposition of fibrin, the formation of
microthrombi, and the shedding of thrombomodulin and heparan sulfate proteoglycan, which lead to
the infarction of the transplanted organ. Destruction of the vascular endothelium can also lead to
oedema and haemorrhage in the tissue. This cascade also causes inflammation, which impairs blood
flow to the new organ. The severity of the hyperacute rejection is dependent on the levels of
antibodies in the blood stream and the type of antibody expression [2, 5].
Due to a need for donor organs, attempts to cross the ABO barrier have been made, especially in renal
transplantation of which failed attempts are not typically fatal [6]. For a successful incompatible
transplantation, many immunosupression techniques must be used such as the administration of
Nanomedicine 4
tacrolimus, mycophenolate, and steroids for pretransplant immunosuppression, administration of

rituximab for B-cell depletion, and plasmapheresis and immunoadsorption for antibody removal. Even
with these measures in place, it is likely that antibodies return due to B-cell memory [6]. Before West et
al. attempted incompatible infant heart transplants in 1996, intentional incompatible heart
transplantation had never occurred due to the obvious fatal consequences of a failed transplant and
the susceptibility of heart transplants to hyperacute rejection. Only eight cases worldwide had been
previously reported of incompatible heart transplantation, all in adult patients [7], and none were
intentional as all of these cases were a result of failure to correctly record either donor or recipient
blood type. Six out of the eight patients died as a result of hyperacute rejection to the incompatible
transplants [7].
The need for donor organs, especially hearts, is high for infants due to high wait list mortality for this
age group. Infants with lethal cardiac disease often die before transplantation due to a shortage of
donor hearts. Figure 1.2 shows that most children who die on heart transplant waiting lists are less
than 10-15 kg, indicating that infants and toddlers have the highest wait list mortality rates. In addition,
infants with type O blood type experience disproportionate compatible donor organs since they can
only receive heart transplants of blood type O. Also, many blood type AB donor organs are wasted
since they are only compatible with AB blood type recipients [8].
In 1996, West et al. successfully attempted to cross the ABO barrier in infants, due to the
underdeveloped nature of their immune systems [4], as infants do not produce isohemagglutinins, and
anti-A and anti-B antibodies exist in very low levels up until 12-14 months of age. In addition, the
complement system of infants is not fully developed at a young age [9]. As a result, it is easier to
manipulate the immune systems of infants and ensure that they do not execute a normal immune
response, or hyperacute rejection, to foreign antigens in the blood. Therefore, it was hypothesized that
the ABO blood barrier could be breached, if done carefully, with the knowledge that the transplanted
organ is of incompatible blood type [4]. Between the years of 1996 to 2000, West et al. commenced
the first study of its kind in which incompatibles hearts were transplanted into young infants whose
situations would be fatal otherwise.
Knowing that the transplanted organs were of incompatible blood type had a distinct advantage over
the 8 adult cases, which were mostly fatal, since preparations could be made in order to suppress the
immune system, keep track of antibody levels, and transfuse the appropriate (donor) blood type into
the recipient [4]. Patients from the transplantation waiting list were tested for the presence of anti-A
and anti-B antibodies using standard agglutination tests. Those with no or very low levels of antibodies
were considered for the study. In addition, antibody levels were tested for each recipient; a.
periodically over the waiting period, b. when a potential donor was identified,c. immediately before
the surgery, d. continuously during the surgery, e. and every six hours for four days after the surgery in
order to ensure that the graft would not be rejected via antibody-mediated hyperacute rejection. In
order to prepare for the bypass surgery, the bypass circuit was primed with the blood type of the donor
organ or with AB-type blood so that no anti-donor antibodies would be present at the time of the
surgery, and the recipient’s blood was removed via the bypass circuit and replaced with the donor
blood type for the duration of the surgery.
Nanomedicine 5
FIGURE 1.2
Number of children who died while on the waiting list between 1999 and 2006 according to weight at listing. The
numbers above each bar denote the total number of children who were listed for heart transplant within each
weight category reproduced with permission from [8].
The patients were also succumbed to intensive immunosuppressive treatment during and post-
operation, which was reduced over time. In addition to testing for antibody levels post-operative to
monitor any possible antibody-mediated rejection, patients underwent biopsies to rule out chronic
rejection within six months after the transplant and then annually after that. All in all, for this study,
ten patients between the ages of four hours to fourteen months old underwent ABO-incompatible
transplantation and were compared to ten patients between the ages of two months to twelve months
who underwent ABO-compatible transplantation from 1996 to 2000 [4].
Initial findings indicated that none of the patients experienced hyperacute rejection. Higher incidences
of cellular rejection actually occurred in the ABO-compatible group than in the ABO-incompatible
group, possibly due to less intensive immunosuppressive therapy or the slightly higher mean age of the
ABO-compatible group. There was also no antibody-mediated chronic rejection observed in either
group [4]. Since the time when the initial study was published in 2001, additional results from a total of
35 patients that have undergone ABO-incompatible transplantation and 45 patients that have
undergone ABO-compatible transplantations have been analyzed and published. It was found that the
freedom from death and transplantation rates were very similar for the two groups at 77% and 74%,
respectively for the ABO-incompatible transplantation group and 84% and 74% for the ABO-compatible
group [10]. These percentages were found to have no statistical difference. Out of the larger group of
Nanomedicine 6
ABO-incompatible transplants, only 2 patients had antibody-mediated rejection and both episodes
were handled easily via immunosuppressive techniques. Cellular rejection episodes were similar
between the two groups. Of the patients that died, none of them did so as a result of ABO-
incompatibility [10]. From these results, it is apparent that this study was a success and that infants’
immune systems are at an immaturity level that allows for the ABO barrier to be crossed in
transplantation.
Not only was the ABO-barrier crossed successfully as a result of incompatible transplantation, but an
induced tolerance to donor antigens was also observed in the infant recipients. It has been known for
many years that induction of antigen tolerance is possible in animals. In 1945, Owen defined the
inherent susceptibility that animals have to immune tolerance [11]. In 1949, Burnet and Fenner linked
this tolerance to developmental events, using twin fetal cows [12], and in 1953, Medawar showed that
this tolerance could be induced intentionally in mice [13]. Up until now, these findings were not
considered to be clinically relevant to humans since human neonates were considered to have an
increased maturity of their immune systems at birth, and therefore, were thought to be beyond
susceptibility to tolerance induction. However, as previously stated, human infants’ immune systems
do show signs of immaturity, such as low or no levels of isohemagglutinins within the first month after
birth. This study showing successful incompatible heart transplants was the first to refute what was
previously thought and provide evidence for neonatally acquired donor-specific immune tolerance in
humans [14]. Fan et al. also provide evidence that suggests persistent exposure to donor antigens is
required for tolerogenesis (induction of immune tolerance), which is also dependent on the degree of
antigen expression [10].
Nanoparticles for Tolerogenesis

Since evidence shows that the occurrence of tolerogenesis is dependent on degree of antigen
exposure, it may be possible to induce tolerance via methods other than graft exposure or
transplantation, thereby possibly reducing mortality rates on transplantation wait lists. If an infant
must wait for an organ past the time of susceptibility to induced tolerance, it may be beneficial to
induce tolerance early in the child’s life to allow for tolerance to both A and B antigens, or essentially
make that infant’s blood type a “universal acceptor”. This would increase the likelihood of
transplantation at a later point in time when a donor organ may be available, thereby decreasing the
likelihood of a fatal outcome.
In order to achieve this goal, it is essential to determine novel methods of tolerance induction. Since
lone antigens in the blood stream would likely be cleared quickly by the renal system, our study
investigates the possibility of injecting nanoparticle-antigen conjugates into the blood stream to induce
tolerance. Ideal particles will be easily detected in order to track them in the blood stream for
characterization purposes and circulate for a prolonged period of time in vasculature to ensure
maximum exposure to blood stream antigens [3]. Therefore, since polyethylene glycol (PEG) coatings
have been shown to increase nanoparticle (NP) circulation time in the blood stream, the majority of the
NPs in this study were silica functionalized on their surfaces with PEG [15]. Silica has demonstrated
biocompatibility [16] and silica NPs are easily tuned for size [17]. Also, most NPs synthesized contain
fluorescent dyes for detection in order to monitor and assess their localization and circulation
behaviour.
Previous NP circulation studies have been performed either in adult animals [18] or in tumor models
[19]. Neither of these are the most relevant to model neonatal blood circulation. Neonates will have
varying degrees of natural angiogenesis, which is modeled poorly by tumor models and not at all by
Nanomedicine 7
adult animals. Desirable is a simple angiogenic model into which NPs can be injected and tracked for
circulation time, aggregation behavior and general NP stability. The chorioallantoic membrane (CAM,
Figure 1.3) of the chicken embryo is an excellent model for angiogenesis. Within the CAM the blood
pressure is normal and the vessels are not tortuous unlike tumor models and most of the blood vessels
in the zebrafish embryo. The CAM serves as the respiratory system for the chicken embryo up until day
19 of the 21 day gestation period, handles any waste products from the embryo, and supplies the
embryo with nutrients from the yolk [20], The CAM has been used as a model to study angiogenesis of
explanted tumours and anti-angiogenic drugs [21-27], tumour vascular targeting [28], metastasis [29,
30], ion transport [31], allergens [32], transplantation [33], contraceptives [34, 35], effects of
hyperglycemia [36], and photodynamic therapy [37]. NPs can easily be injected directly into blood
vessels of the CAM through a window cut into the eggshell (Figure1. 3). The concentration of the
injected particles is then monitored over time using fluorescence correlation spectroscopy (FCS), and it
has been shown that the relative concentration of certain particles decreases exponentially within the
first few minutes of injection [38]. This exponential decrease of NP concentration is due to loss of the
NPs from the blood stream through the angiogenic fenestrations (~500nm in size) [39]. The presence of
angiogenesis in these developing vessels allows us to find ideal NPs that will circulate for a prolonged
period of time, despite the “leaky” nature of the developing vessels. As mentioned above, NP
concentration in the CAM can be monitored through the window in the shell using FCS.
FIGURE 1.3
Day 9 embryo with window for experimentation. NP concentration can be determined through the window cut
into the shell using FCS on the exposed CAM blood vessels above the embryo.
Nanomedicine 8
Two-photon excitation (TPE)-FCS is a non-invasive fluorescence technique commonly used to analyze

fluorescent particles in living organisms, primarily for advantages such as: low phototoxicity, long-term
analysis and ability to distinguish aggregation [38]. TPE-FCS involves analysis of fluorescent intensity
fluctuations resulting from two-photon excitation of fluorescent molecules to obtain data about
diffusion, concentration, and size of the fluorophores. TPE-FCS has been used to gain valuable
information in many applications such as the examination of angiogenic blood vessel formation in
zebrafish; the study of active transport, localization of proteins, and diffusion of receptor clusters in
cells; monitoring drug delivery using photocages; and the study of DNA replication [40].
In this study, TPE-FCS is used as the primary technique to provide minimally invasive monitoring of NP
behaviour in the CAM of the chicken embryo. By tracking the change in concentrations and aggregation
tendencies as functions of size and surface chemistries, we may determine which NPs are most easily
detected, aggregate the least, and circulate in the blood stream the longest. These data will allow us to
predict the NP properties most suitable to provide maximum blood exposure to the synthesized
antigens used to induce immunological tolerance in infants.
Methods
Nanoparticle Design and Synthesis
Various NPs were synthesized with multiple dyes, ratios of dye to silica, surface functionalization, and
dye-incorporation methods (See Table 1.2 for the naming system for nanoparticles, according to their
synthesis). NPs with dye incorporation were synthesized according to the Stöber process [41], which
involves the hydrolysis of alkyl silicates, followed by the condensation of silica acid in alcohol using
ammonia as a catalyst. This synthesis resulted in uniform particles of which sizes can be controlled
from approximately 0.05 µm to 2 µm in diameter [41]. In order to synthesize the core-shell particles, a
modified version of the Stöber process was used, as described by Larson et al. [42], in which the dye-
rich compact core is synthesized prior to the silica shell. The NPs were functionalized by directly adding
polyethylene glycol (PEG) or APTMS to the synthesis flask, which formed covalent bonds from the
polymers to the NPs as the base-catalyzed silanization occurs [3]. Once synthesized, particles were
resuspended in water and stored in scintillation vials covered with aluminum foil to reduce exposure to
light.
Nanomedicine 9
Degree of stability of the dye (relative strength of adhesion of the dye to nanoparticle vs potential
dissipation of free dye?)
TABLE 1.2
Symbols used for naming nanoparticles.
Material Functionalization Dye Incorporation Dye

Symbol Meaning Symbol Meaning Symbol Meaning Symbol Meaning
Si Silica PEG Polyethylene R Random R6G rhodamine
glycol 6G
Fe Iron NR3 Amine C Core- TRITC tetramethyl
oxide shell isothiocyana
te
SPEG Short PEG NP Lg NPs FITC fluorescein
from sm isothiocyana
NPs te
LPEG Long PEG AFX Alexa Fluor®
NF No Funct. Pyr Pyrene-
maleimide
Two-Photon Excitation Fluorescence Correlation Spectroscopy
TPE-FCS measures the fluctuations of fluorescence in an optically-defined interrogation volume over

time. A temporal autocorrelation analysis is performed on the fluorescence data (see Figure 1.4) to
generate the autocorrelation decay, G().
Equation (1)
Where F(t) is the difference between the instantaneous fluorescence at time, t, and the average
fluorescence intensity, <F>. As the lag time, , increases, G() decreases, and an autocorrelation decay
(ACD) curve results. Equation (2) can be used to model the ACD, when the system is dominated by
Brownian diffusion determine the diffusion coefficient, size, and concentration of the NPs [43].
Equation (2)
Where r is the radius of the ovoid of the two photon excitation volume (perpendicular to the direction
of laser propagation), Z0 is the depth of the ovoid of two photon excitation (in the direction of laser
propagation), c is the local concentration, D is the diffusion coefficient, and t is the lag time. It is also
Nanomedicine 10
important to note from Equation 2 that G(0) is equal to 1/N (where N equals the average number of
freely diffusing fluorescent particles in the TPE volume). Thus, N is the equivalent of concentration.
Changes in N over time may indicate the NPs are being taken up into the angiogenic tissue, aggregating
or dissolving.
FIGURE 1.4
a) Count rate fluorescence trajectory, where the red line is equal to the average count rate and b) Autocorrelation
decay curve, where the red line depicts G(0), which is equal to 1/N. Both graphs are examples of typical plots that
result from FCS analysis of nanoparticle injection into the CAM.
To help determine the origin of NP concentration changes we will use the average particle brightness,
. If N decreases and  increases, this suggests aggregation is taking place. If N decreases and 
remains constant, this suggests uptake or dissolution. And finally if N remains constant and decreases
this suggests that the fluorescent dye in the NPs is unstable, which could indicate that the NP surfaces
are changing. It is simple to calculate the particle brightness (kHz/NP):
= 1/N * <F> Equation (3)
Nanoparticle Characterization in Solution
In order to assess the suitability of the NPs for injection into the CAM, initial characterization of the NP
solutions was first performed in various solutions. If the NPs were unstable in simple solution, they
were deemed inappropriate for study in the CAM environment. Particles were diluted to the 10-100
nM concentration range (10 < N< 100). This would be the appropriate range for the CAM studies and
likely the desirable range for tolerogenesis. Particles that were monodisperse, bright, and gave
consistent autocorrelation decay curves in water, were further analyzed in various buffer and sera
solutions.
The monodisperse, bright NPs mentioned above were analyzed for their behaviour in phosphate
buffered saline (PBS), chicken blood serum (CBS), and porcine blood serum (PorBS) over time to
observe possible effects on salts and proteins on the NPs from the buffer and sera, respectively
aggregation. FCS measurements were taken at short intervals for the first 30 minutes post dilution and
at longer intervals after that over the time span of an hour. Autocorrelation decay curves and count
rate trajectories were analyzed for signs of aggregation and changing concentration. Time for complete
loss of fluorescnce?
Nanomedicine 11
CAM preparation
Fertilized eggs were obtained from the Ijtsma Poultry Farm north of Calgary (RR 284 and highway 72,
Mailing address: RR2, Site 11, Box 6, Airdrie, AB, T4B 2A4). Eggs were stored for up to two weeks in a
refrigerator at 4°C, at which time they lose viability. Nine days before experimentation, eggs were
removed from the refrigerator and allowed to sit at room temperature for a few hours, after which, the
eggs were cleaned using a 70% ethanol solution. The eggs were then placed horizontally on egg racks,
and the date of experimentation was written on the top center of the egg to allow for a reference point
later in the incubation period. The eggs were then incubated at 37 degrees Celsius and approximately
60% humidity. On day four and a half of the incubation period, the eggs were windowed to provide
visual observation of the embryo development. In order to do this, eggs were first candled to verify
development of the CAM and embryo. If this was not the case, eggs were disposed of. All viable eggs
were windowed. The first step of this process involved removing 3 mL of albumen using a BD syringe
and 21-gauge needle in order to provide an air sac within the egg, which allowed it to be opened
without damaging the embryo. Next, the egg was rotated 180 degrees to allow for the CAM to develop
over top of the chicken embryo, and cellulose tape was placed on the new top of the egg. Dissecting
scissors were then used to cut a small hole into the tape-covered portion of the egg, creating a window
to allow visual observation of the development of the chicken embryo and CAM. Cellulose tape was
then used to cover the window in the egg, and the eggs were placed back into the incubator. Over the
course of the subsequent incubation period, embryos were monitored. On day nine of incubation, the
eggs were removed once again for experimentation. The windows in the eggs were expanded to allow
for injection of nanoparticles and monitoring of the injectate via FCS. The top portions of the eggs were
further covered with cellulose tape to prevent crumbling of the shell into the amniotic fluid and CAM.
Dissecting scissors were then used to cut the shell away to the line of the fluid within the egg, thus
providing maximum exposure to the CAM for experimentation.
S2.4. Microinjection
Injections were performed using a manual microinjector from Sutter Instruments Co. Needles were
formed using P-30 Puller (Sutter Instruments Co.), beveled using a K.T. Brown Type Micro-Pipette
Beveller, Model BV-10 (Sutter Instruments Co.), and held in place and directed by an x, y, z-manipulator
(Sutter Instruments Co., MM-33).
Undiluted nanoparticles (~10 nM) were drawn up into a 3 mL BD syringe, which was then attached to
the top of the microinjector. The tubing that leads to the manipulator was primed, and a beveled
needle was secured into place at the end of the tubing. The needle was also primed and flow through
the tip was verified. The valve leading from the BD syringe port to the micro syringe was opened and
nanoparticles were drawn into the micro syringe.
Once the microinjecter was set up, an egg was placed in an egg holder on the egg stage of the
microscope (In-house design, University of Calgary, [44]) and viewed through the 5X objective with a 1
cm working distance. The needle, adjusted to inject parallel to blood flow. Approximately 100 µL of
nanoparticles were then injected into CAM blood vessels of approximately 100 – 200 µm in diameter.
After successful injections, the objective turret position was changed to the 20X lens and the laser was
focused in the center of the blood vessel. The focused laser beam was always several hundred microns
downstream from the injection site.
Nanomedicine 12
Chicken Embryo Chorioallantoic Membrane (CAM) Model for Nanoparticle Characterization
The types of NPs that did not aggregate in solution (aggregation analysis described by Clancy et al.) [38]
and were bright enough to be easily detected were injected into blood vessels of the CAM. The NPs
were injected through a window of a chicken embryo egg into the blood vessels of the CAM, which are
contiguous with the vessel system in the embryo, and FCS was used to track concentration changes of
the NPs within the blood vessels. More precisely, 100 µL of undiluted NP solution was injected into a
vessel of approximately 100 to 200 µm in diameter. The injectate was allowed to circulate and
distribute throughout the blood stream (30 s,[38]), after which FCS measurements were taken
continuously from approximately 3 min post injection to approximately 50 min post injection, with the
TPE laser beam focused into the center of the lumen.
FCS Analysis
For all experiments, count rate trajectories and autocorrelation decay curves were plotted and
analyzed using OriginPro 7.
S2.5. Zeta-Potential Measurement

-10
Nanoparticles were diluted to approximately 10 M concentration from original solutions, and a small
amount was drawn into a 3 mL BD syringe. Nanoparticles were then injected into disposable
polystyrene cuvettes and inserted into the cell holder in the Nano ZS DLS instrument (Malvern
Instruments Ltd). Zeta-potentials were obtained in water using averaged data from approximately 80
accumulated data points and pre-assigned settings for silica nanoparticles with the refractive index set
to 1.5, the refractive index of silica.
Results and Discussion

Nanoparticle stability in aqueous solution and blood sera
A series of approximately 50 types of NPs that were synthesized with various combinations of sizes,
dyes, surface functionalizations, polymer materials, and synthesis methods, were analyzed for stability
in aqueous solution and blood sera. For the purpose of this chapter, the key findings for aqueous
solution are presented in Table 1.3, which gives average particle brightness and the percentage of
particles that aggregated and were used in eggs based on synthetic particle variables. To sleuth out
trends in the characteristics of the NPs, all data for specific characteristics were pooled and then the
averages or % displaying the behaviour were calculated. For example, 46 NPs were surface-
functionalized with PEG. Their cumulative average  was 3.5 kHz per particle and 13/46 displayed
aggregation in water and 11/46 we ultimately deemed suitable for further study in the CAM. Thus, the
results displayed in Table 1.3 were used to help determine which NPs were stable and bright enough
for use in the CAM in vitro studies.
Nanomedicine 13
TABLE 1.3
Summary of nanoparticle characterization results in water.
* tetramethyl isothiocyanate, § fluorescein isothiocyanate
Average Percent of Percent
particle particles that injected into
Variable
brightness aggregated in eggs (%)
(kHz/particle) water
Iron oxide core 0 N/A 0
NP Material
Silica 3.2 36 23
PEG 3.5 28 23
Functionalization Amine 0.3 80 20
None 1.3 100 0
Core-shell 4.0 41 32
Dye- Random 1.5 0 0
incorporation Large particles 0 75 0
Method made from
small ones
Rhodamine 6G 4.4 32 29
TRITC* & FITC§ 1.1 33 16
Dye
Alexa fluor 0.8 50 0
Pyramine 0.2 50 0
0-75nm 3.7 60 0
Size 75-200nm 3.9 30 37
>200nm 0.8 25 8
Out of the fifty particle solutions investigated in this water, twelve types of NPs were mixed with buffer
and blood sera to further investigate stability with respect to aggregation in the presence of ions and
proteins. Table 1.4 displays the results of these studies.
There was increased particle instability and aggregation in blood serum in comparison to buffer. This
was also observed in a study by Eberbeck et al., where they studied magnetic particles in different
solutions. It was found that particles had a higher tendency to aggregate in different biological media
[45]. However, the results from mixing the silica NPs in different solutions were very promising as a
large number of the NPs synthesized showed high stability in PBS and two different blood sera, which
was beneficial for the desired purpose of injecting them into the CAM blood vessels.
Nanomedicine 14
TABLE 1.4
Observed time of nanoparticle aggregation in various solvents over the typical 45 to 60 minutes of data collection,
determined from CRT and ACD analysis.
Approximate time in solution until aggregation

Nanoparticle is observed (min)
PBS CBS PorBS
Si_PEG_C_TRITC_89nm >60 >16 Insufficient
data
Si_PEG_C_R6G_105nm >60 >60 24
Si_PEG_C_R6G_120nm >60 >60 <6
Si_PEG_C_R6G_125nm 35 45 2
Si_PEG_C_R6G_142nm >45 45 19
Si_PEG_C_R6G_148nm >60 23 40
Si_PEG_C_R6G_150nm_2 10 9 <8
Si_PEG_C_R6G_177nm >60 60 23
Si_PEG_R_TRITC_179nm >60 15 8
Si_PEG_C_R6G_191nm >45 45 40
Si_PEG_C_TRITC_250nm Insufficient 7 6
data
Si_PEG_R_R6G_350nm_2 20 11 <16
Overall, only seven out of the total of 50 NPs were deemed appropriate to be injected into the CAM
model, which reflects the importance of the specific combination of variables on resulting particle
properties. Interestingly, all of the particles that were determined to be suitable for injection were
core-shell silica NPs; the core-shell particles tended to be brighter, thus more easily detected for the
purposes of this study. All of the NPs injected into the eggs were functionalized with PEG. Five were
synthesized utilizing R6G as the dye, while the other two contained TRITC. Also, all NPs stable enough
to be injected into the CAM had diameters in between 89 to 250 nm.
Nanoparticles in the CAM
Based on the above-mentioned results, seven different NPs were studied in the CAM blood vessels. Out
of those seven particle solutions, five were found to be stable in the CAM vasculature, with little signs
of aggregation over the time periods analyzed (typically around one hour to ensure preservation of the
embryo viability).
Figure 1.5a displays a typical plot of the number of silica NPs in the TPE volume, N, as a function of time
for all of the particles injected into the CAM. As demonstrated in Figure 1.5a, there is no indication of
any systematic change in particle numbers over the time period (33 min) for this particular NP. The
large scatter in the data (average +/- 50%) (Figure 1.5a) results from a number of factors:
inhomogeneities in the concentration in time due to pulsatile flow, movement of the embryo and some
degree of aggregation. Recall that the angiogenic blood vessels of the CAM possess fenestrations that
are 500 nm in diameter or smaller [44]. Therefore, NPs smaller than this size could leave the blood
stream through these nanofenestrations. Figure 1.5b, by comparison shows dioleoylphosphatidyl-
Nanomedicine 15
serine (DOPS) liposomes (=-59 mV and 100 nm diameter) injected into the CAM. In the case of DOPS,
there is clearly a slow decrease in N over time, which we have previously shown is due to uptake into
the angiogenic blood vessel walls [39].
Data on a Hemolysis assay?
a
b
FIGURE 1.5
Number of particles in focal volume versus time post injection into eggs for a) 89 nm core-shell TRITC silica
particles with PEG functionalization. b) 102 nm diameter DOPS liposomes loaded with lissamine. Red curve
-1
represents monoexponential decay fit with rate constant, k=0.002 s .
The lack of verifiable uptake for these NPs in the CAM is quite significant, since this is not the case with
most particles of similar sizes studied by Yaehne et al. [46] In this previous study, we found that many
different NPs were taken up into the angiogenic tissues. These particles included PEGylated quantum
dots, polystyrene fluospheres and liposomes [39]. It is emerging that size, functionalization, and surface
potential all play a large role in the observed uptake rates. We have shown previously that uptake rates
have been found to be dependent on size for particles with zeta potentials in the range 0 < < -40 mV
[39]. The silica NPs in the current study are of similar sizes to these NPs, but do not deposit in the
vessel walls. We therefore propose that there is a negative charge cut-off, which plays a significant role
in keeping the NPs in the blood stream. This cut-off appears to be around -50 to -60 mV as discussed in
[39], and shown in Table 1.5 as particles with high negative charges are not taken up readily. These
findings are likely a result of the large negative zeta potentials of the NPs causing repulsion with the
negatively charged endothelial cells making up the blood vessel walls. Furthermore, large surface
charge could help to stabilize NPs as the NP-NP repulsion would reduce aggregation. Since many of the
non-silica particles in our previous study that displayed uptake are coated with PEG and are of similar
diameters [39], this charge effect is likely a significant reason for the differences in circulation
behaviour. This result is also supported by the contrast in the results for the circulation behavior of
liposomes. The zwitterionic dioleyolphosphatidyl-choline (DOPC, = -40 mV, 100 nm diameter) left the
-1
blood stream with a rate constant of kloss = 0.003 s [39], whereas the DOPS liposomes (= -59 mV, 100
-1
nm diameter) lasted 50% longer in circulation, kloss = 0.002 s (Figure 1.5). The charge-induced, long
circulation time is a very beneficial characteristic for tolerogen carriers, as it would permit maximum
exposure of B-cells to NP-conjugated antigens.
Nanomedicine 16
Lack of uptake does not necessarily mean complete stability of the silica NPs during circulation. Some
were observed to display a degree of aggregation after prolonged exposure to the CAM blood stream.
Table 5 gives the length of time during circulation before aggregation of the NPs is observable. This
represents the onset of 20-20% aggregation of the NPs. From Table 1.5, it is evident that PEG-coated
NPs in the size range 120-150 nm diameter were optimal for better stability against aggregation.
Other PEG-functionalized particles, similar to the silica NPs used in this study, have shown similarly long
circulation times in other animal models. For example, Maldiney at al. studied the effects of diameter
and surface coating on biodistribution within healthy mice [47]. As part of the study, the biodistribution
of intravenously administered silicate-based NPs coated with PEG (chain length approximately 10 times
longer than the ones use in this study) was compared to that of particles with exposed hydroxyl groups.
It was found that PEG-functionalized particles circulated in mice for a longer period of time than the
similar hydroxyl-functionalized particles[47]. This was indicated by the rapid uptake of the hydroxyl-
coated particles into the liver and a high distribution of the PEG functionalized particle throughout the
rest of the organism [47].
TABLE 1.5
Zeta potentials and observed time of nanoparticle aggregation in the CAM over the typical 45 to 60 minutes of
data collection, determined from CRT and ACD analysis.
Approximate time (post (SD, mV)

Nanoparticle injection) until aggregation is
observed (min)
Si_PEG_C_TRITC_89nm 25 -64 (20)
Si_PEG_C_R6G_105nm 45 Insufficient data
Si_PEG_C_R6G_120nm >45 -60 (10)
Si_PEG_C_R6G_125nm >45 -47 (15)
Si_PEG_C_R6G_150nm_2 >45 Insufficient data
Si_PEG_C_R6G_177nm 45 -68 (9)
Si_PEG_C_TRITC_250nm 30 -48 (5)
DOPS liposomes >45 -59 (12)
Indication of flouresent life time? (time taken before complete quench of flouresence?)
Maldiney also found a strong dependence on size with longer circulation times for particles with 120
nm hydrodynamic diameter compared with larger particles (190-230 nm) [47]. Interestingly, their study
found that negatively charged, bare silica particles were rapidly deposited in the liver and spleen. We
saw little evidence of this for the negative particles in the current study, however our particles did have
a degree of PEG coating and the CAM blood volume is very large compared with the organ blood
volume.
PEG has been validated in many previous studies as a coating that increases blood stream circulation
time of NPs [48-51]. Our previously reported work has demonstrated that amino-terminated PEG
coated quantum dots disappear too quickly from the CAM blood stream to measure the uptake rates
(10). Intuitively, the amino functionalization would cause particles to be less negative and therefore be
attracted to the negative endothelia cells of the CAM blood vessels. It therefore emerges that a
combination of PEGylation and large, negative charge promote longer circulation time in angiogenic
blood vessels.
Nanomedicine 17
Conclusion
PEGylated silica nanoparticles were studied in the CAM model as a significant step towards developing
long-circulating, stable tolerogen carriers. Such NPs would allow for an increased exposure of the blood
stream to the antigens they will carry. It was found that PEG-functionalized particles between 90 to 200
nm in diameter with large negative charges produced the desired effects and circulated for long
periods of time in CAM blood vessels. PEGylated silica NPs carry a large negative charge likely because
of incomplete coverage of the silica surface by PEG. These findings suggest that silica NPs with the
above-mentioned qualities would be suitable for tolerogenesis. As a result, these silica NPs will be
conjugated to antigens to determine if the resulting exposure will be persistent enough to induce
immunological tolerance to foreign blood type antigens.
Acknowledgements
The authors would like to thank the Natural Sciences and Engineering Council of Canada and the
Canadian Institutes of Health Research for their financial support.
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Accelerated Blood Clearance Phenomenon by Coating of Nanoparticles with Various
Hydrophilic Polymers. Biomacromolecules. 2010;11(10):2700-6.
51. Wang M, Thanou M. Targeting nanoparticles to cancer. Pharmacol Res. 2010;62(2):90-9.
Nanomedicine 21
2
In Vivo Applications of Quantum Dot
Nanoparticles for Optical Diagnostics
and Therapy
1 2 1
Elnaz Yaghini*, Alexander M. Seifalian, Alexander J. MacRobert
1
Dr. E. Yaghini and Prof. A. J. MacRobert, Division of Surgery & Interventional Science and UCL Institute for Biomedical
Engineering, University College London, London, UK.
2
Prof. A. M. Seifalian Centre for Nanotechnology and Regenerative Medicine, Division of Surgery and Interventional Science,
University College London, London, UK.
Outline:
Introduction……………………………………………….………………………………………………………………………………..22
Quantum Confinement and Size-tunable Properties of qds …………………………………………..……………. 22
Bulk Semiconductor Physics …………………………………………………….…………………………………………………. 23
Synthesis and Bioconjugation of qds ………………………………………….………………………………………………. 24
Photophysical Properties of qds and Comparison with Organic Dyes ………………………………………….. 25
IN VIVO STUDIES of qds ……………………………………………………………………………………………………………… 26
Non-targeted In Vivo Imaging ……………………………………….…………………………………………………………….27
Sentinel Lymph Node (SLN) Mapping ………………………………………………………………………………………….. 27
Passive Tumour Targeting …………………………………………………………….……………………………………………. 29
Targeted In Vivo Imaging …………………………………………………………………………………………………………….30
Active Tumour Targeting using Antibodies ………………………………………………………………..……………….. 30
Active Tumour Targeting using Peptides ………………………………………………………………………..…………… 31
In Vivo Imaging Using Self-illuminating qds (BRET) …………………………………………………………………..... 33
Multifunctional qds: …………………………………………………………………………………………………………………… 34
Dual-modality Imaging ………………………………………………………………………………………………………………. 34
Integration of Imaging and Therapy …………………………………………………………………………………………… 34
Cell Tracking ………………………………………………………………………………………………………………………………. 35
Future perspective ……………………………………………………………………………………………………………………… 36
Improving the Biocompatibility of qds ………………………………………………………………………………………… 36
Potential Toxicity of qds ……………………………………………………………………………………………………………… 38
Conclusions ………………………………………………………………………………………………………………………………… 40
References……………………………………………………………………………………..…………………………………………… 40
Nanomedicine 22
Introduction
Nanotechnology is a new field of interdisciplinary research on the fabrication of materials with
nanoscale dimensions between 1-100 nanometre (nm) [1]. As the sizes of organic and inorganic
materials are decreased toward the nanometre scale, their optical, electronic, magnetic and structural
properties largely vary from that in the bulk and become size and shape dependent. Such size and
shape dependent properties of nanomaterials make them attractive to a diverse range of applications
ranging from energy conversion towards biomedical imaging and nanomedicine.
Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometre scale, which are
emerging as a new class of fluorescence probe for in vivo bimolecular and cellular imaging. These
nanometre-sized crystalline particles are nanocrystals of inorganic semiconductors with a generally
spherical shape. The core of the semiconductor material is synthesised with sizes in the range of 2-10
nm and contains hundreds of thousands of atoms of group II and VI elements (e.g. CdSe, CdTe) or
group III and V elements (e.g. InP). The core is typically enclosed within a shell of another
semiconductor that has a larger bandgap (Fig. 2.1).
FIGURE 2.1
Schematic illustration showing the core/shell structure of quantum dots
QD nanoparticles are highly efficient fluorophores with a size-dependent emission wavelength, which
due to quantum confinement increases with increasing size of the QD. Their extreme brightness and
resistance to photobleaching enables the use of very low light intensities over extended time periods,
making them especially useful for live-cell imaging [2]. Due to the unique photophysical properties of
QDs, progress in synthesis and biofunctionalisation of QDs has generated an increasingly widespread
interest in the fields of biology and medicine. Recently a wide range of methods for bio-conjugating
colloidal QDs in a diverse area of applications have been developed such as cell labelling , cell tracking ,
in vivo imaging , DNA detection , and multiplexed beads [3-10]. This chapter is intended to provide an
overview of the in vivo applications of QDs as well as the future direction for the development of
optimised QD nanoparticles for their clinical translation.
Quantum Confinement and Size-tunable Properties of QDs
One of the most intriguing features of QDs is that by altering the QD size and its chemical composition,
fluorescence emission can be tuned from the near ultraviolet, throughout the visible and into the near-
infrared (NIR) spectrum, spanning a broad wavelength range of 400–2000 nm [11-13]. To understand
these properties, the photophysics of bulk phase semiconductors will be discussed in following
sections.
Nanomedicine 23
Bulk Semiconductor Physics
Based on electron conductivity, solid state physics typically divides materials into three well known
categories: conductors (metals), semiconductors and insulators. The difference in the energy level
between the valence and conduction band determines the conductivity of the solid materials. The
valence band is the highest electronic energy level that is occupied with electrons at room temperature
(Fig. 2.2). Likewise, the conduction band is the lowest energy electronic state that is not occupied by
electrons. An electron in the valence band may gain energy (thermally or by the absorption of a
photon) to enter the conduction band, thus vacating a positively charged hole in the valence band. The
difference in energy between the valence and conduction bands, called the bandgap energy (e.g.
typically expressed in electron volts [eV]), determines the energy that must be gained for an electron to
enter the conduction band [14]
FIGURE 2.2
A schematic representation of the valence and conduction band of QD nanoparticles
The raised electron and the hole taken as a pair is called an “exciton”. Once in the excited state
(electron in conduction band), a conduction-band electron may relax back to its ground state in the
valence band through radiative recombination with a hole, resulting in the emission of a photon with
the same energy as the bandgap. Light emission is only one of the many possible decay processes, but
it is of both fundamental and practical importance.
QDs as semiconductor materials exhibit size-dependent energy state due to the confinement of the
charge carriers (electrons-holes) in three dimensions. As the size of the QD decreases, the band gap
increases, resulting in shorter wavelength of light emission (Fig. 2.3). Because the bandgap determines
the fluorescence emission wavelength of semiconductor materials, considerable effort has been
devoted to engineer fluorescence emitters with precisely tuned bandgaps. An intriguing property of
semiconductors is that the bandgap is not only dependent on size but also on the particle composition
[15]. Therefore, the composition of the material may also be used as a parameter to alter the bandgap
of a semiconductor.
Nanomedicine 24
FIGURE 2.3
Size tunable optical properties of QDs illustrating the increase in emission wavelength with increasing quantum dot
size.
Synthesis and Bioconjugation of QDs
In the design of QDs, the selection of a core QD composition is determined by the desired wavelength
of emission. Among various methods described in the literature, the highest quality QDs are typically
prepared at elevated temperatures (~300 C ̊ ) in organic solvents such as tri-n-octylphosphine oxide
(TOPO) and hexadecylamine, both of which are high boiling point solvents containing long alkyl chains.
These hydrophobic organic molecules perform as the reaction medium (solvent), and the basic
functional groups (phosphine, phosphine oxides and amine) coordinate with unsaturated metal atoms
on the QD surface to prevent the formation of bulk semiconductors. As a result, the nanoparticles are
capped with a monolayer of the organic ligands and are soluble only in nonpolar hydrophobic solvents
such as chloroform and hexane. A shell of another semiconductor such as ZnS or CdS with a wider
bandgap is grown on the core surface to provide electronic insulation. The growth of a semiconductor
shell on the surface of the core has been shown to significantly increase the fluorescence efficiency and
quantum yield of QDs. It has also been shown that a ZnS shell is less susceptible to surface oxidation
and therefore enhances the chemical stability of the QDs and decreases the oxidative photobleaching
of QDs to a great extent [16;17].
For use in biological systems, QDs must be rendered hydrophilic so that they are soluble for aqueous
formulations. Two general strategies have been developed to disperse QDs in aqueous solutions. In the
first approach, the hydrophobic monolayers of the ligands on the QD surface are exchanged with
hydrophilic ligands, such as mercaptoacetic acid [18;19]. However, this method has some drawbacks
such as particle aggregation and precipitation in biological buffers and change in photophysical
properties of QDs [18;20]. Alternatively, the native hydrophobic ligands (TOPO) can be retained on the
QD surface, and rendered water soluble through the adsorption of amphiphilic polymers [4;21]. In this
Nanomedicine 25
method the coordinating organic ligands (TOPO) are preserved on the inner surface of the QD, a
feature which is imperative for maintaining the optical properties of QDs and shielding the toxic
elements of the core from the outside environment by a hydrocarbon bilayer. Although this method
preserves the optical properties of QDs, the overall size of the particles after coating is rather large
which could place a restriction on biological applications. Furthermore, the surface of water-soluble
QDs can be functionalised with biomolecules such as antibodies, oligonucleotides or small molecule
ligands for specific targeting. The surface of QDs may also be modified with hydrophilic molecules such
as polyethylene glycol (PEG) to eliminate possible non-specific binding or to increase their circulation
times in the bloodstream following intravenous injection.
Photophysical Properties of QDs and Comparison with Organic Dyes
Fluorescence techniques are very well suited to study many fundamental cellular processes where
specific fluorescence probes are widely used in cell biology [22;23]. However, traditionally used
fluorophores, in particular organic fluorophores, have their limitations. These molecules generally have
narrow absorptions with asymmetric, broad emission spectra and low photobleaching thresholds [24].
Due to these intrinsic photophysical properties, traditional fluorescence probes have limited
applications with regards to long-term imaging and multiplexing. QDs have several dramatically
different properties compared to organic fluorophores, as was reviewed by Resch-Genger et al. [25].
Table 1 compares photophysical properties of QD nanoparticles versus organic dyes.
QDs have continuous broad absorption spectra that gradually increase in intensity towards shorter
wavelengths, enabling excitation by a wide range of wavelengths (below the first exciton absorption
band). Their emission spectra are symmetric and narrow, spanning the UV to near-infrared.
Consequently, QDs of different sizes can be excited by a single wavelength, with minimal signal overlap.
These differences provide QDs with several distinct advantages over organic fluorophores. Firstly, a
narrow emission spectrum provides the possibility of distinguishing multiple fluorophores
simultaneously. Secondly, the broad excitation spectrum of QDs facilitates the use of a single excitation
wavelength (light source) to excite QDs of different colours. These unique features of QDs allow the
simultaneous detection of different colour QDs, enabling several cells to be tracked in vivo [26;27], and
the simultaneous bio-sensing of multiple molecules in vitro [28]. Furthermore, owing to the efficient
multicolour excitation cross-section of QDs and the ability to synthesis QDs that can emit infrared or
near-infrared light, QDs are highly suited for imaging cells deep within tissues [27;29]. The molar
absorption coefficients at the first absorption band of QDs are generally larger compared to organic
dyes [30]. The large molar extinction coefficient of QDs makes them brighter probes under photon-
limited in vivo conditions where light intensities are severely attenuated by scattering and absorption.
Following light exposure, organic fluorophores can also undergo light-induced reactions leading to
irreversible change in their photophysical properties; a phenomenon known as “photobleaching”. This
limits their application for long-term imaging. On the other hand, QDs are very stable light emitters
owing to their inorganic nature, making them exceptionally resistance towards photo-and chemical
degradation [4;26;31;32]. This excellent photostability makes QDs very effective probes not only for
imaging QD-tagged proteins over long periods [33;34] but also for imaging the growth and
development of organisms for periods ranging from weeks to months [26;29]. In addition, the two-
photon cross-section of QDs is significantly higher than that of organic fluorophores making them quite
well-suited for examination of thick specimens and in vivo imaging owing to deeper penetration of
near-infrared excitation light [35;36]. Another important characteristic of QDs is their fluorescence
lifetime of 10-100 nanoseconds (ns), which is significantly longer than typical organic dyes or auto-
fluorescent proteins that decay in the order of a few nanoseconds [37;38]. The longer excited lifetime
Nanomedicine 26
of QDs provides a means to separate the QD fluorescence from short-lived background fluorescence
using time-gating techniques. It also worth noting that bioconjugated QD probes are similar in size to
fluorescence protein and do not suffer from major kinetic or steric hindrance problems [8;34].
TABLE 1
Photophysical properties of fluorescent organic dyes versus quantum dots.
Property Organic dyes QDs
Absorption spectra Narrow, discrete bands Broad, steadily increasing

toward UV wavelength
4 5 -1 -1 5 6 -1 -1
Molar absorption 2.5x10 - 2.5 x10 M cm 10 - 10 M cm
coefficient
Emission spectra Asymmetric, with red tail Symmetric
Quantum yield 0.5-1.0 (visible),0.05-0.25(NIR) 0.1-0.8 (visible), 0.2-0.7
(NIR)
Fluorescence lifetime 1-10 ns, mono-exponential 10-100 ns, typically multi-
decay exponential decay
-52 -48 4 -1 -47 -46 4
Two-photon cross section 1x10 – 5x10 cm s photon 2x10 – 10 cm s
-1
photon
Size ~0.5 nm, molecule 2-10 nm, colloid
Photochemical stability Low photobleaching threshold High photobleaching
threshold
Toxicity From very low to high, Limited known yet
dependent on dye (potential nanotoxicity)
Spectral multiplexing Not ideal due to the spectral Ideal for multi-colour
overlap experiments
IN VIVO STUDIES of QDs

QD nanoparticles are emerging as a new contrast agent in the biological system and have been
receiving increasing attention for potential use in biology and medicine [10;39]. In vivo fluorescence
imaging visualises the fluorescence emission from fluorophores in whole-body of living small animals.
Although traditionally used near-infrared dyes such as Indocyanine green continue to be used, the
development of fluorescent QD nanoparticles for in vivo fluorescence imaging offers several
advantages [40]. The unique optical properties of QDs for in vivo imaging include: high absorption
coefficient, high fluorescence quantum yield, and high resistance to photobleaching. More importantly,
broad absorption and narrow emission spectra of QDs make them suitable for simultaneous multiplex
imaging. Optical imaging presents itself as a powerful technique in image-guided therapy. However,
optical imaging in live animals remains hampered by the limited penetration depth of visible light in the
body. This limitation stems from the high absorption and autofluorescence that occur in biological
tissue across most of the electromagnetic spectrum. In the near-infrared region (700-900 nm) the
influence of the main tissue absorbing components, oxy and deoxyhaemglobin (ʎmax < 600 nm) as well
as water (ʎmax: > 1150 nm) is minimal. As a result, contrast agents that emit in the near-infrared region
(700-900 nm) of the spectrum can overcome this problem of penetrating deeper into the tissue and
Nanomedicine 27
out than UV, visible, or infrared light do [41;42]. Therefore optical imaging agents should ideally emit in
the near-infrared region (700-900 nm) so that they can be used clinically. Few organic dyes are
currently available that emit in the near-infrared region and they suffer from the same photobleaching
problems as their visible counterparts. One of the greatest advantages of QDs for imaging in living
tissue is their size/composition-dependent emission wavelength and their high two-photon absorption
cross section and high photobleaching threshold. Consequently, development of far red/near-infrared
QDs can overcome the limitation of organic fluorescence dyes. Several groups have thus synthesised
biocompatible near-infrared emitting QDs as a fluorescence probe for in vitro and in vivo labelling [43].
In general, methods for designing fluorescence imaging probes can be divided into non-targeting and
targeting. Due to their high surface area to volume ratio, QDs can be actively targeted to the desired
tissues with antibodies, peptides or passively via the enhanced permeability and retention (EPR) effect.
More importantly, QD nanoparticles offer multifunctionality, such as combining both imaging and drug
delivery or dual modality imaging combining near-infrared and magnetic resonance imaging. The in vivo
applications of QDs are summarised below with future opportunities and directions for their clinical
translation.
Non-targeted In Vivo Imaging
Sentinel Lymph Node (SLN) Mapping
Cancer spreads in a variety of ways, by direct extension, via the vascular system and via the
lymphatics/lymph nodes. The sentinel lymph node (SLN) is the first lymph node that drains from the
primary tumour. There is generally orderly progression of cancer from the primary tumour to the SLN
and then to the echelon lymph nodes. SLN localisation is one of the common methods used to identify
the presence of cancer in a single lymph node and is particularly important for the treatment of
melanoma, breast, cervical and head and neck cancers [44-46]. The techniques currently used for SLN
mapping employ a radioactive tracer, or a blue dye for visualisation of lymphatic vessels. But these
techniques are non-specific during surgery, which leads to unnecessary removal of lymph nodes,
causing unwanted trauma such as lymphoedema and nerve damage. In addition some adverse
reactions has been reported in patient following administration of the blue dye [47]. The potential
radiation hazards and the overall procedure duration are other limiting factors for these techniques.
The ideal method for SLN mapping should be accurate, rapid, and noninvasive. QD nanoparticles could
be ideal probes for SLN mapping because they are highly fluorescent, non-radioactive and easily visible
deep within tissues since QDs with strong red or near-infrared emission can be used where
haemoglobin absorption is minimal. Intraoperative imaging permits removal of the SLNs during
surgery. Using this technique it appears that near-infrared QD nanoparticles have the potential to
revolutionise human cancer surgery by providing sensitive, specific, and real time intraoperative
visualisation of normal and disease processes. Fig. 2.4 displays a schematic depiction of an
intraoperative imaging set-up for real time fluorescence imaging of the surgical field. This enables
surgeon with instantaneous imaging of lymphatic flow and provides real time visual feedback for
image-guided localisation and dissection [48].
Nanomedicine 28
FIGURE 2.4
Multispectral intraoperative camera system. A laser or light-emitting diode is used to excite the fluorescence. The
red QD fluorescence is imaged in one CCD camera channel, and tissue autofluorescence which occurs at shorter
wavelengths with another camera. A colour image of the target field is also obtained using white light illumination
and a third camera.
For the lymphatic system the overall size of the nanoparticle is an important parameter in determining
the biodistribution and clearance of QDs. Frangioni and colleagues used near-infrared emitting
CdTe/CdSe core/shell QDs with a hydrodynamic size of 15-20 nm and demonstrated SLN mapping of
QDs when injected intradermally into the paw of a mouse [29]. They also injected near-infrared QDs
intradermally on the thighs of pigs and followed lymphatic flow towards the SLN in real time. Using
2
excitation fluence rates of only 5 mW/cm , within 5 minutes localisation of QDs in the SLNs was
identified [29]. In subsequent studies, this group has demonstrated QD fluorescence imaging of lymph
nodes draining the lungs [49;50], oesophagus [51] and gastrointestinal tract [52]. In another study,
Zimmer et al. utilised very small PEG encapsulated near-infrared emitting QDs, with a hydrodynamic
diameter of 10 nm [53]. When these QDs were injected into the paw of a mouse a sequence of draining
lymph nodes was labelled instead of remaining trapped in SLNs. The authors concluded that migration
through the first draining lymph node was due to the small size of their QDs. The multiplexing
capabilities of QDs have also been exploited for mapping complex network of lymphatic vessels.
Kobayashi et al. demonstrated utility of simultaneous multicolour fluorescence imaging of five different
lymphatic basins using five types of near-infrared QDs with different emission spectra [54]. This is only
possible with QDs due to their narrow and symmetric emission wavelength and broad absorption
Nanomedicine 29
spectra. Ballou et al. were amongst the first to inject the QDs into the tumours to map SLNs. In their
study PEG encapsulated QDs with various charged groups were injected into the tumours of mice [55].
They demonstrated that tumour injection of QDs resulted in rapid migration of QDs from the tumour
through lymphatics to surrounding lymph nodes which was visible through the skin. In their study,
terminal charged groups had almost no effect on the extent of the drainage and pattern of the
migration of QDs from the tumour to the adjacent lymph nodes. In study by Marchal et al. the
detection of the SLN and the toxicity of the cadmium containing QDs (CdTeSe/CdZnS) were compared
with cadmium free counterparts (CuInS 2/ZnS) [56]. In their work both types of QDs were injected
subcutaneously in to the paw of healthy mice and were localised in the axillary lymph node in few
minutes after the injection. Cadmium-based QDs clearly showed signs of toxicity such as increased
lymph node weight with several inflammation sites. However, Cadmium-free QDs did not show any
features of toxicity under the same conditions. In another study silicon QDs (Si QDs) with hydrodynamic
of 20 nm were injected subcutaneously to the mice [57]. Following the injection QDs were observed in
the axillary lymph nodes and no adverse effect was observed in mice from the injection of the Si QDs
indicating their biocompatibility as a fluorescence probe for imaging.
In conclusion, the non-invasive fluorescence detection of SLN using QDs offers an opportunity to track
lymphatic flow in real time and guide their nodal dissection after intraoperative injection. Using QDs it
only takes a few minutes to detect the SLN. The QDs then slowly leaks from the injection point and the
SLN into the rest of the body via lymphatic and blood circulation. This gives the surgeon a few hours to
resect both the SLN and the injection point. Therefore at the same time the large majority of QDs are
removed from the body which limits concerns regarding toxicity of QDs. Ideally the QDs should be
targeted to the malignant tissue to maximise specificity, and there are several ways of achieving this as
follows.
Passive Tumour Targeting
Under in vivo conditions QDs can be delivered to tumours by both passive and active targeting via the
vasculature. In passive extravasation there is no need for special surface chemistry since nanometre-
sized particles accumulate preferentially at tumour sites through the enhanced permeability and
retention (EPR) effect [58]. This effect is believed to arise from two factors: (i) angiogenic tumours over
express vascular endothelial growth factors that increase vascular permeability in tumours and cause
leakage of circulating macromolecules and small particles; and (ii) lack of effective lymphatic drainage
system, which leads to accumulation of macromolecules and nanoparticles [59;60]. Several studies
have shown that QD nanoparticles can accumulate in the tumour due to the EPR effect [35;61]. For
example, in a study by Gao et al. in vivo passive tumour targeting of QDs was reported and it was
demonstrated that targeting was dependent on the stability of the QD in biological systems and its
circulation time [35]. In their study PEG encapsulated QDs were taken up readily via passive delivery by
tumours compared to carboxyl functionalised QDs. The observed findings were related to the increased
circulation time and reduced non-specific adsorption of serum proteins due to the PEG encapsulation.
The negatively charged nature of carboxylic acid terminated QDs enhanced their reticuloendothelial
uptake and therefore hampered their tumour uptake. In a recent study by Shuhendler and colleagues
[62], near-infrared emitting QDs encapsulated in solid fatty ester (QD-FEN) was used for in vivo imaging
in a breast tumour-bearing animal model. In vivo animal imaging revealed that fatty ester encapsulated
QDs surprisingly did not accumulate in the liver. They found that these QDs mainly accumulated in the
spleen and their accumulation was observed to decrease to nearly preinjection levels by 96 h after
injection. They stated that the encapsulation of QDs in fatty ester nanoparticles enhanced the
biocompatibility of QDs by providing complete clearance of the QDs from the body. Furthermore, the
Nanomedicine 30
results showed that by 1 h after QD-FEN injection fluorescence signal of QDs in the tumour sites was
detected, indicating the passive uptake of QD-FEN within the tumour tissue. The size and shape of the
nanoparticles play an important role in this process and it has been shown that smaller particles
penetrate deeper into the tumour interstitial medium [63]. Thus to obtain efficient tumour targeting
without selective targeting several parameters should be considered in designing the QD nanoparticles.
The design considerations should also focus on the development of highly stable and biocompatible
QDs with long circulation times.
Targeted In Vivo Imaging
Active targeting can be efficient and is not necessarily affected by the degree of tumour vascular
leakiness which differs according to the tumour locations. In active targeting, the QD surface is
decorated with a ligand that selectively binds to surface receptor sites in the extracellular environment
that play key roles in tumour proliferation, angiogenesis and metastasis. Many of these recognition
sites exist in normal tissue but are significantly upregulated in numerous tumour cells of certain cancer
types and offer a means of preferentially targeting the circulating QDs to tumours. The choice of ligand
for active targeting plays a crucial role in the performance of the QD nanoparticles. The degree of
selectivity, the strength of the interaction, the overall probe size, QD charge density and targeting
ligand accessibility are all important factors that should be considered. For instance in a study by
Bawendi et al. it has been shown that a maximum hydrodynamic diameter of about 5.5 nm is required
for renal excretion of QDs within 4 h [64]. They also reported that QDs with high charge-to-
hydrodynamic diameter ratios can cause adsorption of serum proteins and increase the hydrodynamic
diameter [64]. Bawendi et al. have suggested that a zwitterionic coating can reduce nonspecific
adsorption on the QD surface because of the low net charge density [65]. It should also be noted that
the penetration of QDs from vasculature into tumour can be affected by the overall size of the QDs.
Large targeting ligands such as antibodies (Abs) may lead to probe accumulation in tumour vasculature
with little or no tissue penetration, despite the high selectivity and affinity of the antibodies as
targeting agents. Therefore, careful consideration must be given to the design of specific targeting
ligands. The following section describes examples of active targeting of tumour in vivo using peptides
and proteins.
Active Tumour Targeting using Antibodies
Active targeting can be obtained by binding to receptor sites recognised by antibodies and other
proteins such as epidermal growth factor (EGF). The main disadvantages of employing proteins as
targeting agents arise from the size of the proteins as ligands and the resulting size of the QD
conjugates. Some examples of protein-mediated active targeting of QDs are given below.
In a study by Gao et al. PEG encapsulated QDs were bioconjugated with a specific antibody (antibody
against the prostate-specific membrane antigen: PSMA) and were injected into the tail vein of mice
bearing subcutaneous human prostate cancer [35]. They demonstrated that tumour targeting was
much faster and more efficient for the targeted QDs compare to un-targeted QDs, which accumulated
in tumour sites passively via the EPR effect. In a similar approach by Yu et al. QD nanoparticles were
conjugated with a specific antibody against an important marker for hepatocellular carcinoma [66].
Upon intravenous injection of QDs into the mice bearing hepatocellular carcinoma, active tumour
targeting of bioconjugated QDs was observed in tumour sites where their uptake was considerably
higher than that of un-targeted QDs due to passive targeting.
Nanomedicine 31
Tada et al. used QDs to study the biological process involved in active targeting of nanoparticles to the
tumour [67]. In their study, QD nanoparticles were labelled with antibody against human epidermal
growth factor receptor 2 (HER2) and administered intravenously into mice with HER2-overexpressing
breast cancer implanted tumours. Upon systemic delivery of QDs their migration from injection sites to
the tumour sites was monitored, using intravital fluorescence microscopy. The QDs circulated in the
bloodstream, extravasated into the tumour, diffused in extracellular matrix, bound to their receptors
on tumour cells, moved from the cell membrane to the perinuclear region, and then localised in the
perinuclear region of the cells. Therefore, the combination of sensitive QD probes with methods such
as real time microscopy for in vivo animal imaging could provide new insights into the understanding of
tumour biology and processes involved in the transport of drug carriers.
Active targeting of QDs to tumours has also been reported by the use of epidermal growth factor
receptor (EGFR). EGFR is a transmembrane protein that plays an important role in tumour progression
and proliferation. EGFR becomes highly upregulated in many cancer types which provides an
opportunity for designing a receptor targeted approach for the detection and treatment of cancer
[68;69]. In a study by Yang et al. single-chain epidermal growth factor receptor antibody (ScFvEGFRA)
was conjugated with QDs and paramagnetic iron oxide (FeO) nanoparticles to image human pancreatic
cancer in mice [70]. Targeted and untargeted QDs were injected intravenously into mice and their
biodistribution was studied using fluorescence microscopy. Strong QD fluorescence signal was detected
in the cytoplasm of tumour cells from the mice that received ScFvEGFRA-QD but not untargeted QDs.
Strong fluorescence signals were obtained from normal tissues of the mice that were injected with un-
targeted QDs, whereas the QD fluorescence from normal tissues of the mice that received targeted
QDs was negligible. These findings indicated the potential of ScFvEGFRA conjugated nanoparticles for
the detection of EGFR-expressing tumours using in vivo imaging approach.
Overall, these results imply that targeted nanoparticles have the potential to be used as effective
agents for in vivo tumour imaging. Efficient intracellular delivery of targeted nanoparticles can also be
used for specific delivery of therapeutic agents.
Active Tumour Targeting using Peptides
The small size of peptides relative to antibodies makes them attractive as targeting ligands. One of the
earliest examples of active targeting of QDs using peptide conjugates was reported by Akerman et al.
[71]. They demonstrated, for the first time, selective targeting of peptide coated QDs to the vasculature
of normal lung and tumours in vivo. In their work three different thiolated peptides were conjugated
with mercaptopropionic acid (MPA) coated QDs and injected intravenously into mice to target the lung
blood vessels, tumour blood vessels and tumour lymphatic vessels. Using fluorescence microscopy,
they showed that QDs coated with a lung-targeting peptide accumulated in the lungs of mice after
intravenous injection, whereas two other peptides specifically directed QDs to blood vessels or
lymphatic vessels in tumours.
Arginine-glycine-aspartate acid (RGD) peptide is one of the most widely used sequences for targeting
tumours due to its high affinity for integrin [72]. The protein integrin, which binds to RGD containing
components of the interstitial matrix, plays a key role in the progression of angiogenesis and
metastasis. It is highly overexpressed on the surface of angiogenic endothelial cells and tumour cells of
certain cancer types, making it attractive for tumour targeting [73;74]. In vivo targeting and imaging of
tumour vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled QDs was investigated for
the first time by Cai et al. [75]. In their work amine-modified QDs with peak emission at 705 nm
(QD705) was conjugated to the thiolated RGD peptide resulting in the formation of QD705-RGD.
Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumours were administered
Nanomedicine 32
with QD705 and QD705-RGD intravenously. In vivo fluorescence imaging of U87MG tumour bearing
mice showed that when mice were injected with QD705-RGD the fluorescence signal was detectable in
the tumour as early as 20 min postinjection, which reached its maximum at 6 h postinjection. However,
no significant fluorescence signal was observed in the tumour for the QD705 injected mouse.
Successful tumour imaging indicated the specific in vivo targeting of integrin αvβ3 using QD705-RGD. Ex
vivo fluorescence imaging also confirmed the uptake of QD705-RGD into the tumour. Microscopic
images of frozen tumour slices stained for CD31 immunofluorescne staining showed that QDs were
localised inside the vessels and did not extravasate, which was attributed to the large hydrodynamic
diameter of these QDs.
In another study by Gao et al. [76] the specific tumour targeting of RGD peptide labelled near-infrared
non-cadmium QDs was investigated. PEG encapsulated core/shell/shell QDs (QD800-PEG) with a peak
emission wavelength at 800 nm were conjugated with RGD peptide (QD800-RGD). In their study
QD800-PEG were also conjugated with the thiolated RAD peptide c(RADy(ε-acethylthiol)K) to produce
QD800-RAD which was used as the negative control. Dynamic light scattering (DLS) analysis showed
that the hydrodynamic diameter of QD800-PEG, QD800-RGD, and QD800-RAD were about 16.5 nm,
19.6 nm, and 20.1 nm, respectively. Animal experiments were performed on nude mice bearing
subcutaneous U87MG human glioblastoma tumours. After intravenous injection of QD800-RGD,
QD800-PEG, and QD800-RAD, the fluorescence signal of QDs was monitored at various time intervals
using the IVIS Imaging System. Their results showed that after about 1 h postinjection of QD800-RGD,
the fluorescence signal of the tumour reached maximum and then slightly decreased over time, while
there was little to no tumour contrast in the mice injected with QD800-PEG or QD800-RAD. These
results indicated highly specific tumour targeting of QD800-RGD. A bright fluorescence signal of QDs
was observed in the liver, spleen and bone marrow, demonstrating the accumulation of QDs in the
reticuloendothelial system sites which was due to the relatively short half-lives of these QDs. For ex
vivo imaging, tumour and major organs were harvested and immediately were subjected to
fluorescence imaging using the same imaging system. The ex vivo results further confirmed the obvious
fluorescence signal in the U87MG tumour of mice injected with QD800-RGD, whereas there was almost
no fluorescence signal in the tumours of mice administered with QD800-PEG or QD800-RDA. In their
work although the uptake of the QDs in the liver was highest among all of the organs, the difference
between tumour fluorescence signals indicated that only QD800-RGD has the capability to specifically
target and detect U87MG tumour. Through CD31immunofluorescne staining, performed to investigate
the microscopic localisation of the QD in the tumours, fluorescence overlay images, showed the
presence of QD800-RGD in the tumour vessels but no QD fluorescence signal in the tumour vessels of
mice injected with QD800-PEG or QD800-RAD. Furthermore the images showed that QDs were
localised inside the tumour and did not extravasate from the tumour vessels suggesting that QDs of
greater size can only target the vascular integrin but not tumour cell integrin [71;75;77]. However, QDs
with smaller sizes (<10 nm in hydrodynamic diameter) can facilitate their extravasation (due to the EPR
effect), enhance the specific targeting to tumour cells and minimise the reticuloendothelial uptake
[64;65;78;79].
Recently in a study by Gao J et al. [80] dendron-coated non cadmium containing core/shell QDs
(QD710-Dendron) with peak emission at 710 nm were conjugated with RGD peptide (QD710-Dendron-
RGD2). Active tumour uptake of these QDs was investigated in nude mice bearing subcutaneous SKOV3
tumours. In vivo and ex vivo fluorescence imaging showed that the QD710-Dendron-RGD2
nanoparticles resulted in high tumour uptake and long retention of these nanoparticles at tumour sites.
The QD710-Dendron also displayed tumour accumulation, although to a lesser degree, which was likely
caused by passive uptake of nanoparticles due to the EPR effect. In comparison to the QD710-Dendron-
RGD2, the fluorescence signal of QD710-Dendron at tumour sites was relatively low and decreased
Nanomedicine 33
significantly over time, and there was almost no tumour contrast after 24 h. The rapid change of
tumour fluorescence signal in the mice injected with QD710-Dendron was attributed to a weak
interaction of passive targeting. In their study anti-CD31 immunostaining of tumours was used to study
the microscopic location of QDs in the tumour and the results revealed the presence of QD710-
Dendorn-RGD2 both inside and outside tumour vessels. These findings indicate that QD710-Dendorn-
RGD2 not only specifically binds to vascular integrin α vβ3 but also extravasates and interacts with
integrin αvβ3 expressed on tumour cells. The extravasation of QD710-Dendorn-RGD2 was in contrast to
previous work whereby using QDs with a hydrodynamic diameter larger than 20 nm they found no
extravasation was observed [75-77;81]. They attributed this to the extravasation of QD710-Dendorn-
RGD2 owing to their relatively small hydrodynamic diameter in vivo.
In Vivo Imaging Using Self-illuminating QDs (BRET)
The application of QDs in living systems can be limited due to the requirement of an external
illumination source for excitation which produces strong background autofluorescence from
endogenous chromophores. In addition, because of absorption and scattering of optical photons in
tissues, little light could be available for QDs excitation at deep tissues. Moreover, there can be
nonselective tissue damage at the illumination site at high laser powers. Therefore, an ideal QD would
emit light without the need for the external excitation source. Towards these goals new types of QDs
referred to as “self-illuminating” QDs have been explored. These QDs can fluoresce without the need
for an external light source, based on the bioluminescence resonance energy transfer (BRET) process
[82-84]. BRET is a naturally occurring process whereby a bioluminescent compound (the donor)
nonradiatively transfers energy to a fluorescent compound (the acceptor) in close proximity [85;86].
BRET is analogous to Förster resonance energy transfer (FRET), except that the donor emission comes
from a chemical reaction catalysed by an enzyme rather than from absorption of light from an external
source, such as a laser. Efficient BRET occurs when there is an appropriate spectral overlap between
the emission spectrum of the donor and excitation spectrum of the acceptor. BRET efficiency is
inversely dependent on the sixth power of the distance between the donor and the acceptor, and
therefore a short-range effect is typically a few nanometres. The unique photophysical properties of
QDs such as broad absorption spectra, large molar extinction coefficient, high quantum yield and
tunable emission spectra make them ideal acceptors for the BRET studies.
Near-infrared self-illuminating QD conjugates can emit light from red to near-infrared region in living
cells and in living animals for deep tissue in vivo imaging. For instance, new types of self-illuminating
QD conjugates have been developed by So et al. [82]. In their study carboxyl functionalised QDs were
employed as the acceptor and coupled to a mutant of the bioluminescent protein Renilla reniformis
luciferas (Luc8). They showed that the conjugates emit bioluminescent light in cells and in animals even
in deep tissues. Upon the addition of the substrate coelenterazine or the protein emits blue light with a
peak emission at 480 nm. The QD which was in close proximity with protein was excited nonradiatively
via the BRET mechanism. They demonstrated coupling of the QD with the luciferase protein, and the
emission from QDs was detected at subcutaneous and intramuscular sites in a mouse model. In
addition, multiplex imaging was demonstrated by labelling three groups of cells with three different QD
BRET conjugates injected into the same mouse. In another study, C6 glioma cells were labelled by QD-
BRET conjugates and injected through the tail vein into the mouse [84]. Using BRET emission from QDs
their trafficking into the lungs was imaged. In their study, multiplex imaging by labelling two groups of
cells with two different QD BRET conjugates injected into the same mouse was demonstrated.
Nanomedicine 34
In general, by eliminating the need for the external light source for the excitation BRET offers a variety
of advantages, including insignificant photobleaching of the fluorophores, no autofluorescence
background and no direct excitation of the acceptor. With the ability to tune the emission of QDs
simply by changing their size and composition, a variety of BRET pairs could be developed, which would
make it possible for various interactions to be imaged concurrently in the same animals.
Multifunctional QDs:
The large surface of QDs makes these nanoparticles suitable scaffolds to accommodate multiple
imaging and therapeutic agents through chemical linkage or physical immobilization. This may enable
development of multifunctional nanostructures for multimodality imaging and integrated imaging and
therapy.
Dual-modality Imaging
Optical imaging is highly sensitive, though its application in humans and in vivo experimental models is
hampered by limited tissue penetration depth, and poor anatomical resolution and spatial information.
It is also difficult to sufficiently quantify QD signals in living subjects based on fluorescence intensity
alone. To address these limitations, several groups have developed dual modality nanostructure by
combining QD-based imaging with other imaging techniques such as positron emission tomography
(PET) and magnetic resonance imaging (MRI) as described in the literature [87] . The most commonly
used method to build multifunctional QDs are coating QDs or conjugating them with paramagnetic (for
MRI) or radioactive (for PET) ion chelates or molecules. For example, Chen et al. developed a dual
64
function imaging probe for both optical imaging and PET [77]. PET-detectable radionuclide Cu was
attached covalently to the polymeric coating of QDs. Targeted in vivo imaging of a subcutaneous mouse
tumour model was obtained by additionally attaching arginine-glycine-aspartate acid (RGD) peptides
on the QD surface. The ultrahigh sensitivity of PET imaging enabled the quantitative analysis of the
biodistribution and targeting efficacy of this dual-modality imaging probe. This approach can overcome
the tissue penetration depth limitation for QD imaging, therefore allowing quantitative in vivo targeted
imaging in deep tissue and may facilitate future biomedical applications of QDs.
Another method to form multifunctional probes is doping the QDs with transition metals, such as Mn.
For example in a study by Wang et al. water soluble core/shell CdSe/Zn 1-xMnxS nanoparticles were
synthesised and were applied to the cells [88]. Using both MRI and fluorescence microscopy they
demonstrated that QDs produced sufficient contrast in cells, indicating the utility of these
nanoparticles for imaging. In a third approach fluorescence/paramagnetic nanoparticles were made by
assembling QDs with Fe3O4 or FePt magnetic nanoparticles in silica beads, micelles, and polymer
particles or within nanoheterostructures [89-91].
Integration of Imaging and Therapy
Drug-containing nanoparticles have shown great promise for treating tumours in animal models and in
clinical trials [92]. QDs have also been utilised to carry distinct classes of therapeutic agents for
simultaneous imaging and therapeutic applications. Farokhzad et al. developed a ternary system as a
targeted cancer imaging, therapy, and sensing system [93]. In their study the QD surface was
functionalised with an A10RNA aptamer (which recognises the extracellular domain of the prostate
specific membrane antigen (PSMA)) to develop a targeted QD imaging system (QD-Apt). A well-known
anti-cancer drug, Doxorubicin (Dox), was attached to the stem region of the aptamers, taking
Nanomedicine 35
advantage of nucleic acid binding ability of Doxorubicin. In this configuration, two donor-acceptor pairs
of fluorescence resonance energy transfer (FRET) between QD-Dox and Dox-RNA aptamers occurred.
They showed that this multifunctional nanostructure can deliver Doxorubicin to the targeted cells and
monitor the delivery of Doxorubicin by activating the QD fluorescence, which simultaneously images
the cancer cells.
The potential of QDs for photo-induced formation of reactive oxygen species (ROS) has been reported
by several groups which is the basis of photodynamic therapy (PDT). Photodynamic therapy is a
minimally invasive therapeutic modality approved for the clinical treatment of several types of cancer
and non-oncological disorders. Generally, the photodynamic therapy procedure consists of
administrating the photosensitiser systemically (intravenous injection) or topically to the skin. Then,
the diseased tissue is illuminated with light at an appropriate wavelength and energy dose at an
appropriate time (when the photosensitiser concentration corresponds to a maximum accumulation in
target tissue). The photosensitiser is activated by the light and interacts with molecular oxygen (O2) to
produce cytotoxic reactive oxygen species [94;95]. Most of the clinically and experimentally available
photosensitisers have some major drawbacks such as instability in aqueous solutions, prolonged
cutaneous sensitivity, chemical impurity, poor selectivity (in terms of targeting diseased tissue), low
extinction coefficients, low photobleaching thresholds and weak absorption at the therapeutic
wavelength [96]. To address these problems new photosensitisers are being sought together with
improved delivery systems, including nanoparticle carrier systems. There has been an increasing
number of reports on the preparation and utilisation of nanocarriers for photodynamic therapy agents
[97-103]. Among the different nanoparticles that offer great promise in photodynamic therapy
applications are QD nanoparticles. In relation to photodynamic therapy, QD nanoparticles possess
several characteristics which make them attractive as a new therapeutic agent. For instance, their
surface can be functionalised for specific targeting, they have large extinction coefficients, large two-
photon cross sections and are exceptionally resistant to photobleaching [104].
Although targeted delivery of QDs in cancer cells and tumour tissue by using anticancer antibodies and
other biomolecules has become possible recently, the efficiency of QDs on their own to produce ROS
under direct photoactivation appears to be relatively low, compared with conventional photosensitiser
drugs. Thus, in order to utilise the exceptional photostability, broad absorption band and large two-
photon cross section of QDs beside improving the production of ROS, several groups have investigated
the utility of quantum dot-photosensitiser complexes or hybrids as new generation drugs for
photodynamic therapy [105-108]. In this configuration, the QDs act as energy donors and excite
photosensitiser acceptors indirectly via the Förster resonance energy transfer (FRET). Based on the
growing number of in vivo studies of QDs in experimental tumour models, there is clearly considerable
potential for quantum dot-photosensitiser complexes in photodynamic therapy.
Cell Tracking
Cell tracking using QD nanoparticles is also a very active and promising area of research. The ability to
track and target local tumour growth, distant metastasis, tumour associated neovasculature, and stem
or immune cells in live subjects are crucial to many biological studies. For these purpose QDs should be
able to efficiently label target cells in vitro and they should not leave the cells after in vivo injection.
Several studies have shown that QDs remain inside their initial cells during the experiments. Dubertret
et al. demonstrated for the first time the in vivo cell tracking using QD nanoparticles. In their study QDs
were injected into the cytoplasm of single frog embryos. They showed that as the embryos grew, the
cells divided, and each cell that descended from the original labeled cells retained a portion of the
fluorescent cytoplasm, which could be fluorescently detected under illumination [4]. In another study
Nanomedicine 36
by Lei et al. PEG encapsulated CdSe/ZnS were conjugated with the HIV derived cell penetrating Tat-
peptide and were introduced into living mesenchymal stem cells (MSCs) [109]. Using the confocal
microscopy the results show that Tat-QDs could enter the MSCs efficiently. They also injected Tat-QDs
intravenously into the mice and tissue distribution of these labelled stem cells was assessed with
fluorescence microscopy. The results showed that Tat-QDs were observed in the liver, spleen and lung
with little or no QDs in other organs. They demonstrated that labelled stem cells can be visualised at
the single cell level, offering a promising application in stem cell transplantation and stem cell based
therapy. Also Gao et al. loaded cancer cells with QDs and injected these cells subcutaneously into the
mice [35]. They observed that implantation of QD-loaded cells led to normal tumour growth in animal
model and the fluorescence of the QDs was detected through the skin. In another study human
mesenchymal stem cells were loaded with QDs [110]. They showed that these cells could be implanted
into an extracellular matrix patch for use as a regenerative implant for canine heart with a surgically
induced defect. Eight weeks after implantation QD fluorescence were detectable in histological
sections, indicating that the locations of these cells could be determine for at least eight weeks
following delivery in vivo.
The ability to separate fluorescence from different type of QDs using multiphoton and emission-
scanning microscopy provides the opportunity to study the interaction of different population of
tumour cells and tissue cells within the same animal. For instance, in a study by Voura el al. tumour
cells were labelled with QDs and were intravenously injected into mice [111]. In this study QDs and
spectral imaging allowed the simultaneous identification of five different populations of cells using
multiphoton laser excitation.
Future perspective
Improving the Biocompatibility of QDs
For most in vivo imaging studies using QD nanoparticles, systemic intravenous delivery of QDs is the
main route of administration. For this reason, the interaction of nanoparticles with the plasma
components, adsorption to blood cells and the vascular endothelium and the eventual biodistribution
in various tissues are important factors. These parameters are particularly important when considering
tumour-specific imaging applications.
Upon entering the body nanoparticles like other foreign pathogens, encounter multiple lines of
defence that protect the body from invading substances. Adsorption of plasma proteins on the
nanoparticle surface is called opsonisation [112]. This helps immune cells such as macrophages to
recognise and take up the nanoparticles. Opsonisation could lead to a dramatic change in
physicochemical properties of nanoparticles such as increased hydrodynamic size, aggregation and
charge neutralisation. The phagocytic monocytes and macrophages, found around the body and largely
in the liver, spleen, lymph nodes and bone marrow, collectively make up the reticuloendothelial system
(RES). As a result of nanoparticle phagocytosis by macrophages in the blood and via these
reticuloendothelial system organs, nanoparticles are commonly sequestered to these organs. The
immune recognition of nanoparticles and their uptake by the reticuloendothelial system reduces their
circulation time in the blood and can dramatically affect their drug delivery efficacy. This problem is
particularly important for targeted nanoparticles that need time to bind to the tumour receptors and
for non-targeted nanoparticles that rely only on the EPR effect.
There are several factors which influence in vivo QD biodistribution including the nanoparticle size,
hydrophobicity, surface charge, the route of administration, and the physiological environment to
Nanomedicine 37
which nanoparticles are introduced. Most of the in vivo biodistribution studies of QD nanoparticles
have demonstrated the accumulation of QDs in the RES such as liver, spleen. It has been shown that
time-dependent accumulation of QDs in the RES was strongly dependent on the QD size, where the
uptake was higher for larger particles. Furthermore, it has been revealed that the adsorption of serum
proteins on the QD surface is dependent on surface coating, which can significantly increase the
hydrodynamic diameter of QDs and subsequently their RES uptake. For instance, in a study by Bawendi
et al. the hydrodynamic diameter of purely cationic or purely anionic QDs was enhanced by
approximately 15 nm in the presence of serum [64]. In a subsequent study by this group it was
demonstrated that neutral and hydrophilic polymer encapsulated QDs tended to avoid the serum
protein adsorption, while maintaining permeability and retention in tumour sites [113]. In another
study by Fischer et al. hydrophilic ligand exchanged QD, using mercaptopropionic acid exhibited
hydrodynamic diameter growth from 25 nm to 80 nm in the presence of bovine serum albumin (BSA).
However, zwitterionic cysteine encapsulated QDs did not change in size when subjected to fetal bovine
serum [114].
Hence for successful targeted drug delivery and imaging, it is necessary to minimise opsonisation and
prolong the circulation of nanoparticles in vivo. This can be achieved by surface coating of the
nanoparticles with hydrophilic polymer/surfactants such as polyethylene glycol (PEG). Studies show
that PEG confrontation at the nanoparticles surface is of utmost importance for the opsonin repelling
function of the PEG layer. In the case of QD nanoparticles, numerous studies have also shown that PEG
increases the colloidal stability of QDs in blood [64;113;115]. For instance, in a study by Gao et al. QDs
were encapsulated with triblock copolymer and were linked to PEG [35]. In vivo targeting studies of
human prostate cancer growing in nude mice showed an increase in the blood circulation time of these
QDs which was proportional to the length of the PEG chain. In another study, near-infrared emitting
QDs were ligand exchanged with dihydrolipoic acid (DHLA) and conjugated with PEG chains with
various lengths. Intermittent sampling from the tail vein revealed that low-molecular-weight PEGylated
QDs had shorter blood circulation times compared to the larger molecular weight PEGylated QDs [113].
The same trend was reported by Al-Jamal et al. who found that blood retention times for larger
molecular weight PEGylated QDs (PEG 2000-5000) was significantly longer that low-molecular-weight
PEGylated QDs (PEG 750) [116].
It should be noted that the presence of a PEG coating may only delay the RES uptake of QD
nanoparticles, since all nanoparticles are prone to eventually accumulate in the RES, irrespective of
surface coating. The liver is considered the main organ for capturing nanoparticles greater than 10-20
nm [64]. It appears that liver uptake is dependent on the size of the nanoparticles: uptake of larger
particles is greater and they are excreted from the body more slowly. For instance, 4 h after
intravenous injection, the QD515 of hydrodynamic diameter of 4.4 nm were mainly found in the
bladder, with less than 5% located in the liver. In contrast, 25% of the injected dose of the larger QD574
with hydrodynamic diameter of 8.7 nm was observed in the liver [64]. Similar results were reported by
Fischer et al. where 90 min postinjection 99.5% of the injected dose of QDs coated with bovine serum
albumin (hydrodynamic diameter: 80 nm) was found in the liver, whereas only 36% of the injected dose
of QD coated with mercaptopropionic acid (hydrodynamic diameter: 25 nm) was found in the liver
[114]. In another study that examined the effect of size, non-cadmium containing QDs were coated
with two different carboxylate coatings, one with a hydrodynamic diameter of 25 nm (QD800-COOH)
and the other with a hydrodynamic diameter of less than 10 nm (QD800-MPA). QDs were injected
intravenously into mice bearing 22B and LS174T tumours. It was found that tumour uptake with
QD800-MPA was higher than QD800-COOH, likely due to their smaller size. In addition it was revealed
that smaller QDs were excreted from the body more efficiently than larger QDs. Fluorescence signals of
QD800-MPA were detected in the kidney and bladder which decreased with time, indicating renal
Nanomedicine 38
clearance. In contrast, for the larger particles fluorescence signals were mainly found in the liver, bone
marrow and spleen but tumour contrast was very low. The non-specific uptake of QD800-COOH
particles was related to their larger size [79].
Surface coating determines the overall QD size, therefore high molecular weight polymers or organic
coating lead to considerable increases in the QD diameter that can accelerate QD blood clearance and
liver entrapment. From the clinical point of view, it would be possible to inhibit the accumulation of
QDs and avoid potential toxic effects if they are within the size range that enables renal excretion.
Studies have shown that the biodistribution and elimination of QDs are strongly correlated with QD size
[64;65;113]. For example Frangioni et al. demonstrated that the renal clearance of QDs is closely
related to their hydrodynamic diameter and the renal filtration threshold (~ 5.5 nm) [64]. Of equal
importance to the QD size, is that the surface does not promote protein adsorption, which could
significantly increase the QD diameter above that of renal threshold, and promote phagocytosis. They
reported that the surface charge of QDs had an important effect on the overall size of QDs under
physiological conditions. Purely positive or negative QD surface charge was related with an overall
increase in the QD hydrodynamic diameter above 15 nm, due to the interaction with serum proteins.
This increase prevented their renal excretion and led to the enhancement in their accumulation in liver.
In their study only coated QDs with a hydrodynamic diameter of less than 5.5 nm were rapidly excreted
in the urine and eliminated from the body
In general, the metabolism and the excretion route of QDs still remain unclear and have to be
investigated extensively. Ideally the small amount of the QDs remaining in the body should be
ultimately eliminated, either via the kidneys into the urine or via the hepatobiliary pathway. The
mechanisms for complete clearance are however not well understood yet.
Potential Toxicity of QDs
The major concern to the clinical translation of QDs is their toxicity due to their chemical composition
of toxic heavy metal atoms (e.g Cd, Hg, Pb, As). Currently the most commonly used QDs contain
cadmium. Although this element is incorporated into the core of the nanocrystal, surrounded by inert
zinc sulphide, and encapsulated within a stable polymer, it is still unclear if this toxic ions can be used
as clinical contrast agents. Therefore, despite potential biomedical applications of QDs concerns
persists about their safety.
In vitro studies have shown that QD toxicity arises from several factors such as chemical composition,
size, shape, surface charge, surface coating, and dose. Their ability for photo-induced formation of
reactive oxygen species (ROS) and nanoparticle aggregation are other parameters involved in QDs
toxicity [117-122]. Toxicology data derived from in vitro studies may not reflect the response of a
physiological system to an agent. Animal model is the preferred system for the toxicological
assessment of a novel agent. Therefore comprehensive in vivo toxicity evaluation of QDs is crucial for
their clinical translation. In vivo toxicity is determined by several factors including dose, rout of
administration, metabolism, excretion rate, and immune response. Chemical composition, size, shape,
aggregation and surface coating are also involved in QD-induced toxicity. Various animal models have
been used to study in vivo toxicity of QDs and a few examples are described in the following sections
[123].
Generally, mammalian models are used for the in vivo toxicity study, but recently some groups have
used zebrafish for toxicity study of QDs. King-Heiden et al. used a zebrafish model to study the toxicity
of CdSe/ZnS QDs [124]. In their study zebrafish embryos were exposed to aqueous solutions of QDs.
They reported that the toxicity was influenced by the QDs coating. At sublethal doses, many QDs
preparations produced characteristic signs of cadmium toxicity that weakly correlated with
Nanomedicine 39
metallothionein expression suggesting that QDs were slightly degraded in vivo. They also reported that
QDs produced distinctively different toxicity that could not be described by cadmium release. In
another study, Truong et al. evaluated the importance of QDs surface functionalization in both
nanoparticle stability and in vivo biological responses using the embryonic zebrafish [125]. They used
two PbS QDs formulation with the same core sizes but different surface functionalization. Exposure to
these QDs was begun 6 hrs post fertilisation. They showed that QDs toxicity was strongly associated
with the surface ligands.
In vivo toxicity studies of QDs have also been carried out in various mouse and rat models. For instance,
Hauck et al. performed a systemic study to evaluate the effect of dosing and surface chemistry on the
short- and long-term in vivo toxicity of CdSe/ZnS QDs in rats [126]. Animal survival, animal mass,
haematological and biochemical tests, and organ histology showed that QDs did not cause appreciable
toxicity under in vivo condition. Even chronically dosed rats did not experience QDs toxicity. In another
study by Su et al., short- and long-term biodistribution and toxicity of CdTe QDs were investigated after
administration into the mice [127]. They showed that QDs were initially accumulated in the liver after
short-term post-injection, and then were absorbed by the kidney during long-time. Histological and
biochemical analysis, and body weight measurements revealed that there was no advert effect of QDs
in mice even after long-time exposure time. Gao et al. injected dendron-coated InP/ZnS QDs into the
mice [80]. The dendrimer and dendron molecules have unique physicochemical properties and have
great potential for use in a variety of biomedical applications including drug delivery [128]. A pilot
mouse toxicity study by this group confirmed that these dendron coated QDs were not toxic at the
doses tested.
Non-human primates are irreplaceable animal models for biomedical research because of their close
evolutionary relationship to humans. The first study of QDs in primates was reported by Ye et al. [129].
In their study phospholipid micelle encapsulated CdSe/CdS/ZnS QDs were injected into rhesus
macaques. The results revealed that the haematological and biochemical markers were within normal
ranges over 90 days of monitoring. In addition histological analysis of major organs displayed no sign of
inflammation or injury. However, chemical extraction of the tissues revealed that most of the initial
dose of the cadmium remained in the liver, spleen and kidney after 90 days post-injection, indicating
that the breakdown and clearance rate of QDs is slow in comparison to biodegradable polymer
nanoparticles. This suggests that longer-term studies will be essential to determine the eventual effect
of these nanoparticles in the body.
To characterise fully the in vivo toxicity of QDs, further comprehensive investigations are still required.
Some of these assessments include the evaluation of QD composition (cadmium-based versus cadmium
free QDs), surface chemistry, size, and shape. In future for the nonhuman primate studies of QDs
toxicity, the cardiovascular and respiratory measurements would also be valuable. Specific
immunohistochemistry assay can be used to hepatocytes in the liver samples. Genetic analysis such as
polymerase chain reaction (q-RT-PCR) can also be performed on tissue samples to determine the
inflammatory gene regulation. Future in vivo studies should also focus on long-term accumulation of
QDs and on understanding their excretion through the faces and urine. Combining the results from all
of these studies will eventually provide valuable information and guidance for the design and use of
QDs in biomedical field.
Nanomedicine 40
Conclusions
The long-term goal in medical applications of nanobiotechnology is to design and develop
nanoparticles with multiple functions that are capable of targeting diseased tissues, treating the
disease and monitoring progress in real time. One of the most early applications of QDs would be in
areas such as image-guided surgery for tumour removal. QDs can also be designed to develop more
complex multifunctional nanostructure, such as incorporation of paramagnetic metals into the
nanoparticles. In this imaging modality, QDs could be used as MRI contrast agents for the detection of
deep seated tumours, while intraoperative fluorescence imaging of QDs would enable the surgeon to
define the tumour margins to excise the entire tumours more completely. QDs can also been
conjugated to therapeutic agents, which would enable simultaneous diagnostic imaging and drug
delivery monitoring in real time. For biomedical applications, it is important to minimise the overall size
of QDs, to reduce nonspecific protein adsorption, to understand the toxic effect of semiconductor
materials, to develop near-infrared emitting QDs to minimise the problems of indigenous fluorescence
of tissue and enhance penetration depth. For the clinical translation, QD nanoparticles should be
composed of nontoxic materials and should be biodegradable. Cadmium free QDs could therefore offer
the opportunity to fulfil the biological applications of QDs without the toxicity limitations encountered
by cadmium containing QDs. By reaching these gaols, QD nanoparticles will complement organic
fluorophores deficiencies in particular applications as in vivo imaging.
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3
Conjugation of Gold nanoparticles and
liposomes for combined vehicles of drug
delivery in cancer
1,† 1,† 2 2 1,
Sara Figueiredo , Rita Cabral , Daniel Luís , Alexandra R. Fernandes and Pedro V. Baptista *
1
CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de
Caparica, 2829-516 Caparica, Portugal
2
Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica,
2829-516 Caparica, Portugal.
†
These authors contributed equally to the Chapter
* Corresponding Author
Outline:
Strategic targets for cancer therapeutics ……………………………………………….…………………………………… 49
RNAi-based cancer therapy – going nano …………………………………………..………………………………………. 50
Gold nanoparticles: powerful vehicles for RNAi delivery ……………………………………………………………… 51
AuNP-siRNA conjugation ………………………………………….………………………………………………………………… 51
Gold-thiol bond ………………………………………..………………………………………………………………………………… 51
Electrostatic interactions ……………………………………………………………………………………………………………. 53
Biocompatibility, biodistribution and targeting of AuNPs ……………………………………….………………….. 53
Cellular internalisation and active targeting ………………………………………………………………………………. 55
AuNP-RNAi conjugates for cancer therapy: in vitro and in vivo studies ……………………………………….. 56
Toxicity evaluation of AuNPs for therapy ……………………………………………………………………………………. 57
Liposomes for drug delivery - Great things sometimes come in small packages …………………………. 58
Towards optimised properties - size and surface charge …………………………………………………………….. 61
Passively targeted vs. ligand-targeted liposomes ……………………………………………………………………….. 62
Toxicity and bioavailability …………………………………………………………………………………………………………. 63
Combined therapy ……………………………………………………………………………………………………………………… 65
Co-loading of drug and RNAi effector molecules ………………………………………………………………………… 66
Nanoparticle cocktail …………………………………………………………………………………………………………………. 67
Conclusion…………………………………………………………………………………………………………………………………… 67
References……………………………………………………………………………………..…………………………………………… 69
Nanomedicine 49
Strategic targets for cancer therapeutics

In our current understanding, cancer is the result of a multi-step process of sequential somatic
alterations in several oncogenes, tumour-suppressor genes or micro RNA (miRNA) genes that regulate
cell growth and differentiation. These genetic alterations include base substitutions, insertions and
deletions (indels), DNA rearrangements, copy number variations and even epigenetic changes [1]. They
are random, unpredictable and cumulative, and some of them establish the basis for gene
deregulation, which underlies the uncontrolled growth or increased survival of cancer cells [2]. The
evolutionary progression of cancer depends upon a growth advantage within the micro-environmental
constraints, including resource limitations, presented by the surrounding tissues [3]. Typically, cancer
therapy is based on surgery, radiation therapy, and chemotherapy with wide-range cell killing
chemicals. Maybe due to the adaptive evolution of cancer cells, most conventional therapies fail as
tumour heterogeneity within the same diagnostic type results in variable clinical behaviour and
response/sensitivity to treatment [2]. Also, these chemical therapeutic agents subject cancer cells to
new potent form of selective pressure, favouring the survival and proliferation of cells or clones with
growth advantages, which become resistant to treatment [4].
To overcome the limited therapeutic response of some cancers, alternative strategies have been
proposed, where nanomedicine has been playing a pivotal role. Firstly, personalised specific therapies
may be required, bearing in mind that the emergence of genetically favoured resistant clones is always
a possibility. Second, the therapeutic focus may be directed towards understanding and controlling the
evolutionary process of tumours in its early stage, including the genetic principles and the impact of
combinations of mutations on cancer phenotypes [4, 5]. Third, it might be more effective to focus on
combinatorial therapies, designed to the individual cancer genome and simultaneously targeting more
than one component of networked signalling pathways, hitting the primary target as well as the
compensatory mechanisms to overcome resistance to therapy [4]. Other suggested targets include: (1)
components of the self-renewing programme of cancer stem cells regardless of the specific mutant
genotype, especially in cases where these can be distinguished from normal adult stem cells; (2) the
micro-environment surrounding cancer cells, aiming at angiogenesis or inflammation, for instance, or
exploring hypoxic conditions; (3) cell division in general, with the use of cytostatic drugs to control the
cancer and convert it into a chronic disease, by delaying both progression and mortality [4].
Most of these suggestive treatments are still in research and slowly translating into the clinics. Due to
their high versatility, nanotechnology based platforms have been pushing forward novel therapeutic
approaches, mainly by combining the steady advances in the molecular targeting of events in cancer
and the optimised delivery of anti-cancer drugs. Here we shall be focusing on the developments of
combined therapy for cancer using gold nanoparticles and liposomes towards delivery of RNA
interference (RNAi) effector molecules and chemotherapy. Optimisation of these combined strategies,
mainly by introduction of active targeting moieties and increased selectivity of chemical agents against
cancer cells, holds great promise for the introduction of highly effective drug delivery systems for
cancer therapeutics while overcoming drug resistance.
Here we shall discuss trends on the use of gold nanoparticles for vectorisation of anti-cancer drugs, in
particular the possibility of directed targeting and siRNA technologies. Then we will focus on the use of
liposomes for selective drug delivery in cancer. Finally, the conjugation of AuNPs and liposomes for
combined therapeutic strategies against cancer will be discussed.
Nanomedicine 50
RNAi-based cancer therapy – going nano

RNAi is a conserved biological process, thought to have evolved as an innate defence mechanism
against foreign genetic material [6], that plays a crucial role in transcriptional and post-transcriptional
gene regulation [7, 8]. RNAi relies on short antisense RNAs (siRNA) to repress translation or to degrade
cytoplasmic messenger RNA (mRNA) of homologous sequence by post-transcriptional gene silencing
triggered by double–stranded RNAs (dsRNAs) within the cell [7, 9]. Perfect complementarity to the
target mRNA induces cleavage of this transcript at a very precise location [10], followed by its
degradation by cellular exonucleases [11]. Chemically synthesised siRNAs can be introduced into the
cytoplasm of cultured mammalian cells as a means to manipulate gene expression, sometimes able to
bypass the initiation phase and transiently trigger the RNAi biological response, leading to strong and
sequence-specific silencing of gene expression [12]. There are currently two ways to ‘artificially’ trigger
the natural RNAi process in mammalian cells: either by applying siRNAs directly to cells or by
engineering small hairpin RNA (shRNAs) structures and inducing their expression in the cells, where
they get processed into siRNAs.
RNAi-induced gene silencing mirrors the inhibitory effects of protein-based drugs (e.g. antibodies and
vaccines) and small molecules that block the function of their targets, with several additional
advantages. First, it can overcome the problem of “non-druggable” disease targets, such as non-
enzymatic molecules or proteins which conformation is not amenable to conventional drugs or small
molecule compounds [13-15]. It can also limit resistance mechanisms such as those caused by anti-
angiogenic molecules [16]. Second, because a single mRNA molecule translates into many protein
copies, targeting the mRNA is more efficient than blocking the function of its products [15]. Many
siRNAs can dampen target gene expression while simultaneously stimulating the immune response in a
sequence-independent manner, stimulating dendritic cells to respond immunologically to cancer cells,
while reducing blood and lymphatic vessel growth, thus inhibiting angiogenesis [17-20]. Despite these
advantages offered by RNAi-based systems, one of their major challenges stems from the need for
intracellular delivery, unlike some of the protein-based and small molecule drugs.
Besides ensuring the chemical and biological stability of siRNAs, one needs to carefully consider their
effective entry into the target cells and the associated biological processes, as the extent of RNAi-
mediated gene downregulation largely depends upon this step [21]. In addition, naked siRNAs
delivered systemically are quickly cleared by the kidney and eliminated, or degraded by serum
exonucleases [22]. These obstacles have prompted the development of a wide array of gene/RNAi
delivery vehicles and their application through two major delivery technologies: (1) Transfection
systems (siRNAs and non-viral expression vectors), which involve complexing siRNAs or RNAi trigger-
expressing plasmids with a carrier, most often lipid-based vesicles or cholesterol, to allow them to
transpass the cell membrane while protecting siRNA against degradation; (2) Transduction of viral
vectors [20]. A more detailed summary of the various approaches for delivering siRNAin vitro and in
vivo can be found in some recent reviews [23-28]. Among the more traditional delivery systems,
cationic liposomes and viral vectors are the most commonly used, some of which have reached clinical
trials. However, all of them present safety concerns and problems both in vitro and in vivo, such as: (1)
immune recognition for most viral systems; (2) mutagenic integration for some viral systems; (3)
inflammatory toxicity, rapid clearance and low efficiency in primary and non-dividing cells for
liposomes [22]; (4) lack of a cell specific targeting mechanism for all traditional delivery systems; (5)
limited endosomal/lysosomal escape of RNAi-effector molecules for non-viral systems; (6) limited
release of RNAi-effector molecules from the non-viral carrier leading to the need to administer large
amounts of siRNA for efficient gene silencing [29-31]. Nanotechnology has allowed to overcome some
Nanomedicine 51
of these obstacles while providing for effective RNAi delivery to target cells with extremely promising
clinical efficacy.
Gold nanoparticles: powerful vehicles for RNAi delivery
Gold nanoparticles (AuNPs) have emerged as versatile, selective and highly multifunctional anti-cancer
therapeutics due to their unique properties, and represent a powerful alternative to the conventional
RNAi delivery systems [32]. AuNPs can be easily synthesised in a wide range of sizes (e.g. 1 to 100 nm)
and shapes (e.g. nanospheres, nanoshells, nanorods), and their surface functionalised with several
biomolecules, providing specific functions, targeting selectivity and stability in biological environments
[33, 34]. In addition, AuNPs present particular optical and electronic properties arising from their
reduced size, which we can take advantage of for both therapeutic and bioimaging purposes [32, 34].
AuNP conjugates can also be delivered systemically, with long circulatory half-lives while eliciting low
immunogenic responses [32]. In the following sections we will further discuss some of the properties
AuNPs offer, with special emphasis on their application as RNAi delivery systems for improved cancer
therapeutics.
AuNP-siRNA conjugation
When conjugated to AuNPs, siRNAs have been shown to exhibit increased stability, cellular uptake and
efficacy in physiological conditions, retaining the ability to act through the RNAi pathway [26, 35]. The
first demonstration that DNA-AuNP conjugates could be easily internalised into cells, without the need
for transfection agents, and induced gene silencing by an antisense mechanism was reported by Rosi
and co-workers in 2006 [36]. This remarkable study prompted others to use AuNPs as siRNA delivery
systems and contributed to the development of many strategies to improve intracellular siRNA delivery
in vitro and in vivo. These strategies can be grouped into two major categories that are currently used
for tethering siRNAs to AuNPs, namely (1) the gold-thiol bond and (2) electrostatic interactions. Both
categories involve, in some way, the use of poly(ethylene glycol (PEG) or other passivating agents (see
2.2) for stabilisation and to promote endosomal escape of the AuNP conjugates into the cell cytoplasm
[28].
Gold-thiol bond
The gold-sulphur interaction is a strong but dynamic, quasi-covalent bond that can be dissociated
under reductive conditions, such as in the presence of high glutathione concentrations in the cell
cytoplasm, due to oxidation of thiol groups [28]. However, gold-thiol bonds are formed easily and its
dynamic structure has been exploited for the formation of AuNP-siRNA conjugates [28]. Oishi and co-
workers were the first to report the conjugation of thiolated siRNAs (SH-siRNA) with AuNPs for gene
silencing [37]. The authors decorated 15 nm AuNPs with a thiolated poly(ethylene glycol)-block-poly(2-
(N,N-dimethylamino)ethyl methacrylate) copolymer (SH-PEG5000–PAMA7500), followed by
immobilisation of thiolated siRNAs (SH-siRNA) directly onto the AuNPs. With these conjugates, where
the size of siRNAs (~14 kDa) and the co-loaded PEG-PAMA (~12.5 kDa) were equivalent, they reported
~45 siRNA molecules per AuNP. Following 24h incubation with 100 nM of siRNA targeting the firefly
luciferase gene, they observed a 65% downregulation of luciferase expression in HUH-7
hepatocarcinoma cells [37]. Giljohann and co-workers later reported a slightly different approach,
where the sense strands of the siRNA duplexes contained an ethylene glycol spacer and an alkylthiol
group (SH-PEG400-siRNA), the latter used to tether the siRNAs onto 13 nm AuNPs. The remaining AuNP
Nanomedicine 52
surface was further covered with PEG400 molecules for additional stability. The siRNA sequences were
designed to target the firefly luciferase gene and labelling of antisense strands with Cyanine 3 (Cy3)
fluorescent dye was used to determine siRNA loading. In this system, PEG molecules are significantly
smaller that the siRNAs, however the authors report a loading of ~33 siRNAs per AuNP, which is similar
to that of the previous study. A 70% luciferase knockdown was observed following a 4 day-incubation
with 100 nM of siRNA in HeLa cells [35]. These authors made a few other interesting observations: (1)
internalisation of AuNP conjugates was detected 6h after incubation, as revealed by fluorescence
imaging, and confirmed in over 99% of the population by flow cytometry; (2) in stability experiments,
AuNP-siRNA conjugates revealed a 6 times greater half-life than molecular siRNA duplexes in 10%
serum (816 ± 59 min vs. 133 ± 30 min), as determined by fluorescence measurements, indicating a
protective effect on siRNAs owing to conjugation with AuNPs; (3) transfection of the same number of
luciferase siRNA duplexes (100 nM) using the commercial agent Lipofectamine 2000 showed a less
efficient luciferase knockdown compared to delivery of AuNP-siRNA conjugates, 4 days after treatment
(33 ± 2% for lipofectamine-siRNA vs. 73 ± 7% for AuNP-siRNA) [35].
Others have reported comparable silencing of luciferase by 13 nm PEGylated AuNPs functionalised with
thiolated siRNAs and Lipofectamine complexed siRNA, but AuNPs-siRNA elicit lower innate immune
response (about one third less) [38]. IFN-β is a cytokine produced by host cells in response to foreign
nucleic acids or microorganisms that contributes to activation of the innate immune system. Activation
of the innate immune system can result in adverse stress toxicity by triggering signalling events that
induce cell death, sequester immune cells and activate the adaptive immune response. These events
can challenge siRNA therapy, in which cases they should be avoided. In this study, IFN-β levels inversely
correlated with the nucleic acid density at the surface of AuNPs, suggesting that tightly packed siRNAs
on AuNP surfaces can lessen the immune response [38]. Interestingly, increased packing of siRNAs onto
AuNPs also contributes to increased stability and subsequent increased cellular internalisation of the
conjugates [39, 40]. Mirkin and co-workers have used a pharmacological approach to show that, unlike
the common belief that serum proteins aid in the cellular uptake of oligonucleotide-functionalised
AuNPs, their entry is actually mediated by scavenger receptors on the cell surface and increases
proportionally to the density of oligonucleotides on the AuNP surface [40].
In a similar approach, Lee and co-workers have exploited the photothermal properties of near infrared
(NIR) absorbing Au nanorods (AuNRs) for a facilitated and controlled intracellular delivery of AuNP-
siRNA conjugates and knockdown of oncogenic genes [41]. Here, antisense oligonucleotides targeting
the breast cancer biomarker HER2 gene were hybridised with thiol-modified sense oligonucleotides
previously linked to AuNRs. When irradiated with NIR light of appropriate wavelength, AuNP
photothermal heat causes denaturation of the double-stranded oligonucleotides, releasing the
antisense strands. The system was optimised for minimal cellular photodamage while allowing spatial
and temporal control of the effector gene silencing oligonucleotides. Using this approach, they
observed ~10% increase in HER2 downregulation in breast carcinoma cells (BT474) after treatment and
irradiation of AuNRs compared to controls (no AuNRs, no NIR or scrambled sequences) [41]. Braun et
al. employed 40 nm Au nanoshells to improve endosomal escape and achieve spaciotemporal silencing
of a reporter gene (GFP) using a similar NIR photothermal approach, but this time Au nanoshell-siRNA
conjugates were further coated with a TAT lipid cell internalizing peptide (derived from HIV-1 trans-
activator peptide) [42]. In contrast with the previous study, these authors observed laser cleavage of
the Au-sulphur bond with concomitant siRNA release which, however, remained in endosomes unless
these were ruptured by more disruptive nanoshell heating (higher laser power). Upon nanoshell
surface release and endosomal escape, GFP-targeting siRNAs enabled reporter gene silencing
comparable to Lipofectamine transfection [42].
Nanomedicine 53
Another way to covalently link siRNAs to AuNPs is by first loading AuNPs with a SH-PEG-NH2 and then
conjugating the siRNAs to the terminal amine of the PEG using a disulphide crosslinker, N-succinimidyl
3-[2-pyridyldithio]-propionate] (SPDP). Using this approach, Lee and co-workers reported a loading of
~30 siRNAs per AuNP (15 nm), in concordance with the previous studies, which were then coated with
the positively charged, terminally-modified poly(beta-amino ester)s (PBAEs), previously shown to
facilitate intracellular DNA delivery. Two out of 14 screened PBAE coatings showed efficient in vitro
delivery of the corresponding AuNP conjugates, comparable to that of the commercial Lipofectamine. A
120nM siRNA dose for 24h resulted in over 90% downregulation of luciferase expression in HeLa cells
for AuNP conjugates bearing these PBAE coatings, but AuNP-siRNAs without PBAEs did not reveal any
silencing capability [43]. This was probably due to a less efficient cellular internalisation of the AuNP
conjugates lacking PBAE and shows that for each system, the AuNP surface properties need to be
carefully studied and controlled to ensure efficient cellular uptake and subsequent gene silencing.
Electrostatic interactions
As an alternative to the covalent Au-thiol conjugation, siRNAs can be linked to the AuNP surface by
electrostatic interactions. Generally, AuNPs are synthesised via citrate reduction, which makes their
surface negatively charged due to citrate adsorption. Given that siRNA is also negatively charged,
several approaches have used in a layer-by-layer format, incorporating a positively charged polymer,
e.g, poly(ethylene)amine (PEI), between the Au-citrate core and the siRNA. AuNPs within this system
can have either siRNAs or PEI exposed in the terminal layer, with final sizes ranging between 20-25 nm.
Their loading capacity can be ~780 siRNAs per AuNP, which is much higher than that of AuNPs bearing
thiolated siRNAs [44]. While AuNPs bearing siRNAs in the final exposed layer appear to have increased
cellular uptake, they were shown by transmission electron microscopy (TEM) to be trapped within the
endosome and, presumably for that reason, resulted in no reporter gene silencing, even when
considerable amounts of siRNA (~288 nM) were used. AuNPs containing an external PEI layer resulted
in 70% reporter gene downregulation 48h post-treatment [44]. A pH sensitive anionic charge-reversal
polyelectrolyte layer has also been incorporated between PEI layers, allowing disassembly of the
complex upon acidification inside endosomes. This strategy has been shown to improve endosomal
escape and to result in knockdown efficiencies comparable to those of Lipofectamine siRNA
transfections [45].
Other strategies involve changes to the traditional citrate reduction synthesis method, such as: (1)
replacing the citrate by PEI (as the reducing and stabilizing agent) during AuNP synthesis, yielding PEI-
capped AuNPs with a positively charged surface that can be electrostatically covered with siRNAs.
Using this strategy, Song et al have demonstrated efficient knockdown of the oncogene polo-like
kinase 1 (PLK1) followed by induction of apoptosis in MDA-MB-435s breast cancer cells [46]; (2)
incorporating a stabilizing cationic polymer, poly(N-2-hydroxypropyl methacrylamide(70)-block-N-[3-
(dimethylamino)propyl] methacrylamide(24) [P(HPMA(70)-b-DMAPMA(24))], which conveys a non-
immunogenic, hydrophilic and sterically stabilizing shell (similar to the role of PEG in the previously
discussed particles) and a positively charged surface, amenable to siRNA linkage [47]; (3) incorporating
cysteamine hydrochloride to produce amine-functionalised AuNPs to which siRNA-PEG moieties can be
added by electrostatic interactions between siRNAs and the positive AuNPs [41].
Biocompatibility, biodistribution and targeting of AuNPs
Non-functionalised AuNPs are prone to non-specific protein adsorption, to degradation and to

aggregation in biological media [48]. Surface conjugation with chemical functional groups or
Nanomedicine 54
biomolecules serves multiple purposes and confers to AuNP conjugates three major properties that are
crucial for biomedical applications: biocompatibility (and colloidal stability), targeting specificity and
functionality. Au surfaces have affinity for a number of organic molecules (e.g. thiolates, dithiolates,
amines, carboxylates, cyanides, isothiocyanates, phosphines), which self-assemble in high coverage
monolayers through chemical bonds that, in most cases, form rapidly and spontaneously at room
temperature ([32, 48].Proper surface functionalisation of AuNPs determines their interaction with the
environment and can be tailored for specific applications [49].
The biodistribution and pharmacokinetics of AuNPs depend greatly upon their physicochemical
properties, including size, shape, charge and surface coating. Physiological environments, including cell
culture media, can compromise the stabilising capacity of many AuNP ligands (compared to water and
other low ionic strength media) because of their high salt and serum content, where attractive forces
between AuNPs often overrule electrostatic repulsions leading to aggregation [32]. Biocompatibility
and stabilisation of AuNPs in biological fluids is thus achieved by decorating AuNPs with hydrophilic
polymers, such as PEG, polylysine (PLL), polystyrene sulfonate (PSS), starches and polyvinylpyrrolidone
(PVP) [32]. The modification of AuNP surfaces with PEG moieties, or PEGylation, is the most commonly
used approach in biomedical applications, and has been shown to increase AuNP stability in high salt
concentrations and in biological environments [50]. Thiol groups bind with great affinity to noble metal
surfaces, in particular to gold (bond strength ~44 Kcal/mol; [51], and therefore PEG molecules bearing
terminal thiol groups are often used.
When delivered systemically, AuNPs must travel in the blood stream, undetected by the immune
system, long enough to reach the target tumour tissue, before they can extravasate into the tumour
interstitial space. Circulating plasma proteins, known as opsonins, can bind to NPs or other foreign
small molecules and microorganisms and remove them from circulation through the mononuclear
phagocyte system (MPS), composed mainly of monocytes and macrophages. Besides conferring
stability to AuNPs in physiological environments, PEGylation (or functionalisation with other hydrophilic
polymers) results in their increased half-life blood circulation and subsequent tumour accumulation, by
preventing adsorption of proteins and molecules involved in the phagocytic uptake [52-57].
As discussed above, tumours develop by accumulating genetic mutations that confer growth
advantages to certain populations of cancer cells, which progress to divide rapidly and uncontrollably.
In order to sustain this uncontrolled growth, tumours require oxygen and nutrients, which they acquire
by recruiting new blood vessels in a process known as angiogenesis. These new blood vessels are
formed rapidly upon tumour demand and as a consequence exhibit a disorganised, defective and leaky
endothelium with large gaps or fenestrations (600-800 nm) into the tumour interstitial space. In
addition, the growing tumour mass exerts damaging pressure onto the lymphatic system, which
collapses and fails to efficiently drain fluids out of the tumour. These unique characteristics of most
solid tumours contribute to what is known as the enhanced permeability and retention (EPR) effect –
see Figure 3.1. The EPR effect sets the stage for the so called passive targeting of AuNPs and other
nanosized circulating molecules, which gain access to the tumour by passively diffusing through the
large fenestrations on angiogenic vessels and remain at the tumour site due to its defective lymphatic
drainage [34, 58, 59]. Passive targeting via the EPR effect of tumours has been shown in vivo for a
number of nanostructures, including micelles, liposomes, metal NPs and quantum dots, as well as for
several protein/DNA/polymer complexes, and preferential tumour accumulation is expected for
particles with hydrodynamic diameter above the renal clearance threshold and up to two micrometres
[32, 60]. Very small particles tend to easily extravasate through the leaky capillary walls but are often
pushed out of the tumour by blood, thus showing good permeability but poor tumour retention.
Conversely, tumour access of large particles is limited by the pore cut-off size of tumour blood vessels,
Nanomedicine 55
which can be as small as 7-100 nm for tumours such as gliomas and ovarian cancer [61, 62]. The type of
cancer, stage and location also influence the optimal size for EPR of AuNP conjugates [32].
Other biological barriers need to be addressed for AuNPs to reach the target site, e.g. the liver, kidney
and lymphoid organs. They must be small enough (≤ 100 nm) and possess negative or neutral surface
charge to avoid uptake by the MPS, but large enough (≥ 6 nm) and hydrophilic or neutral to thwart
rapid renal clearance. AuNPs bearing positively charged coatings tend to stick non-specifically to cells,
and therefore neutral AuNPs are preferred for extended blood circulation time [63]. Upon intravenous
injection into rats, spherical AuNPs of sizes between 10 nm and 250 nm have been shown to
accumulate preferentially in the liver and spleen, whereas AuNPs smaller than 10 nm were more
broadly distributed [64]. A strong negative charge on the particle surface also leads to accumulation in
the liver due to phagocytic uptake [63].
FIGURE 3.1
The potential of combined therapy mediated by AuNPs and liposomes capable of passive and active targeting.
When administered intravenously, both AuNPs and liposomes tend to extravasate through the tumour
defective endothelium into the tumour interstitial space. With the aid of active targeting moieties at
the surface of both nanoparticles, they specifically bind to the surface of tumour cells and enter them
mostly by receptor-mediated endocytosis. Once inside tumour cells, different strategies can be used to
promote endosomal escape and delivery of effector molecules to the cytosol, where they perform their
therapeutic function.
Cellular internalisation and active targeting
Experimental studies reveal that many macromolecules and nanomaterials are actively internalised
into cells via endocytosis, a vesicular transport mechanism that embraces different pathways,
collectively known as phagocytosis and pinocytosis [65]. Clatherin mediated endocytosis (CME) is the
best-characterised mechanism and, when receptor mediated, it results in the internalisation of several
ligands, such as low-density lipoprotein (LDL), transferrin, growth factors and insulin [66]. Caveolin-
mediated endocytosis (CvME) is the most common clatherin-independent uptake pathway and is
involved in the uptake of smaller molecules. Both CME and CvME pathways have been shown to
Nanomedicine 56
mediate cellular internalisation of AuNPs [67, 68]. However, the route and degree of cellular
internalisation depends on the size [69, 70], shape [69], surface charge [71] and functionalisation [68]
of the AuNP conjugates, as well as on the temperature [67, 72] and cell type [68], among other factors.
One of the major advantages AuNPs offer is the opportunity for active targeting. Taking advantage of
tumour molecular markers as docking sites to concentrate the therapeutic effect at tumours, it is
possible to increase therapeutic efficacy while reducing systemic exposure and off-target effects.
Several of these tumour molecular markers are surface proteins/receptors present in cancer cells and
in tumour vasculature that are not expressed or are expressed at much lower levels in normal cells,
thereby distinguishing tumour masses from the surrounding normal tissues [73]. The surface of AuNPs
can be modified to accommodate targeting biomolecules that allow specific biochemical interactions
with surface proteins/receptors highly expressed on target cells. Tumour-targeting biomolecules
currently in use include monoclonal antibodies, peptides/proteins [e.g. transferrin, epidermal growth
factor (EGF)], folic acid, carbohydrates and DNA/RNA [33, 49, 59]. For example, the anti-epithelial
growth factor receptor (EGFR) monoclonal antibody has been used as an active targeting agent, since
EGFR and its ligands are commonly overexpressed in a variety of solid tumours [74-76]. The targeting of
tumour angiogenic vessels is also gaining increased attention, as it may improve therapeutic efficiency
in cases where tumour cells are less accessible [73]. In combination with passive targeting strategies,
active targeting may enhance receptor-mediated endocytosis of the AuNP conjugates in target cells
[69, 77]. The intracellular localisation of effector molecules can be further directed with the use of cell-
penetrating peptides, which are short peptides that facilitate the delivery of various cargoes to cells, or
with nuclear localisation sequences, which direct cargos to the nucleus [78-80]. Decorating AuNPs with
proton sponge groups or using photothermal heating can further assist escape from endosomal
sequestration/degradation [81]. Active targeting by AuNPs [82-84] has been shown to result in greater
tumour accumulation than passive targeting, when AuNPs are administered systemically in vivo (6-13%
versus 2-5%) [82, 83].
AuNP-RNAi conjugates for cancer therapy: in vitro and in vivo studies
Over the past several years, many studies have demonstrated promising proofs of concept for
therapeutic applications of AuNPs. Specifically, numerous studies have demonstrated the potential of
AuNPs to deliver RNAi effector molecules specifically to target cancer cells, with subsequent
knockdown of tumorigenic proteins [43, 85-87]. However, in vivo studies using this system are still
scarce, alerting us for the need to overcome remaining barriers that prevent its translation into the
clinics. In this section, we summarise some of the milestone studies that hopefully can lead us to
improved therapeutic approaches against cancer.
Zhang and co-workers have developed an anti-metastasis therapy consisting of AuNRs conjugated
electrostatically with siRNAs, which targeted the protease-activated receptor 1 (PAR-1). These
conjugates were then delivered to highly metastatic human breast cancer cells. The authors observed
efficient downregulation of PAR-1 mRNA and protein levels and decreased metastatic ability of the
cancer cells [88]. Another interesting study showed the development of single-stranded DNA-
functionalised AuNPs (via a thiol-Au bond) as a general gene delivery system (AuNP-GDS) for loading
and delivering DNA oligonucleotides and shRNAs [36]. This platform allows any short nucleic acid to be
hybridised to the cargo DNA covalently linked to the AuNP, the former which can be designed for a
specific purpose, such as gene knockdown, redirection of alternative splicing, and modulation of signal
transduction pathways. Using this system, the authors delivered shRNAs targeting the Mcl-1L mRNA to
a xenograft tumour in a mouse model, and showed a ~26% reduction in protein expression which was
sufficient to induce apoptosis of the xenograft tumour cells [89]. These studies did not include a
Nanomedicine 57
targeting strategy because they were performed either in vitro or in vivo by directly injecting the
conjugates into tumours. However, for systemic delivery, an additional targeting moiety is generally
required to improve treatment efficacy and reduce off-target effects. Lu and co-workers used Au
nanocages targeted to folate receptors (overexpressed in many types of cancer) and carrying a siRNA
against the NFkBp65, which encodes a transcription factor highly involved in tumour formation and
progression. They injected these constructs intravenously into in nude mice bearing HeLa cervical
cancer xenografts and observed a significantly higher tumour uptake of the targeted conjugates
compared to the non-targeted ones. They additionally took advantage of the photothermal properties
of the Au nanocages to achieve a controlled cytoplasmic delivery of siRNA upon NIR light irradiation
and observed efficient NF-kappaB p65 downregulation only when tumours were irradiated with NIR
light [81]. Conde et al have recently demonstrated the efficiency of multifunctionalised AuNPs for the
specific silencing of the c-myc proto-oncogene in three biological systems of increasing complexity,
namely in vitro cultured human cancer cells (HeLa), in vivo invertebrate (freshwater polyp, Hydra) and
vertebrate (mouse, C57BL/6j) models. Their AuNPs were functionalised with PEG, cell penetration (TAT)
and cell adhesion peptides (RGD, which binds to the integrin αVβ3 receptor family; *90], and c-myc
targeting-siRNAs [91]. They have also shown in a more recent study that these same nanoconjugates
are capable of targeting tumour cells in a lung cancer murine model and of inducing significant
downregulation of the c-myc oncogene, followed by tumour growth inhibition and prolonged survival
of lung tumour bearing mice [92].
Toxicity evaluation of AuNPs for therapy
As the utility of AuNPs largely depends on the degree of inherent toxicity, studies on the toxicological
profile of AuNPs are of utmost relevance before they can be used for cancer treatment and
management. AuNPs have been considered safe for long due to the biocompatibility and general
inertness of gold in its bulk form [93]. Indeed, a few studies support the safety of these nanocarriers in
vitro and in vivo using animal models [94-97]. However, as more studies on the use of AuNPs for gene
delivery have been reported, evidence of cytotoxicity has increased dramatically [98-101]. On their
review about the toxicity and cell uptake of AuNPs, Alkilany and Murphy addressed a fundamental
issue: the effect of the supernatant in cell viability should always be assessed in order to conclude that
the observed toxicity is really due to the nanoparticles and do not result from any toxic component
used for their preparation [102]. Also, because toxicity experiments typically rely on the use of
colorimetric techniques to measure cell viability, care should be taken to ensure that the dye is neither
being adsorbed on nanoparticles nor reacting with them and that the photochemical properties of the
AuNPs are compatible with the selected methodology, therefore being advisable to compare the
results obtained from multiple methods [103, 104].
Toxicity of AuNPs is generally accepted to be dependent on particle size, shape, and surface charge and
chemistry [105-108]. For instance, very small particles (1.4 and 5 nm in diameter) seem to be capable
to enter the nucleus, where they can interact with DNA and cause molecular disturbance [109, 110].
Larger particles (16 and 33 nm) are retained in endosomes and accumulate in the periphery of the
nuclear region [111, 112]. At least three different studies reported that cellular uptake of AuNPs reach
maximum levels for a particle size of about 50 nm [67, 72, 113]. Also, surface functionalisation seem to
be capable of inducing higher level of apoptotic cell death, probably related to increased cell uptake
when compared to unmodified 40 nm AuNPs [72]. According to data from in vitro studies, AuNP
toxicity is believed to result mainly from the induction of oxidative stress [95, 98, 101]. Indeed,
upregulation of stress related genes was found to result from cell exposure to AuNPs, which also
promoted the downregulation of cell cycle related genes [101, 114, 115]. Nevertheless, most of these
Nanomedicine 58
studies paid little attention to genome damage, such as DNA strand breaks and nuclear abnormalities,
or characterization of protein markers for toxicity. An integrated toxicology evaluation encompassing
DNA damage, stress related enzymes and a proteome profiling approach showed no significant
cytotoxicity of PEGylated AuNPs and no upregulation of proteins related to oxidative damage [97].
Also, both positive and negatively charged AuNPs were found to be similarly more cytotoxic against
human keratinocytes (HaCaT cells) when compared to neutral AuNPs, with LD 50 values of roughly half
of those determined for the latter [116]. Despite the disruption in cell morphology and the dose-
dependent toxicity observed for all three types of AuNPs, both anionic and cationic AuNPs induce
mitochondrial stress and apoptosis in opposition to the necrotic cell death caused by neutral particles
[116]. Another in vitro study comparing positive and negatively charged AuNPs reported that cationic
NPs were far more toxic to Cos-1 cells, human red blood cells and E. coli than anionic NPs, possibly as a
result of cell lysis, as shown by a dye leakage technique [107]. However, Alkilany and co-workers clearly
showed that serum proteins become readily adsorbed to the surface of charged NPs, inducing an
inversion of surface charge in particles that were originally cationic [117]. This would reduce
electrostatic interaction between the original positive NPs and the negative cell membrane, the first
step towards cell lysis mediated toxicity of cationic NPs [107].
Regarding in vivo experiments, several studies have demonstrated that AuNPs of 50 nm and larger
were non-toxic to mice, conversely to what has been observed for AuNPs <40 nm [108, 118]. In fact,
there are concordant data from different studies on the biodistribution and accumulation of AuNPs in
mice showing that most of the intravenously injected nanoparticles are retained in the liver, regardless
of their size [64, 119, 120]. There is also an agreement in that AuNPs have the ability to transpose the
blood-brain barrier and thus reach the brain, with a cut-off limit in diameter of around 20 nm [121],
and that smaller particles have the most widespread organ distribution [64, 119, 120]. Organ
distribution seems to be ruled by a more or less complex relationship with nanoparticle size. For
instance, it is known that renal excretion of AuNPs is maximized for a narrow size range of 6-8 nm,
resulting in an accelerated clearance rate [122]. Despite the valuable use of animal models, the effect
of size on the toxicity of AuNPs in humans is difficult to predict since the size of endothelial cells'
fenestrae is highly variable between individuals, thereby affecting nanoparticle clearance [123].
Therefore, more consistent data on the toxicological profile of AuNPs in vivo is necessary. For a more
complete review on biodistribution, encompassing earlier studies and administration routes other than
intravenous injection, see Khlebtsov and Dykman [121]. Furthermore, core size, charge and surface
chemistry of AuNPs seems to correlate to toxicity on the development of zebrafish embryos, with
positive and negatively charged AuNPs causing mortality and malfunctions to the embryos, respectively
[100]. Adverse effects were also found in the model system Drosophila melanogaster after exposure to
citrate-capped AuNPs, which were shown to reduce fertility in a dose-dependent manner and also the
life span [98, 99].
Nonetheless, long-term studies in higher organisms are necessary to further characterise the safety of
AuNPs as therapeutic agents, so they can be safely administrated to humans without concerns about
late toxicity symptoms.
Liposomes for drug delivery - Great things sometimes come in small

packages
Conventional cancer chemotherapy deals with poor therapeutic efficacy and increased toxicity to
normal tissues, due to reduced drug selectivity. Nanomedicine has provided valuable concepts to
Nanomedicine 59
develop systems that could unambiguously increase drug concentration at the target site and, thus,
increase the therapeutic index of drugs [124]. Nanoparticles have the ability to carry large payloads, to
protect the agent of interest and to improve the bioavailability of water-insoluble drugs. In addition,
incorporating imaging agents into nanoparticles allows the in vivo visualisation of biological processes
occurring at the cellular and/or molecular level, revealing the outcome of a personalised therapeutic.
Lipid base nanoparticles (e.g., liposomes, solid lipid nanoparticles, oily suspensions, lipid microbubbles,
lipid microspheres) [125] have attracted much attention, and nanosystems are currently being
introduced into clinical use and several more under clinical trials or pre-clinical development (Table 3.1)
[126-128].These self-assembling lipid-based nanovesicles can be tailored to meet the individual
biological characteristics of the target and are especially used as drug-delivery carriers, but recently
there has been a growing interest in protocols for image-guided drug delivery [129-131]. Liposomes are
considered highly attractive particles, as they portray low toxicity while permitting controlled drug
release and targeting. Moreover, liposomes present both an aqueous cavity and hydrophobic
membrane, allowing incorporation of both lipophilic and hydrophilic drugs. Furthermore, in a
pharmaceutical perspective, large-scale production of liposomes is feasible.
The use of modified phospholipids allows the introduction of functional groups, such as maleimide,
that prompt conjugation to antibodies or other ligands. Liposomes can be loaded with a variety of
water-soluble and insoluble drugs [132] and the entrapment of the chosen drug can be achieved
through several different processes, including (i) encapsulatation of drugs into the aqueous cavity of
the vesicle, (ii) incorporation of lipophilic drugs in the membrane bilayer, (iii) active entrapment
methods such as pH gradient protocols and (iv) electrostatic interactions between drugs and the
liposome membrane. The characteristics of liposomes can be tuned by changing lipid composition,
pulling off apposite physico-chemical properties, such as size, zeta potential (surface charge),
permeability and stability. Liposomes can be classified according to the number of lamellae (uni, oligo-,
and multi-lamellar vesicles), size (small, large or giant), and net charge (cationic, anionic and neutral
nanoparticles) [126, 133].
Nanomedicine 60
TABLE 3.1
Selected Liposomal formulations clinically approved or under clinical trials.
Composition Trade name Indication Administration

Liposome formulations under clinical trials
Phase I – Acute lymphocytic
Liposomal
L-Annamycin leukaemia, acute myeloid I.V.
annamycin
leukaemia
Phase II - Progressive
Liposomal cisplatin SLIT Cisplatin osteogenic sarcoma metastatic Aerosol
to the lung
Liposomal
Sarcodoxome Phase I/II – Soft tissue sarcoma I.V.
doxorubicin
Phase II – Postoperative
Liposomal fentanyl AeroLEF Aerosol
analgesic
Liposomal
OSI-211 Phase II - Ovarian cancer I.V.
Iurtotecan
Liposomal
Onco TCS Non-Hodgkin’s lymphoma I.V.
Vincristine
Liposomal Paclitaxel LEP-ETU Phase I - Advanced Cancer I.V.
Liposomal
Phase II - acute myeloid
doxorubicin/vincristi CPX-351 I.V.
leukaemia
ne
Liposomal
Phase II - Colorectal cancer
irinotecan CPX-1 I.V.
treatment
/floxuridine
Clinically approved liposomal formulations
Liposomal Abelcet and
Fungal Infections I.V
amphotericin B Ambisome
Liposomal Malignant lymphomatous
Depocyt I.T
cytarabine meningitis
Liposomal
Daunoxome HIV-related Kaposi’s Sarcoma I.V
daunorubicin
Liposomal
Myocet Metastatic Breast cancer I.V.
doxorubicin
HIV-related Kaposi’s Sarcoma,
Liposome-PEG
Doxil/Caelyx Metastatic breast and ovarian I.V.
doxorubicin
cancer
Liposomal IRIV Inflexal
Influenza/Hepatitis A I.M.
vaccine V/Epaxal
Liposomal morphine DepoDur Postsurgical analgesia Epidural
Micellular estradiol Estrasorb Menopausal therapy Topical
Note: I.V.- intravenous; I.M.- intramuscular; I.T.- intrathecal.
Nanomedicine 61
Towards optimised properties - size and surface charge
Liposomes are considered suspensions that can be used in several therapeutic platforms, such as
aerosol and lotion, but intravenous administration is the most common route for drug administration.
As already discussed, in vivo circulation and biodistribution of nanoparticles depends on several
physicochemical parameters, and it is clear that the size and surface charge play a major role in
determining their fate, where small vesicles can be rapidly cleared by the kidneys and larger
nanoparticles may be captured by the MPS [134, 135]. Also, following systemic administration,
nanosystems accumulate differently along the organism – lung tends to capture micron-sized particles,
the MPS engulfs medium sized ones and smaller particles are able to extravasate and accumulate via
the EPR effect and/or cleared by the kidney.
Once in circulation, larger nanoparticles are rapidly opsonised, recognised and captured by the immune
system. Fang and co-workers showed that protein adsorption on smaller particles is much lower than
on larger particles, which correlated with a higher uptake of larger nanoparticle by macrophages [136].
Dexi Liu and co-workers in the early 90’s showed that liposomes’ surface could become highly
hydrophilic via covalent attachment of polymers (such as PEG), helping these vesicles to overcome
MPS, thus increasing their blood circulation half-life [137]. It was demonstrated that 4h after injection
of stealth-liposomes (functionalised with PEG), those smaller than 70 nm tended to accumulate in the
liver, while the larger ones were essentially gathered in the spleen. Additionally, as already discussed,
particle size may affect tumour uptake and also influence the mechanism of cellular internalisation -
clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-
independent endocytosis, or phagocytosis in immune cells -which have tremendous influence on the
fate of the particle and the outcome of the drug [138].
Concerning charge, liposomes can be characterised as cationic, neutral or anionic. Due to the natural
propensity to aggregate, bare neutral lipid-based nanoparticles are generally not used for drug
delivery. Instead, positively or negatively charged liposomes have been continuously used as vectors
for therapeutic proposes. Cationic liposomes enter cells by endocytosis induced by ionic attractions,
which are able to stabilize electrostatic interactions with the negatively charged cell membranes.
Krasnici and colleagues assessed the distribution of neutral, anionic and cationic liposomes and
revealed that cationic liposomes accumulate on the vascular endothelium while neutral and anionic are
promptly cleared after intravenous injection [139]. Hence, cationic liposomes are an efficient tool for
intracellular delivery but care must be taken to prevent non-specific internalisation [140]. The positive
charge of the liposome is often due to incorporation of cationic lipids, such as DOTAP (1,2-dioleoyl-3-
trimethylammonium propane), DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride) or DODAP (1,2-dioleyl-3- dimethylammonium-propane), and tent to be used in association
with nucleic acids (forming lipoplexes). A major drawback of cationic liposomes is the observed
correlation between their positive charge and high toxicity, an aspect that will be further discussed. As
a consequence, neutral and anionic liposomes become the natural alternatives and it has been
established that neutral particles suffer less opsonisation than charged particles. Another rather
remarkable study was performed by Lee and Huang [141], where folate-targeted pH-sensitive anionic
liposomes were prepared, to which DNA complexed to polylysine was added. It was detected that at
low lipid to DNA ratios, the overall charge of the particles waspositive and the cellular uptake was
independent of the folate receptor. On the other hand, using high lipid to DNA ratios (negatively
charged particles) the uptake was folate receptor-dependent. Hence, DNA delivery systems based on
anionic lipids are a possible alternative to cationic lipids with the major advantage of reduced
cytotoxicity (see section ‘Toxicity and bioavailability’) [142].
Nanomedicine 62
Passively targeted vs. ligand-targeted liposomes
To prevent detection by the immune system and increase their circulation half-life, “stealth liposomes”
were developed, in which the liposomal membrane could be functionalised with sialic acid derivatives
(monosialoganglioside (GM1)) that mimic the erythrocyte membrane. Another approach consisted in
shielding the membrane with a hydrophilic coating, generally with PEG, since it is used in several
approved formulations and is FDA approved, and because it prevents interactions with plasma
proteins, probably due to steric hindrance [143-145]. However, although it was thought that PEG was
not immunogenic, soon it was shown that the administration of particles functionalised with PEG
caused IgM production and accelerated blood clearance in a second dose, an effect that will be latter
discussed [146]. What is even more critical, some reports have evidenced that PEG impairs the uptake
of liposomes by the tumour cells, an issue that could compromise the effect of the drug [147]. An
approach used to tackle this issue consists in using “detachable” PEG conjugates (DSPE-S-S-PEG5000)
[148] or PEG-derivatised phospholipids with lengths different than those of the anchor molecules that
are effortlessly uncorked from the liposomal membrane. Although stealth liposomes have favourable
characteristics for clinical applications, there is still plenty to be done before their full potential can be
ascertained.
In order to be internalised by tumour cells, liposomes need to find and bind cancer cells, and active
targeting could enhance this association. In theory, active targeting would improve the therapeutic
index of the drug and would also minimize side effects to healthy cells. Targeting moieties include
antibodies, peptides, glycoproteins, carbohydrates, or specific receptor ligands [149] – see Table 3.2.
Liposomes that have the aptitude to target tumour cells but they still need to escape from the blood
stream and, as already mentioned, extravasation is still the limiting step. For instance, Park and co-
workers showed that liposomes conjugated to monoclonal antibody fragments specific for the HER2
receptor are taken up much more efficiently than are untargeted liposomes [150]. Presence of
targeting ligands on the liposomes’ surface clearly improves internalisation but success is highly
dependent on the proper choice of the ligand. Coupling of the targeting moiety to the liposomes can be
undertaken by covalently attaching the ligands to liposomes, functionalised with PEG-derivatised
phospholipids. Another method consists in the transference of the targeting moiety from a PEG-
derivatised phospholipid micelle to the liposome [151]. A major drawback of adding targeting moieties
to the surface of these nanocarriers is that it often leads to an increased immunogenicity, as peptides
and antibody fragments can be considered by the host immune system as antigens [152].
Nanomedicine 63
TABLE 3.2
Some functionalisation moieties for liposome and AuNP active targeting.
Targeting
Nomenclature Target Ref.
Moiety
Epidermal growth factor
Anti-EGFR (cetuximab) [153-156]
receptor (EGFR)
Anti- HER2 Epidermal growth factor
[157-159]
(Trastuzumab-Herceptin) receptor type 2
Antibody Anti-VEGF Vascular endothelial growth
[160, 161]
(bevacizumab) factor (VEGF)
CD19-expressing B-cell
anti-CD19 [151, 162, 163]
lymphoma
Anti-CD20 Ab (rituximab) B-lymphocyte antigen CD20 [164, 165]
Arg–Gly–Asp-based
αvβ3 integrin receptor [166, 167]
(RGD)
Peptides/
Arg-Lys-Lys-His (RKKH) α2β1 integrin receptor [168]
Proteins
Transferrin Transferrin receptor [83, 169, 170]
VEGF peptide VEGF receptor [171-173]
asialoglycoprotein (ASGP)
Lactose [174]
receptors
asialoglycoprotein (ASGP)
Galactose [175, 176]
receptors
Sugars
Mannose receptor
(scavenger receptors) in
Mannose [177]
Tumour associated
macrophages
Small Folic acid Folate receptor [178-181]
Molecules Hyaluronic acid CD44 receptor [182-184]
Toxicity and bioavailability
Liposomes are designed to improve pharmacokinetics and efficacy of bioactive agents, while reducing
systemic toxicity. Despite being generally considered as nontoxic, biodegradable, and biocompatible,
early release of the drug may not only increase undesirable toxicity against healthy cells but also lower
the amount of drug that will be available at the target site and thus promote the acquisition of
resistance arising from exposure to sub-lethal doses [185]. Drug release is dependent on the chemical
properties of the drug itself and on the liposome lipid composition. The lipid bilayers present low
permeability to hydrophilic molecules that are enclosed in the aqueous core, but are highly permeable
to hydrophobic drugs, such as paclitaxel, thus reducing their appropriate retention [186].
Besides the drug cytotoxicity concerns, several other liposome related characteristics, such as vesicle
size, surface charge, lipid composition, drug-to-lipid ratio and liposome functionalisation may
potentiate toxicity effects [187-190]. Some authors have reported that smaller liposomes are more
damaging to cell physiology in vitro, which may result from the higher curvature of the smaller particles
Nanomedicine 64
[191]. These data have been contradicted by in vivo studies reporting that reduction of liposome size is
accompanied by a decrease in toxicity [192-195].
Conventional first generation liposomes negatively or positively charged have been reported to be
toxic, rapidly removed from the circulation and too leaky to ensure appropriate stability in vivo. As a
result, the fluidity of those vesicles had to be subsequently reduced by incorporating cholesterol or
sphingomyelin [186, 194]. In particular, positively charged liposomes, such as those used to deliver
nucleic acids for gene therapy, were shown to be highly toxic to macrophages, especially when
associated with the fusogenic lipid DOPE [196]. Moreover, cationic DOTAP nanoparticles were shown
to promote hepatotoxicity, as revealed by the release of the liver enzymes alanine aminotransferase
(ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and associated body weight
loss in mice [197]. Also, cationic DOTAP nanoparticles, unlike their uncharged and anionic counterparts,
are able to enhance cytokine and interferon response genes in T-cells, B-cells and monocytes [197]. As
such, cationic liposomes are even more likely to potentiate adverse immune reactions as well as
interact with serum proteins that are negatively charged [198]. Even though an increasing number of
cationic liposomes are used, studies have demonstrated that besides the formation of aggregates in
blood, and the tendency to induce inflammatory response, these liposomes still cause significant
toxicity, such as cell contraction and mitotic inhibition [196, 199, 200]. Cytotoxicity of cationic
liposomes arises from the inhibition of protein kinase C (PKC) activity by their tertiary or quaternary
amines, and from the interaction with other enzymes. Particularly, lipids bearing tertiary amines have
been shown to result in lower cytotoxicity when compared to those with quaternary ammonium
groups, such as DOTAP and DSTAP [201]. Nevertheless, induction of reactive oxygen species (ROS) was
observed in the presence of liposomes bearing either DOTAP or DSTAP, with the amount of generated
ROS correlating with the levels of cationic lipid present [191, 202, 203]. This has prompted researchers
to design new liposomal structures with modified cationic liposome structure [204]. For instance, lipids
whose hydrophilic groups contain heterocyclic rings are associated with lower cytotoxicity and also
higher transfection efficiency by allowing the delocalization of the positive charge [205]. Other
strategies include the transformation of the cationic group into a neutral or negative group by covalent
modification and the use of titratable lipids to formulate liposomes with pH-dependent surface charge
[206]. As such, these pH-sensitive liposomes can be designed to remain neutral at physiological pH,
only becoming positively charged when they reach acidic environments, such as those found in solid
tumours, hence preventing the negative effects of the cationic surface charge on systemic toxicity. In a
study where the in vivo toxicity (in mice) of pH-sensitive liposomes loaded with cisplatin was assessed,
the liposomal formulation was able to increase the LD 50 by 3-fold compared to free drug and no
alterations were found after haematology and histopathology analysis [207]. Also, no clinical symptoms
were detected at doses lower than the LD50, in contrast with the symptoms observed for the free drug
even at low doses, such as diarrhoea, ataxia, and weakness. Changes in drug-to-lipid ratio also
markedly affect the acute toxicity of encapsulated doxorubicin and liposomal amphotericin B [189,
208].
As discussed above, stealth liposome technology was developed to overcome most of the challenges
encountered by conventional liposome technology namely the toxicity due to charged liposomes [209,
210]. PEGylated liposomal doxorubicin, Doxil, represents a step forward in cancer therapy by reducing
the dose limiting cardiotoxicity associated with the free drug [211]. Nevertheless, this formulation is
not completely devoid of undesirable side effects, causing cutaneous and mucosal toxicity [212, 213].
Indeed, the most common side effect associated with Doxil treatment, the hand-foot syndrome, is
dose-limiting [213] and may compromise the efficacy of chemotherapy.
Toxicity of stealth liposomes is likely to be affected by the accelerated rate of blood clearance that
results from repeated administration. This effect increases the accumulation of liposomes in the liver
Nanomedicine 65
and spleen and thus may promote severe hepato- and splenotoxicity when highly toxic drugs are being
transported [191, 214, 215]. Therefore stealth liposomes also induce an immunogenic response
through complement activation that affects their pharmacokinetic behaviour and raises concerns about
safety. Several authors have also observed an increased accumulation of doxorubicin in mice liver,
spleen and lugs for both PEG, lactoferrin- or transferrin-PEG liposomes compared to the free drug [216-
218]. This accumulation, related to the rich blood supply of these organs [219], decreases the amount
of drug loaded liposomes that is available to reach the target tissue. This is particularly relevant when
the delivery of the nanocarrier relies on passive targeting, namely on the EPR effect found in solid
tumours. Furthermore, since the liver and spleen are major organs inthe MPS, the high uptake of
liposomes by the macrophages in these tissues may lead to a depletion of these phagocytic cells and
therefore cause a negative impact onthe immune system [219].
Most of the in vitro and in vivo toxicity studies involve survival and tissue distribution studies, serum
enzyme analysis and ROS assessment [191, 196-200, 202-204, 207]. Thus far, little attention has been
directed to the detection of genome damage, such as DNA strand breaks and formation of nuclear
abnormalities.
Combined therapy
Cancer therapeutics is not only being hampered by barriers to drug delivery (anatomical, chemical and
clinical) but also by tumour heterogeneity and adaptive resistance to treatment, which together lead
cancer cells to evade therapy. The development of a multimodal treatment strategy, through the use of
combined therapy with different mechanisms of action, will likely increase the probability of
eradicating all cells within a tumour. Hence, joint therapies can be achieved through the combination
of the following approaches: i) chemotherapy; ii) immunotherapy; iii) hormonal therapy and iv) gene
therapy. On this basis, multifunctional particles could surmount the greatest challenges and potentiate
new treatments and an ideal platform should be able to encapsulate both conventional anticancer
drugs with the new genetic drugs and present outstanding properties, such as proper retention of
drugs, outstanding targeting efficiency and long circulation lifetime. Similarly, the combination of
distinct types of nanoparticles, including AuNPs, liposomes, polymeric micelles, dendrimers, carbon
nanotubes or quantum dots, could result in new platforms with application in a variety of areas. We
believe that combinatorial drug delivery technology offers the potential to improve the efficacy of
single agents and the hope is that by increasing the amount of research in this field, one can contribute
to the translation of combined technologies into the clinics.
Although combination therapy may seem an expensive therapeutic approach, it could provide greater
savings by decreasing the amount of drug needed per treatment (thus lowering the rate of treatment
failure). Indeed, the use of nanomedicines for combined therapy could even improve the balance
between the efficacy and the toxicity of the used drugs. Co-loading of multiple drugs can be achieved
by i) co-encapsulation of hydrophilic drugs in the inner core of the liposome; ii) encapsulation of
hydrophilic drugs and simultaneous incorporation of lipophilic drugs within the membrane; iii) co-
encapsulation of hydrophilic drugs with gene-therapy based drugs (such as siRNA). Hence, for instance,
combined therapies designed to simultaneously target more than one signalling pathway could lead to
decreased drug resistance and favour better outcome.
Abraham and co-workers anticipated that the therapeutic potential of drugs could be increased by co-
encapsulating doxorubicin and vincristine in liposomes. Indeed, these drugs have been used in several
combination regimens due to their different intracellular mechanisms (doxorubicin (DOX)–
intercalation into DNA and generation of free radicals; vincristine – vinca alkaloid that binds to tubulin,
Nanomedicine 66
inhibiting proper assembly cell cytoskeleton) and several individual liposomal formulations have been
continuously evaluated [220]. The co-encapsulation method itself is challenging and depends on the
complexation of DOX and also on the addition of an electroneutralionophore that creates a pH gradient
which facilitates the accumulation of vincristine into liposomes pre-loaded with DOX. The efficacy of
the formulation was assessed in mice bearing human breast cancer cells, however, they noticed that
the co-encapsulation of these two drugs did not result in a synergistic effect, concluding that the
appropriate ratio of drugs must be taken in account [221]. This knowledge led to the proposal of CPX-
351 (cytarabine/daunorubicin at a 5:1 molar ratio) [222] and CPX-1 (irinotecan/floxuridine at a 1:1
molar ratio) [223], formulations, which are currently in phase II clinical trials.
As already mentioned, multidrug resistance (MDR) is probably the utmost hindrance to successful
cancer chemotherapy and is associated with overexpression of a glycoprotein (Pgp) that acts as an
efflux pump that reduces the intracellular concentration of the drug. Verapamil has been shown to
have Pgp inhibitory activity and additionally, it may induce cardiotoxicity, since it is a known calcium
channel inhibitor. Hence, liposomal delivery of Verapamil may reduce its cardiotoxicity while
contributing to a milder MDR. Wu and co-workers prepared a transferrin-conjugated liposome co-
encapsulating DOX and verapamil and assessed its efficiency in DOX-resistant cells, presenting a
promising approach to reverse cancer drug resistance [224].
The interaction between the tumour and its microenvironment is one of the hallmarks of cancer.
Tumour microenvironment is an array of extracellular matrix components and cell types that together
determine tumour progression. Aiming at the microenvironment is also an interesting approach to fight
cancer. For example, tumours need to generate their own new blood vessels to support their growth
and several studies proposed liposomes as anti-angiogenic therapeutic platforms [225-227]. Of
particular interest are those strategies that use a single liposomal formulation that can target both
endothelial and cancer cells. Moura and co-workers designed sterically stabilised, pH-sensitive
liposomes entrapping DOX targeting the nucleolin receptor, present in both cell types. Mice treated
with this formulation showed a positive outcome, with significant decrease of the tumour and
microvascular density [228]. Luo and co-workers showed similar results using paclitaxel-loaded
liposomes targeting aminopeptidase N receptors, which are expressed both in the tumour endothelium
and on tumour cells [229]. However, the neovascularisation tends to create disorganised and leaky
vessels, which enhances the leakage of nanoparticles to the tumour area by EPR, and therefore
treatment with drugs that inhibit angiogenesis could reduce the actual targeting of the tumour.
Actually, studies performed with an anti-angiogenic drug, sunitinib (VEGFR/PDGFR kinase inhibitor),
have shown that by inhibiting primary tumour growth, tumour invasion and metastasis is promoted, an
effect considered outrageous in the fight against cancer [230].
Co-loading of drug and RNAi effector molecules
Despite all the progresses in the development of new chemotherapeutic agents, conventional cancer
therapy often fails with drug resistance being considered the primary hindrance towards the cure.
Cutting edge multifunctional nanoparticles can provide simultaneous targeted delivery of
chemotherapeutics and gene silencing agents, with the rationale behind the use of combined
treatment being based on the heterogeneity of tumour cells. Drugs and genetic material can be
delivered into the target through the mechanisms already described and this dual therapeutic platform
is a promising approach for the treatment of neoplastic diseases.
Combining strategies that induce increased drug sensitivity could ideally decrease the number of cells
that are unaffected by drug (through evasion or acquired drug resistance). A common example consists
of combined strategies that target the oncogene BCR-ABL (for gene silencing) with widespread
Nanomedicine 67
chemotherapeutic agents (as for example imatinib), that once entrapped in a nanoparticle, induce pre-
sensitisation of tumour cells, followed by gene silencing, as presented by Mendonça and co-workers
[231]. In this report, several formulations encapsulating siRNA and imatinib at different ratios were
prepared and their anti-leukemic activity was also assessed in both imatinib resistant and sensitive
leukaemia cells. The data showed that the liposomal formulation with higher amount of anti-BCR-ABL
siRNA led to higher reduction of imatinib IC50. Furthermore, the researchers assessed the effect of pre-
sensitisation of cells using an administration schedule, in which cancer cells were incubated during 48h
with the pre-sensitisation stimuli, followed by 48h of treatment with the complementary component,
and showed that pre- sensitisation with siRNA followed by treatment with imatinib resulted in higher
therapeutic index, an expected observation as the BCR-ABL oncoprotein is known to be involved in
imatinib-resistance [232]. However, the efficiency of liposomes is still reduced, with low transfection
efficiency being their major drawback [233].
Hence, proper combinatorial regimens should be tested based on the assumption that the pre-
sensitisation of cells with a first therapeutic approach enhances the response to the second drug,
therefore decreasing the probability of drug resistance and therapy failure. Another possibility is to
take advantage of the ability of AuNPs to be readily taken up by cells, combined with the ability of
liposomes to efficiently reach the tumour microenvironment, and to design formulations where
liposomes work as carriers for AuNPs.
Nanoparticle cocktail
New-generation nanoparticles are considered effective systems for cancer treatment and are an
assembly of biomaterials, with improved properties, ideally, with effective dual functions, through the
incorporation of both diagnostic and therapeutic agents. These nanostructures are usually referred to
as hybrid nanoparticles, and combine lipid-based nanoparticles with polymers or AuNPs, with the main
goal of achieving a biocompatible nanostructure which effect is superior to the one obtained from the
mixture of the individual components [234].
Conclusion
Tumours are complex tissues that recruit cells and nutrients in order to enhance their survival and
proliferation. Cancer biology is described through its six hallmarks, that consist of i) immortality; ii)
proliferative signalling - growth factors from oncogenes that stimulate their own growth; iii) evading
growth suppressors - override ‘stop’ signals - anti-growth signals from tumour suppressor genes; iv)
resisting cell death and apoptosis; v) inducing new-angiogenesis; and vi) contact inhibition and evasion.
Tumour complexity arises also from their individual specialised cell types – tumour microenvironment -
that can be either tumour-promoting or tumour-killing agents [235, 236].These hallmarks are potential
targets for drug therapy and have been widely explored over the past few years. However, the
therapeutic doses used in the clinics are also lethal to normal cells, leading to undesirable side effects
and systemic toxicity [237]. Nanomedicine can provide the necessary tools for tackling these issues,
such as the use of nanoparticles as drug delivery agents, minimising side effects and toxicity of the
drugs. Furthermore, the selective targeting of cancer cells or tumour vessels by nanoparticles
containing anticancer drugs and/or therapeutic genetic material are now a recognised strategy for
improving the therapeutic efficacy of conventional cancer treatment and have shown promise in
preclinical models. Some recent advances in ligand-targeted liposomes have started to demonstrate
their promised improvement in cancer therapy but many tumours become resistant to drugs, making it
Nanomedicine 68
challenging to develop drug targeting vehicles that deliver high concentrations of combinatorial
therapeutics. Various attractive approaches have been explored to maximise dual-drug transportation
to the tumour that can be performed by association of lipid-based nanoparticles with hybrid polymers,
systems that tend to have high loading capacity and to be biologically compatible.
FIGURE 3.2
The fate of therapeutic nanoparticles and their characteristics.
The use of multiple nanoparticles that can be used together may overcome current limitations of each
individual nanoformulation alone. For example, AuNPs have proven to be outstanding vectorisation
systems for gene delivery and can be used to target molecular pathways, including those involved in
drug resistance and in survival of cancer cells. Acting together with drug loaded liposomes, suitable for
increased drug delivery with targeting capability, may decrease the surge of drug resistance while
enhancing the chances of a successful clinical outcome. Nanoparticles can be engineered to be
identified after in vivo delivery, working as imaging agents. Furthermore, new developments with
AuNPs have elucidated their ability to transduce optical energy into thermal energy, which can be
employed in thermal ablation, to destroy tumour cells. Here, we have discussed nanomedicine
platforms with enhanced properties, prompt to detect or treat cancer, where new polymeric modalities
have been envisioned, with improved biocompatibility, circulation and therapeutic response. Despite
these improvements and advantages, AuNP based systems are still in the research phase and are
expected to reach pre-clinical and Phase I trials within the next 2-3 years. Liposomal formulations have
been going into the clinics for the last decade and have made an impact on the delivery of standard
chemotherapeutic drugs against cancer. Combination of these two platforms, alone or conjugated, is
still inside the research lab and ought to take at least 5 years before we can see an impact.
Nanomedicine 69
Acknowledgements
The authors would like to thank Fundação para a Ciência e Tecnologia (MEC) for financial support (PEst-
OE/SAU/UI0009).
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4
Multifunctional core-shell
nanostructures
1 2
M. Clara Gonçalves , M. Barbara Martins
1 Chemical Engineering Department, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001
Lisboa, Portugal.
2 Faculdade de Farmácia da Universidade de Lisboa, Avenida Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 84
Nanoparticles design …………………………………………..……………………………………………………………………… 88
Core properties …………………………………………………………………………………………………………………………… 90
Fe2O3/Fe3O4 nanoparticles - a full range of biomedical applications ………………………………………...... 90
Gold nanoparticles – new unexpected properties ………………………………………..……………………………… 92
Shell properties …………………………………………………………………………………………………………………………… 92
Liposome dual character ……………………………………….……………………………………………………………………. 93
Polymer diversity ………………………………………………………………………………………………………………………… 94
Silica versatility …………………………………………………………………………………………………………………………… 94
Multifunctional nps platforms for nanomedicine - selected examples …………………………………........ 96
Optical imaging ………………………………………………………………………………………………………………………….. 96
Plasmonic effect …………………………………………………………………………………………………………………………. 98
Magnetic performance ………………………………………………………………………………………………………………. 98
Lipossome carriers ……………………………………………………………………………………………………………………… 100
DNA vectors ……………………………………………………………………………………………………………………………….. 101
Targeting ……………………………………………………………………………………………………………………………………. 102
Final Remarks …………………………………………………………………………………………………………………………….. 103
References……………………………………………………………………………………..…………………………………………… 104
Nanomedicine 84
Introduction
Nanomedicine is a new attitude towards conventional medicine where challenges are taken over a
bottom-up rather than top-down approach, medical actions are performed at a single cell level, tailor-
made therapeutic prescription are performed and theranosis is promised.
Personalized nanomedicine refers to the use of nanocarriers to elaborate optimized treatment
protocols tailored to each patient. While introducing thousands of times less drug into the body,
nanomedicine scales down the possibility of side effects, like tissue and organs destruction, and at the
same time increases the localization of pharmaceutical drugs in the diseased tissue.
Nanomedicine, the nanotechnology new costumer, is pushing up forward NPs design, where excellent
review articles have recently been published [1-8] (Table 4.1a, 4.1b). NPs refers to all
particulate/nanocarrier with all the three dimensions falling within 1-100 nm (0-D), for nanomedicine
applications the upper limit can be 200 nm, regardless of their shape or structure. A number of NPs-
based products for diagnostics and therapeutics have already been approved by FDA and European for
clinical applications [see ref. 1, 2, 4-8 and wherein references], and even more are currently under
clinical trials. Multifunctional NPs – NPs that are capable of accomplished multiple objectives such as
imaging and therapy (theronostic) or performing a single advanced function through incorporation of
multiple functional units – are a last quest in nanobiotechnology. Multifunctional core-shell NPs can
play a significant role in a near future offering new opportunities for monitoring the response to
therapy in real time.
This review focus the potential of multifunctional core-shell NPs designed to integrate simultaneously
several functions of clinical relevance such as: the delivery of contrast agents for different clinical
imaging tools, like magnetic resonance imaging or radionuclide imaging or fluorescence imaging, the
co-deliver of specific therapeutic agents such a drug or a gene and also to allow the functionalization of
the external shell surface for passive targeting or active targeting or for other functionalities (Fig. 4.1).
In the following sections, we briefly review NPs architecture as well as core and shell options function
of the final NPs performance. We present nanoarchitectures for combining two or more image
functions for multimodal imaging, integrating imaging with drug delivery for image-guided drug
delivery, and combining drug delivery with thermal therapies to take advantage of synergistic effects.
Current clinical trials and future perspectives concerning the application of multifunctional NPs are also
focused.
Nanomedicine 85
FIGURE 4.1
Multifunctional core-shell NPs are design to integrate several functions of clinical relevance.
Nanomedicine 86
TABLE 4.1a
Selected NP-based therapeutics approved or in clinical trials.
Product NP drug Delivery Indication FDA status Company

component route
Doxil PEGylated IV Ovarian Approved Ortho Biotech
liposome/doxorubicin cancer 11/17/1995
hydrochloride FDA050718
Amphotec Colloidal suspension subcutaneous Invasive Approved Sequus
of lipid-based aspergillosis 11/22/1996
amphotericin B FDA050729
Estrasorb Micellar NPs of Topical Reduction of Approved Novavax
estradiol hemihydrate emulsion vasomotor 10/9/2003
symptoms FDA021371
Abraxane Nanoparticualte IV Various Approved American
albumin/paclitaxel cancers 1/7/2005 Pharmaceutical
FDA021660 Partners
Triglede Nanocrystalline Oral tablets Lipid Approved SkyPharma PLC
fenofibrate disorders 5/7/2005
FDA021350
Megace ES Nanocrystal/megestrl Oral Breast Approved Par
acetate suspension cancer 7/5/2005 Pharmaceutical
FDA021778 Companies
Combidex Iron oxide IV Tumor Phase III Advanced
imaging Magnetics
Aurimune Colloidal gold/TNF IV Solid tumors Phase II Cytlmmune
Sciences
NB-00X Nanoemulsion Topical Herpes Phase II NanoBio
droplets labialis
caused by
herpes
simplex I
virus
AuroShell Gold-coated silica NPs IV Refractory Phase I Nanospectra
head and Biosciences
neck cancer
CALAA-01 Cyclodextran- IV Various Phase I Calando
containing siRNA cancers Pharmaceuticals
delivery NPs
Cyclosert Cyclodextran NP IV Solid tumors Phase I Insert
Therapeutics
INGN-401 Liposomal/FUS1 IV Lung cancer Phase I Introgen
SGT-53 Liposome Tf IV Solid tumors Phase I SynerGene
antibody/p53 gene Therapeutics
Nanomedicine 87
TABLE 4.1B
Comparison of commonly used bioimaging techniques.
Technique Typical NP Signal Reso- Depth Sensitivity Throughput Cost Main

label measured lution (moles of label limitation
detected)
NIRF QDs, dye- Light 1-3  1 cm 10-12 high low Poor depth
doped NPs, (near-IV) mm penetration
UpC NPs,
SWNTs
MRI Iron oxide Alteration 50 m No 10-9-10-6 low high Low
NPs, s in limit sensitivity,
Gd(III)- Magnetic cannot follow
doped NPs, field many labels
NPs-based
CEST and
hyperpolar
ized
probes
(e.g.
129Xe)
PET NPs Positron 1-2 No 10-15 low high Can detect
incorporati from mm limit only one
ng radionucli radionuclide,
radioisotop des requires
es (e.g. radioactivity
18F, 11C,
64Cu, 124I)
SPECT NPs -rays 1-2 No 10-14 low high Requires
incorporati mm limit radioactivity
ng
radioisotop
es (e.g.
99mTc,
111In)
CT Iodonated X-rays 50 m No 10-6 low high Poor
NPs, Au limit resolution of
NPs, iron- soft tissues
oxide
doped n-
materials
US -bubles, sound 50 m Severa 10-8 high low Poor image
n- l cm contrast,
emulsions, works poorly
SiO2-NPs, in air-
PS-NPs containing
organs
PAI Au-n- sound 50 m  5 cm 10-12 high low Information
shells, Au- processing
n-cages, and machines
Au-n-rods, still being
Au-NPs, optimized
SWNTs,
dye-doped
NPs
Adapted from [9].
Nanomedicine 88
Nanoparticles design
Various types of engineered core-shell NPs have been developed in outstanding efforts between
academia and industry, in the last decades. Present core-shell NPs may exhibit a wide range of
geometries - from spherical to tubular, through centric, eccentric and star-like - different sizes, core-
shapes and shell-thicknesses, may comprise multiple cores, and may differ in crystallinity and surface
morphology (Figure 4.2). NPs benchmarket demands an accurate core-shell NPs characterization which
absence has been one of the main drawbacks in in-vitro/in-vivo applications. The following set of
properties needs to be addressed:
i) size (actual and hydrodynamic diameter) and size distribution;

ii) shape and surface curvature;
iii) surface area and smoothness/roughness;
iv) surface charge, surface chemistry/reactivity, hydrophobicity/hydrophilicity;
v) coating thickness;
vi) chemical composition of both core and shell;
vii) crystallinity of both core and shell;
viii) porosity (size and size distribution);
ix) identification and levels of any impurities.
FIGURE 4.2
Core-shell NPs may exhibit a wide range of geometries (a) - from spherical to tubular, through centric, eccentric
and star-like - different sizes, core-shapes and shell-thicknesses, may comprise multiple cores, may differ in
crystallinity and surface morphology, alongside with being functionalized (b).
Nanomedicine 89
An appropriate size range is required to run a nanobiomedical system effective. The nanobiomedical
system needs to be capable of targeting, entering, and providing therapy at a single cell level. The core-
shell NPs ideal diameter is between 10 and less than 200 nm. Monosized distribution is required to
make the process reproducible.
The properties of nanomaterials are determined mainly by the properties of their surfaces (opposed to
bulk properties), due to their extraordinary high surface area to volume ratio. As a result, irregular
shapes with higher surface area to volume ratio play a larger role in dictating those properties. Further,
non-spherical NPs do have energetically different surface sites (surface/edge/corner) which may
differently act in chemical processes. Besides, shape may be characteristic of a crystalline structure,
and that is an additional reason to avoid shape distribution.
Surface roughness/smoothness may affect the contact area between NPs and their environment
(biological or other), thus reducing/increasing the number of accessible active surface sites. Chemical
and structural identical NPs not surprisingly may exhibit different behavior depending on surface
roughness/smoothness. Surface hydrophobicity/hydrophilicity, which may be tuned during the
synthesis processes, controls the wettability of a NP in respect to a specific environment, determining
the number of accessible active surface sites.
Shell structure and particularly shell thickness may hinder the core properties. The core-shell
assemblage should thus be optimized/tested according to the designed performance. Depending on
the core-shell NPs application, the characterization is devised; UV-visible, NIR, fluorometry, absorption
cross sections, fluorescence quantum yields, and fluorescence lifetimes for optical applications;
susceptibility and relaxivity for magnetic applications and thermoelastic properties for
temperature/mechanical sensoring applications, as an example. Although sheltered, the core material
should be biocompatible in order to evade the immune response and avoid rapid elimination or
toxicity.
In-vivo toxicity tests are the ultimate screening to clinical use [10]. However, deciding on a set of
standard toxicity assays is difficult as different toxicological tests study different toxicity facets (e.g.,
membrane permeability, apoptosis). Further, commercial testing kits designed for molecular toxins
may not be recommended as they may interfere with core-shell NPs. Additionally, results of in-vitro
analysis may not necessarily be valid in-vivo.
The last but not the least remains the lack of reproducibility in synthesis, functionalization and
toxicological results of core-shell NPs. Batch-to-batch variations within the same laboratory, between
laboratories and even between different protocols employed in both synthesis and decoration
methodologies are frequent, often yielding the same core-shell NPs but with slightly different
characteristics (e.g. size distribution, shape, number of conjugated biomolecules). These difficulties
may arise from scare characterization of the NPs. Toxicological results from different laboratories
where researchers may use different tests to confirm or deny toxicity may not be conclusive.
One of the great challenges in fabrication and processing of NPs is to overcome the surface energy, and
to prevent the nanostructures or NPs from growth in size, driven by the reduction of overall surface
energy. Missing is the discussion of all the mechanisms NPs have to face in order to keep their nanosize
and nanostructure.
Nanomedicine 90
Core properties
The specific properties of the core materials provide distinct monitoring and therapeutic applications. It
is critical to consider the purpose behind selecting a certain core material for the NPs system. For
example, magnetic NPs provide monitoring and localization properties based on their magnetic
susceptibility, fluorescent NPs based on light emission performance and gold NPs on plasmonic effect.
The high surface-to-volume ratio of metallic core structures yields high surface energies, promoting
surface oxidation, and particles aggregation or clustering in physiological environments. Yet, passivated
metal-cores (with satisfactory surface chemistry) exhibited longer plasmatic half-life and slower uptake
by liver and spleen after intravenous administration [11]. The use of a colloidal stabilizing agent, such
as a surfactant or polymer, to prevent agglomeration/coagulation and to increase metal-core
dispersion in various solvents and in the bloodstream is mandatory. Many of the times it is necessary to
invert the surface charge of the metal-core materials through the addition of a surfactant, often
followed by the addition of a stabilizer, prior to the (chemical/physical) bounding to the shell material,
to increase core-shell affinity [12].
Fe2O3/Fe3O4 nanoparticles - a full range of biomedical applications
An intense research work has been devoted to iron oxide NPs, where a large number of publications
have already been reported [13]. As NPs, iron oxides can be classified based on their size, as magnetic
iron oxide nanoparticles (MION, m), superparamagnetic iron oxide (SPION, hundreds of nm), and
ultra-small paramagnetic iron oxide (USPION, 50 nm). SPIONs with appropriate surface chemistry
have been incorporated into a diversity of nanomedicine platforms for in vivo applications such as
negative contrast agents in magnetic resonance imaging (MRI) (Figure 4.3) [14-17], photo thermal
microscopy [18], tissue repair [19] immunoassay [20], detoxification of biological fluids [21],
hyperthermia treatment [22], guided non-viral vectors [23], in particular allowing for magnetic
guidance in drug-delivery nanosystems [24].
SPIONs’ biomedical applications require high magnetization values; narrow NPs size distributions,
chemical stability, and homogeneous dispersion when in (biological) liquid media. Magnetite (Fe3O4)
and maghemite (-Fe2O3) are the most commonly used iron oxides for such applications, with a
preference for magnetite because of its high saturation magnetization value [25]. A large number of
publications report the development of core-shell NPs with a core of superparamagnetic iron oxide NPs
(SIONPs), a contrast agent for magnetic resonance imaging (MRI) and for image guided therapy [13].
SPIONs have been incorporated into a diversity of nanomedicine platforms [26-34].
Nanomedicine 91
FIGURE 4.3
Silica coated SPIONs to be used in vivo as negative contrast agents in magnetic resonance imaging (MRI). Adapted
from [15].
The synthesis of Fe3O4 SPIONs is not straightforward. SPIONS can be fabricated by either top-down or
bottom-up approaches. Chemical routes are better suited to produce SPIONs with uniform composition
and size comprising:
i) classical synthesis by co-precipitation,

ii) reactions in constrained environment,
iii) hydrothermal and high-temperature reactions,
iv) sol-gel reactions and v) polyol methods.
Coprecipitation techniques are rather complex approaches; the shape, size, size distribution and
crystalline structure of NPs being strongly dependent on large number of experimental parameters.
Apart from that, the easy oxidation of Fe(II) on magnetite surfaces becomes particularly important in
NPs, where an extremely high surface/volume ratio is reached. Nevertheless co-precipitation methods
are easy to implement and require less hazardous materials then the other procedures. Among the
hydrolytic synthetic routes, the alkaline co-precipitation of ferrous and ferric salt precursors, in
aqueous medium under inert atmosphere originally present by Massart et al. [35] has been the most
important and widely used. Through a careful control of experimental parameters such as type of salts
2+ 3+
employed (e.g. chlorides, sulfates, nitrates), Fe /Fe ratio, temperature, pH, and ionic strength, quasi-
spherical SPIONs with 8 nm [36], 16.6-4.2 nm [36], 2-15 nm [37-39], 8-13 nm [40], 7 nm have been
produced [41].
Nanomedicine 92
Gold nanoparticles – new unexpected properties
Among the nanocarriers, gold NPs have been actively investigated in a wide variety of biomedical
applications due to its biocompatibility, non-cytotoxicity and non-immunogenicity (Figure 4.4). The
ease of conjugation of gold NPs to biomolecules offers multiple modalities for biological and medical
applications. Not only spherical gold NPs have been synthesized, but a wide variety of geometries
/shapes can be obtained through the appropriate technique.
Gold NPs and gold colloidal suspensions exhibited a nano-property, known as surface plasmon
resonance. At ~520 nm the free electrons (of the conduction band) of the surface of gold NPs
collectively oscillate and scatter/absorb the incident electromagnetic wave, causing a strong
absorption/scattering band. The ruby-red color of gold NPs and gold colloidal suspensions when
exposed to visible light are an evidence of the phenomena.
FIGURE 4.4
Gold NPs
http://www.fdbusiness.com/2013/04/gold-NPs-help-detect-listeria-cheaply/
Shell properties
The use of metals as core always require a coating that inhibits surface oxidation and at the same time
bring better colloidal properties to the new nanostructure. Liposomes are one of the most studied
nanoplatforms for incorporation of SIONPs [29, 30, 32-34]. The role of liposomes as core-shell NPs is
due to their quite specific structure which allows: 1) to carry into its inner compartment a diversity of
Nanomedicine 93
SPIONs or other imaging agents 2) to load therapeutic agents either into its inner space or into its lipid
bilayer 3) to be grafted into its outer surface by a diversity of molecules for targeting, for stealth
properties or for other functions.
Polymer coatings through the presence of amine or carboxyl groups provide a link to functionalize
these metal and QD cores with other biomolecules. Through these functional groups, molecular layers
can be constructed on the core to provide biocompatibility, cell targeting, intracellular localization,
biosensor diagnostics, and drug or gene delivery. Silica is another common coating material, able to be
conjugated through the surface silanol group or the non-hydrolyzed organic group introduced in-situ
during the synthesis procedure.
Liposome dual character
Liposomes are NPs, with a vesicle structure (Figure 4.5), spontaneously formed when phospholipids are
suspended in water in a definite range of molar ratios. The phospholipids become organized in bilayers
that surround an aqueous core. They can also form an onion like structure with concentric bilayers
entrapping water between them surrounding an inner water core. They also can be formed with
several vesicles inside. The final structure of a liposome NPs is a function of the mixture of
phospholipids that form the bilayers, of the method of preparation and of the composition of the water
medium. Liposomes made with natural phospholipids are identical to biological membranes,
consequently they are totally biocompatible. Medicines and other molecules or nanostructures, like
contrast agents for imaging can be encapsulated into liposomes, being located there according with
their lipophilic ties. Strongly lipophilic molecules or nanostructures are completely entrapped in the
lipid bilayer, strongly hydrophilic molecules or nanostructures are sequestered in the aqueous
compartment, and molecules or nanostructures partially hidophobic/hidrophilic are located partially
buried between the lipid and partially exposed to the aqueous phases
FIGURE 4.5
Liposomes are nanoparticles, with a vesicle structure, spontaneously formed when phospholipids are suspended in
water in a definite range of molar ratios. The phospholipids become organized in bilayers that surround an
aqueous core. They can also form an onion like structure with concentric bilayers entrapping water between them
surrounding an inner water core.
Nanomedicine 94
Liposomes were first identified in the 60´s by [42] and were initially used in research related with
biomembranes [43-45]. But the technology of liposomes did considerable progress during the last four
decades [46-47]. The success of liposomes as drug delivery systems with several medicines loaded in
liposomes in clinical use for years [48] and several other in clinical studies, [49-50] bring this class of
NPs a focus of attention for many clinical purposes including theronostics purposes [51]. Furthermore
a number of different molecules can be covalently linked to outer surface of liposomes, for active
targeting or for other functionalities. A review the diversity of reactions was published by [52].
Liposomes have also been used to obviate the tendency of SPION to aggregate when suspended in
water, due to the absence of electrostatic and/or steric stabilization which is of special concern for in
vivo applications. The term magnetoliposome was first mentioned by [53] for small liposomes coating
each IONP by a phospholipidic bilayer, without an internal aqueous compartment. Since then an
increasing number of publications focus on the development of magnetoliposomes obtained by the
incorporation of IONPs previously stabilized with different coatings (polymers, lipids, surfactants) and
incorporated into the inner aqueous space of the liposomes or into the bilayer of the liposomes,
according with the hydrophilic or hydrophobic coating of the IONPs [51, 54-55].
Other works focus the development of liposomes for imaging purposes [56-59].
Polymer diversity
A diversity of polymers has been used to build NPs for drug delivery purposes. Polymeric NPs are
obtained by different processes based on two main approaches: polymerization reactions or the use of
preformed polymers These include polymeric micelles, capsules, colloids, dendrimers, and others. The
term polymeric nanoparticle encloses nanospheres and nanocapsules. The polymers extensively used
are poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly (ε-caprolactone) (PCL) and
their copolymers diblocked or multiblocked with poly(ethylene glycol) (PEG) polycyanocrylate (PACA),
chitosan, gelatin and sodium alginate. A number of recent publications focus the development
multifunctional polymeric NPs for drug delivery [60] and [61]. Several recent publications focus the
development of magnetic core-shell polymeric NPs [31, 60, 62-65].
Silica versatility
Silica-based biomaterials have unique structural characteristics, with a 3D amorphous silica network,
where surface silanol groups render silica a high hydrophilic character, a high biocompatibility and the
further possibility of functionalization and/or bioconjugation [6]. Organically modified silica (ORMOSIL)
[66] is an alternative material for biomedical applications with even better and more versatile
properties than silica; the presence of non-hydrolysable organic groups in the alkoxysilane precursors
behave like glass modifier and reduce the degree of the silica network cross-linking. In addition,
ORMOSIL surfaces will be populated both with silanol and organic groups, allowing an easier chemical
conjugation/decoration of biomolecules at the ORMOSIL surfaces and/or the load with either
hydrophilic or hydrophobic drugs/dyes. A tunable wettability, by a judicious choice of the ratio of
hydrophilic to hydrophobic sol-gel precursor monomers, a tailor-made porosity (size and shape) and a
shell hardness/complacency making ORMOSIL a very competitive material. Mammalian cells take up
and internalize easily silica/ORMOSIL-coated NPs without any cytotoxic effects [67], opening the door
to their use in health science.
Silica may also be mesoporous nanostructured, with pores ranging between 2 and 10 nm in diameter,
by the use of templating agents, such as lyotropic liquid crystalline phases of surfactants (Figure 4.6)
[68-69]. Nanostructured mesoporous silica material is an ideal candidate for host-guest nanosystems to
Nanomedicine 95
confine drugs or biologically active molecules into their mesopores [70]. Nanostructured mesoporous
silica have a high pore volume, an homogeneous size ordered pore network, an high surface area, all
together allowing the hostage of a large amount of drugs, fine control of the drug load and release
kinetics, and high potential for drug adsorption through the interaction between the drug and the pore
walls. The organic functionalization of mesoporous silica walls revealed to be the main factor governing
release kinetics, and it may also influence molecule adsorption by promoting host-guest interactions.
FIGURE 4.6
Mesoporous nanostructured silica [68]
Regarding silica as coating material, the two major approaches include the reverse microemulsion [71]
and the sol-gel methods [72]. In reverse microemulsion, aqueous solution disperses in the organic
phase (in the interior of the self-assembly reverse micelles) and forms a number of monodisperse
nano-droplets. The confined nanoreactor environment within the reverse micelle has been shown to
yield highly monodisperse NPs and increase the incorporation of non-bonded non-polar molecules,
which are often difficult to incorporate into the hydrophilic silica matrix [73]. The principal advantage
of using reverse microemulsion is that particle shape and corresponding size distributions can be
readily controlled by adjusting the molar ratio of water to surfactant, aging time, and reactant
concentration. However the reverse microemulsion synthesis often have low yields and the use of
surfactants and potentially toxic organic solvents demands extensive washing before any biological
application, to avoid disruption or lyses of biomembranes by the surfactant molecules, rendering the
process slow, expensive and low eco-friendly.
Alternatively Stober [74] developed a mild synthetic protocol for growing monodisperse spherical silica
NPs based on the sol-gel of silicon alkoxides. Stober’s method involves the hydrolysis and condensation
of tetraethoxysilane (TEOS) in ethanol solution in the presence of water with ammonia as a catalyst, to
create monodisperse, spherical, electrostatically-stabilized particles. The Stober method is a promising
method for producing surfactant free silica coatings, yet, the final particles size remain in the hundreds
of nanometers to microns regime, which are too large to some of the biologic studies.
Nanomedicine 96
Multifunctional NPs platforms for nanomedicine - selected examples

Medical imaging plays an important role in disease detection, prognosis, follow-up and treatment
planning. Major medical imaging techniques include X-ray computed tomography (CT), magnetic
resonance imaging (MRI), ultrasound imaging, positron emission tomography (PET), single-photon
emission CT (SPECT), optical imaging, and photo acoustic imaging. Biomedical imaging is rolling toward
the development of bimodal and multimodal imaging agents, following a number of relevant
developments on combined imaging equipment for clinical and pre-clinical diagnostic such as the
combination of CT and MRI and of PET and MRI and more recently the combination SPECT and MRI.
The development of multifunctional NPs has greatly expanded the outlook of nanomedicine with
advanced imaging and therapeutic platforms. Contrast agents for MRI, optical imaging, photo acoustic
imaging and on other imaging modalities are discussed by [1] with a focus on the intrinsic quantum
mechanical properties of inorganic NPs.
An overview of the variety of nanomaterials under investigation for diagnosis, imaging, and therapy of
cancer is reported in the review of [75] with a special focus on inorganic NPs suitable for exploitation in
different imaging modalities, their capability for thermotherapy and photodynamic therapy. These
authors present a summary of some of the successfully approved and commercialized nanomedicines
for the treatment and detection of cancers and many others at the various stages of clinical trials. They
also discuss the effective modification and functionalization of these nanoprobes to provide further
control of the localization, biodistribution, biocompatibility, and efficacy of nanomaterial systems in
vivo.
Engineering NPs with more than one type of contrast agent in the same NPs, combining multimodal
detectability consequently multiple components, it is a challenge with impact on imaging, molecular
diagnostics, and therapeutics. However, combining multiple components on a nanometer scale to
create new imaging modalities unavailable from individual components has proven challenging [76].
Before the clinical translation, basic research aimed to gain deeper understandings on imaging agents,
particularly on the connections between their imaging capability and their physicochemical
microenvironments within nanocarriers aspects that will have a key role in developing robust
nanotherapeutic platforms with high-performance imaging capability [77].
Optical imaging
Hollow spheres are utilized for the encapsulation and controlled released of various substances (e.g.
drugs, biomolecules, cosmetics, dyes and inks), as confined reaction vessels, in catalysis and removal of
pollutants as light fillers, acoustic insulation materials, low dielectric constant materials and photonic
band gap materials [12, 78-85] (Figure 4.7). In nanomedicine silica hollow spheres can be used as host
3+ 3+ 3+
for Er enhanced photoluminescence (PL) in visible range and Yb to Er enhanced energy transfer
phenomena [12] as non-invasive in vivo imaging [9, 84-85]. Up-conversion (UpC) effect (which converts
long-wavelength excitation light into short-wavelength emitted light) based on rare-earth (RE) doped
materials [86] is a promising phenomenon, since the exciting source (in the NIR) falls within the
therapeutic window (600-1300 nm) [87] , allowing maximum penetration depth, minimizing photo-
damage and reducing tissue auto-fluorescence [88]. Er/Yb co-doped silica hollow spheres takes
advantage of the high capacity of this platforms to ferry cargo and loads onto them both imaging and
therapeutic functions. Applications such as drug-delivery and bio-label targeting can be combined into
bio-imaging nanosystems creating multifunctional platforms for theronostic [9, 85-86]. When the
matrix is silica, the advantages of biocompatibility and biodegradability [87] combine with silica optical
3+
transparency, allowing the PL emitted light to cross the silica matrix efficiently [88]. Among the RE, Er
Nanomedicine 97
PL at 1.54 m is of particular interest because it corresponds to the minimum absorption loss in silica-
based matrices [79].
FIGURE 4.7
3+ 3+ 3+
Silica hollow spheres can be used as host for Er enhanced photoluminescence (PL) in visible range and Yb to Er
enhanced energy transfer phenomena [12].
Multifunctional nanomaterials with unique magnetic and luminescent properties have broad potential
in biological applications. [89] describe the development of multifunctional core-shell Fe3O4@SiO2 NPs
with the ability to target inflammatory endothelial cells via VCAM-1, magnetism, and fluorescence
imaging, with efficient magnetic resonance imaging contrast characteristics. Superparamagnetic iron
oxide and fluorescein isothiocyanate (FITC) were loaded successfully inside the NP core and the silica
shell, respectively, creating VCAM-1-targeted Fe3O4@SiO2(FITC) NPs that were characterized by
scanning electron microscopy, transmission electron microscopy, fluorescence spectrometry, zeta
potential assay, and fluorescence microscopy.
Focusing on the need of noninvasive and sensitive tumor diagnosis NIR fluorescent probes, which
activate their fluorescence following interaction with functional biomolecules, are desirable. Ref1007
developed a probe with a self-assembling polymer micelle, encapsulating various quantities of NIR dye
(IC7-1) conjugated anti-HER2 single chain antibodies to the micelle surface and examined the probe’s
capacity to detect HER2 in cells and in vivo and the results presented that this core-shell NPs would be
a useful NIR probe that is applicable for use in noninvasive in vivo optical imaging for specific detection
of target biomolecules expressed in tumors.
3+
The development of novel core-shell α-(NaYbF4:0.5% Tm )/CaF2 NPs with efficient NIRin/NIRout UpC for
high contrast and deep bioimaging and their applications for high-contrast in vitro and deep tissue
bioimaging are reported by [90]. Whole-body imaging of a BALB/c mouse, intravenously injected with
an aqueous dispersion of that core-shell NPs (700 pmol/kg), showed a signal to back ground ration
about 10-fold higher than that previously reported for in vivo imaging by these UpC NPs. The retention
3+
of the NIRin-NIRout (NaYbF4:Tm )/CaF2 NPs on a synthetic scaffold surrounding a rat femoral bone
under centimeter-deep soft tissues was successfully visualized, demonstrating potential of these NPs
3+
for image-guided tissue engineering applications. Also UpC PL from a (NaYbF 4:Tm )/CaF2 NPs
suspension was imaged through a 3.2-cm pork tissue, with a high contrast against the background. The
authors conclude that the observed capabilities of these engineered NIR in-NIRout UpC NPs, provide
promise for their wide application in biomedical imaging.
Nanomedicine 98
Plasmonic effect
Gold NPs and gold-based multifunctional core-shell NPs are the subject of intensive studies and
biomedical applications. Applications of engineered gold-based NPs and nanocomposites in analytical
and theronostic by using plasmonic properties and a diversity of optical techniques are reviewed by
[91], specifically bioimaging of bacterial, mammalian, and plant cells; photodynamic treatment of
pathogenic bacteria; and photothermal therapy of xenografted tumors. In addition to recently
published reports, the authors discuss new data on dot immunoassay diagnostics of mycobacteria,
multiplexed immunoelectron microscopy analysis of Azospirillum brasilense, materno-embryonic
transfer of gold NPs in pregnant rats, and combined photodynamic and photothermal treatment of rat
xenografted tumors with gold nanorods covered by a mesoporous silica shell doped with
hematoporphyrin.
Plasmonic gold-shell-magnetic core star shape NPs developed for the early detection of circulating
tumor cells are reported by [92], using magnetic/plasmonic NPs with the surface conjugated with SK-
BR-3 breast cancer S6 aptamer able to perform magnetic separation of cancer cells from whole blood
sample by fluorescence imaging, followed by separation and phototermal destruction.
The development of a theronostic plasmonic shell−magnetic core star shape NPs for the targeted
isolation of rare tumor cells from the whole blood sample, followed by diagnosis and photothermal
destruction is reported by [92]. These authors demonstrate that the plasmonic star shape NPs
developed are capable of isolating rare cancer cells from whole blood samples, followed by imaging
and photothermal destruction, using the SK-BR-3 human breast cancer cell line, which over expresses
the epidermal growth factor receptor HER2/c-erb-2/Neu (HER-2) on the cell surface pointing to
improve early detection of cancer in personalized medicine
The development of a multifunctional plasmonic shell–magnetic core nanotechnology-driven approach
for the targeted diagnosis, isolation, and photothermal destruction of cancer cells is reported by [92]
with experimental data showing that aptamer-conjugated plasmonic/magnetic NPs can be used for
targeted imaging and magnetic separation of a particular kind of cell from a mixture of different cancer
cells. A targeted photothermal experiment resulted in selective irreparable cellular damage to most of
the cancer cells. The authors showed that the aptamer conjugated magnetic/plasmonic NPs-based
photothermal destruction of cancer cells is highly selective. They discuss the possible mechanism and
operating principle for the targeted imaging, separation, and photothermal destruction using
magnetic/plasmonic nanotechnology.
Magnetic performance
A summary on different core materials based on ferrite and ferrite doped magnetic NPs and on shell
coating is reported by Karimi et al. 2013 [93] with an emphasis on suitable magnetic core-shell NPs with
chemical/biological functionalization to be used in nanomedicine. A comparison of different properties
of shell materials such as dextran, polyethylene glycol, chitosan and silica to improve the performances
of magnetic materials is performed. Their advantages and disadvantages and properties such as bio-
adhesive, charge, functional groups, increase in blood circulation time are focused.
Superparamagnetic NPs with Fe3O4/Fe2O3 core and inorganic and organically modified silica as shell
were produced through a non-reverse emulsion method. The superparamagnetic nanostructures
exhibited several magnetic cores and a highly porous external shell. No significant modification of the
magnetic properties of the core iron oxide NPs were detected, conferring the core-shell nanostructures
strong magnetic behavior and making them appropriate to biomedical applications [15-17, 34] (Figure
4.8).
Nanomedicine 99
FIGURE 4.8
Core-shell SPIONS-silica as MRI negative contrast agents
A. Carvalho, M.Clara Gonçalves, unpublished work.
Folic acid conjugated on the surface of FePt@Fe 2O3-PEG NPs loaded with the chemotherapy drug,
doxorubicin (DOX) via hydrophobic physical adsorption, were developed by Liu et al 2013 [94] for
targeting to folate receptor (FR)-positive tumour cells, targeted intracellular drug delivery and selective
cancer cell killing acting as a multifunctional theronostic nanoplatform in imaging guided cancer
therapy.
The use of superparamagnetic core shell multifunctional NPs, as novel drug delivery vehicles, to be
guided with the help of an external magnetic field to its target is discussed by [95], stressing the need
of monitoring the burst release effect of SPIONs. The authors focus the need of an adequate shell
matrix controlling the drug release rate and incorporating the SPIONs within a polymer matrix that
allows retention of their magnetic properties, thus enabling them to be guided by an external magnetic
field. The matrix would regulate the release of drug as desired. Guidance by external magnets enables
a third targeting mechanism, giving them an advantage when all three mechanisms work in unison.
Carcinomas near the body surface, like squamous cell carcinoma, malignant melanoma, Kaposi’s
sarcoma, and breast carcinoma, are expected to benefit the most from SPION-based therapy.
Development of transdermal patches containing magnetic circuitry using supermagnets capable of
generating highly penetrating localized magnetic fields could result in enhanced and efficient
accumulation of drugs carrying SPIONs at desired sites [95]. This would be particularly beneficial for
ambulatory patients and could also increase patient compliance with treatment. Additionally, the
complex dosing regimen according to the individual needs of the patient can be carried out by
modulation of the magnetic field strength used.
Iron oxide and gold coupled core-shell NPs with defined structural characteristics (e.g., size, shell
thickness, and core-shell separation) and physical properties (e.g., electronic, magnetic, optical,
thermal, and acoustic) were developed by [76]. The reported NIR responsive magnetic-gold core-shell
nanostructures were obtained by creating a gap between the core and shell, as the core and shell of
our particles are spatially separated with a dielectric polymer layer. The resulting NPs show highly
Nanomedicine 100
integrated properties including electronic, magnetic, optical, acoustic, and thermal responses, which
allow multimodality imaging. Additionally the surface of the particles will also allow conjugation with
targeting ligands to develop all-in-one nanostructures for non-invasive imaging, molecular diagnosis,
and hyperthermia-based treatment of complex diseases.
Lipossome carriers
The development of nanotheronostics using lipid- and polymer-based formulations, with a particular
focus on their applications in cancer research are reviewed by [77] with an emphasis on recent
advances in nanotechnology aimed to combine therapeutic molecules with multimodal imaging agents
for magnetic resonance imaging, radionuclide imaging, or fluorescence imaging and with a primary
focus on platforms using liposomes and polymers. Liposomes and polymer-based NPs are both
established drug delivery platforms for cancer treatment. Combine both therapeutics and imaging into
a single carrier, results in a number of novel theronostic platforms engineering lipid and polymer based
nanotherapeutic platforms with multiple imaging modalities incorporated into increasingly
sophisticated architectures [77], not only oncological applications but for other fields like cardiology
where nanotherapeutic platforms will find numerous potential applications.
Magnetoliposomes assume a special place among the diversity of paramagnetic core-shell iron oxide
NPs used as contrast agents to improve MRI sensitivity. Several works focus the effect of the NPs on
magnetic properties [16-17, 54-55, 96-99]. Other works focus the increasingly relevant role of
magnetolipossomes in the targeting of MRI contrast agents [28, 30, 34, 97, 100] and for the co-delivery
of medicines and imaging agents [1, 27, 29, 31-32, 98, 101-103].
The magnetic properties of long circulating magnetoliposomes sterically stabilized by PEG (PEGylated)
were first studied by [104] that rank them among efficient MR T2 contrast agents. More recently [96]
demonstrated that the magnetic properties of PEGylated magnetoliposomes are dependent on the
mol% of PEG lipid. PCA applied to FTIR data can successfully differentiate magnetoliposomes from the
empty liposomes as reported by [17] using magnetoliposomes loaded with different concentrations of
SPIONs (Figure 4.9).
Long circulating magnetoliposomes designed for the load of PEG-coated SPION and for passive
targeting to liver ischemia-reperfusion injuries were developed by Martins et al. [34]. The authors
demonstrated that the passive targeting of the optimized magnetoliposomes improved the sensitivity
of MRI to visualize inflamed tissues.
A review on the design and multifunctional properties of lipid bilayer coated presented by [102]
evidences how lipid bilayers are now being utilized as excellent carriers for drug-loaded and solid core
NPs such as iron oxide, mesoporous silica and calcium phosphate and polycation-based solid NPs with a
focus on their design as well as their multifunctional role in cancer therapy are discussed.
Nanomedicine 101
FIGURE 4.9
Magnetolipossomes for MRI contrast enhancement [17].
The state of the art of magnetic resonance imaging (MRI)-guided nanorobotic systems associated to
drug delivery (nanorobotic-MRI DDS) addressing the novel concept of guiding core shell NPs in the
human vasculature for drug delivery purposes using an MRI scanner is reviewed by [105]. The authors
discuss the potentiality of these platforms, to perform diagnostic, curative, and reconstructive
treatments in the human body at the cellular and subcellular levels in a controllable manner. The
concept of an MRI-guided nanorobotic system is based on the use of an MRI scanner to induce the
required external driving forces to propel magnetic multifunctional core-shell NPs to a specific target.
Some perspectives on the successful realization of the nanorobotic-MRI DDS are expected to produce a
significant increase in therapeutic efficiency and a decrease in side effects on healthy tissue [105].
Polymeric core-shell NPs produced by a non-emulsion technique, were fabricated to carry iron oxide
within the shell and the chemotherapeutic agent, temozolomide (TMZ) are described by [106] and
results on the endocytosis-mediated uptake by glioma cells using intracranial delivery through rodent
brain and tracked in vivo by standard MR imaging were demonstrated [106] and reduction of the
growth of glioma xenografts and extend survival of tumour bearing animals was rerported.
DNA vectors
One focus in nanobiotechnology is the development and use of nonviral vectors for safe and efficient
gene delivery. Eco-friendly inorganic and organically modified silica NPs were prepared through a non-
reverse micro-emulsion technique and successfully complexed to DNA plasmids, at different ratios
(Figure 4.10) [107].
Nanomedicine 102
FIGURE 4.10
Ormosil nanoparticles as nonviral vectors for safe and efficient gene delivery
[107] and J. C. Matos, A.R. Soares, I. Domingues, G. A. Monteiro and M. C. Gonçalves unpublished results.
The use of external magnets for nonviral gene vectors that facilitate the introduction of plasmids into
the nucleus with a performance improved when compared with the routinely available standard
technologies is also reported. SPION-induced hyperthermia has also been utilized for localized killing of
cancerous cells [95].
Targeting
The possibility of selective delivery of VCAM-1-targeted Fe3O4@SiO2(FITC) NPs to sites of inflammation

and their accumulation or uptake by targeted cells give them high potential in vascular magnetic
resonance imaging for clinical diagnosis of cardiovascular disease, eg, atherosclerosis and thrombosis is
discussed by the authors [89].
Photothermal ablation is a minimally invasive approach, which typically involves delivery of
photothermal sensitizers to targeted tissues. [101] demonstrated that the use of gold shell–iron oxide
core hybrid NPs (Fe3O4/Au) for both imaging and laser irradiation. They demonstrated that the core-
shell NPs are up taken by pancreatic cancer cells, permitting magnetic resonance imaging (MRI) of
sensitizer delivery and photothermal ablation with NIR laser irradiation. An exposure to these NPs and
subsequent laser irradiation led to significant reductions in pancreatic cancer cell proliferation.
A review from [108] focuses on the fundamentals and strategies that are used to develop diverse
functional PLGA nanoparticulate carriers with a focus on recent research trends with multifunctional
Nanomedicine 103
PLGA core-shell hybrid NPs that provide temporal drug delivery, enable imaging and drug targeting in a
single utility, and achieve synergistic therapeutic outcomes to develop highly effective nanoparticulate
carriers that can deliver a spectrum of chemotherapeutic, diagnostic, and imaging agents for various
applications.
The development of a surface engineered magnetic core-shell NPs-based drug delivery system and
PLGA core-shell NPs designed for aerosol therapy of lung diseases are reported by [109]. These authors
developed NPs by coating of Fe3O4 magnetic NPs (MNPs) with poly(lactic-co-glycolic acid) (PLGA). The
polymeric shell of these engineered NPs was loaded with a potential anti-cancer drug quercetin and
their suitability for targeting lung cancer cells via nebulization was evaluated. The quercitin loaded
PLGA-MNPs were applied to the human lung carcinoma cell line A549 following a single round of
nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which
was comparable when applied either by nebulization or by direct pipetting.
New magnetic-based core-shell NPs (MBCSP) were developed by [110] to target skin cancer cells while
delivering chemotherapeutic drugs in a controlled fashion. MBCSP consist of a thermoresponsive shell
of poly(N-isopropylacrylamide-acrylamide-allylamine) and a core of poly(lacticco- glycolic acid) (PLGA)
embedded with magnetite NPs. To target melanoma cancer cells, MBCSP were conjugated with Gly-
Arg-Gly-Asp-Ser (GRGDS) peptides that specifically bind to receptors of melanoma cell. MBCSP consist
of unique multifunctional and controlled drug delivery characteristics. Specially, they can provide dual
drug release mechanisms (a sustained release of drugs through degradation of PLGA core and a
controlled release in response to changes in temperature via thermo-responsive polymer shell), and
dual targeting mechanisms (magnetic localization and receptor-mediated targeting). The particles
exhibited excellent cytocompatibility to healthy cells and efficient uptake by the targeted cancer cells.
Moreover, the particles displayed a high potential as imaging probes for MRI and optical imaging
modalities. Finally, the tumour-targeting capabilities of GRGDS-conjugated NPs are promising, as they
are effectively recruited by static magnets to the tumor site in melanoma mice models.
Over the past decade, positron emitter labeled NPs have been widely used in and substantially
improved for a range of diagnostic biomedical research. A variety of NPs has been engineered and
explored for diagnostic and therapeutic potential in various diseases. A review of [111] summarizes the
major applications of NPs labeled with positron emitters for cardiovascular imaging, lung diagnosis and
tumour theronostics taking into account that for personalized medicine and translational research, a
major challenge in the field will be to develop disease specific nanoprobes with facile and robust
radiolabeling strategies and that provide imaging stability, enhanced sensitivity for disease early stage
detection, optimized in vivo pharmacokinetics for reduced non-specific organ uptake, and table
improved targeting for elevated efficacy.
The examples presented in this review focus on NPs labeled with PET isotopes for cardiovascular,
pulmonary and tumour imaging, as well as for pharmacokinetic evaluation. So far, significant progress
has been achieved in NPs structure/design, in vitro trafficking, and in vivo fate mapping by using PET.
More effort will be necessary to achieve development of approved biocompatible and biodegradable
NPs for personalized medicine and translational research [112].
Final Remarks
The research innovations in the field of design and concept of multifunctional core-shell NPs reached a
level that allows the identification of some critical parameters.
The core materials can be improved, for example magnetic NPs biocompatible, biodegradable, and
have improved magnetic characteristics needed to be developed.
Nanomedicine 104
The shell materials can be focused to maximize their functionality in vivo to allow the implementation
of real-time feedback control of the targeting process.
The assemblage of a diversity of functions, forming complex systems in one nanoparticle, need to be
refined. Although a number of biomedical systems are in the borderline of prototyping.
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Nanomedicine 111
5
Biomaterial surface modification of
titanium and titanium alloys for medical
applications
1,2 2 2 1
Mukta Kulkarni , Anca Mazare , Patrik Schmuki , Aleš Iglič
1
Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana SI-1000, Slovenia
2
Department of Materials Science and Engineering, University of Erlangen–Nuremberg, Erlangen, Germany
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 112
Titanium and titanium alloys in medical applications …………………………………………..…………………….. 112
Biocompatibility of medical devices and the need for surface modification ……………………………….. 114
Surface modification of titanium and its alloys ………………………………………….……………………………….. 116
Overview of surface modification methods for titanium and its alloy …………………………………………. 116
Mechanical methods ………………………………………………………………………………………………………………….. 117
Chemical methods ……………………………………….…………………………………………………………………………….. 118
Chemical treatment ……………………………………………………………………………………………………………………. 119
Sol-gel deposition ………………………………………………………………………………………………………………………..120
Chemical vapour deposition (CVD) ……………………………………………………………………………………………… 120
Electrochemical methods ……………………………………………………………………………………………………………. 120
Biochemical methods …………………………………………………………………………………………………………………. 120
Physical methods ……………………………………………………………………………………………………………………….. 121
Selectively modified method: Anodization …………………………………………………………………………………. 123
General aspects of electrochemical anodization …………………………………………………………………………. 123
Biomedical applications of tio2 nanotubes …………………………………………………………………………………. 124
Nanotube diameter and cellular response ………………………………………………………………………………….. 124
Nanotubes and protein interaction ……………………………………………………………………………………………..126
Nanotubes for orthopaedic and dental implants ………………………………………………………………………… 126
Nanotubes for bladder stents ……………………………………………………………………………………………………… 127
Nanotubes for blood-contacting applications …………………………………………………………………………….. 127
Nanotubes for antibacterial activity …………………………………………………………………………………………… 128
Nanotubes for drug delivery ……………………………………………………………………………………………………….. 128
Summary …………………………………………………………………………………………………………………………………… 129
Acknowledgement ……………………………………………………………………………………………………………………. 129
References……………………………………………………………………………………..…………………………………………… 130
Nanomedicine 112
Introduction
The biomaterials research domain is a multidisciplinary one and includes various aspects of materials
science, chemistry, physics, biology and medicine. A biomaterial is a non-viable material used in
medical devices intended to interact with biological systems in order to evaluate, treat, augment or
replace any tissue, organ or function of the body [1]. The performance and applications of biomaterials
in biological systems are of critical importance for the development of biomedical implants and tissue
engineering. There are numerous biomaterials that can be used in the human body, such as metals
(e.g. stainless steel, cobalt alloys, titanium alloys), ceramics (aluminium oxide, zirconia, calcium
phosphates), and synthetic and natural polymers [2]. Among these, titanium (Ti) and titanium alloys are
considered to be some of the most significant biomaterials, due to their resistance to body fluid effects,
great tensile strength, flexibility and high corrosion resistance and this specific combination of strength
and biocompatibility [3] makes them suitable for medical applications. For example, commercially pure
Ti (c.p.Ti) is the dominant material used for dental implants while for orthopaedic applications Ti-6Al-
4V alloy is used [4]. Here, in this chapter various methods of surface modification of titanium and its
alloys are reviewed, including promising methods of obtaining specific nanotopography (e.g. titanium
nanostructures) such as electrochemical anodization, together with the latest research evaluating the
use and importance of nanotubular structures on Ti and its alloys for biomedical applications, as well as
future perspectives.
Titanium and titanium alloys in medical applications
Metallic materials have been used in medical applications (orthopaedics or dentistry) for more than 50
years. Titanium and its alloys received extensive attention in dental applications so that nowadays
commercially pure Ti (c.p.Ti) is the dominant material for dental implants and is used as an alternative
to Ag-Pd-Au-Cu alloy – not only because of its excellent properties but also due to the increasing cost of
Pd. Other reported representative dental titanium alloys are Ti–6Al–7Nb, Ti–6Al–4V, Ti–13Cu–4.5Ni, Ti–
25Pd–5Cr, Ti–20Cr–0.2Si etc. [5]. For hard tissue replacement, the low Young´s modulus of titanium
and its alloys is generally viewed as a biomechanical advantage because the low elastic modulus can
result in smaller stress shielding compared to other implant materials, and thus inducing healthier and
faster bone regeneration [6]. Besides artificial bones, joint replacements and dental implants, titanium
and titanium alloys are often used in cardiovascular implants, for example in prosthetic heart valves,
protective cases for pacemakers, artificial hearts and circulatory devices. Because of their inert, strong
and non-magnetic properties, some alloys like nickel-titanium alloy (Nitinol, shape memory alloy) have
received more attention in magnetic resonance imaging (MRI), which is a very powerful diagnostic tool.
Currently, nickel-titanium alloy stents are often used in treatment of cardiovascular disease and are
usually coated with a thin carbon film to enhance blood compatibility [7].
Since the focus of biomaterials has shifted towards tissue engineering, complex medical applications
and biotechnology, there is a need to better define and evaluate the specific interaction between
biomaterials and tissue components. After a thorough evaluation of the biomaterials field, Williams
proposed a unified concept of biocompatibility [3], which states that “Biocompatibility refers to the
ability of a biomaterial to perform its desired function with respect to medical therapy, without eliciting
any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating
the most appropriate beneficial cellular or tissue response in that specific situation, and optimising the
clinically relevant performance of that therapy.”. Titanium and its alloys remain essentially unchanged
when implanted into the body and this is a result of their excellent corrosion resistance, so that such
materials are referred to as bio-stable or biologically inert [4].
Nanomedicine 113
The widespread and successful application of titanium and titanium alloys in biomedical devices
(implants) is clearly due to the combination of its high corrosion resistance and appropriate mechanical
performance, which in turn makes it biocompatible. The outstanding corrosion resistance of titanium
and titanium alloys in vivo environments is actually due to their ability to form a chemically stable,
highly adherent and continuous protective oxide layer on their surface [8, 9]. Although this protective
oxide layer is thermodynamically stable; nevertheless, metal containing species can still be released
through a passive-dissolution mechanism.
Titanium is a reactive material and has an very high affinity for oxygen, which means that the
protective oxide film forms spontaneously and instantly and its disruption or damage is repaired
immediately [9], if the metal is in the presence of air or oxidizing media, as is the case in a biological
system when a bioliquid surrounds the metal [10]. This is generally valid for all metals used in surgical
implants, as these metals obtain passivity from the oxide films of the alloying elements – except for
applications where there is no oxygen present or in a reducing medium, which could occur in a crevice
where titanium could not form the passive film and as such would not be corrosion resistant [11].
The nature, composition and thickness of the protective oxide layers formed on titanium and titanium
alloys depend on the environmental conditions. Usually, the composition of the protective oxide film is
based on TiO2, Ti2O3 or TiO [9,11]. It could be that the oxide film results from bulk titanium alone and
that alloying elements (e.g. Mo, Nb, V, Cr, etc.) are probably not present in the passive film to any
significant extent [11]. From a microscopic point of view, it was shown that the passive film is
continuously dissolved and reconstructed in aqueous solutions [12]. As such, dissolution of alloying
elements is possible, as well as incorporation of different elements from solution into the film – e.g.
repassivation of titanium in biological liquid led to the adsorption of calcium and phosphate ions into
the film and at the outermost surface calcium phosphate and calcium titanium phosphate were formed
[13].
The most frequently mentioned mechanical properties of titanium and its alloys are summarized and
compared to those of stainless steel and Co-Cr based alloys in Table 5.1. These latter are the major
classes of selected metals and alloys used in the manufacture of dental, maxillofacial, orthopaedic,
cardiac and cardiovascular implants. Compared to other metallic materials, titanium is more suitable
for orthopaedics due to its high specific strength and low elastic modulus. Because the Young’s
modulus is smaller, less stress shielding can be expected leading to, as previously mentioned, healthier
and faster bone regeneration [6]. A low elastic modulus is desirable, as the metal should tend to
behave a little more like bone itself, which from a biomechanical perspective is essential. Titanium and
its alloys have a significantly lower density than other metallic biomaterials, so that Ti implants are
lighter than similar items fabricated from stainless steel or Co-Cr alloys. However, because of its low
hardness, titanium exhibits a low wear and abrasion resistance, which can result in a reduced service
life of the implant. By applying a suitable surface modification method, this problem can, to a large
extent, be overcome [5].
TABLE 5.1
Mechanical properties of c.p. Ti grade II and titanium alloys [14, 16]
3
Material Density (kg/m ) Young’s modulus, E (GPa)
c.p. Ti grade II 4200 100-110
Ti-6Al-4V 4500 100-130
Ti-6Al-7Nb 4520 110-130
Stainless steel 316L 7800 200
Co-Cr alloys 8500 210-230
Nanomedicine 114
As a result of the properties shown above, titanium is considered to be one of the most promising
biomaterials for implants, especially in orthopaedic applications such as joint replacements and bone
pins, plates and screws for repairing broken bones. One important aspect is that the fate of the implant
material is not only governed by the bulk of the material (critical in determining the biological
performance), but also by its surface properties (surface chemistry and structure) which are crucial
factors in the interactions of the material with the surrounding tissue. The material chosen as bulk
material should withstand stresses which are too high for ceramic or polymeric materials, but
acceptable for metallic materials. However, the human body is able to recognise implant materials as
foreign and tries to isolate them by encasing in fibrous tissues. Such is the case if the surface properties
are not capable of leading to formation of a stable bonding between the surface of the implant and the
surrounding tissue, but result in the formation of a fibrous layer which would undermine the load
transmission between bone and implant, and would favour micro movements, eventually leading to
implant failure [17].
Biocompatibility of medical devices and the need for surface

modification
Depending on the intended implant location, namely the desired application of the biomedical device,
there are different factors to be considered. For example, if the biomedical device is intended to be a
blood-contacting device (catheter, graft and stent), blood compatibility (haemocompatibility) of the
biomaterials is crucial, whereas for bone applications osseointegration is the key parameter. For both
types of applications, the host response and its severity are strictly related to the surface properties of
the biomaterial.
Biomedical devices for use in contact with blood must not activate the intrinsic blood coagulation
system, nor attract or alter platelets or leucocytes. From this point of view, biocompatibility is more
difficult to achieve as it covers aspects such as thrombogenicity, complement activation, leukocyte
activation and changes in plasma proteins [18].
After the implantation of a blood contacting biomaterial, the first event that rapidly takes place is blood
protein adsorption at the solid-liquid interface. The proteins undergo conformational changes allowing
biological interactions and depending on the exposure time, the composition of the adsorbed protein
layer varies and proteins with stronger adsorption are favoured. In time, a resident protein layer is
formed which influences the interaction of platelets, activation of intrinsic coagulation, adhesion and
aggregation of platelets and activation of the complement system [18]. Furthermore, at the implant or
platelet adhesion surface, some blood coagulation factors are triggered and this could lead to
formation of thrombin, converting fibrinogen into insoluble fibrin, from which a fibrin network and
thrombin can be produced [19]. Several studies have reported the haemocompatibility of titanium
[18,19] but less data is available on the haemocompatibility of nanobiomaterials [20,21]: the current
status of research is further discussed in next sections.
The clinical goal and most critical factor in the success of bone implants (orthopaedics and dentistry) is
achieving osseointegration, particularly by establishing a strong and long-lasting connection between
the implant surface and peri-implant bone, leading to a stable mechanical attachment of the implant at
the site of the implantation [22].
In bone, titanium is integrated in close apposition to the mineralized tissues under the proper
conditions. However, titanium and bone are generally separated by a thin soft-tissue layer as a result of
a weak foreign body reaction that prevents titanium from being in direct contact with the bone [23]. As
Nanomedicine 115
soon as the implantation procedure occurs, several biological reactions take place in a specific order.
Initially, there will be wetting of the implant surface and rapid adsorption of biologically active
molecules (such as proteins), followed by enlisting of the osteoprogenitor cells that would regenerate
the tissue [17]. It is obvious that the two factors affecting osseointegration are the mechanical
properties of the implant and the biological interactions with the metal surface, of which the latter is
more relevant. These interactions could lead to:
I. Successful osseointegration as a result of the osteoconductive process of healing of

the peri-implant bone, when newly formed bone has direct contact with the implant
surface as a result of bone cell proliferation and differentiation.
II. Rejection, due to an acute foreign body response caused by the inflammatory
response reaction of the body to the implant.
III. Micromovements of the implant, favouring the formation of fibrous tissue instead of
a bony interface, due to the lack of stability between the surrounding tissue and the
implant surface. Micromovements can lead to implant failure.
IV. Bacterial infection at the surface of the implant that might lead to biofilm formation
and thus to short-term or long-term failure of the implant.
Usually, the steps occuring in the interaction between a biomaterial and the body, i.e. the healing
response, consist of acute inflammation, chronic inflammation, granulation tissue formation, foreign
body reaction and fibrosis [24]. Regardless of the type of biomaterial used or of the injury location, the
initial inflammation response is always present and will progress to acute inflammation (which usually
lasts only a few days). If the inflammatory responses do not subside, chronic inflammation sets in
followed by granulation tissue formation (the amount of granulation tissue determines the extent of
fibrosis). The foreign body response is next and the most important factor at this point is the surface
properties of the biomaterial as they influence the presence and magnitude of the foreign body
response. The last step in the healing process is fibrosis, which consists of encapsulation of the fibrous
tissue of the implant and depends on the proliferation capacity of the cells in the respective tissue [24].
It should be pointed out that for both orthopaedic and dental implants, fibrous encapsulation is not
desirable as it cannot withstand the same physical stresses as bone, thus leading to micromovements.
Recruitment of parenchymal cells (specifically osteosblasts) on the implant surface is desired.
Considering the above-mentioned factors, it is obvious that there is still room to improve the implant
surface, especially to enhance tissue engineering and to decrease implant failure or rejection. Firstly,
bone regeneration is a slow process so improvements were and are currently being made in order to
achieve faster osseointegration, either by morphological modifications or by various coatings, as will be
discussed in detail in the following sections. Secondly, a common cause of implant failure is bacterial
infection and the possibility of a bacteria-repellent surface modification is worth investigating.
Currently, the most common method of achieving improvement is by modification of the implant’s
surface properties, either morphologically and/or by biochemical coatings. It follows that there is a
major need for surface modification of implants in order to increase tissue adhesion, implant
integration, decrease bacterial adhesion and decrease inflammatory response or to avoid the foreign
body response.
Nanomedicine 116
Surface modification of titanium and its alloys

It is evident that the response of a biomaterial depends entirely on its biocompatibility and surface
properties. Therefore, in order to improve the performance of biomaterials in biological systems, there
is an urgent need for their surface modification [25]. The structures encountered by the cells present in
the human body are not only on the micrometre scale but also on the nanometre scale since bone is
made up of nanostructures. Consequently, it is necessary to produce better implant materials, i.e with
nanometre roughness, especially since the influence of the surface roughness on cell attachment was
evaluated mainly on the micrometre scale.
There are various approaches possible to modify the surface of metallic materials (including nanophase
materials) that improve their cellular activities when compared with conventional microrough
materials. Generally, nanoscale surfaces possess high surface energy leading to increased initial protein
adsorption which is very important in regulating the cellular interactions on the implant surface.
Surface properties also have an impact on adhesion, together with charge distribution and the
chemistry of the material [26,27]. It was recently observed that the roughness of titanium
nanostructures alone influences the adhesion of osteoblast cells and their spreading and proliferation
[28].
In the present section, surface modification methods for titanium and titanium alloys will be reviewed,
including the mechanical, chemical and physical methods used for morphological modifications
(increasing roughness, shifting topography from the micro to the nanoscale, tailoring the nanoscale
morphology), or for obtaining different coatings on the surface of the implant. These include
hydroxyapatite, biomimetic calcium phosphate coatings, biomolecule functionalized coatings, as well
as a mixture of morphological changes and coatings for a combined synergistic effect. The goal is to
improve bioactivity, biocompatibility, blood compatibility, and the wear and corrosion resistance of
titanium and titanium alloys for their respective applications.
Overview of surface modification methods for titanium and its alloy
The assumption that increasing the surface roughness of a metallic implant will result in higher
micromechanical retention than for a smooth or as-machined implant was proven to be correct [29].
Nowadays, the implant surface is modified in commercial products by mechanical methods (grit
blasting with various types and sizes of abrasives, attrition for obtaining nanophase materials),
chemical methods (acid etching, electrochemical processes) and physical methods (plasma-sprayed
titanium coatings) or by their combination [30,31]. In vitro results indicated a correlation between
surface roughness and cellular attachment and osteoblast activity [32,33], as well as with other factors
influencing implant osteointegration (selective protein adhesion, collagen synthesis, and chondrocyte
maturation) [34, 36]. The results were conclusively supported by in vivo tests [37, 39]; namely,
microstructured surfaces provided a better implant surface–bone contact and an increased mechanical
retention after implantation. Material roughness modification influences other physicochemical
properties, e.g. the higher the roughness, the higher the local surface electrostatic charge density
[40,41] and adhesion energy. It is possible to design metallic surfaces with high surface energy and
superhydrophilic properties through different methods [40,42], which are shown to accelerate the
early stages of bone healing [42,43], possibly by preferential adsorption of fibronectin, osteocalcin or
other growth factors and by favouring bone growth [44]; however, the mechanism of this higher
osseointegration has not yet been elucidated.
Nanomedicine 117
Roughness modification does not alter the bioinert nature of titanium and titanium alloys and hence
further chemical modifications were developed to ensure rapid osseointegration (rapid bone
regeneration). Starting from simple inorganic coatings containing apatite (with the potential to actively
signal the cells at the implant surface after implantation) up to more complex coatings, much research
was performed both in vitro and in vivo to evaluate the optimal method of deposition (e.g. plasma-
spray, electrodeposition, biomimetic precipitation of calcium phosphate by immersion in a simulated
body fluid, protein adsorption, etc.), as well as investigation of their mechanical properties. Recently,
more emphasis was put on biomolecule functionalization of the implant surface with different
molecules (natural extracellular matrix proteins such as collagen, fibronenctin, etc.; peptides;
engineered protein fragments). Nevertheless, the critical steps are the actual binding of the bioactive
molecule to the implant surface and the selection of the immobilization method.
A tremendous amount of research has been invested into surface modification of microrough and
nanorough titanium and titanium implants [45]. If the “somewhat” compact surface of the implant is
replaced with a nanostructured surface (nanotubes, nanorods, etc.), it follows that the possibilities of
structural, surface and chemical modifications are numerous and their resulting synergistic effects
could lead to significant improvements in the field of tissue engineering.
Mechanical methods
The mechanical methods widely used to obtain either a rough or a smooth surface are subtraction or
attrition processes (as shown in Table 5.2) [46]. The main objective of mechanical modification is to
obtain specific surface topographies, to clean or roughen the surface, which could lead to improved
adhesion in bonding, as the roughness of the structure would be more favourable for bio-
mineralization due to the increased surface area [40]. These methods involve external action by the
application of physical forces to modify the surface characteristics. Common mechanical surface
modification methods, such as machining, polishing, and grit-blasting involve physical treatment by
shaping or removal of the materials surface. In the case of metallic materials, machining usually
produces deformations: crystalline grains disappear, surface properties are changed and, in general,
surface hardness increases. In order to obtain finer degrees of the finishing, surfaces can be exposed to
a smoothing process, by means of polishing. Blasting process consists of the impact of a jet of abrasive
particles against the surface by compressed air; this could also increase the surface reactivity of the
metal and the blasting particles can induce abrasive pollution on the implant surface [7].
The currently used micron sized surfaces of biomaterials do not perfectly replicate the surface or the
mechanical properties of the replaced bone, which can lead to failure due to insufficient bonding with
juxtaposed bone, bone loss, implant loosening or fracture.
Nanomedicine 118
TABLE 5.2
Overview of mechanical methods used for surface modification of Ti and Ti alloys [7]
Mechanical methods [7] Modified layer Objective

Grinding Produce specific surface
Rough or smooth
Polishing topographies;
surface formed by the
Machining Clean and roughen surface;
subtraction process
Blasting Improve adhesion in bonding.
To fabricate nanophase
surface layers on Produce materials with
titanium of commercial nanometre size grains (1-100nm);
Attrition purity which improve To produce rough morphology
the tensile properties and higher hydrophilicity.
and surface hardness of
the titanium
Nanophase materials are unique materials that simulate the dimensions of the components constituing
bone since they possess particle or grain sizes less than 100 nm [33]. Interestingly, osteoblasts were
observed to adhere specifically at particle boundaries. Since a nanophase metal has a higher
percentage of particle boundaries at the surface, this could explain the greater number of osteoblast
cells on nanophase metals compared to conventional ones [33]. Nanophase materials possess unique
surface and mechanical properties similar to bone and are thus considered to represent the future
generation of orthopaedic biomaterials (human bone consists of inorganic minerals of grain size varying
from 20 to 80 nm in length and 2 to 3 nm in diameter) [33]. Variations of the adhesion energy due to
variation in the nanosurface roughness lead to desirable cellular responses on nanostructured titanium
and other materials, resulting in high osseointegration. Studies [33-41] investigating cell adhesion on a
submicron, nanometre structured titanium surface compared to a flat smooth titanium surface
suggested that both nanometre and submicron surfaces have very high adhesion energy, thus resulting
in a high adhesion of bone cells.
Chemical methods
The main objective of using chemical methods is to improve biocompatibility, bioactivity and bone
conductivity, corrosion resistance and removal of contamination. These methods provide titanium with
bioactive surface characteristics. Some of the commonly used chemical methods are reviewed in this
section (Table 5.3). The most widely used chemical methods are acid and alkaline etching,
electrochemical anodization, chemical deposition and biochemical surface coating methods [7,47,48],
and these will be further discussed in what follows.
The chemical methods described here include chemical treatments consisting of soaking in NaOH
followed by heat treatment, or etching in HCl [49] and subsequent NaOH treatment, electrochemical
56
treatment (anodic oxidation) [50, 53], sol–gel deposition [54,55], hydrogen peroxide treatment [ ],
chemical vapour deposition (CVD) [57,58], and biochemical modification [17,23]. During chemical
treatment, electrochemical treatment or biochemical modification, chemical, electrochemical or
biochemical reactions occur, respectively, at the interface between titanium and the solution.
Nanomedicine 119
Chemical treatment
Chemical treatments of titanium and its alloys are mainly based on chemical reactions occurring at the
interface between titanium and the solution. Common chemical treatments include acid, alkali, H2O2,
heat, and passivation treatments [56,59,60]. Such treatments are generally performed to remove the
oxide scales and contamination present on the surface and can also improve biocompatibility,
bioactivity or bone conductivity.
TABLE 5.3
Overview of chemical methods used for surface modification of Ti and Ti alloys [7,47,48]
Chemical methods Modified layer Objective

Chemical treatment
Acidic treatment [59] <10 nm of surface oxide layer Remove oxide scales and
contamination.
~1µm of sodium titanate gel
Alkaline treatment [60] Improve biocompatibility,
bioactivity or bone
conductivity.
~5 nm of dense inner oxide
Hydrogen peroxide and porous outer layer Improve biocompatibility,
treatment [56] bioactivity or bone
conductivity.
~10µm of thin film, such as Improve biocompatibility,

Sol-gel [54] calcium phosphate, TiO2 bioactivity
and silica or bone conductivity
~1µm of TiN, TiC, TiCN, Improve wear resistance,
CVD [56] diamond and diamond-like corrosion resistance and blood
carbon thin film compatibility
Produce specific surface

~10 nm to 40µm of TiO2 topographies; improve
layer, adsorption and corrosion resistance; improve
Anodic oxidation [50-53] incorporation of electrolyte biocompatibility, bioactivity or
anions bone conductivity
Coating deposition
Modification through silanized Induce specific cell and tissue
Biochemical methods titania, photochemistry, self- response by means of surface-
[23,61,62] assembled monolayers, immobilized peptides,
protein-resistance, etc. proteins, or growth factors
Nanomedicine 120
Sol-gel deposition
In the sol–gel method, chemical reactions occur not at the interface between the sample surface and
solution or gel, but rather in the solution. Generally, the sol-gel method is widely used in order to
deposit thin (<10µm) ceramic coatings. When compared to conventional thin film processes, the sol-gel
method ensures better control regarding of the chemical composition and microstructure of the
coating, preparation of homogeneous films, reduction of the densification temperature, and finally
simpler equipment and lower cost [7]. Many coatings, such as titanium oxide (TiO2), calcium phosphate
(CaP), TiO2–CaP composites and silica-based coatings have been prepared on titanium and its alloys for
biomedical applications [7,54].
Chemical vapour deposition (CVD)
On the other hand, chemical vapour deposition (CVD) is a process involving chemical reactions
between chemicals in the gas phase and the surface of the substrate, resulting in the deposition of a
non-volatile compound on the substrate and is quite widely used as a chemical surface modification
method. Untill now, many different CVD processes have been developed [57], such as atmospheric-
pressure chemical vapour deposition (APCVD) – good uniformity of the coating; low-pressure chemical
vapour deposition (LPCVD) – increased hardness and corrosion resistance; laser-enhanced chemical
vapour deposition (LECVD) – improved wear and corrosion resistance; plasma-enhanced chemical
vapour deposition (PECVD) – improved wear and corrosion resistance; and plasma-assisted chemical
vapour deposition (PACVD) – improved biocompatibility, chemical stability and corrosion resistance.
More details about each of these chemical vapour deposition procedures and their advantages can be
found in the literature [7,57].
Electrochemical methods
Electrochemical processes are performed by connecting the metallic device to the positive pole of an
electrical circuit and immersing the entire device into an electrolyte solution containg ionic substances
or oxidants. This methodology can lead to incorporation of some ions on the material surface, and
includes the possibility of changing the surface finish. One method that can be identified is anodization,
which is obtained by using strong acids (e.g. H2SO4, H3PO4, HNO3, and HF) as the electrolyte solution.
The end results of the anodization process are the thickening of the oxide layer from the usual 5-10 nm
oxide layer formed due to atmospheric oxidation, or by modifications in the microstructure and the
crystallinity of the titanium oxide layer [50-52]. Another electrochemical method which has been
extensively used for deposition of ceramic coatings on the surface of metals is micro-arc oxidation
(MAO), and high micro-hardness coatings with good adhesion, strength and wear resistance are
attained [63]. Furthermore, if MAO is performed in the presence of an electrolyte containing calcium
and phosphorus, a bioactive coating with reduced osseointegration of the implant is obtained [63].
Biochemical methods
Some processes are based on the deposition of foreign chemical substances on the implant surface by
electrodeposition, biomimetic precipitation of calcium phosphate through immersion in simulated
body fluid, protein adsorption, etc. [17,62]. For example, hydroxyapatite (HA) is considered one of the
most effective bioactive materials and can be easily deposited by immersion of the implant surface in
different simulated body fluids.
Nanomedicine 121
Biochemical methods [23] provide the possibility of adding and bonding specific biomolecules at the
implant surface. These treatments are focused on controlling and guiding the complex sequence of
biochemical phenomena that take place at the interface between an implanted device and biological
tissue, such as osseointegration.
There are three major methods available for the biochemical treatment of a metallic surface: a)
physico-chemical adsorption; b) covalent binding of a biomolecule on the surface and c) peptide
inclusion into a carrier material. Physico-chemical adsorption of an active molecule to the surface is
based on the simple process of immersing the sample into a bioactive peptide-containing solution.
Despite the ease of this method, the main drawback is that it does not ensure controlled deposition,
which is essential for directing the interactions with biological tissues. Furthermore, the adsorbed
molecules can be displaced and diffused from the adsorption site. Covalent attachment uses the
chemical properties of the material surface to covalently bind the bioactive molecule. Here different
strategies are focused on increasing the number of reactive -OH groups. However, covalent binding
does not result in control of the surface density of peptides, which can affect the biological response.
Hence, peptide inclusion into a carrier molecule has the advantage of enabling control of the amount
of bioactive peptide introduced and used to coat the implant surface. Carriers, mostly polymers, can be
either simply impregnated with the active biomolecules or covalently bound to the carrier structure.
An elegant method frequently used to render the metallic substrate responsive to various stimuli is
based on using self-assembled monolayers (SAMs) as coatings. The advantage of SAMs lies in their
structure, which is that of a bifunctional molecule with a head group that is able to interact strongly
with a metal, oxide or polymer and arrange itself on the surface of the material; the attachment
mechanism has been extensively discussed [64] and is based on immobilization of the biomolecule on
the biomedical implant surface [65].
The biomechanical performance of an implanted device largely depends on the properties of its
surface, in terms of both chemical composition and roughness. The anchored or adsorbed
biomolecules are normally present on the cell membrane and extra-cellular matrix. Among other
proteins, many studies have been focused on the family of beta transforming growth factors (TGF-β)
and bone morphogenetic proteins (BMPs) [61]. However, the use of a protein by itself is not very
reproducible since it will have both a low chemical stability and solubility in the biological environment.
As an alternative, smaller biologically active sequences, namely peptides, which are part of the total
aminoacid sequence of a protein have been isolated/synthesized and attached to the desired substrate
[17]. One of the most investigated peptides is the Arg-Gly-Asp (RGD) amino acid sequence, which is
known to be the minimal cell-recognisable sequence in many adhesive proteins [66,67].
Physical methods
Physical surface modification methods include processes such as thermal spraying, physical vapour
deposition, ion implantation and glow discharge plasma treatment, where chemical reactions do not
occur. The surface modified layer, film or coating obtained on the titanium substrate is mainly a
product of the thermal, kinetic, and electrical energy involved. Some of these methods and their
objectives are listed in Table 5.4.
In thermal spraying methods, the coating is obtained by thermally melting the coating materials into
liquid droplets and spraying them onto the substrate at high speed – in this case the coating is due to
kinetic energy. Depending on the maximum temperature used, thermal spraying may be separated into
flame and plasma spraying, in which plasma spraying can provide very high temperatures. Other
spraying techniques include high velocity oxy-fuel (HVOF) spraying, detonation gun (D-GUN) spraying,
arc spraying and so on [7,68,69]; for the milestones achieved in thermal spray coating of functional
Nanomedicine 122
titanium dioxide, as well as a thorough review of their applications in bone implants, see Gardon et al.
[70]. Recently, cold gas spray for deposition of titanium dioxide coatings has gained interest [70,71]
Physical vapour deposition (PVD) consists of evaporating or sputtering the target materials in a vacuum
to form atoms, molecules or ions that are then transported to the surface of the substrate where film
growth takes place by condensation or by reaction with the surface of the material [7].
TABLE 5.4
An overview of physical methods used for surface modification of Ti and Ti alloys [7,47,48,]
Physical methods Modified layer Objective
Thermal spray [70,72] ~30 to ~200µm of Improve wear resistance,

Flame spray, plasma coatings, such as titanium, corrosion resistance and
spray, high velocity oxy-fuel HA, calcium silicate, Al2O3, biological properties.
spray, etc. ZrO2,TiO2
Physical vapour depositon ~1µm of TiN, TiC, TiCN,
Evaporation [73] diamond and diamond-like Improve wear resistance,
Ion plating [74] carbon thin film corrosion resistance and
Sputtering [75] Hydroxyapatite coating by blood compatibility.
sputtering
Modify surface composition;
Ion implantation and ~10 nm of surface modified improve wear, corrosion
deposition [76] layer and/or um of thin film resistance, and
biocompatibility.
Cleaning, sterilizing or
Glow discharge plasma ~1nm to ~100 nm of oxidizing the surface; surface
treatment [77] surface modified layer nitridation; removal of the
native oxide layer
In evaporation, the thermal phase change occurs from solid to vapour and is successfully used for
obtaining TiC and TiN coatings by evaporation of Ti in the presence of an acetylene and nitrogen
plasma [73]. Similar TiN or TiC coatings can also be obtained by ion plating, through energetic
bombardment of particles on the surface of the substrate influencing film formation; there are several
procedures such as arc ion plating and plasma immersion ion plating frequently used [7,74]. Of all the
PVD methods, sputtering is preferred for deposition of thin films due to its simplicity and flexibility. For
biomedical applications, sputtering was mainly used to deposit thin films on titanium and its alloys so
as to enhance their bioactivity, biocompatibility, wear and corrosion resistance. Recently, sputtering
was also used for deposition of hydroxyapatite nanocoatings for biomedical applications [78].
Ion implantation is performed via a bombardment, which introduces ions into the surface layer of a
solid substrate and depending on the shape of the sample, conventional beam-line ion implantation or
plasma immersion ion implantation are used. For more complex shapes, the latter is used as it
circumvents the line-of-sight restriction inherent to conventional ion implantation [7]. For more
detailed information about the advantages of ion implantation and the state-of-the-art in titanium
based biomaterials, see Rautray et al. [79].
Nanomedicine 123
Another physical method for implant modification is glow discharge plasma treatment, which is
extensively used for surface modification of titanium or titanium alloys, so as to increase the
adsorption of extracellular matrix proteins on the implant surface [80].
Selectively modified method: Anodization

General aspects of electrochemical anodization
In recent years, several methods have been developed to produce nanoscale structures on titanium
surfaces. While irregular nanomorphologies can be easily established by chemical methods,
electrochemical anodization of titanium is one of the most popular and novel strategies to produce
controlled structures (including nanotubes, pillarlike nanostructures, and nanodots) on implant
surfaces.
In order to improve the biological, chemical, and mechanical properties of biomaterials, significant
research has been aimed at finding more suitable biomaterials with nanotopography which could offer
improved bioperformance. TiO2 nanotubes can be easily grown by electrochemical anodization of Ti or
its alloys. Over the last two decades, the electrochemical formation of self-organized nanotube layers
in dilute fluoride-containing electrolytes has been studied intensively [81, 84]. Wei et al. [85] showed
that on anodization of Ti in organic electrolytes of low water content, the formation of ordered TiO2
nanoporous structures could be observed. I.e., the water content in the electrolyte is the critical factor
that determines whether self-ordered oxide nanotubes or nanopores are formed. This supports the
concept that tube formation originates from ordered porous oxide by a “pore-wall-splitting”
mechanism [85].
By tailoring the anodization conditions [86,87] (applied voltage, anodization time and concentrations of
chemicals) TiO2 nanotubes of different diameters from 15 nm up to 300 nm and different lengths can
be obtained [86]. Recently, Roy et al. [87] and Kowalski et al. [88] reviewed the mechanisms involved in
anodization and the recent advances in the formation of nanostructured oxides in the form of
nanotubes, nanopores with hole morphology, mesosponges, nanochannels and microcones grown on
titanium or titanium alloys.
The general nanotubular and nanoporous morphologies obtained for titanium in classical organic
electrolytes, similar to [85], are presented in Figure 5.1 with emphasis on the ability to tailor
nanotubular morphologies (e.g. diameter controlled), as well as indicating the difference between
nanotubular and nanoporous structures (see Figure 5.1 e) and f)).
For more details about the mechanism of anodization and all the factors involved in controlling the
morphology of the nanostructures through the anodization conditions, see the comprehensive review
by Roy et al. [87]. Moreover, the possibility of using amorphous or crystalline nanostructures, and the
effect of crystallinity on the hydrophilic character or other morphological or structural properties
should be considered.
TiO2 nanotubes can be obtained on almost all titanium alloys containing valve metals (transition
metals), such as Ti-6Al-7Nb [89, 91], Ti-6Al-4V [89], TiZr alloys with different Zr concentrations [92,93]
or on other biomedical alloys developed or tested for biomedical applications (Ti-6Al-4Zr, etc.).
Nanomedicine 124
FIGURE 5.1
Top view SEM images of different diameter TiO2 nanotubes obtained in classical ethylene glycol electrolyte: a) 15
nm, b) 50 nm, c) 100 nm, and of TiO2 nanopores: d) 15 nm. Cross-sectional images and top view of the
nanostructures along the cross-section of e) TiO2 nanotubes, and f) TiO2 nanopores.
Biomedical applications of TiO2 nanotubes

Nanotube diameter and cellular response
Titania nanotube arrays are one of the most promising candidate of titanium or titanium alloy
nanostructures for implant applications, e.g. dental implantology. Several in vitro studies [18,94, 96]
have demonstrated that cells cultured on nanotubular surfaces possessed higher adhesion,
proliferation, ALP activity and bone matrix deposition.
Nanomedicine 125
The influence of the nanomorphological features of titania nanotubes on the cellular response is
particularly striking, especially the finding that there is a clear effect of the diameter, and that
diameters of 15-20 nm are optimal for increased cell adhesion and proliferation [96,97]. Examples of
the nanotubular structures used for checking the influence of diameter size on cell adhesion and
proliferation are presented in Figure 5.2, showing the remarkable control achieved over the diameter
of such structures.
FIGURE 5.2
Top-view SEM images of self-assembled layers of vertically oriented TiO2 nanotubes of six different diameters
ranging between 15nm and 100 nm formed in 1 M H3PO4 containing 0.3 wt.% HF, at potentials between 1 and 20 V
for 1 h. Scale bars: 200 nm. Reproduced with permission from [97]. Copyright 2007. American Chemical Society.
Besides the clear diameter size effect showing that a diameter of approximately 15 nm significantly
increases the adhesion, proliferation and differentiation of mesenchymal stem cells, higher tube
diameters of approximately 100 nm led to programmed cell death (apoptosis) [96, 98]. This size-effect
was confirmed for different substrate materials such as TiO 2 and ZrO2 [98], for different states of
crystallization (amorphous and annealed) [98], and for different fluoride contents in the tubes [98].
Furthermore, the size effect was confirmed for several types of living cells, i.e. mesenchymal stem cells,
haematopoietic stem cells, endothelial cells, osteoblasts and osteoclasts [87]. The size effect is
explained by the specifically tailored nanotubular morphology, because integrin clustering in the cell
membrane leads to a focal adhesion complex with a size of about 10 nm in diameter, this being a
perfect fit to nanotubes with diameters of about 15 nm [97].
One aspect often ignored concerns the toxicity of TiO2 nanostructures. In this respect in 2010 Feschet-
Chassot et al. used the ciliated protozoan T. pyriformis to predict the toxicity of titanium dioxide
nanotube layers towards biological systems [99]. Titanium surfaces do not show any characteristic in
vitro toxicity effect [100].
Nanomedicine 126
Nanotubes and protein interaction
Irrespective of the location of the implant (blood-contacting, orthopaedic or dental implant) the first
step taking place after implantation is the adsorption of proteins from the surrounding tissue or
medium. The amount and type of protein adsorbed further influences the fate of the implant– as
pointed out in section “Biocompatibilty of medical devices”.
Gongadze et al. [40,41] proposed a mechanism for the adhesion of cells to a nanorough titanium
implant surface with sharp edges. The basic assumption was that the attraction between the negatively
charged titanium surface and a negatively charged osteoblast is mediated by charged proteins with a
distinctive quadrupolar internal charge distribution. Similarly, cation-mediated attraction between
fibronectin molecules (present in the extracellular matrix) and the titanium surface is expected to be
more efficient for a high surface charge density, resulting in facilitated integrin-mediated osteoblast
adhesion. Osteoblasts could be more strongly bound along the sharp convex edges or spikes of
nanorough titanium surfaces where the magnitude of the negative surface charge density is the
highest. It is therefore plausible that the nanorough regions of titanium surfaces with sharp edges and
spikes could promote the adhesion of osteoblasts. A small diameter nanotube surface has on average
more sharp convex edges per unit area than a large one, leading to a strong binding affinity on the
surface of small diameter nanotubes [40,41].
Nanotubes for orthopaedic and dental implants
Implant topography is critical to the clinical success of bone-anchored implants, yet further research
needs to be done on how nanomodified implant topography affects osseointegration. Previous in vitro
studies [98] reported that the topography of TiO2 nanotubes improved osteoblast proliferation and
adhesion compared to normal titanium surfaces. The increased in vitro cellular activities for titania
nanotubes also translated into in vivo bone bonding [96,98]. Nanotubular surfaces significantly
improved bone bonding strength by as much as nine-fold compared with grit blasted surfaces [44], and
histological analysis revealed greater bone-implant contact and collagen type I expression, confirming
the better in vivo behaviour of titania nanotubes. In vitro tests performed on nanotubes obtained on
different alloys such as Ti-6Al-7Nb [90] or TiZr alloys [101] suggest their use in orthopaedic cellular
therapy.
Covalent immobilization of different biomolecules can be used as a tool for differentiation of
mesenchymal stem cells (MSCs) from specific cells. For example, immobilization of epidermal growth
factor (EGF) [102] on 100nm diameter TiO2 nanotubes enabled the seeded cells to avoid apoptosis and
to become attached and promote proliferation (as shown in Figure 5.3a). Immobilization of bone
morphogenetic protein-2 (BMP-2) [103] on nanotubes of varying diameter showed higher osteocalcin
and osteopontin levels on 30 nm diameter TiO 2 nanotubes. When BMP-2 was covalently immobilized
via carbonyldiimidazol (CDI) [104] the differentiation of MSCs was observed to depend on diameter,
namely, an enhanced osteogenic differentiation occurred on 15 nm nanotubes (see Figure 5.3b), and
chondrogenic differentiation on 100 nm nanotubes.
Nanomedicine 127
FIGURE 5.3
a) Schematic illustration of EGF immobilization via carbonyldiimidazole chemistry; cell proliferation rates after 7
days for unmodified and EGF immobilized surfaces with compact oxide, 15 nm and 100 nm diameter nanotubes (
cell proliferation was measured by a colorimetric WST-1 assay 7 days after cell plating (*p = 0.0021; **p = 0.0120;
***p = 0.0005) Reproduced with permission from Ref. [102]. Copyright 2011. The Royal Society of Chemistry. b)
Schematic illustration of BMP-2 immobilization by covalent reaction of an amino group of the protein with the
grafted CDI; osteocalcin staining indicates that differentiation of MSCs to osteoblasts was strongly supported on 15
nm BMP-2-coated nanotubes, but much less on uncoated nanotubes. Reproduced with permission from Ref.
[104]. Copyright 2012. WILEY-VCH Verlag GmbH & Co.
Nanotubes for bladder stents
The materials currently used for bladder applications often suffer from incomplete coverage by
urothelial cells, leading to continuous exposure of the underlying materials so aggravating the immune
response [105]. Complications with ureteral stents usually comprise infection and/or blockage due to
encrustation [106]. Such complications could be avoided by promoting the formation of a monolayer of
urothelial cells on the surface of the stent, since the urothelial cells would prevent immune cells and
bacteria from interacting with the stent [107]. Nanotechnology may aid in urothelialization of bladder
stents, since the unique surface energies of nanostructures could promote the adsorption of proteins
important for urothelial cell adhesion and proliferation. Comparing bladder stents coated with 20nm or
80 nm diameter nanotubes to normal titanium bladder stents [108], the 20 nm diameter nanotubular
titanium stents enhanced human urothelial cell adhesion and growth up to 3 days in culture. Despite
these promising results, there are few studies on the use of TiO2 nanotubes for bladder stents, where
nanotubular structures should be further explored for such applications.
Nanotubes for blood-contacting applications
As discussed previously in section “Biocompatibility of medical devices”; the first event taking place on
the surface of the implant immediately after its implantation is adsorption of blood proteins at the
Nanomedicine 128
implant-liquid interface. What happens to the adsorbed protein layer governs the interaction of
platelets and their adhesion or activation, leukocyte recruitment, activation of intrinsic coagulation and
of the complement [109]; moreover, all four processes are capable of eliciting a thrombogenic
response in vivo. Stepwise, first the adsorbed protein layer will lead to adhesion and activation of the
platelets, which is fundamental in forming the fibrin clot and recruiting leukocytes (as monocytes and
neutrophils). Second, the platelets will trigger an inflammatory immune response which will lead to
either thrombosis and/ or fibrous encapsulation of the implant [109,110]. Studies [111, 113] indicated
increased blood serum protein adsorption, platelet adhesion and activation and whole blood clotting
kinetics on titania nanotube arrays. Furthermore, a decrease of thrombogenic effects and surface-
induced fibrin clot formation was observed on nanotubes when compared to titanium surfaces. This
was evident from the slightly decreased levels of complement activation and slightly increased degree
of free fibrinogen on nanotubular surfaces [21,111,112]. When the structure of the nanotubes was
modified from amorphous to crystalline, a decrease of the haemolytic index was observed, indicating
an increase in the haemocompatibility; although amorphous nanotubes were also non-haemolytic with
a haemolytic activity below 2% [114]. Overall, titania nanotubular surfaces seem a promising interfacial
material for the long-term success of blood-contacting implants.
Nanotubes for antibacterial activity
Bacterial infection of in-dwelling medical devices is a growing problem that cannot be treated by
traditional antibiotics due to the increasing prevalence of antimicrobial resistance and biofilm
formation [115,116]. Controlled diameter nanotubes (amorphous or crystalline) displayed significantly
changed responses to both Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S.
aureus) – pathogens relevant for orthopaedic and other medical device-related infections [115,117]. It
is obvious that a similar size-effect also exists for bacteria, where use of larger diameter nanotubes
decreased the number of live bacteria (S. aureus and S. epidermidis) as compared to lower diameter
ones (20 nm) or Ti [115,118].
The antibacterial activity of titanium or titanium alloy nanostructures could be due to: i) the
nanostructuring of the implant surface (the diameter size-effect); ii) the use of alloys with inherent
antimicrobial properties or by decorating the nanostructures with antimicrobial nanoparticles; and
finally iii) by the functionalization of the nanostructures with anti-inflammatory agents or by loading
drugs inside the nanostructures (as will be discussed in the following subsection).
When using titanium alloys containing elements which could inhibit bacteria (eg. zirconium) such as
Ti50Zr alloy [119], smaller diameter nanotubes showed an increased antibacterial effect against E. coli.
Other examples of such alloys include Ti-Nb-Zr-Mo [120], Ti-Al-Nb and so on [87]. Antimicrobial
nanoparticles used for decorating the nanostructures include Ag [121] and Zn [122], while less
investigated antimicrobial agents include copper, fluorine and calcium.
Nanotubes for drug delivery
Current orthopaedic implants have functional lifetimes of only 10–15 years for a variety of reasons
including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged
bonding of the implant to the juxtaposed bone) [4]. To improve properties of titanium for orthopaedic
applications, it is possible to coat the nanotubular structures with infection-reducing drugs
(penicillin/streptomycin) or inflammation-reducing drugs (dexamethasone) by simple physical
adsorption or deposition from simulated body fluid (SBF). For example when drugs where deposited
from SBF, a drug elution times of up to 3 days was registered [123].
Nanomedicine 129
Shrestha et al. [124] showed that TiO2 nanotubes can be filled with magnetic Fe3O4 particles and thus
be magnetically guided to desired locations. Such a structure can be used directly for photocatalytic
reactions with cells or tissue, such as the site-selective killing of cancer cells. UV light can also be used
for killing cancer cells though the use of nanotubes but there is the disadvantage of needing direct
access for the UV light to the TiO2 nanotubes.
More recent work [125,126] focused on using an amphiphilic nanotubular structure consisting of
nanotubes that provide a hydrophobic cap (using a monolayer coating) which does not allow body
fluids to enter the nanotubes unless the cap is opened by a photocatalytic interaction. Based on the
same principle, drug-loaded nanotubular layers can also be capped with biopolymers [127].
Summary
Titanium and its alloys continue to be some of the most promising biomaterials used for biomedical
devices. Despite their outstanding properties (good mechanical resistance, corrosion resistance,
biocompatibility) since they are bioinert materials surface modification is necessary to improve
osseointegration, haemocompatibility or other properties important in their respective biomedical
applications. In this work, an overview of the generally used surface modification methods for
improving the properties of titanium and titanium alloys for biomedical applications was presented,
also taking into account the current shift of research from the micrometre to the nanometre scale.
Mechanical methods (grinding, polishing, attrition, etc.), chemical methods (chemical treatments, sol-
gel methods, anodic oxidation, etc.) and physical methods (thermal spray, ion implantation and
deposition, etc.) were discussed with respect to their effects on the implant surface and its
biocompatibility. One of the most promising recently emerging methods for obtaining nanometre scale
surfaces, namely electrochemical anodizing leading to nanotubular structures with controlled
diameters in the range of 15 nm up to 250 nm, was discussed. General aspects of electrochemical
anodization were presented, as well as the use of such nanostructures in the biomedical field where
cellular interactions and protein adhesion in orthopaedic, dental and blood-contacting applications or
drug-delivery applications were discussed. With the development of current surface engineering
techniques, cutting edge morphologies in the nanometre scale for implant applications, bladder stents
and for specific biomedical applications. Visualizing biointerfaces and biomaterials with nanometre
precision in a three-dimensional scale, could reveal new fundamental information on material
properties and bone response, enabling better design of biomaterials for the future.
Acknowledgement
This work was supported by Deutsche Forschungsgemeinschaft (DFG), the DFG Cluster of Excellence
(Engineering of Advanced Materials, EAM) and Slovenian Research Agency (ARRS) grants J1-4109, J1-
4136, J3-4108 and P2-0232.
Nanomedicine 130
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6
Experimental and Clinical Therapeutic
Uses of Low-Molecular-Weight
Heparin/Protamine
Micro/Nanoparticles
1* 2 1 1 2
Masayuki Ishihara , Makoto Takikawa , Hidemi Hattori , Masanori Fujita , Miya Ishihara and Shingo
3
Nakamura
1
Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
2
Department of Medical Engineering, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
3
Department of Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 138
Preparation of LMW-H/P M/nps …………………………………………..……………………………………………………. 139
Protein-Delivery Micro /Nano Particles ………………………………………………………………………………………. 141
FGF-2-Containing LMW-H/P M/nps ………………………………………........................................................ 142
HGF-Containing LMW-H/P M/nps ………………………………………..…………………………………………….......... 144
PRP Containing LMW-H/P M/nps ……………………………………………………………………………………………….. 145
Cell-Delivery Micro/Nano Particles ……………………………………….……………………………………………………. 147
LMW-H/P M/nps as Stromal Cell Carriers …………………………………………………………………………………… 147
LMW-H/P M/nps as Tumor Cell Carriers ………………………………………………………………………………………147
A Cell Culture System Using LMW-H/P M/nps-Coated Plates …………………………………...................... 149
Various Types of Cell Cultures Using LMW-H/P M/nps-Coated Plates …………………………………………. 149
Proliferation of bone marrow-derived mesenchymal stem cells (bmscs)
and ascs on LMW-H/P M/nps-Coated Plates ………………………………………………………………………………. 150
Proliferation of CD34+ Hematopoietic Progenitor Cells (CD34+ hcs)
on LMW-H/P M/nps-Coated Plates …………………………………………………………………………………………….. 150
Conclusions ………………………………………………………………………………………………………………………………… 151
Acknowledgments ……………………………………………………………………………………………………………………… 151
References……………………………………………………………………………………..…………………………………………… 152
Nanomedicine 138
Introduction
Polyelectrolyte complexes (PECs) are generated by electrostatic interactions between oppositely
charged polyelectrolytes. When this interaction occurs at non-equivalent ratios, nonstoichiometric
PECs are produced, causing each PEC particle to carry an excess charge [1,2]. Proteins interact with
both synthetic and natural PECs [3,4]. These binding characteristics, along with a simple preparation,
allow PECs to be an excellent model for studying the in vivo behavior of charged biopolymers as well as
having potential applications in medicine and biotechnology. Reported studies indicate that polyanions
and polycations can bind to proteins below and above their isoelectric points, respectively. These
interactions can result in soluble complexes, complex coacervation and/or the formation of amorphous
precipitates. Main aspects studied by different authors are compositions of PECs obtained under
various experimental conditions, such as the strength and position of ionic sites, charge density and
rigidity of polymer chains as well as chemical properties such as solubility, pH, temperature and
concentration [1,5-8].
Electrostatic interactions are also important because of their similarity to biological interactions.
Interactions between proteins and nucleic acids, for example, play a role in the transcription process
[3]. DNA/chitosan PECs [9], chitosan/chondroitin sulfate PECs and chitosan/hyaluronate PECs [10]
function as gene and drug carriers. Moreover, PECs that are insoluble also have potential applications
as membranes, microcapsules, micro/nanoparticles and scaffolds for tissue engineering [10].
Particulate drug delivery systems have become widely employed in both experimental and clinical
setting to address a range of applications. Their popularity can be attributed to ease of application as a
suspension and ease of manufacture. The size of the particles plays a substantial role in determining
the properties of the final product and its potential applications. When injected into tissue, large
particles (microparticles: approximately 1－5 µm in diameter) tend to stay where placed, while smaller
particles (nanoparticles: about 50－200 nm in diameter) will circulate for a period of time determined
by size, surface chemistry and other factors [11]. In general, microparticles will be useful if the particles
are delivered locally. Examples include local anesthetic microparticles and microparticles including
growth factor (GF), antibiotics and chemotherapeutics [12].
Heparin interacts with a variety of functional proteins, including heparin-GFs, cytokines, extracellular
matrix components and adhesion molecules [13-15]. Thus, heparin may be useful as a therapeutic
agent in various pathological conditions that involve functional proteins. However, high-dose heparin
cannot be used because of the excessive risk of bleeding [16]. In contrast, low-molecular-weight
heparin (LMW-H) has pharmacological and practical advantages compared with native heparin. The
lower protein binding activity of LMW-H produces a low, stable and predictable anticoagulant response
[16]. On the other hand, protamine neutralizes heparin and LMW-H by forming a stable complex that
lacks anticoagulant activity [17]. Protamine is also in clinical use to reverse the anticoagulant activity of
heparin following cardiopulmonary bypass as well as in cases of heparin-induced bleeding [18].
We previously prepared water-insoluble particles by mixing non-anticoagulant heparin with chitosan
and by mixing fucoidan with chitosan, and investigated the ability of the resulting insoluble
heparin/chitosan and fucoidan/chitosan microparticles to protect fibroblast growth factor-2 (FGF-2)
activity [20, 21]. Water-insoluble micro/nano particles (100 nm－3 μm in diameter) were then
prepared by mixing LMW-H with protamine, and reported the ability of the resulting injectable low-
molecular-weight heparin/protamine micro/nano particles (LMW-H/P M/NPs) to protect and activate
FGF-2 and hepatocyte growth factor (HGF) activity [21-23]. Furthermore, GFs released from platelets
that were involved in cell proliferation, migration and angiogenesis could be adsorbed onto LMW-H/P
M/NPs [24].
Nanomedicine 139
We also reported that LMW-H/P M/NPs bind to cell surfaces of adipose-derived stromal cells (ASCs)
through specific interactions between LMW-H/P M/NPs and cell surface heparin-binding proteins such
as some integrin. The interaction of the cells with LMW-H/P M/NPs resulted in cells/LMW-H/P N/MPs-
aggregate formation. The ASCs/LMW-H/P M/NPs-aggregate formation substantially promoted cell
viability in vitro. Furthermore, the aggregates induced vascularization and fibrous tissue formation in
vivo [25,26]. The LMW-H/P M/NPs, in combination with ASCs, are a new convenient cell carrier and
may be a promising novel therapy for inducing vascularization and fibrous tissue formation in ischemic
disease by transplantation of the ASCs/LMW-H/P M/NPs-aggregates.
As a coating matrix, LMW-H/P M/NPs were efficiently bound to tissue culture plates. With the ability of
LMW-H/P MPs to retain GFs, LMW-H/P M/NPs could be very useful in cell culture. Human
microvascular endothelial cells (hMVECs) and human dermal fibroblast cells (hDFCs) adhered to LMW-
H/P M/NPs-coated tissue culture plates [27,28] and grew optimally in low fetal bovine serum (FBS)
(1－2%) medium supplemented with FGF-2 (5 ng/mL). This protocol could make it possible to use low
autologous serum (1－2%) for the culturing of human bone marrow-derived mesenchymal stem cells
(BMSCs) and ASCs [29,30]. Furthermore, CD34+ hematopoietic progenitor cells (CD34+ cells) derived
from bone marrow exhibited a comparatively higher proliferation on LMW-H/P M/NPs-coated plates in
hematopoietic progenitor growth medium (HPGM) supplemented with appropriate cytokines than
those on uncoated plates [31]. Thus, we mainly describe in this Chapter on the LMW-H/P M/NPs which
we originally prepared as PECs, its characterizations and its potential medical applications as protein
carriers for GFs such as FGF-2, HGF and GFs in platelet-rich plasma (PRP), as a cell carrier for cell
transplantation and as coating matrix for human cell cultures.
Preparation of LMW-H/P M/NPs

Heparinoids (heparin, heparin sulfate, low-molecular-weight heparin, and heparin-like polysaccharides)
specifically interact with a variety of heparin-binding functional proteins with high affinity, including
heparin-binding GFs, cytokines, extracellular matrix components and adhesion molecules [13-15]. Thus,
heparin may be useful as a therapeutic agent in various pathological conditions that involve functional
proteins. However, high-dose heparin cannot be used because of the excessive risk of bleeding [16]. In
contrast, LMW-H (MW: approximately 5000 Da) has pharmacological and practical advantages
compared with native heparin. The lower protein binding activity of LMW-H produces a low, stable and
predictable anticoagulant response, thereby bypassing the need for laboratory monitoring of drug
levels to adjust the dosage [16]. In addition, one or two subcutaneous injections per day are sufficient
to maintain therapeutic concentrations because of its longer plasma half-life [16].
On the other hand, protamine, a purified mixture of proteins obtained from fish sperm, neutralizes
heparin and LMW-H by forming a stable complex that lacks anticoagulant activity [17]. Protamine is
also in clinical use to reverse the anticoagulant activity of heparin following cardiopulmonary bypass as
well as in cases of heparin-induced bleeding [18]. Furthermore, protamine is used as a carrier for
insulin (protamine Hagedorn (NPH) insulin) [32].
We previously prepared water-insoluble particles (2–10 μm in diameter) by mixing non-anticoagulant
heparin with chitosan and by mixing fucoidan with chitosan. The abilities of resulting insoluble
heparin/chitosan and fucoidan/chitosan microparticles were evaluated to protect and activated the
activity of FGF-2 in vitro and in vivo [19,20]. Water-insoluble micro/nanoparticles (100 nm－3 μm in
diameter) by mixing LMW-H (6.4 mg/ml) with protamine (10 mg/mL) at a ratio of 7:3 (vol:vol) (see
Figure 6.1) was reported the ability of the resulting injectable LMW-H/P M/NPs (0.1－3 μm in
Nanomedicine 140
diameter) to protect and activated FGF-2 [21,22] and HGF activity [23]. Furthermore, GFs from PRP that
were also involved in cell proliferation, migration and angiogenesis were able to adsorb onto LMW-H/P
M/NPs. The studies indicate that LMW-H/P M/NPs may serve as an effective micro/nano-carrier for
various GFs, particularly for the local application of GFs. GFs containing LMW-H/P M/NPs show a
substantial effect to induce vascularization and fibrous tissue formation because of the gradual
controlled release, protection and activation of GF molecules from GFs-containing LMW-H/P M/NPs
[24]. In another study, we used diluted LMW-H (≤0.32 mg/mL) as an anion molecule and the diluted
protamine (≤0.5 mg/mL) as a cation molecule to synthesize LMW-H/P NPs (approximately 100 nm in
diameter) [22].
FIGURE 6.1
Preparation of LMW-H/P M/NPs.
In order to produce of the nanoparticles, equally diluted LMW-H and protamine (100-fold, 50-fold and
20-fold diluted) were mixed in a ratio at 7:3 (vol:vol) (see Figure 6.2). The diameter of generated LMW-
H/P NPs by mixing 100-fold, 50-fold and 20-fold diluted protamine to equally diluted LMW-H in the
ratio of 3:7 (vol:vol) were 84.6 ± 26.8, 95.0 ± 27.0 and 112.5 ± 46.1 nm, respectively (see Figure 6.2)
[22+. And no microparticles (>1 μm in diameter) were observed in the mixtures. In contrast,
generations of small amount of microparticles (approximately 1 μm in diameter) were observed by
mixing of 10-fold diluted protamine (1 mg/ml) to LMW-H (0.64 mg/ml) in the ratio of 3:7 (vol/vol).
When non-diluted protamine (10 mg/ml) was added to non-diluted LMW-H (6.4 mg/ml) up to ratio of
3:7 (vol:vol), maximal LMW-H/P M/NPs (100 nm－3 μm in diameter) was produced and the high
turbidity was observed. When 10-fold concentrated protamine (100 mg/ml) was added to the equally
Nanomedicine 141
10-fold concentrated LMW-H (64 mg/ml) up to ratio of 3:7 (vol:vol), mixtures of larger LMW-H/P MPs
(3－10 μm in diameter) and larger cotton-like precipitates (>10 μm) were immediately generated and
those products were insoluble [22]. Cotton-like compounds were generated after lyophilizations of
both LMW-H/P M/NPs (100 nm－3 μm in diameter) and LMW-H/P NPs (approximately 100 nm in
diameter) solutions without dextran, and they were hardly resoluble in water. However, both the
freeze-dried LMW-H/P M/NPs were easily dessolved in water by adding 0.5% and 0.2% dextran,
respectively, before their lyophilizations. In addition, aggregation of LMW-H/P NPs in solution to LMW-
H/P MPs was prohibited in the presence of dextran [22]. Thus, the addition of dextran is effective to
stabilize the LMW-H/P M/NPs and to prepare stable and resoluble freeze-dry LMW-H/P M/NPs. On the
other hand, LMW-H/P NPs in suspension 20-fold diluted with saline was stable for at least 6 weeks at
room temparature.
FIGURE 6.2
LMW-H/P M/NPs generated by mixing diluted LMW-H and Protamine.
Protein-Delivery Micro /Nano Particles

We previously reported the ability of the injectable LMW-H/P M/NPs to adsorb and to protect and
activate FGF-2 [21,22], HGF [23] and GFs in PRP [24] that were also involved in cell proliferation,
migration and angiogenesis. The studies suggested that LMW-H/P M/NPs serve as an effective
Nanomedicine 142
micro/nano carrier for various GFs, particularly for the local application of GFs. Thus, GFs containing
LMW-H/P M/NPs show a substantial effect to induce vascularization and fibrous tissue formation
because of the gradual controlled release, protection and activation of GF molecules from GFs-
containing LMW-H/P M/NPs [24] (See Figure 6.3).
FIGURE 6.3
FGF-2-Containing LMW-H/P M/NPs

-9
FGF-2 binds heparin with high affinity (Kd of 8.6 × 10 M). The polysaccharides can prolong the
biological half-life of FGF-2 as well as protect FGF-2 from heat, acid and proteolytic inactivation [21].
-9
Similarly, the LMW- H/P M/NPs have high affinity for FGF-2 (Kd = 2.4 × 10 M) [21], and this interaction
of FGF-2 with the LMW-H/P M/NPs can substantially prolong the biological half-life time of FGF-2. The
protection of FGF-2 against heat inactivation and trypsin degradation by the LMW-H/P MPs was
effective in a concentration-dependent manner [21]. FGF-2 molecules were released in vitro from the
FGF-2-containing LMW-H/P M/NPs with half-releasing time of about 6 days. Those results
demonstrated that FGF-2 molecules are bound and stabilized on the LMW-H/P M/NPs, and that the
FGF-2 molecules incorporated into the LMW-H/P M/NPs will be gradually released upon
biodegradation of the hydrogel in vivo.
When the FGF-2-containing LMW-H/P M/NPs were subcutaneously injected into the backs of mice,
neovascularization was induced near the injection site after 3 days. Neovascularization induced by the
FGF-2-containing LMW-H/P M/NPs reached a maximum at 1 week, after which a slight decrease in the
neovascularization rate occurred. No significant vascularization was observed after either the injection
of FGF-2 alone or the LMW-H/P M/NPs alone [21]. Figure 6.4 shows prevension of limb loss in hindlimb
ishchemic model (nude mice) by FGF-2-containing LMW-H/P M/NPs. Five ishchemic hindlimbs out of 8
treated with FGF-2-containing LMW-H/P M/NPs were normally recovered after 2 weeks and two of 8
were maintained normal hindlimbs for at least 3 months [33]. Thus the intramuscular injection of FGF-
2-containing LMW-H/P M/NPs into ischemic hindlimbs promotes vascularization and reduces limb loss
associated with local ischemia. FGF-2-containing LMW-H/P M/NPs thus provide an excellent
biomaterial to immobilize, retain and gradually release FGF-2 for the optimal induction of
vascularization and granulation tissue formation. The present approach of using FGF-2-containing
LMW-H/P M/NPs would be a valuable option for therapeutic angiogenesis that is targeted for tissue
regeneration and for treating ischemic disease.
Nanomedicine 143
FIGURE 6.4
Prevention of limb loss in hindlimb ishchemic model by FGF-2^containing LMW-H/P M/NPs.
Another study demonstrated advanced fat survival and capillary formation in FGF-2-containing LMW-
H/P M/NPs-assist subdivided free fat-grafting groups in rats [34]. Furthermore, our study demonstrated
the ability of FGF-2-containing LMW-H/P M/NPs to induce both arteriogenesis and angiogenesis in
rabbit models of ischemic limbs [35] (see Figure 6.5). The primary conclusion is that FGF-2-containing
LMW-H/P M/NPs-treatment effectively induces the development of collateral vessels, which can
provide sufficient blood flow to the pre-existing vascular network in ischemic tissue. Since all
components used in the FGF-2-containing LMW-H/P M/NPs are also used clinically, we feel safety in a
clinical setting is probable [35].
Nanomedicine 144
FIGURE 6.5
Quantification of visible collateral arteries under angiographic viewing on day 28.
HGF-Containing LMW-H/P M/NPs
HGF can accelerate the regeneration of damaged tissue in animal models of hepatic and renal failure,
but the local biological activity of exogenous HGF may be limited in vivo without an adequate protein
delivery system that acts to maintain HGF at the target site and prevents rapid clearance by the liver
[36,37]. Without such a system, very large doses are usually required to exert a significant regenerative
effect [38]. To enhance the repetitive efficacy of HGF, we developed LMW-H/P M/NPs for high-affinity
absorption and controlled release of HGF. LMW-H /P M/NPs prolonged the biological activity of HGF
and protected HGF against heat and proteolytic inactivation. Furthermore, these HGF-containing LMW-
H /P M/NPs were injectable and biocompatible, and triggered significant angiogenesis at the site of
injection. Thus, the LMW-H /P M/NPs are a reliable, efficient and safe protein delivery system to
enhance and stabilize HGF activity at local administration sites for subcutaneous or muscular injection
[23].
More than 5 μg of HGF bound to 1 mg of LMW-H /P M/NPs and was gradually released from HGF-
containing LMW-H /P M/NPs in vitro. HGF-containing LMW-H /P M/NPs appeared to be bioactive since
they stimulated human micro-vascular endothelial cells (hMVECs) proliferation. In fact, HGF-containing
LMW-H /P M/NPs was more mitogenic than HGF alone, reducing the cell doubling time from 45 to 25 h,
possibly because bound HGF is more resistant to biodegradation and inactivation under physiological
conditions. Indeed, proliferation of MVECs was substantially stimulated by preloaded LMW-H /P M/NPs
even after 10 days at 37°C, while HGF alone did not stimulate MVEC proliferation at all after 7 days of
Nanomedicine 145
pre-incubation at 37°C. Furthermore, LMW-H F/P M/NPs could effectively protect HGF against heat
inactivation and trypsin degradation [23].
When HGF-containing LMW-H /P M/NPs were subcutaneously injected into the back of mice, large and
medium vessels were induced near the injection site after 8 days. The number of small vessels induced
by the HGF-containing LMW-H /P M/NPs reached a maximum on days 8–11, after which a slight
decrease in the neovascularization rate occurred. No significant induction in large vessels was observed
after injection of HGF or LMW-H /P M/NPs alone, although a minor induction of medium and small
vessels was observed. We suggest that free HGF diffused away too rapidly to induce arteriogenesis and
that inactivation of HGF remaining at the injection site within a few days also led to less efficient
vascularization. The modest vascularization (mainly small and medium vessels) induced by LMW-H /P
M/NPs alone result from the binding of various endogenous angiogenic growth factors around the
injection site, leading to local accumulation and controlled release [23].
PRP Containing LMW-H/P M/NPs
PRP contains a high concentration of thrombocytes (platelets). When the platelets are activated,
various GFs and other bioactive proteins in α-granules of platelets are released and those proteins
augment tissue repair and regeneration processes [24,39-41]. Platelets contain over 20 GFs, including
platelet-derived growth factors (PDGFs), FGFs, HGF, transforming growth factors (TGFs), and vascular
endothelial growth factors (VEGFs), almost all of which are known to bind to heparin and to LMW-H/P
M/NPs. Recent studies suggest that GFs in PRP not only influence the viability of transferred cells but
may also play bioactive roles in the regulation of proliferation and differentiation in various types of
cells [24]. Any treatment aiming to mimic the critical aspects of the natural biological process should
not be limited to the provision of a single GF, but rather should release multiple GFs at an optimized
ratio, at a physiological dose and in a specific spatiotemporal pattern. Those results indicated that the
LMW-H/P M/NPs also activate the platelets to release the GFs, and that in turn the released GFs from
the platelets can be immobilized, be stabilized and be activated on the LMW-H/P M/NPs [24].
The GFs in PRP are stably bound to LMW-H/P M/NPs in vivo. The GFs adsorbed onto LMW-H/P M/NPs
may be gradually diffused and released upon biodegradation of LMW-H/P M/NPs. When PRP-
containing LMW-H/P M/NPs were subcutaneously injected into the backs of mice, significantly higher
neovascularization and granulation tissue with enhanced filtration of inflammatory cells were observed
compared with the mouse groups injected with PRP alone, LMW-H/P M/NPs alone and the control [24].
Compared to either PRP alone or LMW-H/P M/NPs alone, locally administered PRP-containing LMW-
H/P M/NPs augmented the wound bed and substantially increased viability of rat dorsal paired pedicle
skin flaps [42]. The improved flap survival was noted if PRP-containing LMW-H/P M/NPs was
administered 2 days before the flap elevation [42]. PRP-containing LMW-H/P M/NPs thus represent a
promising new biomaterial for improving skin flaps, particularly in the field of reconstructive surgery.
Clinical research was performed using autologous PRP-containing LMW-H/P M/NPs and PRP alone in 26
patients with thin hair (including 10 women) [43]. Hair growth and thickening following administration
of both PRP-containing LMW-H/P M/NPs and PRP alone was observed in all patients compared with the
control, but PRP-containing LMW-H/P M/NPs appeared to provide the most substantial change in the
hair (see Figures 6.6 and 6.7) [43]. Because of the use of autologous materials, this method using PRP-
containing LMW-H/P M/NPs is simpler and cheaper, and has no side effect compared with
conventional methods.
Nanomedicine 146
FIGURE 6.6
PRP-containing LMW-H/P M/NPs treatment for Alopecia Areata.
FIGURE 6.7
PRP-containing LMW-H/P M/NPs-treatment for Alopecia Areata.
Nanomedicine 147
Cell-Delivery Micro/Nano Particles

LMW-H/P M/NPs as Stromal Cell Carriers
LMW-H/P MPs as cell carriers can enhance cell viability as well as control the release of GFs. LMW-H/P
MPs could substantially enhance the cellular viability of various suspension cultures, including hMVECs,
hDFCs and ASCs [27,28]. In particular, ASCs have the potential to differentiate into skin, bone, cartilage,
fat, myocardium, skeletal muscle and neurons [44]. Several reports indicate that the transplantation of
human ASCs-cultured constructs significantly stimulates angiogenesis, wound repair and re-
epithelialization in athymic mice when compared with the corresponding human fibroblast-cultured
constructs [45]. ASCs can be easily harvested with lower donor site morbidity compared with other
pluripotent stem cell sources. Furthermore, ASCs can easily attach and proliferate in culture, and
therefore, are available on a large scale even for autologous grafting in small animals such as rodents
[46]. However, an application of ASCs for therapeutic angiogenesis and vasculogenesis requires
microcarriers to act as injectable vehicles necessary for transplantation of ASCs. It was observed that
LMW-H/P M/NPs could bind to the surface of the cells. The interaction of these cells with LMW-H/P
M/NPs induced ASCs/LMW-H/P M/NPs-aggregate formation, and substantially promoted cell viability
for at least 3 days in cell suspensions (see Figure 6.8) [25]. The ASCs/LMW-H/P M/NPs-aggregates
adhered and grew on suspension culture plates, and the aggregates similarly grew on type I collagen-
coated plates. Furthermore, cultured ASCs secreted a significant amount of angiogenic GFs such as FGF-
2, HGF, PDGF and VEGF. These secreted GFs could have been retained within the ASCs/LMW-H/P
M/NPs-aggregates. When the ASC/LMW-H/P M/NPs-aggregates were subcutaneously injected into the
back of nude mice, a significant increase in neovascularization and fibrous tissue formation was
observed near the injected site from 3 days to 2 weeks [25]. Taken together, these data indicate that
ASCs/LMW-H/P M/NPs-aggregates are an useful and convenient biomaterial for angiogenesis and
wound repair cellular therapy. The interaction of adhesive cells with LMW-H/P M/NPs induced
ASCs/LMW-H/P M/NPs-aggregate formation, and substantially promoted cell viability in cell
suspensions in vitro, and injections of the aggregates significantly enhanced vascularization and fibrous
tissue formations in vivo.
FIGURE 6.8
LMW-H/P M/NPs as Tumor Cell Carriers
Tumor cell transplantation models offer great strategy for cancer research. They include allograft
transplantation and xenograft transplantation models. Allograft mouse tumor systems, also known as a
Nanomedicine 148
syngeneic model, consist of tumor tissues derived from the same genetic background as a given mouse
strain. The xenograft transplantation method involves actual human cancer cells or solid tumors which
are transplanted into a host mouse [47]. Therefore, to effectively utilize tumor cell transplantation, the
host mouse must possess an impaired immune system similar to nude mice, thereby allowing foreign
tumor cells to survive and not be rejected by the host. In both cases of tumor cell transplantation,
effective tumor cell carrier systems are required to improve cellular viability and growth as well as
decreasing tumor cell rejection by the animals [47].
The LMW-H/P M/NPs also bind to the surface of tumor cells such as Lewis lung cancer cells (3LL), B16
melanoma cells (B16) and human hepatoma cells (Huh7). They promote cell-to-cell interaction, and
increase the aggregation of the tumor cells with the LM-H/P M/NPs. These tumor cells/LMW-H/P
M/NPs-aggregates substantially promote cell survival and proliferation of the tumor cells in vitro as
well as reliably induce tumor formation and rapid tumor growth in vivo (see Figure 6.9) [48]. Taken
together, these data indicate that LMW-H/P M/NPs constitute a new, convenient and effective
biomaterial that functions as a tumor cell carrier in vivo. The application of LMW-H/P M/NPs as a tumor
cell carrier offers a more reliable model in both allograft and xenograft transplantation for cancer
research [48].
LMW-H/P M/NPs bind to the surface of Huh7 cells, promote cell-to-cell interaction, and increase the
aggregation of the tumor cells with the LMW/P M/NPs. These tumor cells/LMW-H/P M/NPs-aggregates
substantially promote cell survival and proliferation of the tumor cells in vitro as well as reliably induce
tumor formation and rapid tumor growth in vivo (see Figure 6.9).
FIGURE 6.9
Huh7/LMW-H/P M/NPs aggregates and tumor formation and growth in nude mice.
Nanomedicine 149
A Cell Culture System Using LMW-H/P M/NPs-Coated Plates

Various Types of Cell Cultures Using LMW-H/P M/NPs-Coated Plates
The LMW-H/P M/NPs are able to attach to polymeric surfaces such as plastic and glass. The LMW-H/P
M/NPs generate a stable paste-like coating through complete drying. It is probable that polypeptides,
such as FGF-2, interleukin (IL)-3 and granulocyte/macrophage-colony stimulating factor (GM-CSF), once
bound to the LMW-H/P M/NPs-coated plates, are gradually released from the coated surface in vitro
with a half-life of 4－6 days [27]. Furthermore, LMW-H/P M/NPs-coating could optimally stimulate
growth of hMVECs and hDFCs in low FBS (1%) containing culture medium with FGF-2 and growth of
hematopoietic cell line (TF-1) with IL-3 and GM-CSF [27] (see Figure 6.10).
Heparinoids bind various GFs and cytokines including FGFs, HGF, VEGF, heparin-binding epidermal
growth factor (HBEGF), PDGF, TGF-β, GM-CSF, interleukins (i.e., IL-1, IL-2, IL-3, IL-4, IL-6, IL-7 and IL-8),
interferon γ and macrophage inflammatory protein-1 [13-15]. These GFs and cytokines can potentially
be immobilized on the LMW-H/P M/NPs-coated plates. Actually, in addition to FGF-2, IL-3 and GM-CSF
described above, we have already observed that FGF-1, HGF, HBEGF, TGF-β, human stem cell factor
(SCF), thrombopoietin (Tpo) and Flt-3 ligand (Flt-3) could be efficiently immobilized on the LMW-H/P
M/NPs-coated plates [31]. Furthermore, the bound GFs to the LMW-H/P M/NPs-coated plates
appeared to enhance and to stabilize those biological activities. Thus, LMW-H/P M/NPs-coating
provides an excellent biomaterial to immobilize and retain GFs and cytokines for optimal growth of
various types of cells with low (no) serum medium (see Figure 6.10).
FIGURE 6.10
Preparation of LMW-H/P M/NPs-coated plates.
Nanomedicine 150
Proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) and ASCs on LMW-H/P
M/NPs-Coated Plates
Cell-based therapies such as tissue engineering will benefit from a source of autologous multipotent
stem cells, including BMSCs and ASCs. There are two stem cell lineages in bone marrow cell
populations, i.e., hematopoietic progenitor cells (HCs) and BMSCs. The BMSCs and ASCs are
multipotential, indicating that in culture [49,50] or after in vivo implantation these cells can
differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, myoblasts [51]
and neuronal cells [52]. Furthermore, cultured ASCs secreted significant amounts of angiogenic growth
factors such as FGF-2, HGF, PDGF and VEGF at levels that are bioactive [53]. Thus, LMW-H/P M/NPs
serve as an effective matrix for cultures of BMSCs and ASCs. The safe and effective expansions of
BMSCs and ASCs represent a promising option for tissue engineering strategies.
Most protocols for the expansion of BMSCs and ASCs include high concentrations (10%－20%) of
animal serum such as FBS as a nutritional supplement. In some cell cultures, this involves multiple
doses of FBS, which raises concerns over possible contamination as well as immunological reactions
caused by medium-derived FBS proteins, sialic acid derivatives, etc. [54,55]. Patients may experience
problems when undergoing autologous cell-based therapies if a serum other than an autologous serum
is used during the culturing of the cells. However, it would be difficult to obtain large amounts of
autologous serum from the patient for large-scale autologous cell culture [27-30]. It should be noted
that the growth of cultured BMSCs or ASCs on LMW-H/P M/NP-coated plates in combination with FGF-
2 and FBS (1－2%) was significantly stimulated, and similar stimulation was coated plates with FGF-2
and 1－2% human serum (HS) prepared from adult bloods instead of FBS [27-30]. Thus, LMW-H/P
M/NPs may serve as an effective matrix for cultures of BMSCs or ASCs. The safe and effective
expansions of BMSCs or ASCs represent a promising option for tissue engineering strategies.
Proliferation of CD34+ Hematopoietic Progenitor Cells (CD34+ HCs) on LMW-H/P M/NPs-Coated

Plates
HCs proliferate and mature in semi-solid media when stimulated by exogenous hematopoietic cell
growth factors (HCGFs) such as SCF, Tpo, Flt-3, IL-3 and GM-CSF [31,56,57]. These cells also proliferate
in association with BMSCs [29,58,59], although biologically active amounts of HCGFs cannot be
detected in stromal culture supernatants [59]. It is possible that HCGFs are synthesized by the stromal
cells but remain bound to the stromal cells and/or their extracellular matrix. In fact, it was
demonstrated that both natural and recombinant HCGFs, such as IL-3 and GM-CSF, could be adsorbed
by heparan sulfate, which is the major sulfated glycosaminoglycan of bone marrow stroma [29,58,59].
Serum-free medium supplemented with large amounts of SCF, Tpo and Flt-3 was reported for
expansion of CD34+ HCs [31,60,61]. Although such medium is commercially available (HPGM, Lonza
Japan Corp., Tokyo, Japan), it is prohibitively expensive. We demonstrated that recombinant HCGFs
such as SCF, Tpo, and Flt-3 were immobilized onto LMW-H/P M/NPs-coated plates, and the
immobilized cytokines were stabilized, were activated, and were gradually released into the medium.
Those cytokines, once bound, can be presented in the biologically active form to HCs [58,59].
Furthermore, only one-fourth of the concentration of the cytokines recommended by the manufacture
was required for maximal expansion of CD34+ HCs on the LMW-H/P M/NPs-coated plates [31,60,61].
These findings have important implications for the use of heparinoid as an artificial matrix for ex vivo
expansion of HCs with adequate cytokines. The LMW-H/P M/NPs-coating matrix in the presence of
lower concentrations of SCF, Tpo and Flt-3 is a convenient and safe material for stable expansion of
CD34+ HCs using HPGM without any animal serum [31].
Nanomedicine 151
Conclusions
It is recognized in polymer chemistry that positively and negatively charged polymers interact ionically
[1,3]. Through these ionic interactions, basic protamine molecules can bind with acidic molecules
(LMW-H) to form micro/nano particle complexes. We previously reported that GF-containing LMW-H/P
M/NPs, which are 100 nm－3 μm in diameter, can be easily injected *20-22]. Furthermore, the LMW-
H/P M/NPs were observed on the protection of FGF-2 and GFs in PRP activity from heat and proteolytic
inactivation. These results indicate that LMW-H/P M/NPs serve as an effective microcarrier for various
GFs, particularly for the local application of GFs. GFs-containing LMW-H/P M/NPs show a substantial
effect to induce vascularization and fibrous tissue formation because of stabilization, activation and
gradual release of GF molecules from GFs-containing LMW-H/P M/NPs [20-22].
LMW-H/P M/NPs rapidly bound to adhesive cell surfaces such as ASCs through specific interactions of
LMW-H/P M/NPs and various cell surface heparin-binding proteins, can promote cell-to-cell interaction
and increase cellular aggregation. The cells/LMW-H/P M/NPs-aggregate formation substantially
promoted cell viability in vitro. The ASCs/LMW-H/P M/NPs-aggregates induced vascularization and
fibrous tissue formation in vivo [25]. The LMW-H/P M/NPs, in combination with ASCs, are a new
convenient cell carrier and may be a promising novel therapy for inducing vascularization and fibrous
tissue formation in ischemic disease (Figure 6.5). LMW-H/P M/NPs also bind to the surface of various
adhesive tumor cells, promoting cell-to-cell interaction and increase cellular aggregation with the
LMW-H/P M/NPs. The tumor cells/LMW-H/P M/NPs-aggregates substantially promote cell survival and
proliferation of those tumor cells in vitro, and reliably induce tumor formation and rapid tumor growth
in vivo [25,48]. Taken together, LMW-H/P M/NPs constitute a new convenient and effective biomaterial
that function as a tumor cell carrier in vivo. The application of LMW-H/P M/NPs as tumor cell carrier
offers a more reliable model in both allograft and xenograft transplantation for cancer research.
The presented method for the optimal proliferation and differentiation of ASCs and BMSCs on LMW-
H/P M/NPs-coated plates in low concentration HS (1－2%) supplemented with FGF-2 (5 ng/ml). No
animal serum is required in the culture of those cell types. The bound GFs to the LMW-H/P M/NPs-
coated plates appeared to enhance and to stabilize those biological activities. The proliferated cells
maintained their potential to differentiate into adipocytes and osteoblasts [25,29,30]. Furthermore, the
LMW-H/P M/NPs-coating matrix in the presence of lower concentrations of SCF, Tpo and Flt-3 were
convenient materials for stable expansion of CD34+ HCs using HPGM without any animal serum [31].
These results suggest a promising cell source, particularly for the preparation of large amounts of ASCs,
BMSCs or CD34+ HCs required for cell-based therapies in several clinical fields.
LMW-H, protamine, several GFs and cytokines, and autologous PRP are already in clinical use. Since
autologous ASCs, BMSCs or CD34+ HCs are available, the clinical safety of LMW-H/P M/NPs as protein-
carrier and as cell-carrier is possible. Furthermore, ASCs, BMSCs or CD34+ HCs can be efficiently
expanded as cell sources for regenerative medicines with the use of LMW-H/P M/NPs-coated plates as
a matrix without animal serum or feeder cells.
Acknowledgments
We acknowledge the expertise and advice of Associate Prof. Koichi Fukuda and we thank the personnel
of the Institute of Laboratory Animals, National Defense Medical College for expert care of animals.
This study was partially supported by the Ministry of Education, Culture, Sports, Science and
Technology of Japan under grant no. 23300181.
Nanomedicine 152
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7
Nanophysical approach to diagnosis of
epithelial tissues using Opto-magnetic
imaging spectroscopy
Lidija Matija, Branislava Jeftić, Gorana Nikolić, Aleksandra Dragičević, Ivana Mileusnić, Jelena
Munćan, Djuro Koruga
Nanolab, Deparment of Biomedical Engineering, Faculty of Mechanical Engineering, University of Belgrade, Belgrade, Serbia.
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 157
Epithelial tissue …………………………………………..……………………………………………………………………………… 157
Optical methods …………………………………………………………………………………………………………………………. 160
Method ………………………………………................................................................................................... 162
Physical background ………………………………………..……………………………………………............................... 162
Operational setup for opto-magnetic imaging spectroscopy ………………………………………………………. 164
Material ……………………………………….…………………………………………………………………………………………….. 167
The cervix …………………………………………………………………………………………………………………………………… 167
The colon …………………………………………………………………………………………………………………………………... 168
Skin …………………………………............................................................................................................... 168
Results ……………………………………………………………………………………………………………………………………….. 169
The diagnosis of colon epithelial tissue using the opto-magnetic imaging spectroscopy ……………. 172
The diagnosis of skin epithelial tissue using the opto-magnetic imaging spectroscopy ………………. 176
Statistical analysis ………………………………………..…………………………………………….................................. 177
Summary statistics ………………………………………..……………………………………………................................. 177
The classification results ………………………………………..……………………………………………........................ 180
Conclusion …………………………………………………………………………………………………………………………………. 183
Acknowledgment ……………………………………………………………………………………………………………………….. 184
References……………………………………………………………………………………..…………………………………………… 184
Nanomedicine 157
Introduction
Cancer is a broad group of diseases involving unregulated cell growth. Inside the tissue where the
cancer occurs (a malignant neoplasm) cells divide and grow uncontrollably and form malignant
tumours, which invade nearby parts of the body. Cancer also spreads to distant parts of the body
through the lymphatic and blood circulatory system [1]. The incidence and mortality of tumours are on
the increase from year to year. For example, cervical cancer is the seventh most common cancer in
both sexes and the third most common cancer among women, which is caused by high-risk HPV
infections. HPV types 16 and 18 are responsible for 70% of all cervical cancers. High-risk HPV also
causes anal, vaginal, vulvar, penile cancer and cancer of the oropharynx [4]. In 2008 an estimated
530,000 women across the world were diagnosed with cervical cancer, which accounted for nearly one
in ten (9%) of all cancers diagnosed in women and resulted in 275,000 deaths, around 8% of all female
cancer deaths. Colorectal (including anal) cancer is the third most common cancer and the fourth cause
of cancer deaths worldwide. Statistic data for 2008 showed that 1.24 million people had been
diagnosed with colorectal cancer, accounting for 10% of all cancers and responsible for almost 610,000
deaths [2]. Skin cancer is the most common cancer in Australia, New Zealand and the United States.
More than 3.5 million skin cancers (non-melanoma cancer) in over two million people are diagnosed
annually in the USA [3]. Melanoma accounts for less than five per cent of skin cancer cases and the
majority of skin cancer deaths [4]. In the UK, in 2011, there were 2,209 deaths from malignant
melanoma skin cancer, in 2010 around 100,000 people were diagnosed with non-melanoma skin
cancer and in 2011 there were 585 deaths from non-melanoma skin cancer [5].
A new approach to the detection of abnormalities in epithelial tissue will be presented in this chapter.
Several types of epithelial tissue such as skin, cervix and colon were investigated in these studies due to
their increasing incidence and mortality rate which is still unacceptably high, even though the existing
diagnostic tests have greatly improved in recent years. Various techniques are in use for the detection
of cancer of epithelial tissue (Pap test, HPV testing, colposcopy, curettage, histopathology, cytology,
colonoscopy as a “gold standard’’, etc.), but some of them are very expensive, some of them cannot be
used for some patients because of contra-indications (for example, patients with stent cannot use NMR
as a diagnostic method), etc. We designed and made a prototype optical device for non-invasive use,
which does not cause (to our knowledge) any contra-indications.
Epithelial tissue
Epithelium, along with connective tissue, muscle, and nerve, is one of the four primary tissue types in
the human body. It is commonly found as a covering or lining for other types of hollow organs, as in the
case of the epidermis and the lining of the entire gastrointestinal tract, and constitutes most of the
glandular tissue. Epithelial tissues are avascular (no blood vessels), composed of layers of tightly
packed cells connected by intercellular nexus and with a minimal content of extracellular matrix and
interstitial fluid. The amount of extracellular matrix and interstitial fluid is extremely small and
therefore invisible under a light microscope. Beneath the epithelial cells lies a very thin glycoprotein
layer of extracellular matrix, the basement membrane, which serves to anchor the epithelium to the
connective tissue below and is made up of two distinct layers called basal and reticular lamina. Basal
lamina is very thin, and also invisible under a light microscope, while the basement membrane is just
barely visible in specially-stained sections. This membrane contains collagen, adhesive glycoproteins
Nanomedicine 158
called laminin and fibronectin, and a large protein-carbohydrate complex called heparan sulphate,
which gradually blends with collagenous and reticular fibres on the connective tissue side.
Histological differences among the various epithelia are based on the shapes of the cells, the number of
cell layers present, various modifications to the surfaces of the cells that can be seen under a light
microscope (microvilli, stereocilia, cilia), and characteristic patterns of reaction with particular dyes
(acidophilia, basophilia, special stains). Epithelial tissues have numerous particular functions; in
general, their functions are to mediate absorption, excretion, protection, secretion, sensory reception
and transport.
Epithelial tissues are divided into 1) simple, which is with one layer and one type of cells (squamous,
cuboidal, columnar), 2) stratified, two and more layers and one or more types of cells and 3)
pseudostratified, one layer that appears to be more than one layer. In this chapter, several types of
epithelial tissues will be presented, with their optical and biophysical characteristics, which are parts of
organs such as the skin, the large intestine, and the cervix of the uterus [6-10].
FIGURE 7.1
Examples of main types of epithelial tissue.
The skin: covers the entire outer surface of the human body, except for one part of the eye and the
area of natural openings (nasal, oral, anal and genital), and becomes appropriate mucosa. It is the
largest organ and weighs from 5 to 17 lbs (16% of the total body weight) and with a total area of 10 to
25 ft². It consists of three main parts: the epithelial tissue – the upper layer of the skin, the dermis - the
middle layer, and the fat connective tissue (hypodermis) - the deepest layer of the skin, which connects
with the muscles and bones. The epidermis is keratinized stratified squamous epithelium and is the
surface layer of the skin. It is made of keratinocytes (95%), melanocytes, Langerhans cells and Merkel
cells. Keratinocytes are the main and most numerous cells that produce keratin and also synthesize:
specific proteins (that form the inner lining of keratinized cells), products such as cytokines, enzymes,
proteins, inflammatory mediators (that are important when it comes to the inflammatory process).
Melanocytes are the cells that synthesize pigment melanin, which protect the skin from the harmful
Nanomedicine 159
effects of UV rays. The number of melanocytes is constant per unit volume, and one melanocyte serves
4-10 keratinocytes.
The layers of epidermis are: stratum basale, stratum spinosum, stratum granulosum, stratum lucidum
(only in the epidermis on the palm and the sole of the foot) and stratum corneum. Stratum basale or the
basal layer is the deepest layer of epidermis, consisting of one line (row) of cylindrical cells.
Ultrastructural analysis shows that the basal cells transform into keratinocytes, which means that the
basal cell is a stem cell from which multiplication begins. Stratum spinosum or the spinous layer has
several layers of large polygonal cells which are tightly interconnected with the nexus, called
desmosomes. In this layer the transformation of the keratinocytes continues. Stratum granulosum or
granulosa layer consists of several flat, polygonal cells. In this layer, the transformation of a
keratinocyte continues. Stratum corneum or the horny corneal layer consists of 15-20 layers of flat,
dead cell, called corneocytes. In this layer the keratinization process is completed. Corneocytes
separate from each other and gradually the whole process ends with desquamation (peeling), and the
cells of basal layer renew the epithelium. It takes about 30 days to restore the stratum corneum. This
means that during this time the cells of the basal layer divide, multiply, and go through all the layers of
skin to become corneocytes in the stratum corneum. The dermis or the loose connective tissue is just
under the epidermis. Components of the dermis provide mechanical support, rigidity and thickness (1–
4 mm). The dermis is composed of two layers: papillary and reticular. Loose connective tissue and
extracellular matrix consist of collagen fibres (collagen type I and III) and elastin, which form the basis
of the papillary layer of the dermis. Irregular loose thick connective tissue is the basis of the reticular
layer. The dense network of the thick erratically positioned collagen bundles, which lies parallel to the
surface of the skin in the direction of the force stretching the skin, provides mechanical stability. The
hypodermis is the deepest layer of the skin, composed of lobules of adipose tissue separated by
connective tissue. It is a thermal insulator, the depot of energetic materials and binds the skin to the
muscle and bones, providing its mobility. The boundary between the dermis and the epidermis is the
basement membrane, which is important because of its prognostic significance of skin tumours. If it is
intact, then it is an indication that the disease is in the early stages, located only in the epidermis and
that a cure is almost 100%. However, if it is compromised, the outcome depends on which tissue is
affected, the biological potential of the tumor spreading to other parts of the body, i.e. secondary
deposits. [9, 11-17].
The Cervix: The womb (uterus) is the internal female sex organ. It is located in the pelvic region called
the cavum pelvis. The uterus has four major parts: a curved upper area in which the fallopian tubes
connect to the uterus (fundus uteri), the body (corpus uteri), the narrow neck region (isthmus uteri),
and the lowest section is the cervix (cervix uteri), which ends in the cavity of the vagina and is the
connection between the uterus and vagina. The uterus is 2.4 to 3.1 inches long; its wall is
approximately 0.8 to 1.2 inches thick. The width of the organ varies; it is 2.4 inches wide at the fundus
and half at the isthmus. The uterus is located between the bladder (front) and the large intestine
(back). The cervix is the narrowed lowest part of the uterus. It has the shape of a cylinder and consists
of two parts: an upper (portio supravaginalis) and lower (portio vaginalis) part. Inside the cervix is a
canal (canalis cervicis) which leads into the uterus.
Histologically, the uterus is composed of three layers: perimetrium myometrium and endometrium.
The perimetrium is a serous membrane which envelops the uterus. The myometrium consists of three
layers of muscle tissue (longitudinally, circularly and obliquely). Between these layers is a connective
tissue, blood vessels, collagen and elastin fibres. The endometrium lines the cavity and changes its
width during the menstrual cycle and during ovulation. The cervix is covered with two types of cells:
squamous cells which are the outer part, called the exocervix (the part which is connected to the
Nanomedicine 160
vagina) and glandular cells which are present in the cervical canal (endocervix). The squamous
epithelium is composed of 15 to 20 rows of cells and does not possess blood vessels or connective
fibres, therefore the nutrients are transferred through the capillary system by diffusion. The glandular
epithelium is composed of one row of cells which produce mucus. These two types of epithelia
converge at a place called the transformation zone. This zone proliferates under the influence of sex
hormones, particularly estrogen when the menstrual cycle begins. The transformation zone moves
during the lifetime and does not always match with the external orifice of the cervix and is the place
where all precancerous and cancerous lesions begin [18, 19].
The Colon: The digestive system includes the alimentary canal and its principle associated organs such
as tongue, teeth, salivary glands, pancreas, liver and gallbladder and the anatomical parts are: the oral
cavity, the esophagus and the gastrointestinal tract (the stomach, the small intestine and the large
intestine) [20]. The main functions of the digestive system are chewing and swallowing food and saliva,
absorption of nutrient material and the elimination of undigested food and waste. Our region of
interest is the large intestine, especially the colon and the rectum. The colon, which is the biggest part
of the gastrointestinal tract, histologically consists of four layers: the inner layer - the mucosa, the
connective tissue - the submucosa, the muscle tissue – the muscularis externa and the outer layer - the
adventitia or serosa. The epithelium of the colon is the simple columnar, made up of several different
types of cells. The main functions of these cells are: absorption, protection and secretion. Numerous
tubular glands extend through the full thickness of the mucosa and they consist of the same epithelium
as the colon. Stem cells are in the deepest part of the intestinal gland and because of their capability to
proliferate and differentiate, the epithelial cells can renew themselves. The cells of the mucosal
epithelium are: the columnar absorptive cells (whose role is the reabsorption of electrolytes and
water), goblet cells (which continuously secrete mucus), caveolated “tuft” cells - a form of exhausted
goblet cells and, enteroendocrine cells (which produce various paracrine and endocrine hormones).
The connective tissue or submucosa contains a thick layer of collagen and proteoglycans, pericryptal
fibroblast sheaths, immune defence cells, nerve plexus, lymphatic and blood vessels. This layer of
collagen and proteoglycans regulate water and transport electrolytes from the intercellular
compartment to the vascular compartment. The muscularis externa contains two concentric layers of
smooth muscle (cells of both layers form a tight spiral): the inner layer is a circularly oriented layer, and
the outer layer is a longitudinally oriented layer. The muscularis externa produces contractions. Two
different types of contractions exist: segmentation (does not result in the propulsion of contents) and
peristalsis (results in the distal mass movement of colonic contents). The outer layer, called adventitia,
exists when the large intestine is in direct contact with the other structure, otherwise the outer layer is
called serosa. Serosa consists of a layer of simple squamous epithelium and connective tissue, with
large blood vessels, lymphatic vessels and nerve trunks [20, 21].
Optical methods
Conventional screening methods used in clinical practice are time limiting and expensive. As such,
these medical techniques are available only to a certain section of the world's population. Screening
and early detection of cancer could significantly improve the optical methods which are present in
modern scientific research. Some of them even allow visualization of the tissue structure at subcellular
level, thus providing the necessary information for identification of precancerous lesions.
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Optical spectroscopic methods such as Raman spectroscopy, FTIR (Fourier Transform Infrared) and
fluorescence spectroscopy proved to be a good alternative method for the detection of neoplastic
changes in cervical tissue [22-24], different types of colon cancer [23, 25, 26] and skin cancer [23, 25,
27]. These methods are fast, objective, and can be developed for in vivo screening of diseases, which
would exclude the need for painful biopsies. The main advantage of these methods is the possibility of
providing information about the biochemical, structural and pathophysiological changes which occur in
the tissue, but these methods are not routinely used in clinical practice, since they are still only
research tools.
FTIR (Fourier Transform Infrared) spectroscopy is a method which has only recently been applied in the
detection of various types of cancer, and as such is being tested in research of the screening of cervical
cancer [22]. FTIR detects irregularities in the cells on a molecular level. This distortion preceded the
development of morphological changes that are available to detect under a microscope, showing the
great importance of FTIR spectroscopy for early detection of precancerous lesions. In the case of colon
cancer detection, FTIR showed good results, especially during an operation. Experimental results
revealed that the spectral characteristics of normal and malignant tissues found in vivo and in situ were
similar to those obtained from in vitro measurements in our previous fundamental research [23]. For
this kind of cancer, it is very difficult to apply any spectroscopy in vivo, which is why it is important to
compare the results between in vivo and in vitro spectroscopy.
The advantages of Raman spectroscopy over the other optical methods include high spatial resolution
(up to 1 μm), the use of less harmful NIR radiation, less demanding preparation of the samples and its
application to in vivo / in situ measurements. Over the years, various algorithms have been developed
for the differentiation of tissue in order to examine the feasibility of using Raman spectroscopy for the
detection of cervical precancerous conditions. Some investigations used empirically selected peak
intensities and the intensity of these relationships to distinguish precancerous conditions from other
tissues. Other algorithms for differentiating diseased from healthy tissue used statistical methods
based on PCA (Principal Component Analysis) [24]. Near-infrared Fourier Transform Raman
spectroscopy is an analytical, non-destructive technique that provides information about the molecular
structure of the investigated sample. The molecular structure of proteins and lipids differ between
neoplastic and normal tissues and therefore Raman spectroscopy has been considered promising for
the diagnosis of cancer. In the Raman spectra, the secondary structure of the proteins was reflected by
the amide vibrations of peptide bonds. It is the most frequently used spectroscopy for the detection of
skin lesions [25]. Raman spectroscopy is also suitable for the diagnosis of colon carcinoma because of
the sensitivity of the method in detecting small molecular changes that are characteristic for cancer,
such as the ratio between an increased nucleus and cytoplasm, disorganized chromatin, increased
metabolic activity and changes in the level of fats and proteins [26].
Infrared spectroscopy is widely perceived as a future clinical technology for cancer detection and
grading for all types of epithelial lesions. For example, melanoma could be differentiated from the
epidermis using parameters derived from absorbance bands originating from molecular vibrations of
nucleic acids and/or their bases. Additionally, absorbance from tyrosine and phosphate that are
abnormally elevated in malignant melanoma could be used as markers [27].
Despite all the benefits of the stated spectroscopy which these methods could bring to future clinical
trials, there are still some issues concerning the implementation of these methods in clinical practice.
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Method
In order to meet the objectives for a more successful method for early detection of epithelial tissue
cancer, with a lower cost price, we have developed an Opto-magnetic image spectroscopy (OMIS). The
OMIS was first introduced as a non-invasive method for skin characterization, in 2009 [28]. Since then,
it has evolved and has been adjusted for a wider area of applications in biomedical engineering;
polymers, liquids and epithelial tissues (healthy and cancer) [29-32.
The method obtains paramagnetic/diamagnetic properties of materials (unpaired/paired electrons)
based on their interaction with visible light. The basic tool is the light of wavelength in a range between
400 nm and 700 nm provided by white light emitting diodes (LED, Nichia, 150 mA, 3V, maximum
relative emission intensity at 450 nm wavelength, chromaticity x=34.4 and y=35.4, ambient
0
temperature 25 C). Since energy of valence electrons and photons of visible light has the same value,
imaging by visible light is non-invasive and provides an examination process that can be repeatedly
conducted without presenting any risks to the patient or sample material damage. Finally, due to the
numerous advantages that the digital image acquisition offers, the design of this technique and
customized hardware solution used in the studies have been further developed. The OMIS technique
has yielded positive results in early detection of cancer of epithelial tissues such as the cervix, the
colon, the skin and other biological samples, which are presented in this chapter. The method is
applicable both in vitro and in vivo.
Physical background
Opto-Magnetic Imaging Spectroscopy (OMIS) is a nanophysical diagnostic technique based on the

interaction of electromagnetic radiation with valence electrons within the sample material, and
therefore examines the electronic properties of the matter (covalent bonds, hydrogen bonds, ion-
electron interaction, and Van der Waals interaction). Bearing in mind that the orbital velocity of a
6 -4
valence electron in atoms is approximately 10 m/s, the calculated ratio is FM/FE ≈ 10 between the
magnetic force (FM) and the electrical force (FE) of matter. Since force (F) is directly related to action
(A= F x d x t, where F is the force in the range 0.01 – 1.0 nN, d is the displacement in the range 0,1 – 5.0
-8 -10
nm, and t is time in the range 10 -10 s) [31,33], it can be concluded that the magnetic force of matter
is four orders of magnitude closer to the quantum state of matter than the electrical force. This opens
an opportunity to detect the conformational states and changes in the matter on a nanoscale level
using a light-matter interaction method.
The fact that the magnetic force of valence electrons is more closely related to the quantum states of
matter, it primarily influences the choice of magnetic interaction as a main measurement modality. The
optical modality was chosen because the photons of visible light are perfect probes for states of
valence electrons of matter with respect to their sufficiently low energy levels and sensitivity.
The light as an electromagnetic phenomenon consists of electric and magnetic waves that are
perpendicular and can be split under specific conditions, which means that the light can be polarized.
One particular type of polarization of light occurs during the interaction of light and matter at a specific
angle, known as Brewster’s angle. Each type of matter has a unique angle value of Brewster’s angle, for
example, Brewster’s angle for water-air interface is 53° (refractive index n=1.33) [34]. When a sample is
illuminated under this specific angle, the reflected light will be polarized. Reflected polarized light
contains only an electrical component in a longitudinal wave, and a magnetic component in a
transversal wave (perpendicular to longitudinal) of light-matter interaction. Since the longitudinal wave
directly influences CCD/CMOS sensor, while the transversal has a neglected influence, then by
Nanomedicine 163
subtracting the reflected polarized light (electrical properties) from the reflected white diffuse light
(electromagnetic properties), the result will provide information about the magnetic properties of
matter based on light-matter interaction.
If a digital image of a material surface is acquired using classical optical microscope equipped with a
digital camera, it represents the reflectance of the sample and thus contains information about it. If the
sample is illuminated with the white diffuse light, the reflected light waves will have an
electromagnetic nature. However, if the sample is illuminated with white light under Brewster’s angle,
specific for that type of sample material, the reflected light will have an extremely dominant electric
nature (Figure 7.2). Advancements in the field of digital image processing have made it possible to do
various operations with digital images. This means that as much information as possible can be
extracted from the properties of the sample. Theoretically, if a digital image of a sample is acquired
using illumination under Brewster’s angle, and subtracted from the image of the same sample acquired
using illumination with diffuse white light, then the resultant image would represent a sort of
composite image or a map of the magnetic properties of that sample.
FIGURE 7.2
The experimental arrangement sketch shows the relative positions of light sources for white (a) and reflected
polarized light (b). The degree of light polarization is 95.4%, while angular diffusion of the light source (six white
LEDs arranged in a circle) is ±1:6° (the difference between the angles θ and θ 1) [28,30].
The digital images acquired using OMIS method are RGGB (red, double green, and blue) images
because the camera is adapted for the human eye (the visual system). The algorithm for the image
processing is thus based on the “Maxwell triangle” chromaticity diagram and it allows the conversion of
the digital image to Opto-magnetic spectra through several operations, starting with the creation of
histograms for each colour channel and subsequent conversion of the histograms to spectra [28]. A pair
of digital images of the sample acquired under white diffuse light, and white diffuse light under
Brewster’s angle will result in three spectra, blue, red and green, for each image. When blue, green or
red spectra for the image of the sample taken under Brewster’s angle are subtracted from the blue,
green or red spectra for the image of the sample taken under white diffuse light, the resultant
composite spectrum will represent an opto-magnetic spectrum of the sample. This resultant spectrum
is presented in the coordinate system, where the x axis presents the wavelength difference (WD)
measured in nanometers, and the y axis as the intensity in normalized arbitrary units. The spectrum is
given a designation, made from several abbreviations, which describes what kind of transformation is
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performed. For example, the designation (R-B)&(W-P) means that the Opto-magnetic spectra of the
imaging matter is developed by subtracting the blue colour channel spectrum (B) from the red colour
channel spectrum (R); where each colour channel spectra were first developed by subtracting the
colour channels spectra for image under Brewster’s angle (P, as in reflected “polarized”) from the
image under diffuse white light (W). Similarly, various combinations can be performed. Since blue light
is reflected from the surface (and a very/very thin layer) and red light from the deep tissue layers, then
the most commonly (R-B)&(W-P) type of Opto-magnetic spectra is used for tissue samples. In this case
we avoid the reflection from the surface (like a natural reflected mirror effect), because we need
information about the tissue characteristics.
In cancer research, the application of the Opto-magnetic imaging spectroscopy method is used to
discover the differences between the paramagnetic/diamagnetic state of water in healthy and
cancerous tissues. Therefore, Brewster's angle of 53° for air-water interface is used whenever this
method is applied in cancer research. Cancer cells contain more free water than normal cells, about
21% , [35] and the degree of malignancy increases with the degree of cell hydration [36, 37]. The
disrupted state of water in cancer cells is used as a parameter for cancer detection and even for
treatment [37].
The described properties of Opto-magnetic imaging spectroscopy provide a basis for an excellent
environment for screening testing, where speed, ease of use, accuracy and low cost equally contribute
to the successful early diagnosis of cancer. The measuring repeatability of OMIS for the same sample is:
98.8±1.2 %, 97.8±2.2% and 96.9±3.1%, for solid state matter, viscoelastic matter (tissues) and liquids,
respectively.
Operational setup for Opto-Magnetic Imaging Spectroscopy

Device and software for Opto-Magnetic Imaging Spectroscopy are developed at the Faculty of
Mechanical Engineering, University of Belgrade at the Department of Biomedical Engineering.
The basic operational setup for Opto-Magnetic Imaging Spectroscopy (OMIS) consists of a customized
housing for a Canon digital camera (model IXUS 105, 12.1 MP) with a system of emission diodes at the
appropriate angle and a sample holder (Figure 7.3). The illumination system consists of six LED diodes
(Nichia, STS-DA7-3195) arranged in a circle and placed in front of the camera lens, providing
illumination of the sample with white diffuse light and white diffuse light under Brewster’s angle.
Due to specific sample requirements, the basic setup was further modified. Thus, there are three types
of devices presented in Figures 7.4, 7.5 and 7.6. The first system was designed for acquiring digital
images of cytology samples spread on microscopic slides (smears), (Figure 7.4), while the second
system provides the same, but with the possibility of sample magnification using an optical microscope
(Keyence magnifying system, model VH-Z100) (Figure 7.5). The third system enables the acquisition of
digital images of tissues for conditions in vivo and in vitro (Figure 7.6).
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FIGURE 7.3
The basic operational setup of the Opto-Magnetic Imaging Spectroscopy (OMIS) (NanoLab, Faculty of Mechanical
Engineering, University of Belgrade)
FIGURE 7.4
Opto-magnetic imaging spectroscopy system for in vitro applications (NanoLab, Faculty of Mechanical Engineering,
University of Belgrade)
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FIGURE 7.5
Opto-magnetic imaging spectroscopy system for in vitro applications with the possibility of sample magnification
using an optical microscope (NanoLab, Faculty of Mechanical Engineering, University of Belgrade)
FIGURE 7.6
Opto-magnetic imaging spectroscopy system for in vitro applications (NanoLab, Faculty of Mechanical Engineering,
University of Belgrade)
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The OMIS procedure consists of:

1. Illuminating the sample with white diffuse light.
2. Acquisition of the first digital image.
3. Illuminating the sample with white diffuse light at Brewster’s angle.
4. Acquisition of the second digital image.
After acquiring images, which lasts approximately 5-10s per sample for both digital images, they are
processed using a code developed in the MATLAB package [28].
Material
The Cervix
Sample preparation varies depending on the method used for cancer detection and diagnosis. The
main screening test for cervical cancer detection is the Papanicolaou test (Pap test or Pap smear). The
cervical cells are taken with a wooden scraper and a cervical brush and are then placed on glass
microscopic slides. Cells scraped from the surface of the cervix are usually placed on one microscopic
slide and cells scraped from the inside of the cervical canal are placed on another slide. After adding a
fixative, cells are stained and then analysed under a microscope. This conventional Papanicolaou
procedure is used to detect precancerous and cancerous changes in cervical cells. The procedure
includes sample staining with haematoxylin, orange and polychrome stain, and ethanol washing after
each of these stains has been added to the sample. The Pap test categorizes samples into four groups:
PAP group 2 (atypical cells with no evidence of malignancy), PAP group 3 (cells suspicious of
malignancy), PAP group 4 (few definitive malignant cells) and PAP group 5 (large number of malignant
cells). Liquid-based Cytology (LBC) is a slightly different cervical sampling method and involves
obtaining cervical cells with a spatula and immersing the spatula in a liquid solution. The samples are
prepared through an automated process [38]. LBC is supposed to increase sensitivity and specificity
achieved with conventional Pap cytology and improves specimen quality and is widely used in
developed countries. However, unsatisfactory results are still present. There is a possibility of
overcoming the problems that lead to unsatisfactory results by reprocessing the samples [39], but it is a
part of our current investigations.
In the studies examining different types of optical spectroscopy (FTIR, fluorescence, Raman
spectroscopy), applied as diagnostic tools, spectra are usually obtained from unstained cytology
(usually LBC is used) and/or tissue samples (samples cut by microtome) [22, 40, 41].
Opto-magnetic Imaging Spectroscopy uses conventionally prepared cervical samples stained according
to standard Papanicolaou procedure. Slides are first screened by a cytotechnologist, and then using
OMIS. Ten cervical cell OMIS spectra are averaged per each patient to obtain a single spectrum for
every microscopic slide, i.e. cervical smear. The study of the stained cervical cytology samples obtained
from 140 patients (280 microscopic slides with cervical cell samples), which included 35 cases from
each Pap group, showed a good potential of OMIS to characterise samples from different Pap groups
[42]. Authors reported problems concerning inadequate sample preparation, i.e. overstraining of the
samples [43].
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The Colon
The primary aim of the use of the Opto-magnetic imaging spectroscopy is to provide a non-invasive
method for the differentiation between healthy and tumour tissue. In vitro optical properties were
investigated in two kinds of tissue, healthy tissue and colonic adenocarcinoma. Tissue samples were
obtained from 70 human patients with colonic adenocarcinoma, with histopathology proof. Tumours
were of various grades and stages. The group included patients of both sexes, aged 19 to 85 years.
After surgical resection, each removed colon sample was first rinsed with aqua purification to exclude
surface blood and then placed on equipment especially designed for OMIS. Digital images of healthy
tissue and neoplasms were taken ten times per each sample under white diffuse and polarized light, 1
hour and 4 hours after removal of the tissue. The imaging of healthy tissue was at least 8cm from the
tumour. After imaging, the tissue sample was fixed in formalin for further histopathology examination.
FIGURE 7.7
Colon sample and regions of interest for imaging (healthy and tumour)
Skin
Optical properties of skin in vivo condition were investigated for two different types of skin lesions that
exist or occur on the skin due to damage. These changes were divided into two basic categories, benign
and malignant pigment changes, and each category was further divided into appropriate subgroups.
The device was put directly onto the lesion. Pictures were taken both with white diffuse and polarized
light. In 97% of cases, the results obtained using OMIS coincided with dermoscopy and histopathology
findings. Samples were obtained from 96 patients (48 samples belong to the melanoma group and the
other 48 to the nevus group).
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Results
The diagnosis of cervical epithelial tissue using the Opto-magnetic imaging spectroscopy
When all of the digital images of the cervical samples were obtained using the OMIS device (Figure 7.8),
the data were processed using an algorithm developed to differentiate normal from cancerous cells.
The differentiation is based on the OMIS spectra and considers two components from the spectra: peak
intensity and the wavelength difference range where peaks appear.
FIGURE 7.8
OMIS digital images of cervical samples belonging to II, III, IV and V Pap group (from left to right)
Figures 7.9, 7.10, 7.11 and 7.12 show the OMIS spectra of normal cervical cells (II Pap group), cells
suspicious of malignancy (III Pap group), malignant cells (IV Pap group) and a large number of malignant
cells (V Pap group), respectively. Spectral differences observed between these four categories reflect in
intensity variations. Peak intensities of malignant cells are lower than peak intensities of normal
cervical cells. There is also a noticeable shift in peak positions. Figure 7.13 shows the OMIS spectra of
samples from all four Pap groups.
Peak intensity variations in the spectra of different cervical samples within each Pap group are assumed
to be present due to inadequate sample preparation and over staining. In order to overcome these
problems, OMIS was conducted on non-stained cervical samples. This study is still in progress and so
far gives good results, but still needs a larger group of patients from all four Pap groups.
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FIGURE 7.9
The OMIS spectra of cervical samples collected from 5 different patients diagnosed with Pap group II
FIGURE 7.10
The OMIS spectra of cervical samples collected from 5 different patients diagnosed with Pap group III
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FIGURE 7.11
The OMIS spectra of cervical samples collected from 5 different patients diagnosed with Pap group IV
FIGURE 7.12
The OMIS spectra of cervical samples collected from 5 different patients diagnosed with Pap group V
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FIGURE 7.13
The OMIS spectra of cervical samples from all four Pap groups
The diagnosis of colon epithelial tissue using the Opto-magnetic imaging spectroscopy
The obtained data were processed and the results are presented with diagrams and tables with the
characteristic values. By analyzing the intensities of the peaks that occur at specific wavelength
difference, and the results obtained by histopathology analysis, it was observed that the OMS
method shows sensitivity between healthy tissue and tumor tissue. Evidently, there are shift
differences of wavelengths where the characteristic peaks occur. It is also apparent that there is a
difference in the activity of tissues in the paramagnetic and diamagnetic zones.
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FIGURE 7.14
Image processing and the obtained characteristic diagram
FIGURE 15
The diagram of the three examined colon tumour samples
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TABLE 7.1
Characteristic peaks, wavelength differences at which they occur and the intensity of the three tumour
samples
I MAXIMUM I MINIMUM
NUMBER OF TUMOR
SAMPLES
WD (nm) I WD (nm) I
sample 1 25.356 112.083 -17.754 116.036
sample 2 32.808 111.117 -8.146 112.65
sample 3 27.574 109.196 -14.795 111.502
FIGURE 7.16
The diagram of the three examined healthy colon tissues
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TABLE 7.2
Characteristic peaks, wavelength differences at which they occur and the intensity of the healthy tissue samples
I MAXIMUM I MINIMUM II MAXIMUM II MINIMUM

NUMBER
OF
HEALTHY
TISSUE
WD I WD I WD I WD I
SAMPLES
(nm)
(nm) (nm) (nm)
sample 1 22.417 113.614 -0.602 114.149 43.698 115.341 - 116.87

33.36
sample 2 17.277 116.036 -3.309 118.455 119.136 5.103 - 123.832

27.07
sample 3 22.871 115.341 - 116.423 6.044 118.189 - 119.48

32.503 4.957
FIGURE 7.17
Differences between tumour and healthy colon tissues
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The diagnosis of skin epithelial tissue using the Opto-magnetic imaging spectroscopy
FIGURE 7.18
OMIS spectra for three melanoma en masse
By looking at the diagram above it can be seen that the intensity of the three melanomas is very small,
practically unreadable and the wavelength difference vary from 120 up to 200 nm. The possible reason
lies in the biophysical properties of the malignant cells and their activities.
FIGURE 7.19
OMIS spectra for three nevi en masse
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The comparison of these three nevi showed similarity in the shape of the curves (with some shifts),
characteristic peaks and wavelength differences. There are two characteristic peaks on each diagram,
one positive (132.263/20.706, 139.979/14.317, 140.372/21.827) and one negative (121.224/-1.491,
142.302/-31.299, 151.328/-29.893). Small variations exist probably because of the presence of many
subtypes of nevi. One of the next steps in our study is to define the subgroups of nevi and melanoma
using OMIS.
FIGURE 7.20
OMIS spectra for both melanoma and nevus (mole)
Statistical analysis
Summary statistics
Opto-magnetic imaging spectroscopy was developed with the purpose of serving as a non-invasive,
economic, readily available, additional method for quick screening in outpatient conditions. It is based
on tracking changes in paramagnetic/diamagnetic properties of tissues or cells which arise from the
changes in the water matrix.
In the longitudinal study, the opto-magnetic spectra of tissues and cytological samples were collected
over time for three types of healthy or benign changes of tissues and cells (healthy colon mucosae,
nevus and cervical cytological samples of II Pap group) and malign changes (colon cancer, melanoma
and cervical cytological samples of V Pap group). Cervical cytological samples were also collected for
transition disease states: Pap group III and Pap group IV. However, in order to simplify the problem
only two extreme groups – Pap II and Pap V were analysed because it was expected that the difference
between them would be more evident and therefore easier to observe.
The types of tissues or cytological smears and the corresponding number of cases for which the opto-
magnetic spectra were acquired, are presented in Table 7.3. The collected data comprise of a database
of opto-magnetic spectra for healthy and cancerous changes in colon tissue, skin and cervical cells.
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In order to estimate an overall tendency of changes in opto-magnetic spectra, a mean opto-magnetic

spectrum for each study group (from Table 7.3) was calculated and presented in Figures 7.21-7.23.
TABLE 7.3
Types and sizes of study groups
Sample type Number of cases Sample type Number of cases

Healthy colon
55 Colon cancer tissue 57
mucous tissue
Naevus 48 Melanoma 48
Stained Pap smears Stained Pap smears

on microscopic 67 on microscopic slides 70
slides – II Pap group – V Pap group
The mean opto-magnetic spectra of malignant changes compared to the healthy state or benign
changes in tissues and cells show decreased intensity values. This decrease is consistent in all cases of
malignancy – in skin, colon and cervix cells.
-6
A water molecule is composed of an oxygen atom with a paramagnetic value of 1.34 x 10 and two
-8
hydrogen atoms with a diamagnetic value of – 2.48 x10 for each of them. A single isolated water
-9
molecule is diamagnetic, with a mass magnetic susceptibility of -9.05 x 10 . Bulk water, in its pure
form, makes dimer, trimmer and higher water clusters, showing both paramagnetic and diamagnetic
properties (the average value of paramagnetism is 1.2 nT, and diamagnetism 2.3 nT), and in between
the water constantly oscillates [44,45]. The opto-magnetic imaging spectrum based on an absorption
difference (in order two, 100 times), of blue (B) and red ® channels of visible light may identified this
dynamic.
In tumour cells and tissues, the state of water is altered [35-37, 46] and from the opto-magnetic
spectra point of view for cancerous cells and tissues, it is evident that the paramagnetic/diamagnetic
dynamics of water in diseased cells and tissues is disrupted compared to healthy tissue. This altered
state of water is expressed through changes in the paramagnetic/diamagnetic properties of water and
can thus be used as a classification criterion between healthy and cancerous states.
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FIGURE 7.21
Comparison of the mean opto-magnetic spectra for the dataset of 48 melanoma cases and the mean opto-
magnetic spectrum for the dataset of 48 nevus cases
FIGURE 7.22
Comparison of the mean opto-magnetic spectrum for the dataset of 55 cases of healthy colon mucosa and the
mean opto-magnetic spectrum for the dataset of 57 cases of colon cancer
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FIGURE 7.23
Comparison of the mean opto-magnetic spectrum for the dataset of 67 cases of II Pap group stained cervical
smears and the mean opto-magnetic spectrum for the dataset of 70 cases of V Pap group stained cervical smears
The classification results

Generally, classification is a data mining function whose goal is to accurately predict class labels of
instances whose attribute values are known, but class values are unknown. In this study the goal of the
classifier is to correctly predict the class – healthy or cancer, based on the opto-magnetic spectra of
tissue or cytology sample.
This study was concerned with the identification of three types of cancer: colon cancer, skin cancer
(melanoma) and cervical cancer (PAP group V), therefore the classification was performed for three
independent datasets of opto-magnetic spectra for cancer and healthy cases. For colon cancer and
melanoma, opto-magnetic images were acquired from the tissue directly, while in the case of cervical
cancer the images were acquired from stained cytological smears on microscopic slides. All these cases
were assigned to a class of cancer cases. For the control group, the opto-magnetic images were taken
of healthy colon mucosae, skin nevi (benign skin changes) and of stained cytological smears on
microscopic slides (II Pap group). These cases were assigned to a class of healthy cases. The types of
samples and the corresponding number of opto-magnetic spectra, used as input in the classification
algorithms, are presented in Table 7.4. Two opto-magnetic spectra from the cervix database were
detected as outliers and removed from further analysis.
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TABLE 7.4
The types and sizes of three datasets of opto-magnetic spectra used for classification
For these three datasets, several classifying methods were applied (Soft Independent modelling of class analogies
– SIMCA, k- nearest neighbours – kNN, support vector machine – SVM etc.) in order to distinguish differences
between healthy and cancer cases, but so far, the most promising results were obtained using Naïve Bayes
classification with kernel density estimation. All analysis of data was performed using statistical software R [46].
Total number of Number of Number of

Material Sample type
cases cancer cases healthy cases
Colon Tissue 112 55 57
Skin Tissue 96 48 48
Stained
Cervix cytological 135 66 69
smear
A naïve Bayes (NB) classifier is an important classifier for data mining and applied in many real world
classification problems because of its high classification performance. It is a simple probabilistic
classifier based on: (a) Bayes theorem, (b) strong (naive) independence assumptions, and (c)
independent feature models [48]. Naïve Bayes classification with kernel density estimation, or so called
“flexible Bayes” is an extension of the naïve Bayes classifier which uses a kernel density estimation
where the density of each continuous variable is estimated averaging over a large set of kernels. The
method performs well in domains that violate the normality assumption and, in general, this flexible
Bayesian classifier generalises better than the version that assumes a single Gaussian [49].
In this study, to estimate the performance of the classifier, a 10-fold cross-validation approach was
used. In the 10-fold cross-validation, the entire dataset is divided into 10 mutually exclusive subsets.
Each fold is used once to test the performance of the classifier that is generated from the combined
data of the remaining nine folds, leading to 10 independent performance estimates and thus ensuring
optimal utilization of the available data. Given the relatively small size of available samples, this choice
seemed to be the best solution.
Evaluation of the classifier performance was done using three performance measures: specificity,
sensitivity and accuracy:
TN
Specificity: (1)
TN +FP
TP
Sensitivity: (2)
TP +FN
TP +TN
Accuracy: (3)
TP +TN +FP +FN
where TP, TN, FP and FN denote numbers of true positives, true negatives, false positives and false
negatives, respectively.
A true positive (TP) is the number of cases correctly identified as cancer, while a true negative (TN) is
the number of cases correctly identified as healthy. A false negative (FN) is the number of cancer cases
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incorrectly identified as healthy, and a false positive (FP) is the number of healthy cases incorrectly
identified as cancer.
As an additional measure of classifier accuracy, Cohen’s kappa *50+ was calculated. Cohen’s kappa
measures classifier accuracy, while compensating for successes due to chance. Cohen’s Kappa is
defined as:
p 0 −p c
K= , (4)
1−p c
where p0 is the total agreement probability, and p c is the ‘‘agreement’’ probability which is due to
chance. It is more convenient to calculate Kappa using counts from the confusion matrix, rather than
probabilities:
𝑁× 𝐼𝑖=1 𝑥 𝑖𝑖 − 𝐼𝑖=1 𝑥 𝑖. 𝑥 .𝑖
𝐾= , (5)
𝑁 2 − 𝐼𝑖=1 𝑥 𝑖. 𝑥 .𝑖
where xii is the count of cases in the main diagonal, N is the number of examples, and x i, xi are the
columns and rows total counts, respectively. Cohen’s kappa statistics range from -1 (total
disagreement) through 0 (random classification) to 1 (total agreement) [51].
Table 7.5 gives a summary of the classification results for the three datasets of inputs – colon samples,
skin samples and cervix smears samples. Detailed prediction results are presented in the form of
confusion matrices (Tables 7.6-7.8).
TABLE 7.5
Summary of classification results using Naïve Bayes classifier with kernel density estimation
Performance measures Specificity [%] Sensitivity [%] Accuracy [%] Kappa

Skin 87.04 97.62 91.67 0.833
Colon 100 92.98 96.43 0.9286
Cervix 78.16 97.92 85.18 0.7018
TABLE 7.6
Confusion matrix for Cervix dataset
Predicted
Confusion matrix Healthy Cancer
Healthy 68 19
Actual
Cancer 1 47
TABLE 7.7
Confusion matrix for Skin dataset
Nanomedicine 183
Predicted
Confusion matrix Healthy Cancer
Healthy 41 1
Actual
Cancer 7 47
TABLE 7.8
Confusion matrix for Colon dataset
Predicted class
Confusion matrix Cancer Healthy
Cancer 53 0
Actual
Healthy 4 55
As it is shown in the results above, the chosen data mining method provide models that possess a high
degree of accuracy. Minimal accuracy was achieved for the cytological samples of cervical cells, but
85.18% of accurately predicted cases is still a very high number. The best results were achieved for the
classification between healthy and cancerous colon tissue, where the accuracy was 96.46%. It should
be noted that the opto-magnetic spectra developed for the images of cytological samples are quite
complex compared to the images of colon tissue, where even untrained personnel can easily
distinguish between an image of colon cancer and colon mucosae, while in the case of cytological
samples it is rather difficult. The achieved accuracy results are therefore very logical given the fact that
the task for the classifier is the simplest in the case of colon tissue, more complex in the case of skin
changes, and very complex for cytological samples.
Kappa statistics for all three datasets is high, close to 1, which excludes the possibility that this
classification result is due to chance. The results of prediction based on the opto-magnetic spectra
confirm the potential of the Opto-magnetic imaging spectroscopy method to discriminate between
healthy and cancer cases.
However, even though these results are promising, it should be noted that the size of the datasets is
still limited for final conclusions. It is also possible that other data mining methods could be more
successful in discriminating between the opto-magnetic spectra of healthy and cancer cases.
Conclusion
Opto-magnetic Imaging Spectroscopy was applied in vitro and in vivo on cervical, colon, and skin
samples. Research included 280 cervical samples, 112 colon samples and 96 skin samples. The
opto-magnetic spectra showed a good differentiation between healthy and cancerous samples
based on characteristic OMIS spectra intensities and peak positions. It is shown that spectra
intensity decreases towards lower values in cases of precancerous and cancerous tissues in all
three kinds of epithelial tissue. Classification results proved a high degree of accuracy in cancer
Nanomedicine 184
detection (skin 91.67%, colon 96.43%, cervix 85.18) using the OMIS method. Research efforts are
underway and are geared towards expanding the databases of the opto-magnetic spectra of cancer
and healthy cases, and also research of an optimal classification method in order to develop a
method which would complement the efforts of medical professionals in quick screening and early
cancer diagnostics.
Acknowledgment
This research has been funded by the Ministry of Education, Science and Technological Development of
the Republic of Serbia, through Project III 41006: Development of methods and techniques for early
diagnostic of cervical, colon, oral cavity cancer and melanoma based on a digital image and excitation-
emission spectrum in visible and infrared domain. Also, this research is partially funded by Tempus
project: „Studies in Bioengineering and Medical Informatics - BIOEMIS“, 530423-TEMPUS-1-2012-1-UK-
TEMPUS-JPCR. We are particularly grateful to Zoran Krivokapid (School of Medicine, Clinical Centre of
Serbia, University of Belgrade), Jadran Bandid (ORS Hospital, Belgrade) and Milena Papid-Obradovid
(Clinic for Gynecology and Obstetrics “Narodni Front”, Belgrade) who provided samples, and therefore
made it possible for us to realise this research. Also, we are thankful to the staff at each clinic who
helped and supported this research. In the initial stage of research MySkin,Inc, USA partially supported
this investigation.
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Nanomedicine 187
8
Solid Lipid Nanoparticles – A Promising
Drug Delivery System
Hristo Svilenov, Christo Tzachev
Faculty of Chemistry and Pharmacy, Sofia University, Bulgaria
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 188
Composition …………………………………………..………………………………………………………………………………….. 189
Preparation ………………………………………………………………………………………………………………………………… 192
Characteristics and characterization ………………………………………................................................... 199
Stability and stabilization ………………………………………..…………………………………………………………………. 205
Safety ………………………………………………………………………………………………………………………………………… 207
Current applications ……………………………………….………………………………………………………………………….. 209
Perspectives and conclusion ………………………………………………………………………………………………………. 211
Acknowledgment ……………………………………………………………………………………………………………………….. 212
References……………………………………………………………………………………..…………………………………………… 213
Nanomedicine 188
Introduction
The lipids are a large group of organic compounds that has a fundamental role in life on Earth.
Whether they act as the energy storage in our bodies or as the building blocks of the cell
membranes they play a key role in different physiological and biochemical processes. Due to their
intrinsic properties lipids can dissolve water-insoluble substances, pass through biological
membranes and undergo digestion by enzymes. In the pharmaceutical industry lipids are used
mainly as vehicles in different formulations intended for all routes of administration – as emulsions,
ointments, pellets, suppositories etc. The emulsions (type “oil-in-water”) are one of the most
important systems because they enable the administration of fat-soluble active pharmaceutical
ingredients (APIs) throughout various routes. For example, the i.v. administration of lipids and lipid
soluble substances wouldn’t be possible if the first parenteral nanoemulsions weren’t invented in
the 1950s. However, the small size of the lipid droplets in the nanoemulsions results in very high
surface area and rapid release of the drug. In addition, the liquid nature of the droplets favors
oxidation and specific instability processes (i.e. Ostwald ripening).
FIGURE 8.1 Comparison between micelles, liposomes, nanoemulsions and solid lipid nanoparticles. (Micelles
with hydrophobic core which is formed by the tails of the surfactant molecules. Liposomes with aqueous core
surrounded by a double phospholipid layer. Nanoemulsions droplets with hydrophobic liquid core composed
of the oil that is dispersed in the water and stabilized by a surfactant monolayer. SLN and NLC with
hydrophobic core of solidified lipid; often the solidification/crystallization of the lipid results in non-spherical
shape of the particles).
The demand to broaden the area of application of oil (lipid) in water dispersions and their
continuous optimization resulted in the development of the first solid lipid microparticles in the
1980s by Speiser and coworkers [1]. In the following years, extensive work and experiments with
solid lipids resulted in the invention of lipid based solid particles in the submicron range by the
groups of Westesen, Müller and Gasco [2 - 4]. This system called Solid Lipid Nanoparticles (SLN) is
defined as drug carrier in the submicron size range made of biocompatible and biodegradable lipids
solid at room and body temperature.
Nanomedicine 189
Some authors describe SLN as nanoemulsion in which the liquid lipid core of the droplets is
substituted by a solid one [5]. Such “substitution”, however, is related to the formation of a
distinctly different system that exhibits its own specific properties and such a comparison of SLN to
nanoemulsions is inappropriate.
In the following years SLN proved as a blockbuster in nanotechnology because many believed that
SLN “offer some of the advantages of polymeric nanoparticles, fat emulsions and liposomes along
with the possibility to successfully resolve problems related to drug physical and chemical stability,
drug delivery and absorption” *6+. Logical sequel was the development of Nanostructured Lipid
Carriers (NLC) which, still being solid at room and body temperature, consist of solid and liquid
lipids. NLC have met the expectations and managed to resolve some of the problems related to
crystallinity and poor drug loading [7].
The goal of this chapter is to summarize the most important information about Solid Lipid
Nanoparticles for the past two decades. It includes examples of the most frequently used
excipients and explanations of the production techniques. Moreover, we have given a short
overview of the analytical techniques that are considered the most important in the process of
characterization of SLN. Finally, we have discussed the current and future applications of SLN from
our point of view. We believe that this chapter will be useful to students and researches who want
to learn more about this type of nanoparticles. In addition, the chapter contains many tables and
citations which will be helpful to anyone who wants to know what research has been done so far in
this area.
Composition
SLN and NLC are composed of lipids and of stabilizers – in most cases surfactants, co-surfactants
and coating materials. Antioxidants, electrolytes, preservatives, viscosity enhancing agents,
adhesives, absorption enhancers and other excipients also find application. Most of the formulation
ingredients are safe and under the Generally Recognized as Safe (GRAS) status issued by the Food
and Drug Administration (FDA).
Lipids
In the following years SLN proved as a blockbuster in nanotechnology because many believed that
SLN “offer some of the advantages of polymeric nanoparticles, fat emulsions and liposomes along
with the possibility to successfully resolve problems related to drug physical and chemical stability,
drug delivery and absorption” *6+. Logical sequel was the development of Nanostructured Lipid
Carriers (NLC) which, still being solid at room and body temperature, consist of solid and liquid
lipids. NLC have met the expectations and managed to resolve some of the problems related to
crystallinity and poor drug loading [7].
Lipids can be defined as fatty or waxy organic compounds. Generally, they are soluble in nonpolar
and insoluble in polar solvents. Their typical constituents are free fatty acids, free fatty alcohols,
glycerol esters of fatty acids and waxes. More complex structures as phospholipids, glycolipids and
sphingophospholipids are also referred to this group. Lipids “build” the core (the lipid matrix) of
SLN/ NLC. The lipid matrix itself determines the particles’ pharmaceutical properties as it is the
structure that stores, transports and releases the drug.
Nanomedicine 190
Often the combination of two and more lipids TABLE 8.1 Commonly used lipids in the
is favorable because it enables adjustments in preparation of SLN and NLC.
the physical state (melting point, crystallinity
and polymorphism), drug loading and drug Group Reference
release kinetics of the particles to be made. Triglycerides
Examples of different groups of lipids used in Glyceryl tristearate [8-13,38]
the preparation of SLN and NLC can be found
Glyceryl tripalmitate [8,9,13,15,17,38]
in Table 8.1.
Glyceryl trimyristate [8,9,12,13,16,38]
Surface active compounds (SACs) Medium chain triglycerides [18,20,48,49]
Glyceryl trioleate [21]
When one of two immiscible phases (e.g.,
lipid and water) is dispersed into the another Monoglycerides
an interfacial boundary is formed. The surface Glyceryl monostearate [12,13,23,24–26,39,89]
energy at this boundary is expressed as Gibbs Mixtures
free energy (G). At constant temperature and
Glyceryl behenate [12,16,18,19,26,27]
pressure G depends on the surface area (A)
and the interfacial tension (γ) (Eq.1) *62+: Glyceryl palmitostearate [22,29,45]
Witepsol grades [28,30,31,88]
2𝛾
∆𝑝 = 𝑃𝑖𝑛𝑠𝑖𝑑𝑒 − 𝑃𝑜𝑢𝑡𝑠𝑖𝑑𝑒 = (Eq. 2) Free fatty acids
𝑟
Behenic acid [33,34]
Where Pinside is the pressure inside the
Stearic acid [13,24,27,33,34,37,39,41]
droplet, Poutside is the pressure outside the
droplet, γ is the surface tension and r is the Palmitic acid [33-36]
radius of the spherical droplet. The lower the Myristic acid [33,34]
value of the Laplace pressure is the less Oleic acid [22,37-40]
energy to break the droplets is needed.
Free fatty alcohols
However, as the droplets become smaller
their radius decreases which results in Stearyl alcohol [42,43,47]
proportional increase in the pressure (e.g. Cetyl alcohol [38,44-47]
tenfold reduction in droplets diameter Myristyl alcohol [47]
increases the Laplace pressure 10 times).
Lauryl alcohol [47]
Therefore, more energy will be required to
produce smaller and smaller particles. Waxes
increases the Laplace pressure 10 times). Still, Cetyl palmitate [41,42,48,49,75]
smaller droplet formation at constant amount Beeswax [50-52]
of energy applied on the system can be Carnauba wax [52,53]
facilitated by lowering the surface tension.
Others
Surface active compounds, due to their
amphiphilic structure, exhibit a tendency to Castor oil [14]
accumulate at phase boundary and form Hydrogenated castor oil [54–56,85]
monomolecular layer around the Hydrogenated palm oil [57,58]
droplets/particles. This normally results in
Cacao butter [59]
system stabilization by lowering the surface
tension, decrease in the Gibbsfree energy and Goat fat [60]
the Laplace pressure. Anhydrous milk fat [61]
Nanomedicine 191
TABLE 8.2 Commonly used surfactants and co-

surfactants in SLN and NLC formulations.
However, surfactants properties are
Surfactant Reference
predetermined by their chemical structure
Nonionic
and they lower the surface tension only to a Polyoxyethylene sorbitan fatty esters
certain limit. Further decrease can be Polysorbate 20 [23,57,78]
achieved with the incorporation of co- Polysorbate 60 [17,43,44]
surfactants [65]. Polysorbate 80 [12,13,15,17,18,24,26,57]
Polysorbate 85 [57]
The choice of SAC(s) is essential to the final Polyoxyethylene alkyl/aryl ethers
properties of the formulation. Surfactants Polyoxyethylene(20)cetyl ether [75,76]
Polyoxyethylene(20) isohecadecyl [75,76]
from almost all groups that find application in ether
the formulation of SLN and NLC are presented Polyoxyethylene(20)oleyl ether [75,76]
in Table 8.2. Elevation of the temperature also Polyoxyethylene(20)stearyl ether [43,76–79]
decreases the surface tension and can be Tyloxapol [80,81,86]
used to produce hot-emulsions with low Ethoxylated castor oils
PEG-35 castor oil [131]
energy input [64].
PEG-40 hydrogenated castor oil [83,84]
Poloxamers
Beside the main components constituting the Poloxamer 188 [14,16,18,19,33,69,78]
SLN/NLC (i.e., the lipids and surfactants) a Poloxamer 407 [30,87,90]
variety of different additives can find place in Others
each particular formulation. Good illustrations Polyoxyethylene- glycerine monostearate [68]
of such examples are the following: Macrogol(15) hydroxystrearate [14,16]
PEG caprylic/capric triglycerides [29]
cryoprotectants in freeze dried formulations –
Polyglyceryl-3 methylglucose distearate [46]
d-sorbitol, d-glucose, d-fructose [28]; Polyglyceryl-6 distearate [48]
polysaccharide coating materials – chitosan Anionic
[30,34]; protein coating materials – silk fibroin Sodium dehydrocholate [24]
[72]; alternative polymeric emulsifiers – Sodium taurocholate [27,59,67]
polyvinyl alcohol (PVA) [35] and poly(lactic-co- Sodium glycocholate [66,68,86]
Sodium cholate [69,70]
glycolic acids) (PLGA) [85]; tonicity adjusting
Sodium lauryl sulphate [71,72]
agents – electrolytes and glycerol [22]; Cationic
preservatives – parabens [69], thiomersal [58, Cetrimonium bromide [42-44,73]
66], imidazolidinyl urea, chloromethyl- DOTAP [38,82]
isothiazolinone and methylisothiazolinone DOTMA [73]
[75]. Other relevant examples are the Chlorhexidine salts [74]
Dimethyldiocta- decylammonium bromide [37]
combinations of SLN and NLC with different
Amphoteric
viscosity enhancing excipients to produce L-α –phosphatidylcholine [21,59]
carbopol [26] and dextran [70] hydrogels. Soya lecithin [10,14,24,29,39,66,89]
Egg lecithin [52,88]
A bright demonstration of the endless and Co-surfactants
diverse applications of the excipients is the 1-Butanol [13,37,59]
innovative approach to obtain Low molecular weight PEG [15,89]
Diethylene glycol monoethyl ether [23]
polyvinylpyrrolidone/tristearine Propylene glycol [26]
microparticles with electrospraying which Ethanol [27]
upon dissolution form SLN [11]. Sorbitan monostearate [71]
Nanomedicine 192
Preparation
The techniques for SLN and NLC preparation can be grouped into three main branches – high-
energy approaches, low-energy approaches and methods employing organic solvents.
High-energy approaches
High pressure homogenization
High pressure homogenization is one of the first techniques used in the preparation of SLN [3].
Nowadays, it is well-established in the production of nanoemulsions for parenteral foods,
homogenization of milk, ice cream and others. In this technique a liquid is pushed at high pressure
through a narrow gap. Both the high pressure (in the range of 100-2000 bar) and the small size of
the gap (in the range of few microns) cause a very high acceleration and pressure drop. As a result
a very high shear stress and cavitation forces disrupt particles/drops in the liquid. An increase in the
temperature during the process (usually around 10 °C for every 500 bar) is also possible due to the
high acceleration and friction. The method is easy accessible and scalable.
Two approaches to produce SLN and NLC are utilized via this technique – homogenization of hot
pre-emulsion and homogenization of cold pre-suspension (Figure 8.2.and Figure 8.3.).
FIGURE 8.2 Mechanism of high pressure homogenization of cold pre-suspension and hot pre-emulsion
respectively in the "cold“ and "hot“ technique.
Nanomedicine 193
In the “hot” method the procedure is carried at temperatures above the melting temperature of
the lipid. A pre-emulsion is formed usually with the help of high shear mixers. This emulsion is
passed through a narrow orifice – valve or nozzle. Usually several cycles are applied to achieve
submicron size with low polydispersity. The product obtained after the homogenization is a hot
microemulsion. The latter should be cooled fast so that the liquid lipid droplets can solidify and
form the intrinsic structure of the SLN and NLC.
The first stage of the cold homogenization approach is the formation of a hot lipid melt mixture of
the substances that form the lipid matrix and the APIs. Then the melt is cooled down to a solid
state and grinded in powder mill to obtain particles in the size range of 50 to 100 micrometers. The
obtained lipid powder is dispersed in aqueous solution of surfactant to form a pre-suspension. This
pre-suspension is then passed through a homogenizer. Usually, more cycles and higher pressure
(compared to the hot approach) are required because the particles are more rigid and harder to
break. The cold approach is desirable in formulations with drugs that are not stable at high
temperatures or can distribute in the aqueous phase during the preparation.
FIGURE 8.3 "Hot" and "Cold" high pressure homogenization technique in the production of SLN/NLC.
High shear homogenization
This is a simple and widely used technique in the production of aqueous dispersions. It is usually
processed in chamber with a rotor-stator homogenizer. The procedure starts with placing the lipid
ingredients and the water phase into the homogenizer followed by applying high shear mixing
(5 000 – 25 000 rpm) at temperatures above the melting temperatures of the lipids (Figure 8.4.).
After homogenization, the formed hot microemulsion is cooled to form SLN and NLC. Although it is
very simple procedure to perform the properties of the final product are usually poor and particles
Nanomedicine 194
in the micrometre range are detected. Higher shearing rates can result in smaller droplets only until
a certain critical value due to processes of coalescence. Although higher shearing rates cannot
decrease particle size beyond that certain value they can reduce the polydispersity [91]. The poor
properties of the final product are the cause why this technique is preferably used as a pre-
homogenization step in high pressure homogenization and ultrasonication.
FIGURE 8.4 High shear homogenization and ultrasonication techniques in the

production of SLN/NLC
Ultrasonication
Ultrasonication is based on the cavitation in aqueous dispersions caused by powerful ultrasound

with wave frequency usually around and above 20kHz. In the production of SLN and NLC a mixture
of pre-emulsion from melted lipid and hot surfactant solution is first prepared (Figure 8.4.). Then
the ultrasound is applied with a sonotrode that is in contact with the liquid. The cavitation causes
disintegration of the lipid phase into smaller droplets. The obtained hot microemulsion is then
cooled to form the solid particles (Figure 8.3.). Advantages of this technique, without doubt, are
the possibility for scale up with flow cells, the low number of wetted and moving elements (easier
cleaning) and the possibility to control the process by controlling the sound wave amplitude. Main
drawback, however, is the risk of metal contamination, which increases with longer sonication
times.
Electro-spray technique
In this relatively new technique an electrodynamic atomization is used to produce SLN directly in
powder form [92]. The obtained particles by this method have narrow distribution and size below
one micrometre. However the method is still under investigation for its applicability in the
production of larger quantities of dispersions.
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Low-energy approaches
Microemulsion method
Microemulsion formation is used as a stage in the production of SLN and NLC since the early 90s
[4]. In this method the microemulsion is spontaneously formed due to the high surfactants/lipid
ratio. The proportions of the excipients are essential and in most cases pseudo ternary diagrams
are used to study and describe the areas of microemulsion formation. This method is simple and is
performed by several common steps. Initially the lipids are melted and mixed with hot surfactant
solution. Gentle stirring is applied until the microemulsion is formed. In the second stage the hot
microemulsion is dispersed in high volume of cold water (2-3 °C) under moderate stirring. This
causes the liquid droplets to solidify. SLN or NLC obtained by this technique are spherical in shape
and have narrow size distribution. However, the method suffers from several drawbacks - the final
dispersion is very diluted (rangingbetween 1:25 up to 1:50 with respect to the hot emulsion). This
may require further concentration by ultrafiltration, lyophylization or other method. The high
concentration of surfactants/co-surfactants used is another major disadvantage of this technique.
Membrane contactor
Membrane contactor technique is a method which allows a gaseous and liquid phase to come in
direct contact without dispersing one of the phases into the other and is used for purposes which
require mass transfer between them. The two phases are separated by a hydrophobic membrane
with typical pore size of 0,05 microns. This membrane doesn`t allow water to pass because of the
high breakthrough pressure needed. In the production of SLN and NLC this technique is modified
and the gaseous phase is replaced with a melted lipid blend [93]. This blend is forced to pass
through the membrane. Small droplets are formed spontaneously (Figure 8.5.). On the other side of
the membrane a hot surfactant solution is circulating and swapping away the droplets. The liquid
lipid droplets are enveloped and stabilized by the surfactant molecules. Then, after cooling down
the dispersion the droplets transform into solid particles. The method is scalable and the particle
size can be tuned by using membranes with different pore size.
FIGURE 8.5 Membrane contractor technique in preparation of SLN/NLC.

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Phase inversion temperature (PIT) method
Phase inversion of O/W to W/O emulsions and vice versa induced by temperature change is a long
known method to produce microemulsions stabilized with non-ionic surfactants [94]. The
technique is based on the change in the properties of polyoxyethylated surfactants at different
temperatures. The hydrophillic-lipophillic balance (HLB) value of surfactants defined by Griffin is
valid at 25°C. At this temperature the hydrophilic parts of the SAC molecules are hydrated to a
certain extent. An increase in the temperature causes dehydration of the ethoxy groups. As a
result, the lipophilicity of the molecules of the SAC rises with corresponding decrease in HLB value.
At a certain point the affinity of the SAC to the aqueous and lipid phase is equal - this temperature
is defined as the phase inversion temperature. This particulate state is characterized by very low
surface tension and presence of complex structures in the system. If the temperature is further
increased the SAC’s affinity to the lipid phase becomes higher enough to stabilize emulsions of W/O
type (Figure 8.6).
FIGURE 8.6 Phase inversions of hot emulsion with variations of the temperature above and below PIT.
If cooled down the system goes through the reverse process. Due to the specific properties of the
system around the PIT very small particles are spontaneously formed just below that
temperature.If rapid cooling is applied at this point stable particles with desirable size and
polydispersity can be obtained [95]. In the last decade different groups report the feasibility of the
method in the production of lipid nanocapsules, SLN and NLC [75, 76, 95, 96]. Several modifications
to improve the final product are also developed e.g., cycling around the PIT to achieve better
distribution of SAC at the phase interface and therefore smaller size and lower polydispersity [97].
Coacervation method
The coacervation method is relatively new, low-energy and solvent free technique. It is based on
precipitation of free fatty acids from their sodium salt micelles in presence of surfactants [33].
Particles obtained by this technique are usually spherical in the range from 250 to 500 nm.
However, the feasibility of the method is still under investigation.
Double emulsion method
The double emulsion technique in the preparation of SLN and NLC is suitable for hydrophilic APIs
and peptides [98]. In this method an aqueous solution of the drug is emulsified in melted lipid
blend to form primary W/O emulsion stabilized with suitable excipients (Figure 8.7.). The primary
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W/O emulsion is dispersed in aqueous solution of

hydrophilic emulsifier to form a double W/O/W
emulsion. Then the double emulsion is stirred and
isolated by filtration. Relatively large particles are
obtained with this technique but beside the
incorporation of hydrophilic molecules it offers the
possibility of surface modification, e.g. with PEGs.
Approaches with organic solvents
Emulsification-solvent evaporation
This technique is based on precipitation of the lipids

from O/W emulsions [99, 100]. The lipid material
and drug are dissolved in organic water-immiscible
solvent (e.g., chloroform). The organic solution is
emulsified in an aqueous phase with the help of
suitable surfactants to form O/W emulsion. Then
the organic solvent from this emulsion is
evaporated at low pressure. This causes the lipid
and API(s) to precipitate in the form of SLN or NLC
(Figure 8.8. and Figure 8.9.). The particles obtained
by this method have optimal properties – small size
(25-100 nm) and narrow size distribution. Main
FIGURE 8.7 Double emulsion technique in
disadvantages are the limitation of lipid
preparation of SLN/NLC.
concentration in the organic solvent to form
desirable particles and the use of organic solvents per se. The method is appropriate especially in
the encapsulation of thermo sensitive APIs because of the absence of any thermal stress. It is
notable that identical formulations don`t achieve the same results when they are processed by
other technique.
Emulsification solvent diffusion
This method is based on the emulsification of partially water miscible solvent (e.g. butyl lactate,
benzyl alcohol) solution of a solid lipid in an aqueous solution of suitable surfactant [101]. The
aqueous phase of the obtained emulsion is saturated with the organic solvent and the excess of the
solvent is in the form of emulsion droplets. A dilution with fresh water causes the organic solvent,
driven by the concentration gradient, to diffuse from the droplets to the water. As a result the
solubility of the lipids decreases until they precipitate (Figure 8.9.). SLN and NLC below 100 nm with
narrow size distribution can be produced by this technique. The solubility of the water miscible
solvent in water and the solubility of the lipid in the organic solvent are crucial to the final results.
However, the type and the concentration of the lipid, surfactant and organic solvent would require
a substantial optimization work. Noteworthy advantage is the low processing temperature.
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Solvent injection
The method is similar to the emulsification solvent diffusion method but the organic solvents used
are selected from the group of the very miscible with water solvents (DMSO, ethanol) thus
eliminating the chance for emulsion to be formed [102]. Firstly, the lipid(s) and API(s) are dissolved
in the organic solvent. Then the organic solution is injected in aqueous solution of surfactant under
stirring. This causes a rapid migration of the organic solvent in the water and precipitation of the
lipids (Figure 8.9.). The obtained particle size depends strongly on the velocity of extraction
respectively on the lipophilicity of the solvent. The more hydrophilic the solvent the smaller the
particles but the less its capacity to dissolve lipids. The method offers advantages such as low
processing temperatures and low shear stress.
Supercritical fluid (SCF) technique
A SCF is defined as a substance above its critical temperature (T C) and critical pressure (PC). The
critical point represents the highest temperature and pressure at which the substance can exist as a
vapour and liquid in equilibrium. The supercritical fluid has unique thermo-physical properties
which can be finely tuned by small changes in the pressure. As the pressure raises the density and
the ability of the fluid to dissolve compounds increases while the viscosity remains relevantly
constant. Accordingly under high pressure and appropriate temperature in the supercritical range
the fluid can act as an alternative to organic solvents and dissolve different APIs and lipids [103].SCF
like carbon dioxide are safe, cheap, non-irritable, relatively inert and has a low critical point.
However, the method often yields particles in the micrometre range and is often combined with
other homogenization technique like ultrasound [104].
FIGURE 8.8 Emulsification solvent evaporation technique in the preparation of SLN/NLC

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FIGURE 8.9 Organic-solvent based methods for preparation of SLN/NLC.
Characteristics and characterization

Size and shape
Size, size distribution (polydispersity) and shape of the particles are important to the
physicochemical and biopharmaceutical properties of the SLN and NLC formulations. The accurate
analysis of these parameters is crucial to the complete understanding of the formulations. Still,
particle characterization in the nanometer range can be a serious challenge due to the presence of
concomitant colloidal structures (e.g., liposomes, micelles, nanocrystals etc.) that are often formed
alongside with the SLN and NLC [105]. These structures may disappear after dilution or drying of
the system [106]. Therefore, it is highly recommended that the analysis should be performed on
samples with minimal additional treatment.
Two of the widely used techniques to measure particle size and particle size distribution in aqueous
suspensions are Laser Diffraction (LD) and Dynamic Light Scattering (DLS). LD is based on the
phenomenon that when a laser beam is passed through a sample with dispersed particles the
larger ones scatter light at small angles while the smaller ones scatter light at large angles [107].
The data collected by LD is used to calculate the equivalent sphere diameter of particles according
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to the Mie scattering solution (also known as the “Mie theory”). The method is effective in
measuring particles in the range from dozens of nanometers to thousands of micrometers
depending on the laser source. Blue lasers are used in the analysis of smaller particles while red
lasers are more suitable for larger particles. However the method lacks accuracy for particles in
colloidal suspensions with diameter significantly smaller than the wavelength of the laser. DLS, also
known as Photon Correlation Spectroscopy (PCS) or Quasi-elastic Light Scattering (QLS) is based on
time-dependent fluctuations in the scattering intensity caused by small particles in suspension
when a laser beam is applied to the sample [108]. These fluctuations are result of the Brownian
motion of the dispersed particles. Smaller particles have higher velocity of Brownian motion while
larger particles have less velocity. Using the Stokes-Einstein equation and the data from these
fluctuations the hydrodynamic diameter of the particles can be calculated. The hydrodynamic
diameter is that of a sphere with the same translational diffusion coefficient as the particle
beingmeasured. The translation diffusion coefficient may depend on additional surface structures
attached to the particles and ions in the medium. Therefore, the size measured with this technique
is usually larger than the one obtained by microscopic techniques. DLS is very sensitive and can
detect particles below 1 nm. However, there are some limitations in the micron range (particles
above 6-10 microns are not suitable for analysis by this technique). The size distribution can also be
calculated using DLS or LD. Size distribution can be presented either as graphic or as the
polydispersity index (PDI). PDI values range from zero to one. Monodisperse samples have PDI
equal to zero and very polydisperse samples have PDI close to one. The priorities and drawbacks of
LD and DLS suggest that the two techniques may supplement each other. In that manner more
accurate information for colloidal systems with several particle populations can be achieved. Both
methods don’t measure particle size directly, instead they use light diffraction and scattering to
compute diameter, assuming that the particles are spherical. Therefore, for example, particles with
irregular shape that is quite different from spherical may result in misleading data. Particle
concentration in the sample is also critical. Analysis of concentrated dispersions may result in lower
average hydrodynamic diameter and higher polydispersity. The Information obtained from LD and
DLS is important but not sufficient for characterization of SLN and NLC dispersions [105]. The
particle size data should be confirmed with another suitable method. Microscopic techniques are
very helpful at this point. Even the simple light microscopy can provide information about the
presence of artefacts and particles in the micrometre range. However, to visualize particle in the
nanometre range more powerful methods are need.
Both Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) find
application in SLN/NLC analysis [109]. These techniques employ electrons instead of light to
visualize particles in the nanometre range. SEM detects the scattered electrons from the surface of
the particles and visualizes the surface. TEM detects transmitted electrons and allows the
researcher to “look beyond” the surface. Both techniques suffer from disadvantages related to the
processing conditions (e.g., vacuum, heating) and sample preparation which may include
dehydration, staining, conductive coating etc. Sample preparation and analysis itself can trigger
changes within the nanoparticles thus giving invalid results [110]. Here Atomic Force Microscopy
(AFM) offers some advantages compared to TEM and SEM. AFM uses calculations based on the
force that acts between the surface of the particles in the sample and probing tip. The method
provides adequate resolution with simplicity of sample preparation and fast image capturing. In
AFM no vacuum is needed and the sample does not need to be conductive. However the measured
particles should be immobilized before visualization. This is easily achieved for larger particles due
to their sedimentation. For smaller particles that undergo Brownian motion immobilization is a
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serious issue which is resolved with removal of the aqueous media. Still, dehydration can cause
changes associated with cluster formation, shrinkage, crystallization of the lipid etc. [111]. A strong
advantage of AFM is that it can give important details which other microscopic techniques are
unable to provide i.e. information about soft surfactant layering around the particles [112]. Often
the information obtained by the latter microscopic methods and LD and DLS can differ significantly
[113]. As we have stated earlier this can be result of water removal and dehydration of the
particles. Contemporary method that can visualize particles in their native state is the Cryo-Electron
Microscopy (Cryo-EM). Cryo-EM allows observations in samples in their native environment at
cryogenic temperatures. The sample is frozen according to specific procedure and exposed to a
very low electron dose. The electron dose affects image resolution and usually a compromise is
settled. The 2D image shows the SLN and NLC particles in their hydrated state [114].
Zeta potential
Zeta potential gives information about the magnitude of the electrostatic repulsion or attraction
between particles in the aqueous suspension of SLN and NLC. Zeta potential can serve as an
important parameter in the predictions for long term stability of the formulations [115]. High
values of zeta potential (e.g., more than +30 mV or less than -30 mV) can stabilize the colloidal
suspension by electric repulsion [115] (Figure 8.10.). Electric repulsion generally results in less
contact between the particles and less aggregation. Particles charge can also influence their
interaction with tissues and cells. Both positive and negative values of zeta potential can be
induced with the help of ionic surfactants and charge inducers [66-68, 73, 74, 116, 117]. Although
zeta values around 0 mV cannot stabilize the suspension by the means of electric repulsion, lack of
particles charge doesn`t necessarily result in particle aggregation. For example colloidal systems
that contain steric stabilizers can express good long term stability even in cases when zeta potential
is as low as around 0 mV [115].
FIGURE 8.10 Influence of zeta potential on the repulsion/coalescence of particles.

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Electrophoretic Light Scattering (ELS) or other appropriate methods are usually used to determine
particle velocity in electric field. This information allows the zeta potential to be calculated. Modern
instruments typically combine the analytical methods for particle size measurement by DLS and
zeta-potential by ELS [118].
Melting point and crystallinity
The melting point of the lipid depends on its TABLE 8.3 Melting points of commonly used
chemical nature (e.g., double bonds, free saturated and unsaturated fatty acids and
hydroxyl groups, chain length). Saturated triglycerides in SLN/NLC
fats have a uniform shape that allows them
Saturated fatty acids
to pack together in a crystal lattice.
Unsaturated fats have double bonds that Name
Number of Double Melting
introduce kinks into the hydrocarbon chain C atoms bonds point
making crystal formation more difficult Lauric acid 12 - 45 °C
which results in lower melting
Myristic acid 14 - 55 °C
temperatures. The melting point of the fatty
acids also increases proportional to the Palmitic acid 16 - 63 °C
chain length and triglycerides of the Stearic acid 18 - 69 °C
constituent fatty acids express a similar
trend. In Table 8.3 are shown the melting Arachidic acid 20 - 76 °C
points of some of the most frequently used Unsaturated fatty acids
fatty acids and triglycerides. The melting
point (or in some cases the melting interval) Palmitoleic acid 16 1 0 °C
does not depend only on the chemical Oleic acid 18 1 13 °C
nature of molecules but it is also determined
by the degree of crystallinity or crystal Linoleic acid 18 2 -5 °C
modification of the substance. For example Linolenic acid 18 3 -11 °C

triglycerides have three main crystal
Arachidonic acid 20 4 -49 °C
modifications, namely α (alpha), β′
(betaprime), β (beta). The α-modification is Triglycerides
in hexagonal subcell loosely packed in
Tricaprylin 8* - 9 °C
random order in which acyl groups are
oriented at 90° to the plane of the glyceryl Tricaprin 10* - 33 °C
group and are assumed to be oscillating with
Trilaurin 12* - 46 °C
a high degree of molecular freedom. The β′
orthorhombic subcell is closely packed Trimyristin 14* - 56 °C
whereas acyl groups are tilted 68-70° from Tripalmitin 16* - 66 °C
plane of the glyceryl group. The β subcell is
triclinic with highly ordered and most closely Tristearin 18* - 68 °C
packed molecules with acyl groups tilted
about 59° from the plane of the glyceryl * Number of C atoms in the fatty acid
groups.
Beside these three main modifications sub-modifications exist. Cooling down a SLN/NLC
formulation below the melting point of the lipids not always results in crystalline structure [119].
Such a state of the particles is defined as a supercooled melt [120]. The supercooled melts are
considered similar to the emulsions prepared with liquid at room temperature lipids. The
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phenomenon of supercooled melts can be explained with the lower chance of a sufficient number
of crystallization nuclei to form in the small volume of the particles in the colloidal dispersion.
Polymorphism and supercooled melts are considered major problems in the production of SLN and
NLC; they seriously affect their long term stability and final characteristics which are later discussed
in the Stability and stabilization part. Therefore the characterization of the state of the particles
and degree of crystallinity is necessary to fully understand the formulations.
Differential scanning calorimetry (DSC) is a widely used technique that measures differences in the
amount of heat required to increase the temperature of a sample compared to a reference [121].
Differences in heat flow may be positive or negative and are presented as function of the
temperature. At phase transition there are differences in the sample compared to the reference.
Owing to the different melting points and melting enthalpies of different lipid modifications DSC
can give information about the sample structure and interactions between the components. It is
highly recommended to confirm the results from DSC by another technique, especially in the
presence of high melting APIs, which can dissolve in the melted lipid blend but tend to crystalize in
the solid lipids. This may lead to the false interpretation that the API is in dissolved state in the solid
core of the particles.
Powder X-ray Diffraction (PXD) is a technique in which the sample is illuminated with X-rays of fixed
wave-length and the intensity of the reflected radiation is recorded. This data is used to calculate
the inter-atomic spacing. PXD is commonly used technique by scientists to analyze the crystal
structure of the SLN and NLC [122]. However, it is performed on powders and the removal of the
water from the colloidal suspension is required. Different polymorph modifications can be formed
after dehydration of the sample. Thus PXD is not suitable to analyze crystallinity of particles which
are originally formulated as colloidal suspension during storage. Nonetheless PXD is useful in the
characterization of lyophilized and spray dried SLN/NLC.
X-Ray scattering is another X-Ray technique that finds application in lipid lattice structure
examination of the particles. The X-Ray beam passed through the sample is obtained by a
synchrotron. A main advantage is the possibility to perform the experiment on colloidal
suspensions in their native state. Both small angle X-ray scattering (SAXS) and wide angle X-ray
scattering (WAXS) find application in the characterization of SLN and NLC [123-125].
Alongside the above described methods, Nuclear magnetic resonance (NMR) can be used to detect
fast and easy supercooled melts [126]. NMR measures the mobility of the molecules and the
different relaxation time indicated by the width of 1H NMR line. Low molecule mobility results in
broader 1H NMR lines. NMR also allows characterization of liquid nanocompartments in NLC and
examinations on drug mobility [127].
Other valuable methods used to determine the physical state of SLN/NLC are Electron Spin
Resonance (ESR), Infrared-, Raman- and External Reflection Spectroscopy [128-131].
Entrapment efficiency (EE) and drug loading capacity (DL)
The amount of the drug incorporated in the particles depends on the capacity of the lipid to
dissolve/disperse it. Two parameters expressing the efficiency of drug loading are most widely
used. Entrapment efficiency is the amount of the drug incorporated in the particles divided by its
overall amount in the formulation. EE is expressed in % (Eq.3):
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𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑡𝑕𝑒 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

𝐸𝐸% = ∗ 100 (Eq.3)
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑 𝑡𝑜 𝑡𝑕𝑒 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛
Entrapment efficiency is influenced by the characteristics of both the lipid and the API. Lipophilic
APIs distribute preferably in the lipid particles and have inherently higher entrapment efficiency
while the more hydrophilic drugs tend to distribute in the aqueous media. Drug loading capacity
(DL) is another parameter that expresses the amount of drug in the particles divided by the weight
of total carrier system (all ingredients taken together). DL is also expressed in % (Eq.4):
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑡𝑕𝑒 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
𝐷𝐿% = ∗ 100 (Eq. 4)
𝐴𝑚𝑜𝑢𝑛𝑡𝑜𝑓𝑑𝑟𝑢𝑔 + 𝑒𝑥𝑐𝑖𝑝𝑖𝑒𝑛𝑡𝑠𝑎𝑑𝑑𝑒𝑑𝑡𝑜𝑡 𝑕𝑒𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛
Challenges with the determination of these parameters are related to the correctness of drug
analysis. A combination of suitable separation and analytical method should be used. Usually
dialysis, ultracentrifugation, gel filtration or membrane filtrations are utilized. Drug concentration
can be measured either in the separated aqueous media or in the particles.
Drug localization and drug release
APIs can be localized either in the SLN/NLC or

in co-existing structures - micelles, liposomes,
drug nanocrystals. The drug state and kinetics
in the co-existing structures can be different
from these of the NLC/SLN. It is necessary to
determine correctly the presence/absence of
co-existing structures before examining drug
localization and release. Even when the drug
is entrapped in the SLN/NLC it is not always
homogenously dissolved in the lipid matrix
[132] and can be localized in different regions
of the particles (Figure 8.11.).
Localization can affect drug release and

biological properties of the product. In some
FIGURE 8.11 Possible drug localization in SLN/NLC
cases drug release kinetics of the drug from
the SLN or NLC can be altered by varying the
production method and/or its parameters. In other cases a burst release is observed independently
of the production technique [133,134]. However, controlled release is achievable and reported for
some formulations [135]. The type of in-vitro dissolution technique itself may also influence the
drug release kinetics of SLN/NLC. Careful evaluation of the release method should be performed
and the components of the dissolution media and conditions should be optimal for the specific
formulation. For example, one of the most preferred methods is with a dialysis bag. The bag,
however, can sometimes retard the diffusion and interact with drug molecules. Dissolutions studies
on SLN/NLC cannot always correctly predict the release behavior in vivo because of possible
enzyme degradation and interaction with cell organelles and lipid membranes in the body [136].
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Stability and stabilization

Chemical stability
The chemical stability of the lipids in formulations is often set aside by researches. However it is
important for the feasibility of the final product. The mechanism of lipid oxidation in water
dispersion differs from bulk lipids due to the aqueous phase that may contain prooxidants and
antioxidants [138].
Major prooxidants in the aqueous phase are the transition metals [139]. In emulsions the transition
metals promote oxidation by decomposing lipid hydroperoxides located at the droplet surface into
free radicals [140]. Free fatty acids are also promoters of the lipid oxidation in water dispersions
[141]. The rigid structure of the SLN and NLC is supposed to protect lipids from oxidation by
trapping them in the solid core [142]. In any case an investigation of the components should be
performed. Radomska-Soukharev studied the chemical stability of different combinations of
glycerides and surfactants during high pressure homogenization and storage. The tested excipients
have shown a very good stability during the production and have exhibited good long term stability.
Degradation of the lipids at ambient temperature was reported to be less than 10 % for over two
years in all formulations and less than 5 % in most of them. Further data place the triglycerides as
more stable than mono- and di- glycerides [143].
Physical stability
Physical instability in most of its part is related to the crystalline state of the lipids in the
formulation. As we have already discussed, lipids express several crystal modifications. These
modifications are α, β` and β. Supercooled melts are also possible. These different states of the
lipids express different properties directly related to the stability presented in Table 8.4.
TABLE 8.4 Properties of supercooled melt and different lipid crystal modifications
State Drug Lipid molecules Stability Crystal size [144] Shape of particles [145]
loading mobility
Supercooled Highest Highest Lowest Not crystalline Sphere
melt
α-modification High High Low Few microns Spheroids
β′-modification Low Low High Less than5 μm Spheroids
β-modification Lowest Lowest Highest 20–100 μm Platelet- and needle -like
When the formulation is rapidly cooled the lipids crystallize favorably in the alpha modification or
in some cases form a supercooled melt. During storage a transition from the less stable crystalline
modifications or supercooled state towards the more stable beta prime and beta modifications
occurs [146]. This leads to formation of more ordered and stable crystalline structures in the lipids.
As a general rule the stable modifications of the lipids usually exhibit lower EE and DL. For that
reason, drug expulsion may occur after transitions of the lipid crystalline structure. Moreover, the
transformation to more ordered crystalline structures cause a shape change from spherical to
platelet-like or needle-like. The shape transformation results in increase in the overall surface area.
Thus surfactant insufficiency can take place in the system (Figure 8.12.).
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FIGURE 8.12 Changes in lipid modification and consequent instability processes of SLN during storage
Transition from one modification into another is hard to predict and many factors may interfere.
For example, presence of liquid phases may promote crystal changes by dissolving less stable
crystals and further recrystallization in more stable modification [147]. Sometimes the transition
may be rapid upon contact with different surfaces, stress conditions or evaporation of the solvent
[148]. Rapid crystallization may result in gelation of the system and substantial difference in the
properties [149]. However the colloidal state of the particles and the high amount of surfactants
usually retard the crystallization into the more stable modifications [146].
Particle size and distribution should be controlled to evaluate long term stability [146]. Still, the gel
formation is considered a more serious problem. Gel formation or gelation is described as the
transition of the low-viscous colloidal suspension into a viscous gel. It is suggested that gel
formation is connected to the crystallization of the lipids and the newly formed unstabilized
surfaces (Figure 8.12.). Gelation is promoted upon high temperatures, light exposure and
mechanical stress [150]. This is valid especially for polyethoxylated surfactants which have
substantial difference in the properties at different temperatures. Other factors that influence
stability are high concentrations of the particles and electrolytes [151].
Sterilizationand antimicrobial preservation
Microbial stability can be achieved by different sterilization techniques. Formulations with small
particle size and narrow distribution can be filtered through 0,22 micrometer filters. Other
formulations are autoclavable. However, autoclaving causes melting of the particles and favors
possible aggregation [152]. Formulations stabilized with polyethoxylated surfactants are not
suitable for autoclaving as the high temperature may significantly decrease their HLB and
stabilization ability. Still, promising results have been achieved with formulations containing
lecithin and ionic surfactants [153]. Gamma-sterilization is not recommended in SLN/NLC
formulations. Free radicals are formed which may undergo secondary reactions and result in
chemical modification of the sample. Such changes were observed in lipid bilayer components
[154].
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Stabilization
Stabilization of the particles can be achieved by removal of the water as the powders are generally
more stable than the suspensions.
Lyophylization is suitable technique to remove the water from SLN/NLC formulations [155]. During
lyophylization the protective properties of the surfactant can be altered and the redispersion of the
powder results in larger particles [156]. Modification changes, drug expulsion and change in the
zeta potential are possible during the process [157]. Inclusion of suitable cryoprotectant is
mandatory before lyophylization. Glucose, mannose, maltose and trehalose in concentration
between 10 in 15 % find application in SLN/NLC [157]. The choice of the cryoprotectant is critical to
the properties of the redispersed powder [155-158].
Spray drying is another possible process that is evaluated to form powders from SLN dispersions.
Due to the high temperatures it is preferable that the lipids in the formulation have melting points
higher than 70 °C. Particle aggregation can occur during the process. This restricts its uses to more
diluted dispersions. Carbohydrates and ethanol mixtures instead of pure water are used during the
drying process to optimize the powder properties after redispersion [159].
Another approach to stabilize SLN suspensions is to increase the viscosity of the formulation
because this will reduce sedimentation and collision of the particles [160].
Despite all the problems that can be observed in the time during storage of SLN and NLC it has
been shown that these system may express stability up to 3 years in their colloidal state [146].
Safety
Most of the excipients used in the preparation of SLN and NLC can be found listed in
Pharmacopoeias (USP, Ph.Eur. JP etc.), Codex General Standard for Food Additives (GSFA) issued by
FAO/WHO or in the current European and US cosmetics regulations [162]. Some are registered as
excipients in pharmaceutical products only for certain administration routes. For example, palmitic
acid is part of many food and cosmetic products but according to the FDA Inactive Ingredients
Database it is only registered for oral application in pharmaceuticals. In addition, many of the lipids
and especially surfactants may receive approval for certain route of administration but in lower
concentrations than the ones used in SLN and NLC formulations. These circumstances reveal the
limitations related to the development and the registration of these products. A careful
reevaluation of safety and toxicity should be held for the new formulations. This is related to long
process of development, testing and high costs. The toxicity of bulk excipients is a good starting
point in the evaluation of toxicity and safety.
As was mentioned in the introduction, the lipids are physiological compounds which are presented
in the everyday life. They are characterized by high safety profile and high LD50 values. Most of
them are components of food sources and metabolic pathways for their degradation naturally
exist. In addition, toxic effects from their degradation products are unlikely [162]. Surfactants may
be a larger issue in terms of toxicity evaluation. Most of them are synthetic or semisynthetic
Nanomedicine 208
products and not all find application in pharmaceutical products. Often the toxicity and the failure
of the final formulation is result of inappropriately selected combination of excipients.
Although the bulk excipients may have high LD50 values and GRAS status a detailed examination on
the cytotoxicity of the final formulations should be performed before any in vivo tests. Proof of the
safety of the lipid nanoparticles can be found in the scientific literature. Unloaded SLN composed of
tripalmitin and stearic acid had no cytotoxic effect in vitro [163]. The low cytotoxicity was
confirmed for SLN composed of various lipids [164]. SLN proved to be less toxic formulation
compared to polylactide (PLA) and polylactide/glycolide (PLA/GA) nanoparticles [165]. Polylactide
and butylcyanoacrylate nanoparticles have shown respectively 10 and 100 times higher cytotoxicity
than SLN composed of glycerolbehenate and cetylpalmitate stabilized with poloxamer 407 and
poloxamines [166].
Prior to in vivo tests an understanding of the possible interaction with the components of the
biological fluids and tissues is mandatory. After administration in the body SLN and NLC are subject
to different metabolism pathways, protein binding, distribution and elimination. Enzymes that take
part in the degradation of lipids are mainly lipases and in lesser extent alcohol dehydrogenase.
Non-enzymatic hydrolytic processes are also possible [167, 168]. Lipases break the ester bond in
glycerol esters and partial glycerides and free fatty acids are formed. The activation of most lipases
requires an oil/water interface to open the catalytic center [169]. Therefore the covering layer of
the particles (the type of surfactant and its concentration) can affect the process of degradation by
lipases [170]. Chemical structure of the lipids can also affect the degradation rate (e.g., longer
length of the fatty acid chains in the triglycerides results in slower rates of degradation [171];
degradation of SLN composed of waxes is slower compared to SLN based on glycerides [166]).
Degradation rate can also be influenced by the size of the particlesbecause smaller size results in
higher contact surface area.
The biodegradation of the SLN and NLC causes surface erosion and subsequent reduction in particle
size until sufficiently small size for excretion is reached [163]. Protein binding is likely to occur and it
is affected by the type of surfactant [172]. Plasma proteins that can be involved in this process are
fibrinogen, IgG, IgM, apolipoproteins, transthyretin and albumin [173]. However, appropriate
modifications of the particles can prevent interactions with proteins – for example, modification of
the particle surface with PEG chains prevents interaction of the particles with human serum
albumin.
Further fact concerning the safety is that within their size range SLN and NLC do not trigger
macrophage activation and cytokine production [174]. Conclusions about the safety of the
formulation cannot be based only on toxicity studies. Problems related to the physical instability of
the particles (i.e., aggregation) may arise. Unexpected and uncontrolled gel formation during
parenteral administration is considered extremely dangerous. Presence of aggregates and
microparticles is possible in both microemulsions and SLN/NLC formulations. However, while the
liquid nature of the droplets in nanoemulsion allows their deformation and lowers the chance of
obstructions in small blood vessels, the rigid structure of the solid lipids increases this risk
enormously. Therefore the development and testing of formulations for topical administration are
much more favorable than those for parenteral.
Nanomedicine 209
Current applications
Parenteral
SLN were originally designed to resolve some of the problems of the parenteral nanoemulsions.
However, for the past twenty years SLN and NLC formulations extended to a variety of new
applications. SLN have solved some of the problems related to the fast drug expulsion from
parenteral nanoemulsion. Upon appropriate surface modification (e.g., stabilization with
poloxamers and poloxamines) SLN may express controlled and prolonged effect after i.v.
administration [175]. Besides achieving controlled release for APIs like camptothecin the nature of
SLN/NLC may increase the drug chemical stability after administration [176]. Reduction in the
toxicity of parenterally administered drugs is also reported for some substances (i.e., docetaxel
[39]). Through different superficial modifications SLN and NLC may be turned into the so called
“stealth” particles for long blood circulation [177] or into targeted delivery systems – to selectively
reach lung [178], spleen [179], brain [179, 180] , liver [179, 181, 182 ], tumors or other damaged
organs and tissues. SLN and NLC proved to be a suitable carrier to increase the cellular uptake and
targeting efficiency and thereby to improve the therapy in cancer [183]. They can provide new
solutions to problems associated with parenteral treatment of significant diseases like malaria with
drugs that express low solubility [184]. A bright example is the poorly soluble anti malaria drug
artemether which upon incorporation in NLC had lower hemolytic potential and increase in the
antimalarial activity [185].
Oral
Problems related to the poor solubility and bioavailability of variety of drugs after oral
administration can be solved with SLN and NLC formulations owing to the “Trojan horse effect”
[110]. After intake the lipid matrices composed of triglycerides are normally digested by pancreatic
lipases into mono- and di-glycerides. The monoglycerides may form micelles and mixed micelles
(with bile salts) that still contain the drug. Then these lipids may undergo absorption together with
the drug via chylomicron formation primarily into the lymphatic system. This transportation
surrounds the liver and the first pass effect [186]. The lymphatic uptake of SLN was confirmed with
fluorescent dyes [187]. Lymphatic uptake can be controlled with particle size as smaller size results
in higher uptake [188]. In addition SLN and NLC may enhance drug absorption via other
mechanisms – increased uptake by M-cells of Peyer`s patches in the gut [189] and adherence of the
particles to the gut wall [190]. SLN and NLC show encouraging results in the improvement of the
bioavailability and stability of proteins after oral administration [5, 191, 192]. In recent study insulin
loaded SLN have shown improved stability against proteolytic enzymes in vitro [193].
Dermal application and cosmetic products
The dermal application of SLN and NLC gained the biggest interest among researchers. Lipid
nanoparticles offer increased chemical stability of the APIs, film formation, higher occlusion,
increased skin hydration and modulated drug release (Figure 8.13.).
Nanomedicine 210
FIGURE 8.13 Tighter distribution and better occlusion of SLN/ NLC applied on skin compared to
microparticles.
SLN can also be administered on damaged and inflamed skin, because they comprise non-irritant
and non-toxic lipids [197].
Enhanced penetration with reduced side effects was reported [198]. Improved safety and
therapeutic characteristics due to the inclusion in SLN/NLC have been reported with calcipotriol
[199], coenzyme Q10 [200], celecoxib [83], ketoprofen [201], vitamin A [202] and many more. In
cases when systemic absorption must be avoided after dermal application SLN can provide
epidermal targeting. Epidermal targeting via SLN was confirmed with fluorescence microscopy for
podophyllotoxin [203]. Further SLN was found to decrease the irritant potential of retinoic acid and
improved its stability [204]. NLC also offer possibilities in the treatment of significant diseases as
psoriasis which was confirmed by acitrecin loaded NLC in a clinical study [40]. The broadest area of
application of SLN and NLC, however, is in the cosmetics [205]. Indeed, this is expected as
cosmetics are subject of less regulatory restrictions with shorter registration times compared to
pharmaceuticals and the introduction of innovative technologies has become fundamental for the
marketing success of the big companies. SLN and NLC are used in different anti-aging creams,
sunscreen products and others. NLC loaded with Q10 show better long-term physical and chemical
stability compared to nanoemulsion [206]. Tests showed that the NLC based cream has no skin
irritation potential and possess a higher hydration effect compared to a conventional cream with
the same composition [207].
Pulmonary, nasal and ocular application
SLN and NLC are suitable carriers with good tolerability and low toxicity for pulmonary application
[208]. Their small size enables their incorporation in microparticles and drops which can effectively
reach the alveoli. Moreover SLN and NLC can improve the pharmacokinetic parameters after
administration via the lungs [208]. Various examples include itraconazole [22],
Nanomedicine 211
phenethylisothiocyanate [209], celecoxib [7], beclomethasone [210], thymopentin [211]. Most of

these formulations are intended for use in the treatment of infectious diseases and cancer.
Promising results are reported for anti-tuberculosis drugs after incorporation in SLN for pulmonary
administration [213]. The lungs also offer alternative route to application of peptides and proteins.
SLN showed to be a suitable carrier of insulin [212].
Nasal application is also promising for variety of drugs. SLN proved to be a suitable alternative
carrier for the nasal administration of the antiemetic drug ondansetron [214]. Other more recent
examples are the SLN formulations for intranasal administration with budesonide [215], ropinirole
[216], alprazolam [217] and many others. Brain targeting of the SLN was achieved by intranasal
administration. An example are the risperidone loaded SLN which showed promising brain
bioavailability after nasal administration in mice [90].
In ocular formulations SLN and NLC exhibit increased retention time and increased absorption for
different drugs - tobramycin [194], cyclosporine A [195], and timolol [196]. Diclofenac loaded SLN
showed sustained release and higher permeability after tests on bioengineered human cornea [60].
SLNs formulations proved better than simple solution in the treatment of ocular infections with
tobramycin [194].
Gene therapy
In recent years SLN and NLC have gained increasing attention in the field of gene therapy as
promising alternative carriers to cationic lipids because of their rapid uptake by cells and protection
of the incorporated compound against chemical degradation [218]. Furthermore SLN can be
prepared by low mechanical force methods that will not damage DNA and RNA strands [110].A
positive charge that will promote the interaction with the negatively charged cell membrane and
the nucleic acids can be easily induced with suitable cationic surfactants. Cationic SLNs with
protamine as transfection promoter were tested and showed promising result [219].
Perspectives and conclusion

For more than 20 years of research the current and future applications of SLN and NLC seem well
shaped. In parenteral formulations they will offer more possibilities for many drugs with poor
aqueous solubility, short half-life and low chemical stability. Moreover SLN and NLC are likely to
find more applications as targeted drug delivery systems which will “direct” the drug molecules to
specific organs of interest and to reduce the systemic toxicity. Thus they can provide solutions for
APIs that failed clinical tests due inappropriate tissue localization. The safety and toxicity of the
excipients after oral administration is well represented by the low LD50 values of the substances
making the testing and registration procedures of peroral formulations lighter. The possibility of
SLN and NLC to improve bioavailability of many newly synthetized or old molecules from BCS
Classes II, III and IV is a realistic challenge. Further they can resolve problems related to unpleasant
taste, irritation and interactions in the gastrointestinal tract. New combinations of SLN/NLC with
traditional dosage forms (e.g., tablets, pellets, capsules) are likely to be studied in terms of
resolving stability, bioavailability and toxicity issues. The oral administration of peptides and
proteins may also be achieved with suchlipid-based formulations. Dermal application will probably
continue to be the area of the biggest interest as it is the main field of practical application of SLN
and NLC at the moment. Poor skin penetration of APIs may be improved due to the better
Nanomedicine 212
occlusion of the nanoparticles compared to traditional creams and gels. SLN and NLC may also offer
improved chemical stability towards oxidation of various lipid soluble APIs for dermal application
due to the rigid nature of the particles. SLN and NLC have shown to express reduced skin irritation
and reduced systemic absorption when targeted to the skin for various drugs as we discussed
earlier. Similar application is likely to be used with more APIs that have irritation potential and
undesired systemic effects. In the cosmetic industry SLN and NLC have already entered the market
by various anti-aging and sun protection products and their number will likely continue to grow.
Other routes of topical application for SLN and NLC that are less studied will induce bigger interest.
Pulmonary and nasal application appear to be suitable in terms of improved safety and
bioavailability. The lung as an “entrance gate” to the body is not only of particulate interest for
various anti-cancer drugs and antibiotics loaded in lipid nanoparticles but also for proteins.
Alongside the pulmonary administration the nasal application of SLN/NLC formulations is also
promising. The reports of achieving high drug concentrations in the brain after nasal administration
of SLN/NLC are very encouraging. Such approach can overcome limitations like low blood/brain
uptake for certain drugs. This approach may be reliable in treatment of diseases related to CNS
disorders like Parkinson`s, Alzheimer`s, multiple sclerosis and others. The increased interest in gene
therapy and the suitability of cationic SLN/NLC will also expand the scope of their application.
Future work will probably be focused on the improvement of the loading of the RNA and DNA and
decreasing the toxicity of the final formulation. Still, SLN and NLC should not be considered as
infinitely applicable. For example the highest standards set for physical stability during the storage
(e.g., absolute lack of aggregation and presence of microparticles) and the probability of
uncontrolled changes (e.g., gelling or aggregation) upon administration may suggest that for some
routes (especially the parenteral) their administration could be an issue.
As a conclusion, we can summarize that solid lipid nanoparticles and nanostructured lipid carriers
are promising drug delivery systems. Their low toxicity is one of their strongest aspects together
with the advantages they offer in almost all administration routes. In addition, their production
methods can be transferred to large scale without organic solvents and the dispersions can be
prepared at relatively high concentrations. However, SLN and NLC cannot be regarded as the
“panacea” to all drug delivery problems. They proved to have some drawbacks mainly related to
the polymorphic modifications of the lipid matrix, physical instability (gelation, aggregation, drug
expulsion) and coexisting structures (micelles, liposomes). Sterilization and antimicrobial
preservation could also be problematic for many of the formulations. In future a better
understanding of the colloidal state of the lipids as a result of the more sensitive and modern
analytical techniques will help the researches to overcome some of the limitations we have
discussed in this paper.
Acknowledgment
The authors would like to express their respectful gratitude to Mr. Vesselin Lazarov, for the
creation of the beautiful artworks presented in this paper.
Nanomedicine 213
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9
Quantum Developments in Nanomedicine:
Nanocurative Actions by Soft Photons
Sources and their Path Integrals
*1 2
Francisco Bulnes , F H. Bulnes-Gonzalez
1
Department of Research in Mathematics and Engineering, Technological Institute of High Studies of Chalco, Federal Highway
Mexico-Cuautla s/n Tlapala “La Candelaria”, Chalco, State of Mexico P. C. 56641, Mexico
2
Quantum and Homeopathic Medical Center “Keter”, Avenue ‘Las Aguilas’ 1863, Col. Axomiatla, P. C. 01820, Mexico City, Mexico
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 239
Soft Photons Sources in Nanomedicine …………………………………………..…………………………………………. 240
Healing the Mind-Body ………………………………………………………………………………………………………………. 242
Electromagnetic potentials and their meanings in the spectral encoding …………………………………… 244
Programming the Combination of quantum factors and their path
integrals to nanomedicine ………………………………………………………………………………………………………….. 246
Monopharmacists in Nanomedicine: Sources of Soft Photons ……………………………………………………. 248
Evolutive and Involutive Quantum Insights by Nanomedicine in Preventive
and Corrective Developments ……………………………………………………………………………………………………. 261
Conclusions ……………………………………….……………………………………………………………………………………….. 263
Nomenclature …………………………………………………………………………………………………………………………….. 263
Units and Abbreviations ……………………………………………………………………………………………………………… 264
References……………………………………………………………………………………..…………………………………………… 265
Nanomedicine 239
Introduction
The quantum methods of medicine into of the class of alternative medicine, fundament their methods
of cure in to health and reactive the vital field X, of the human body B , the regeneration of the centres
of energy of B , and the corrections and restoration of the flux of energy Flux, in each organ B , of the
human body B , taking constant of gradient of their electromagnetic current, voltage and resistance,
obtaining of this manner, the balance of each organ in sunstone with the other organs to characterize
to B, like complete synergic system in equilibrium and harmony as we can to see in the reference [1].
Now we can to establish that the cure that could be realized to nano-metric scale must be executed
with a synergic action of constant field equal to effect in each atom of our body to unison of real
conscience of cure (duality mind-body [2]). Of this form the conscience of B , is the obtained synergy by
the atoms in this sense and that will come reflected in the reconstitution of the vital field X . The vital
field X , is the field that acts on all organism to do so living organism. Also this define through of other
parameters (as the immunity, regeneration, etc) their vitality and health.
Then under this reinterpretation, the sickness is only an effect of the fragmentation of this real
conscience of cure of B , that is deduced by disconnections and disparity of atoms [3]. The quantum
methods of the medicine help to recover the continuity of this conscience through of the electronic
memory of health of the proper body. Remember that B , is the entire human body. The B , is a part
or organic system of the human body.
Diverse studies on medical similitude of many sufferings and their evolution 4 (mutations of virus,
and others), permit establish that the cause of great part of this sickness is only one. The sickness go
taking the form of others sufferings due to the place where it is alien in B , accord to the vitality or
mental/organic state or genetic nature of each person, due to the geographical latitude or longitude,
and due to the trajectory that the microbe take in their hide for to be annihilated in the process of cure
[3]. But, we can to analyze that in all these effects called sickness is replayed only a effect of reaction of
the self body to a dystrophy more deep that is happened in a plane of conscience of body for obtain
the health (auto-cure power of body, 1, 2). But all this, in mind-body is located in the quantum zone of
the transformation of the vital field, such and as is realised in the cosmological plane in the space-time.
Likewise we can to talk of the correspondence between mind-body and space-time like an isomorphism
into the quantum context [5] considering the nano-metric components of the mind-body as the objects
that must be transformed through organized transformations. This defines our characterization on the
quantum methods in nanomedicine.
Conjecture. Exist only one sickness, the atomic disconnection or dissociation in X (B) 6. The different
affections, sufferings or sickness that are presented in B , are only effects of a same cause, that
depending of the relation mind-body [2], and of the equilibrium of B , in their entire, these rise with
different aspects and characteristics.
Actually in the development in which is found the quantum medicine (type of alternative medicine
derived from the homeopathy), the pharmacists are designed to can re-establish the electronic capes
of the atoms using a simple combination of potable water and pharmacist of the quantum medicine
7. But the process of cure must be developed in a major order of precision thus is necessary the
Nanomedicine 240
design and creation of photon devices that under the same principles that govern the process of
quantum medicine realise the cure of all illness and sufferings.
The capsule of integral medicine is dissolved in a glass with potable water and is taken for the patient.
The water has the property of fortalice the covalent links among atoms, and establishes restoration of
cells and fabric of B . In the future developments of our research we want to obtain photonic and
spintronic devices that spill codes of intelligence of cure through of a special ray that includes all the
properties of action of cure (correction and restoration of field X ), and that only by means of a
resonance election treat every one of the sufferings and illnesses. This capacity of cure by photon
devices will get considering the qualities of some mono-pharmacists and supplements as sources of
these soft photons that must be reproduced in these devices.
The next quantum development of the nanomedicine depends of the results and determining to create
these quantum devices to obtain the universal cure that will eliminate that atomic collateral damage
[3, 8, 9].
Soft Photons Sources in Nanomedicine
Results obtained in previous researches and studies [5, 9, 10] establish that the mind and body are not
disconnected parts, these are connected in the deep atomic existence of the quantum zone. Then one
fundamental concept in the quantum methods of the nanomedicine [8] is consider the mind-body as
complete space-time of the organism and the cure actions will be focused to conciliate and solve the
inconsistence of these two parts using the same nature of binary codes [1, 3], from the programmed
path integrals (Feynman-Bulnes integrals [1, 11]) to spill the quantum codes of the cure intelligence
defined by these path integrals [3]. But these binary codes belong to the same source of “soft photons”
(theorem to show the consistence of a spintronic device [12]) that is required to the design of the
“drain-spill” *12+ interchange process required in the quantum cure from the atomic regenerating (see
Figure. 9.1).
FIGURE 9.1
Possible device with a rich source of photons based in the theorem given in the reference [12], which establish
that through of certain soft photon sources and managing the photon rays from these sources as quantum waves
with information of cure, we can design a spinor technology, that is to say spintronic device to cure the
corresponding sickness.
Nanomedicine 241
We need identify some basic elements that we have to the scope to create our quantum methods of
nanomedicine, by quantum devices:
(i) To talk of the mind-body is necessary to talk of a transition zone or energy process
between the Q-zone (or quantum zone) (mind) [13], and the material zone
(organic).Then we can define the energy process of mind-body by the corresponding
energy states and their correlations across the path integrals and their encoding [3,
14].
(ii) The photon type used to cure must be the same that has been used to shape the
organic self-regenerating, immunization and self-correcting of the body
understanding that these processes are the energy process of the mind-body
described in (i). The corresponding correct energy state produced by these
correlations is a binary code of the intelligence spilled in the affected organ [8].
(iii) The interchange of photons must produce charge, gain electrons and steady electron
states [15] to establish the links and alignments of the vital field from the atomic
level.
(iv) The device source that produces the photons described in (ii) and (iii) must be similar
to the space-time, for example the given by certain crystals rich in soft photons.
These are all elements that we have able to cure all illness and diseases if we establish that in the
transition zone only we have energy transformation that is provoked from quantum phase.
A study in spintronics and neurosciences (using the principle: the intention is that defines the field
action *16+) we have been able evidence that the nature photons must be the same that the “good
thoughts” generated by our will of our organic system to risk the cure, which is called self-cure power
of the body. Then we can conclude that the photons that must be produced must have the same
characteristics that these photos characterized by the same quantum resonance. Then one direct
method could be correct the quantum field in the mind through of strengthen the will (Figure 9.2 A).
A B
Nanomedicine 242
C
FIGURE 9.2
A). By the properties recognize  displacement   (1)(0 - 1)   (1)u(0), with an evolution change given by
exp(tX ), we have the curve of recovering of the will. This graph corresponds to the action of Pulsani. The
continuous strengthen of the will. B). Spintronic device of “drain-spill” process necessary to the re-composing of
the vital field. C). Fermi distribution of the Gelsem, of the energy displacement to eliminate the anxiety. Also is
analyzed to this displaced energy of anxiety the action of Gelsem and their optimum energy displacement more
than the Benzodiazepine monopharmacists [9].
Healing the Mind-Body

The thoughts shape the perception of the reality. These there are feed by the great source of soft
photons provided by the space-time to quantum level. These photons interact with the different fibers
called “skies” to compose the cosmic perceptions as light holograms in the mind and are transmitted to
the brain as micro-charges that are conducted through neuronal framework. After, all these signals are
transmitted to other organic parts with their specific instructions in every case. These signals go
enriched with the quantum codes generated from the mind and organic encoding by the brain to active
our peripheral nervous system through the neuro-transmitters to realize the organic activities in every
organic system from the cellular level.
When the codification comes with error codes (existence of symptom in the mind) or incomplete codes
(fractured conscience), these are spilled in all the mentioned processes and received for the different
organs as free radicals that have been resulted of the atomic instability and that through the different
amino-acids, peptides and other substances have to place along with the feeding and food. In this last
point, is necessary pronouncing the following principle of the mind-body that have been published in
[5, 14] and presented in [9] that has to do with the assimilation of the food: "All effects are produced by
the stomach and all causes are produced in the mind" [9, 14].
Nanomedicine 243
In the digestive system are produced and synthetised nutrients, as metabolic substances as proteins
and organic metals in specific quantities and modalities. The organic metals are to establish crystaline
frameworks of compounds consisting of metal ions or clusters coordinated to often rigid organic
molecules in different dimensional material structures that can be porous and of this way promote the
ionic interchange and biochemical information of the organic composes strengthening their covalent
links, example the organic Germanium [17].
Then we need realize quantum actions that include those actions that are effects of global sufferings
(anxiety factors [5, 14]) that begin in the mind, but that for the connection of the mind-body must be
integrated in only one action: correcting + restoring. This action involves the binary codes of the
organic cure [14].
There are mono-pharmacists with quantum actions well-determined that realize this integration of
actions.
Using the soma theory we can conclude that share of the toxins and free radicals that are eliminate in
natural form by the our physiologic system obeys to the direct action of the organism to recognizes the
damaged factors in the body. But this soma is a natural conscience of the healthy state of the organism
that is transmitted by the mind through quantum codes of intelligence that are formatted in our ADN.
This encoding is printed in our ADN and their codes are transmitted to whole body in the nature way
using intelligence codes that have designed in our soma.
A) B) C)
FIGURE 9.3
A) In the mind the sufferings appear when there is de-phasing between of the emotional E , and rational
R , curves in the space-time of Q-zone [13]. In the figure we show a Bose-Einstein distribution that do visible
an anomaly that could be generating begins of the illness. B) De-phasing between emotional and rational
curves in the conscience flux [9]. C) Effectiveness distribution of medication by Gelsesium group: Gelsem,
Ignamara, Medigin and Pulsani. Psychoanalytic effectiveness of the mono-pharmacist of integral medicine
versus depth. This energy was calculated with the following experimental equation M  R aEl , from the
quantum integral transform studied in [16] considering the minimum energy of each mono-pharmacist, which
is according with it predicted by the theorem 1, from [16]. The formula is a short manner of the integral
a l
transform where R , is dim(), and E , this is in between brackets:
 1   
x(t )  correction  restoring   O c (( s ))w( s , t ) dt  dim  ( )    {   (n j )F (n j )dx(t )}.
C 
X(C)
A j 1 
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Electromagnetic potentials and their meanings in the spectral encoding

In quantum mechanics, the spectral and vibration knowledge of the field of particles in the space
facilitates the application of corrective and restorer actions on the same field using their space of
energy states through of the meaning of their electromagnetic potentials studied in quantum theory
(Aharonov-Bohm effect [18]). Thus these electromagnetic potentials can be re-interpreted in a spectral
and vibration space that can be formulated in a set of continuous paths or re-walked, with the goal of
realize corrective and restorers applications of the field, stretch to stretch, section for section, and that
subsists in this combined effect of all the possible trajectories to carry a particle from a point to other.
By gauge theory is licit and consistent to manipulate the actions of correction and restoration of the
field through of an electromagnetic field, which ones are gauge fields of several types of interactions
both strong and weak. In this last point is necessary mention that in the class of equivalence of the
electrodynamics potentials, these can be precisely re-interpreted like a connection on a trivial bundle
of lines of SU (2) (non-abelian part of the gauge theory using electromagnetic fields [19]), and
admitting non-trivial bundles of lines with connection provided with more general fields (as for
example, the of curvature, or the corresponding to SU (3) (the strong interactions)) [6, 19]. In both
cases there are considered to be the phases of the corresponding functions of wave local variables and
constant actions that can be established across of their correspondents Lagrangians [6]. The path
integrals to these cases are of the same form except the consideration of the potential states in each
case. Into of this electromagnetic context and from the point of view of the solution of the wave
equation through of alignments of lines of field, we can use the corresponding homogeneous bundle of
lines that are used to give adequate potentials (potential module gauge, for example, those who come
from the cohomology of O(n  2), n  1 [19, 20]). Of this manage we can establish that {set of fields of
particles}  I  ( H 1 ( PM , O(n  2))), where {potentials}/{gauge}  H 1 ( PM , O(n  2)) [6, 20]).
Here I  ( H 1 ( PM , O(n  2))), is the cohomological class of the spectral images of the integrals of
line on the corresponding homogeneous bundle O(n  2).
To eliminate the distortions of the field is necessary to remember the paths and to continue them of a
systematized form through of certain path or route integrals (that belongs to a class of integral of the
type (1)), that re-establish normal course of the particles, re-integrating their vital field (realizing the
sum of all the trajectories that conforms the movement of the particle), eliminating the deviations (that
can derive in knots or ruptures in the space-time of the body [16]) that previously provoked this
disequilibrium and the born of sufferings and illness. These knots or ruptures in the space-time of the
body, we will call singularities of the vital field [1]. Using of the class of integrals of line followed in the
cohomological study of the Feynman integrals, we can to find the spectral encoding of the singularities
of a vital field [1], by their electrodynamics characteristics:
Theorem 1 (Bulnes, F) 8, 20. If the Radon transform (tomography on X (B) ) is not defined, is infinite
or have the value of zero, the corresponding pathologies are: not have current, voltage or resistance
(death point unless polarity due to the degradation of cells and fabrics), have a node with variation not
bounded of current, voltage or resistance (is lost or is much (ponds of energy)) due to a bad organic
function), have a peak or is a node, due to that not have unique value or this is indeterminate (not have
determined direction, can have a pathologic node with disordered increasing).
Proof. [20, 21].
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These three anomalies can combine to give resulted to distinct sufferings, doing also in different
quantities, given as the multiple sufferings and sickness that is known and diagnosed in the actuality.
But in reality as is affirmed in the conjecture, only exist one cause to all suffering and this is due to the
disconnection between atoms, that which produce distortions in X (B) , and for which is produced the
suffering in B .
In consequence we can calculate the electronic current used in the criteria mentioned by the theorem
1, and to the development of an quantum or micro-electronic device to diagnosis and cure of all
sickness and sufferings:
Corollary (Bulnes F) [22, 23]. Like operator of execution [3, 24], the electronic current due to density of
current j e , in their direct form comes given for
 
t t1
Electronic Current  I e   2 (2 ) 3 n s  {   e i ( k  l ) ~kl n1 (0)dp d }dt
2
0 0  P 3 
2
  2t (2 ) 3 n s   kl n1 (0)  ( k   l )dp, (1)
P3
where n1 (0), is the density of electrons [1].
Proof. [3, 25].
The detection of this anomaly to quantum level can be realised using a inverse method that determines
the state of boson distribution described in the Figure 9.3 A), using micro-electronic resistance signals
through of inverse methods (see the Figure 9.4 a) and b)).
FIGURE 9.4
a) Resistance spin model to explain detection of anomalies. b) quantum binary codes. c) Feynman diagram that
realizes a distinction between anti-electrons and electrons. d) Fundamental cell or 000-box used in the alignment
of a vital field. e) Boson distribution studied to design a boson device to detect organic anomalies. f) Photon
detector with beam of bosons.
Nanomedicine 246
We must be first having understood what a quantum nanocurative action is that is produced on the
vital field that is quantum field. These actions must give it to a nano-scale and only have that establish
it through of very fine energy obtained by a source of photons that restoring and complete the holes or
corrects the incorrect binary codes in the organ system in Bose-Einstein model of their photon
distribution (Figure 9.4 A and B).
Programming the Combination of quantum factors and their path

integrals to nanomedicine
The relation between mind and matter is realized through of a quantum jump and only to this level
succeed. In the quantum mechanics, all the particles like package of energy works like points of
transformation (states defined by energy densities). The vital field in the matter of an organ of B , is
evidenced like answer between these energy states, as is explained in the Feynman diagrams. Due to
that exist duality between wave and particle, a duality also exist between field and matter in the nature
sense. Both dualities are isomorphic in the sense of interchange of answers of interaction of a field. The
states of mind have energy densities that respond to the state changes of others. There is one of the
central ideas of the quantum medicine.
‘The quantum medicine applies treatments of quantum medications that produce quantum
transformations “appropriate” to establish an answer of the states of energy of the part B , to cure
establishing an image of the field X (B) , adapted in same resonance, vitality and bio-rhythm of the
healthy points of the whole body B . This last for the synergic principle of the actions of the field
(postulate 3, in [1]), and due to the metabolic program that has the body with tendency to an auto-
regeneration’.
The answers between densities are realized in accordance with the correlation densities established in
certain commutative diagrams that can be shaped by spaces L2 , on the space-time of the particles [3].
Coding this region of transition states of the corresponding Feynman diagram on a logic algebra
A (, , -) (like full states or empty of electrons like particle/wave, that is to say,  (0)  0, (is not the
particle electron, but is like wave)  (1)  1, (is not the wave electron, but is like particle) and their
complements), where the given actions in (2), are applied and re-interpreting in the region of the
space-time of the particles like a electronic complex of a hypothetical logic nano- floodgate (that is to
say, like a space L2 , with a logic given by A (, , -) , with values in M0,1 , on their transition states
(Figure 9.5)), we can define the Feynman-Bulnes integrals, [1], as those that establish the transition
amplitude of our systems of particles through of a binary code that realize the action of correction and
restoration of the field [8].
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FIGURE 9.5
The Quantum logic algebra and their symbolic reticule [J2].
Likewise a Feynman-Bulnes integral [1, 26], is a path integral of digital spectra with the composition of
the fast Fourier transform of densities of states of the corresponding Feynman diagrams. Thus, if
1 ,2 ,3 , and 4 , are four transitive states corresponding to a Feynman diagram top holes of the field
X (B) , then the path integral of Feynman-Bulnes is:

I    n1 F (n1 ) n2 F (n2 ) n3 F (n3 ) n4 F (n 4 )  0001101001 , (2)

The integrals of Feynman-Bulnes, establish the amplitude of transition to that the input of a system
with signal x(t ), can be moved through of a synergic action of electronic charges , doing through of
pre-determined waves functions by L( x(t )), and encoded in a binary algebra (pre-defined by states
 (0), and  (1) ), (in the kernel of the space of solutions of the wave equation
x  AA '
F  AA '
( x x'  6) of a point to other into of a circuit  j . Their integral it is extend to all
k  k 
space of paths or re-walked included into of the region of Lagrangian action      j
 with a

j  j 
topology of signals in L (,,), [3, 25]. If we want corrective actions for stretch
2
 j , of a path
, these we can realize them using diagrams of strings of corrective action using the direct codification
of path integrals with states of emission-reception of electrons (by means of one symbolic cohomology
of strings [3, 27]). Then the evaluation of the Feynman-Bulnes integrals it reduces to the evaluation of
the integrals: I (, )   () , where , is the orientation of C (),  , is the corresponding

0
C0 (  )
model of graph used to correct after of identify the singularity of the field X, that distorts it. For
Nanomedicine 248
example, observe that it is can to vanish the corrective action of erroneous encoding through of a sub-
graph:
 ()  [0   (1)]  [0   (1)]  [0   (1)   (1)]   (1)  1  1  1  1  1  0,

0
The corresponding equation in the cohomology of strings is [3, 28]:
where the member conformed for one string graph satisfies
How to help to the conscience of cure (intelligence) of the mind-body to risk in any person the total
cure? Have that spill a code of intelligence like the given in (5).
If we have said that there is only a collateral damage that defines all sickness and sufferings then there
exists only one solution of electronic type of deep level of cure to this bad. This transformation of cure
exists only in the quantum region where there is the cause of collateral damage. The cure require the
action in this level of deep and this action only can to define by logic defined in M0,1 , based in path
integrals belonging to the cohomological space H 1 ( PM , O(k )), and whose action organize and
correlating all movements of particles x(t ), in a beautiful symphony that devolve of healthy state that
vibrate in all the scales of harmony in the body, since these vibrations exist in the propel body, and
many times are sleeps.
Monopharmacists in Nanomedicine: Sources of Soft Photons

From the analysis on the re-composition and restoring of field X realized in the theorem 1 (F. Bulnes)
[1, 11] where is explained the way in which it is applied that restoration and re-composition and as a
direct consequence of the postulate of mind/body given in [14], we have:
Corollary. (F. Bulnes, and F. H. Bulnes, Sr) All illness or sufferings of the organic system begins in the
digestive system which is the first in be directly affected for the mind.
Nanomedicine 249
The digestive system is one of the most sensitive parts of the organism, which receives straight the
affective signs of the concomitant psyche on the nutrients that provide with energy the body [14]. In
the deepest level, the food in itself takes codes of quantum intelligence [14] that collaborates to the
electronic memory of the organs of the digestive system to realize their digestive functions. Then their
interaction is realized straight between the psychological and material part of the organism.
On the other hand, the fundamental organ of the digestive system is the liver, which also is an organic
recipient of the emotions, producing enzymes and chemical components jointly with the concomitant
actions of the psyche at the moment of the ingestion, deriving also, the useful intelligence codes for
the whole body. This confirms that the cause that it contains to all other in a suffering of the digestive
system is of nervous type and therefore mentally [9, 14].
Studies realized in integral medicine have led to the design and production of medical patents [7, 10]
that are going to reinforce the restorer action of the liver using a mental stabilizer that helps to the
liver to recover their electronic memory, spilling a code of intelligence that contains the corresponding
code of intelligence of the electronic memory of the liver, being a narrow relation with the digestive
system, as for the production of neuro-peptides that control some bacterial systems as the helicobacter
pilori taken charge of the treatment of lipids and some chemical derivatives of the lipids obtained by
the pancreas and liver in the region of the duodenum which changes its helicity when the individual has
stress.
Psychological factor are responsible of the expectation of the bad functioning of the pancreas doing a
deficient digestion in the many cases. Basing on the principle of mind - body, and on the conjecture of
anything suffering is the loss of healthy conscience of the organ [9, 14], which is an effect of the
fragmentation of the mind-body of the one that presents a suffering and of which the registered
dispersion is their spectral effect (see the anomalous diagram of string in Figure 9.6). Is necessary to
apply a medicine to correct the underlying neurosis in the visible effects at organic level, together with
a diet adapted with a restorer of type supplement (not vitamin nutrients because there can be
presence of sarcomas).
FIGURE 9.6
The corresponding anomalous string graph [28, 29].
The action of applied integral medicine it might have to be one that is anticipated to the negative
actions in the digestive system, product of the somas that realized actions on the thin intestine at a
level mind - body, inside the specific region of the pancreas. Then the action of integral medicine that
would be applied is:
Corrector + restoring = Bervul + [Nutrali-9 + mineral salts], (3)

Nanomedicine 250
Where the supplement Nutrali - 9 + mineral salts, [4, 8], will correct the subjacent energy in the
pancreatic-duodenal zone whose charges have be reversed the polarity of bacterial groups of the
intestinal flora (like the helicobacter pilori), doing that aggressors turn these reagents of protective
type, altering the pH, of the patient. This mechanism is the promoter to the carcinogenic cells. The
restorer in this case must contain an opposite helicity to the anomalous one in these bacterial agents,
reorganizing their electronic field code. Nevertheless the anticipatory action of Bervul, on having
corrected pancreatic fabric and the elimination of the gastric infections and gastric pains that were
making alike the symptoms of others infections like diarrheas, they make possible the reversion of the
sarcoma, which, on not having had degraded products of a pancreas regenerated by Bervul, cannot
gestate anomalous cells, clearly, submitting also the patient inside this treatment, to the systems of
feeding adapted in schedules and light nutrients that promote the helicity initiated for Nutrali-9 +
mineral salt in the helicobacter of the intestinal flora. Nevertheless, the observation of the nervousness
system of many patients that presents irritability and neurasthenia, they become necessary the
application of a mono-pharmacist of integral medicine that was correcting the nervous disorder of the
patient, which collaborates for the anomalous state of the duodenum. In fact in a study on the route
continued for the gestation of the duodenal anomaly in a patient with these emotional characteristics,
one concludes that the disorder in his duodenum is stemming from the loss of healthy memory of his
liver and pancreas (loss of their normal functions), to assimilate the necessary nutrients and the
elimination of the unnecessary substances for his body B.
To their total cure, is required the application of a mono-pharmacist of quantum medicine with deep
corrective action I0 , defined by the correction and restoration actions, and with the action of
electronic memory emptied 01111010110101111010111110, [8] (binary code obtained to the mono-
pharmacist Gelsemium 2006, of their corresponding transition graph G , [1, 30]), the which it was sick
to their mind-body their healthy psycho-somatic state, simulating the action of Gelsem in their organic
similar [14, 30], (that is to say, in the liver).
It is invite to the patient to realize some practices with cold water, like baths of seat, and establish
adequate horary to eat healthy food (full in fiber, avoiding irritants and fat animals). To respect in last
point, also it is necessary the application of the corrective action given by Ignamara + Restoring, since is
necessary correct the function of metabolic cell, such as some bacterial groups of the intestinal flora.
The questions were: Which is the order of application of these quantum medicines? Will these be able
to be applied at the same time or with alternation of the quantum medicines? An analysis of cause-
effect, and the diagnostic route observed in the diagnosis device used in a patient, it makes to
conclude:
eNeurosis  eLiverpancreas  eDuodenum, (4)
Really the effective energy ( ”out” ”in” value of energy used in the process of action of
restoration by half-contours) comes given for [3, 31]:
E  E 0  E  , (5)
where  i1/ 2( )  1/ 2, [3], is the effective energy value necessary to reverse the increase
of soupiest sarcoma in B (possibility). The conclusion of this case is the possible not existence of
Nanomedicine 251
sarcomas and the apparent infection of digestive system is an altered state in the conscientious level of
the mind-body. The diagram of strings included the diagram of strings given by Bervul +Ignamara [8,
14].
FIGURE 9.7
A) The code given in (14), include the codes of change of polarity, where these are recovered by the restorer [7,
32], given for Change polarity = Nutrali9 + Mineral salts, the which are the specific mineral salts [7, 32], of the
immunity system to this case. The code 101101, is more short but with equal action that the intelligence code
given by Gelsemium (the line shortest in path is given by [14]). So only this last stay include in the action of
restoration of liver of the patient given by the code the which have twelve digits of the deep action
01111010110110110110111110. Total Action due Gelsem including the action de Bervul and Ignamara. The
corresponding quantum intelligence code of this total action is given by 01111010110110110110111110, [8]. The
corresponding code given in black include de codes of Gelsem and Bervul. The code in blue is the code of
Gelsemium equal to code of Gelsem. This quantum code was used to cure a patient with a digestive illness [8]. B)
The second string graph shows that can include, in the treatment of a patient a supplement, for example, as the
organic Germanium (which is not medicine) to give the electrons to the atoms of the cells and satisfy the electron
demand of the protons of these atoms. Observe that is obtained a more large binary code with additional chain
101101, that is to say the binary code 101101101101101101, [26, 28, 33].
Other example of mono-pharmacist that is soft source of particles in this case, electrons and photons
1
(that is to say, we have a organic-metabolic source of particles) is the organic Germanium GE-132 ,
which is a compose of type sesqui-oxide bicarbo-exetil of germanium which includes Germanium,
Carbon, Hydrogen and Oxygen being the carbon the element that enrich with covalent links the
electronic share giving it the characteristic to the organic Germanium as powerful metabolic re-
constituting, since to cellular level provides of electrons to the cells that could have defect of them for
the presence of free radicals (see possible string graph to this quantum action Figure 9.7 B).
1
This mono-pharmacists is not medicine as such, this is a preventive, restoring monopharmacists that safeguards the cellular
oxygen when realizes reposition or stability of electrons, that is to say, not wants realize some aliphatic action himself or that be
“contraire” to the illness symptom.
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TABLE 9.1
Some resolving mono-pharmacists more important [14].
# Mono-pharmacists Organic System F (nJ )

Psychological
1 Gelsem and Nervous 1.15
System
Digestive
2 Bervul 0.87
System
Skeletal and
3 Colocuc Muscle 1.17
Systems
Immunological
4 Bryal 0.38
System
Electrical
5 Histol 1.21
Charge System
TABLE 9.2
Some representative groups of mono-pharmacist [10].
Group of Quantum Medicine Mono-pharmacists

Pulsatilla D12 Pulsani
Allium Cepa D6 Allicep
Phytolacca D6 Phytodec
Ignatia D12 Ignamara
ArnicaD6 Armont
Calcarea D12 Colocuc
An example of nono-pharmacists that involves intelligence codes against the increasing cancer cells,
and not only realise an action to eliminate these, also recognise between bad cells and good cells
through of a differencing action when localize decreasing of antigens in a affected region, is the
gavriola (annona muricata) [34]. From a point of view of the quantum medicine we could say that the
gavriola haves a differencing action that satisfies the principle of annulling of scattering states only
though the annulling of their Fermi scattering (scattering that bring the free electrons) using a correct
interchange of photons. The quantum mechanism of the gavriola could be as a soft photon source as
the illustrated by the spin interchange behaviour that is necessary in the drain-spill process (see the
Figure 9.2 B)) with the unique difference of that in the followed photon process by the gavriola is
discriminating on the cancer cells (attack only these cells).
An action that involves the reversion of the anomalous actions given inside genome in some patients
(for example: certain patient in him genealogic tree have diabetic antecedents then is waited that also
him haves this predisposing) appear in some mono-pharmacist as organic Germanium or
Gelsemium2006  special restoring [3]. Of fact, this integral medicine (that is quantum extract
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of homeopathic medicine Gelsemium sempervirens [13]) includes codes of Gelsem (psychological

correct action) and the organic correction of some mono-pharmacist with organic action to cure some
organic sufferings, for example Bervul (hepatic system) [8, 30] in the table 9.1. The reversion is given
from a deep level of the mono-pharmacists Gelsemium
The corresponding Fermi energy is eliminated through a process
  1/ 2  1/ 2    1/ 2  1/ 2, obtaining 0, or 1, (boson states) to stable states of
electrons or themself electrons. Then are completed the covalent links of atoms or are strengthen
them.
A) B)
FIGURE 9.8
A). Inhibition and destroying actions of cancer cell by gavriola. B). Modulator action of Pulsani. The Fermi energy
represents the scattering energy that was eliminated versus advance in nanometers by Pulsani. To their realization
is used a model electromagnetic potential  ( x)  x 2 (1  e  x ) log( x)[3], accord with (1).
The same inhibition analysis of increasing of cancer cells is realised to the gavriola, considering
different extract concentration versus the rate of cytotoxity of neoplastic cells. Furthermore as has
been mentioned the actions are directed only to these cells. Based in the butanolic extract of Annona
muricata through HPLC-analysis (high performance liquid chromatography analysis) let to see that to
certain characteristics of the wavelength in the range 320-370nm ( max , retention time, determining
 , slope and limit calibration) of more than 100 phenolic standards established in the HPLC-analysis
confirm that potent compounds protect normal cells and selectively destroy cancerous cells. The
parameters of the wavelength have variations very differenced to both groups of cells, being the
determining substance to the cytotoxity of neoplastic cells the n-butanolic extract of the gavriola.
Compare the energy of the will (Figure 9.2 A)) to Pulsani (as modulator) that is needed to recover of
health in the mind-body (with the different mono-pharmacists to the different sufferings) with the
curve of different concentrations of n-butanolic extract that take to the eliminating and destroying of
cancer cells (Figure 9.8 A) [35]). Both medicines have similar intentions that are recover the health
gradually gaining place in the affected organ (combats the bad cell only). The gavriola haves
intelligence codes to quantum level that do similar this behavior as the given to correct to quantum
level the will of health in the mind-body to any suffering.
Nanomedicine 254
FIGURE 9.9
A) Energy increasing of the scattering modeled by the Fermi-Dirac distribution to the mono-pharmacists group
given by the table I. Decay of muon   ,   . he curves of decay behaviour of the mono-pharmacists (Gelsem,
Bervul, etc) are included in the blue area. The physical modelling is given in nm versus nJ [13-15]. B) Through of
pharmaceutics study on the inhibition of colony of cancer cells with different concentrations of Ge  132, is
show efficacy of these mono-pharmacists. The effect of organic germanium on cell proliferation was tested by a
clonogenic assay. Incubation of MCF 7 cells for 7 days in Ge  132, showed a dramatic inhibition in the colony
forming ability of the cells in a dose dependent manner [35]. Observe the similar support given by the quantum
decay in the Fermi-Dirac distribution given in A), which can be reflected to macroscopic level in the decaying of
fabric of cancer cells An similar Fermi-Dirac model can be constructed that gives support of the curve in the figure
B). The curve is the distinctive quality of the efficacy of organic Germanium to be organic restoring and a powerful
cytotoxic cancer cell.
Nanomedicine 255
We consider the following clinical case that resumes what is discussed and explained in this section,
where was used the combination in different soft photon sources in different stages of cure for the
complexity of same medical case.
We have the clinical case of the patient Rominia Juárez [4] with symptoms Bleeding from nose and
gums without apparent reason, several times in the night and day, very little energy and pain on the
right side starting at the mouth of the stomach, liver and side, some fainting and general weakness
(lack of energy) and low assimilation of proteins and food with iron. In addition Mrs. Juarez suffers
from poor circulation due to a problem of varicose veins associated with your metabolic change during
multiple pregnancies. An analysis of initial lab based on the initial medical diagnosis on her
concomitant weakness and lack of assimilation of the food mentioned, threw a low production of white
blood cells that are produced in the liver. Conducting an investigation on your medical history focusing
on history of suffering associated with her digestive system was found that the Mrs. Rominia had
suffered a typhoid or enteric fever during one of her pregnancies. It had weakened her digestive
system already that her liver had been disrupted by the treatments at the time, by means of
chloramphenicol in moderate proportions. Due to her state of pregnancy and natural way is alerted her
immune system created by the body of the mother to protect the product and made the production of
amino acids for the manufacture of red blood cells will focus on the manufacture of the different blood
cells for the development of the fetus. However this natural deviation produced by the immune system
protecting the product, filtered certain derivatives from the catalysis realised by the mother's liver to
assimilate the clorafenicol. The treatments of chloramphenicol and this circumstance led in the Mrs.
Rominia to the partial disappearance of cells that act as precursor cells of the different leukocytes.
Then a second diagnosis based on this analysis revealed a state of plastic anemia but with a latent
inflammation of the liver, leading to a frame of hepatitis type C. The usual clinical treatments take to
control (not eliminate) this chronic illness. However, the Mrs. Rominia wanting to feel more energy is
subjected to a clinical treatment with quantum mono-pharmacists that bring greater vitality to
strengthen your liver. In the first instance is proceeded to the cure of the evil hepatic and after we
would proceed then be the cure of Angio-phlebitis of the legs. Likewise is applied to the patient the
quantum treatment using the Epsilon2005 formula, which integrates the quantum mono-pharmacists
for the liver system of Bryal, Bervul and Bellatrop, having each of them a specific medical action for
hepatic system on the production of leukocytes (Bervul) and attack a possible infection (Bryal),
(considering the necessity of protein for the complete synthesis of organic compounds that the liver
must perform) given by:
correction + restoring = Bervul + Bryal + (Bellatrop + supplement of proteins), (6)
In addition was recommended to the patient a specific diet. It is recommended that the intake of food
without fat, without chile and abstention from drinking wine or any drink that would have alcohol
staying all the treatment for two months. To provide the patient of proteins required by the organism
for different processes is recommended that a quantum protein complement defined by the action
Protein Restoring = Histol Revisited + Bellatrop revisited, (7)
where this last Bellatrop version is designed through a soft photon source that is defined in this case by
the use of a photon sensor, which must irradiates to the water solution of 125ml with Bellatrop to
enrich to this solution of photons and strengthen the electrons that will be spilled in the affected part
Nanomedicine 256
by other mono-pharmacists, for example to this clinical case Bervul (see Figure 9.10 C). The photon
sensor spill more photons to the Bellatrop solution increasing the photon account to strengthen and
stabilize the correcting action by Bervul.
In this period is happened a fact that complicates the direct application of the treatment, since the
patient is beaten in a leg and produces an ulceration to burst the skin of the leg presenting three
bleeding wounds and with endodermic fabric exposed. Without interrupting the hepatic treatment
comes to perform a treatment using dermal components and Bellatrop, Bryal with Dermo, the latter to
attack a bacterial infection in the wounds of the leg which being exposed are infected. The quantum
action that in this case could have to be done alternately with (6) is
corrector + restoring = [Dermo + Bryal] + Bellatrop, (8)
with an interval of 30 minutes each medicine once a day for 6 days of the week during the time until
disappear the wounds and restore the dermal fabric. To fix the applications on endo-dermal corrective
cells (equipping of electrons to the mitochondria and greater vitality to cells) and promote the creation
of antibodies to the liver problem is prescribed as a supplement the organic germanium (preferable to
selenium and zinc to raise defenses and with the greatest breadth and depth of spectrum (see Figure
9.11 B)) which re-force the atomic links both in the liver as the wounds in her leg. But it also had to be
corrected the angio-phlebitis without the loss of the immunization of the cells of the endodermal
fabrics (since the patient re-iterated times had resorted to allopath drugs and antibiotics to relieve the
pain caused by the infection, which had continued unabated by the liver problems latent (not had an
adequate synthesis of vitamins on the part of the liver, which was supplying Bervul and Bryal))
increasing the temperature in the area of the skin without have a systolic increase adequated.
The action of the organic germanium succeeds establish the ion equilibrium of the actions given by
Bervul in the hepatic problem and for other side the integration of the circulatory problem filling of
oxygen atoms to leukocytes and electrons to the mitochondria of endo-dermal cells.
As an integration of the actions given in (7) and (8) and in consequence to the body-mind of the
patient, is preceded to stabilize the soma of As a integration of the actions given in (7) and (8) and in
attachment to the body-mind of the patient, to stabilize the soma of the patient influencing properly in
her immune system, achieving their re-affirmation to the cure through re-enforce the will of cure of the
organism creating a state of emotional stability by the production of histamine and serotonin in the
patient.
We remember that the treatment to the inflammation of the liver with the specialized
monopharmacists Bellatrop and Bervul, this last of the homeopathic group Berveris D12, realizes a
correction of the functions of the liver in regard to the synthesis of compounds avoiding the
production of endoxines and free radicals.
On the other hand, the action of Bryal was aimed at the suppression of the infection factor for infection
eliminating coercive charges of the digestive system by a poor dietary intake or digestion of food
promoted by incorrect signals from the nervous system (neuropeptides incorrect [2, 13] ). This
conducive to consider the mental factor as concomitant part for the correct assimilation of the food
and the correct extraction of their nutrients by the liver, in addition to the vitamin assimilation
Nanomedicine 257
necessary for the dermal fabric, thus is proceeded to the implementation of two mono-pharmacists to
correction and restoring of the mind: Gelsem from Gelsesium D12, and Ignatia from Ignamara D12,
which is realised the action [8]:
Correction + Restoring = Gelsem + Ignamara, (9)
These mono-pharmacists increase the serotonin and histamine in the central nervous system and the
production of neuropeptides is done properly eliminating overcharges in the central nervous system
produced by a disordered superposition of quantum states [9, 13], which generate the factors of
anxiety in the space dimension of the symptom [5]. A modulator is required, in this case Pulsani,
quantum extract of Pulsatilla D6, to establish a synchrony between the quantum propagators of the
mono-pharmacists destined to cure organic (liver and gallbladder) and the elimination of the anxiety
factors own of the discomforts of the patient obtaining a modulation for a quantum average frequency
 M , (see scream of the Figure 9.11 E)[4]). Finally is found the following analysis of actions and their
quantum states of energy corresponding to the clinical history of the patient considering that
lines}  {1-lines (contours)}  {vanishing singular points}  {Strong joints}, is the corresponding
graph of the joint action given from (6) to (8) [3, 8, 14]:

where the action of alignment given by    , is for each one of the three restoring mono-

pharmacists of hepatic, dermo and psychological systems given by the Bellatrop, Bellatrop + Histol, and
Ignamara that must be applied simultaneity. Here  [x] ,
U
is the code of the singular state in the
0000-box of the Feynman-Bulnes integral [1]. Due to (13), the new route given by the corrective and
restoring actions is:
To form conscience of cure  To vanish erroneous routes and anomalous codes  To revert (inversion)
 To displace and alignment paths (To form alignment trams)  To form covalent links,
The real action is realised under the deep action of Gelsem in their version of the formula 
2006, which
have their binary code given by the path integral [1, 14] (see the Figure 11 F)):
 [ x]  101011110101101  01  1101, (11)

U
Nanomedicine 258
C
FIGURE 9.10
The left screen proves the electronic propagators of every component mono-pharmacist that are modulating and
corrective included in E2005. The curve in blue is the supported action of E2005, due to the entire action of their
mono-mono-pharmacists in synergy (Figure A and B).This is an example of the principle of combination of quantum
factors. The area under the curve is the energy used for this action, which is bounded by
log ( ) log  ,[Cuba] . The figure C), is a multi-physics modeling of the photon crystal
2 2
functioning.
where 1  0 represents the electron sent with the other string extreme      , to have
particle  . This is repeated two times (to each Germanium atom) and other two electrons of the
 

organic Germanium valence are sent to the other string graphs, before    , and after  ,
 

in (10) where  is the quantum corrective wave. Then the eight electrons from Organic Germanium
(with two atoms of Germanium element) are shared with the three Oxygen atoms and carbon (Figure
9.9B)).
One of the major conclusions of this clinical case is the necessary medical management of the patient
Nanomedicine 259
through the mind-body, considering that for a real and deep correction must be realised the patient
from states of anxiety propitious of the disease, which all the time acts contrary to the recovery of the
patient (Figure 9.2A)), eliminating the subjacent anxiety factors, which transmitting to her liver
producing a state of stress in the hepatic region producing a fatty liver to not perform a proper
synthesis of proteins and lipids.
A B
D
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FIGURE 9.11
A). The usual solution in water of the Bellatrop monopharmacist and the obtaining of Bellatrop Plus. B). Figure
of spectra amplitude (Fourier transform of the energy signals) of Ge-132 comparing with the spectra of other
restoring PGG and CGS [35- 39]. C). The initial action realized in the cure of the clinical case. This is the more
important of treatment because invest the major energy to correct the anomalous of the channels of energy

of the vital field. Their path integral is
 [ x]    1 F (n1 ) 2 F (n 2 ) 3 F (n3 ) 4 F (n 4 ). D). Eliminating
U  
action of the fermion scattering produced by the tumor in an arm. This device removes the particles through a
drain-spill process that is carried by transceptor defined for soft photon source. The elimination of the “bad
energy” is through of destruction anti-particles camera. E). Effectiveness of average frequency:
 Scope Effectiveness  average frequency  Eff  M ( nJ / nm  cycles / seg ). This
measurement establishes the action frequency of the quantum mono-pharmacists by every nanometer.
Compare this spectral curves with the curve of effectiveness versus depth (figure C). In both cases the depth is
invariant. The variations are the cycles per seconds applied in the advances of the mono-pharmacists restoring
vital energy (vitality).
This causes a constant irritation of the vesicle due to coercive charges of the peripheral nervous
terminals ending in the stomach. Some measurements taken during the treatment are consignee in this
clinical analysis using an electronic device for detection of micro-charges is shown in Figures 9.12 A
and B.
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A B
FIGURE 9.12
Electronic device of illness detection by micro-charges.
In all this time is enforced the immunity system with the organic Germanium having positive
consequences in the recovering of the endodermic and hepatic cells.
In the case of Bervul, their action is the reversion of the helicity of the particles that shape the vital field
of the human body [1] doing that the liver realizes the correct synthesis of compounds and chemical
derived. The called restorers contain a double potency measured in (= hundred-thousandth/assets
unit), and these are overawed for resonance in modulating intervals to correct electrical ambiences
(micro-charges in the nervous system) in which Gelsem is a corrector who gets together with restoring
mono-pharmacists of the organic system of which is wanted realize the above mentioned restoration
(hepatic restoring). Also are wanted the restorers to correct the impedance, deformations in the
cellular micro-hologram (quantum intelligence codes engravings in the organism) and/or cellular micro-
circuits (Figure 9.11 C)) [8].
Evolutive and Involutive Quantum Insights by Nanomedicine in

Preventive and Corrective Developments
In some medical procedures of nanometric level, the combination of quantum factors is realized by a
lot of success for the prevention of concomitant illnesses. Such is the case of some mono-pharmacists
of immuno-modulation [3], which through a combination of groups of mono-pharmacists realizes an
elimination of concomitant factors and is an assistant for the medication of the immunological system.
Some of these mono-pharmacists have aliphatic actions because their action is correcting and are given
in special medication. But these haven’t the power anticipative that is needed in the immunological
system and that in much gets lost in the quantum re-compositions of the modulators that could
present small de-phasing [9] that finally the mind-body can to correct himself, but in the majority of the
cases the people aren't ready to understand it, dedicating themselves to antibiotics or remedies little
foundation. Then is required an anticipatory action to cover of possible de-phasing states to prevent
these little disagreements between the organism (which not wants leave their accustomed state, for
example, in the alcoholism the organism takes said state as normal or tending to normal being false to
the soma of the patient (quantum cure conscience)). In this case the organism feels anxious and
appears a symptom that can be organic or mental (see Figure 9.9 A)) then the anxiety of the organism
must be eliminating with one action that must be preventive and anticipatory. The before action needs
a characteristic of quantum evolution and quantum involution proper of the mind - body, since the
human body for one side is a dynamic system and always changeable (even with proper regeneration)
and for other side, the atomic reconstitution must survive intact. The mono-pharmacist Pulsani from
Pulsatilla D12, whose action of displacement turns out to be reflected in the strengthening of the will
of the patient to be treated duplicating the auto-cure factor of B . Also combining it with other
Nanomedicine 262
quantum mono-pharmacists cans realize tasks of psychological healing (as has been seen Figure 9.2A)).
Their modulator action with Gelsem correct de-phasing energy defined by the patterns of leptons
(photons responsible of the charge in the vital field) eliminating the particles of their decaying in some
very short time interval (See Figure 9.13 A)). Here is where the involution of the monopharmacists acts.
In the case of the organic Germanium their actions are directed to the cure of bio-degenerative
illnesses and appearing of cancer (including the reversion when there exists predisposing), through their
capacity of apprehension of predator protons that give free rein to the theft of electrons and their few
disposal to allow covalent linkage between atoms [38, 40] (see Figures 9.13 B), and 13 C)).
C D
FIGURE 9.13
A). This is of energy density on radius in space-time (depending dimension of radius), to arbitrary units. Observe
the behaviour predicted in the Figure 9.9 B, when all the radial energy patterns are considered to the same time.
Also appear in nature form the Fokker-Planck model. This show that the anxiety lives in a lepton space [5, 13]. B).
Actions by organic Germanium to capture electrons and fix them. Here p, is positron, e, electron, e, photon
wave (boson),  , muon and n, anti-muon. C). The preventive action in this string graph corresponding to a
quantum mono-pharmacist is given in the string graph of recovering of particles. This is action is a reversion that
replay to the scattering in the first graph. D). Direct Method: A device shoots the photon beam on the quantum
mono-pharmacist to increase their power of cure. Their administration is followed by the traditional medication.
Inverse Method: A device shoots the photon beam to transform the part of body that is wanted cure, realizing an
automatic correction and restoration interchanging the anomalous particles (anti-particles and leptons of high
decay) for new photons and particles (in this case electrons) with information cures [12, 16]. In the direct method
there exists natural sources that give electrons and soft photons as organic Germanium, Gavriola, and all quantum
mono-pharmacist with corrective and restoring actions. But, what can be done when there are no substances
available? We need an organized transformation of the body energy.
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Conclusions
Imagine that in the coming years we have that to realize prologued travels across of the Universe to
establish space colonies with the goal of establish that stay alive of the human race. In these space
travels we cannot carry with us all the substances and pharmacists with their components, or that it
will be much more difficult the obtaining of all chemical elements in their appropriate state to be used
in the synthetising and formulating of mono-pharmacists to cure of different and possible strange
sickness that appear.
The unique component that will have will be the energy and atoms of proper space-time and their
quantum interactions of these atoms. Then will be necessary the use of quantum devices with
programmed actions that include all the binary codes of their energy states of the photons to create
the cure actions and regeneration of cells, fabrics and organic systems of the body, under only one
principle the correction of their vital field through correct their atomic collateral damage that is
primigenial cause of all illness [3].
The development of new formulae in integral medicine that determine new shorter and effective
intelligence codes will be able to establish more direct actions for the remedy of illnesses and
sufferings. The case described in the section 4, shows the possibility of reduction of the ways continued
in the space - time of the body to establish a more direct curative action and of short re-establishment
time. Although all the ways, routes and trajectories continued in the use of a mono-pharmacists of
quantum medicine to treat a specific suffering are the same way valid since a lot of ways exist to
generate a solution.
The future projection of the quantum medicine is to cure all the illnesses and sufferings under the
same corrective action of photonic type [12], making to change the binary codes of the quantum states
of the medical similarity of their mono-pharmacists to spill an photonic bundle with properties of
strengthening of atomic linkage and reversion of anomalous actions of antigens and specific cells that
must fulfil with property the annihilation of bacteria and external aggressive agents.
It is necessary to mention that from the point of view of the principles that govern the integral
medicine the underlying cause of all the illnesses is only one, which consists of a loss of the healthy
conscience of the body due to a fragmentation of his memory for a deviation in mind - body. The codes
of intelligence spilled by the quantum medicine strengthen this space - time connection in the human
body establishing the suitable paths of emission - reception of healthy energy by means of bundles of
energy that take and bring these intelligence codes in all the places and corners of the body. The last
one creates then a synergic healthy action due to their vital, generated field for the atomic linkage
created by the pharmacists of nanomedicine performance.
Nomenclature
B  Space-time of the human body (mind-body). All the body.

B  Organic part of the human body. Specific region or part of human body.
X  Vital field.
i  ith  Energy state of the field.
  Action of the field.
x(s)  Particle at the time t  s.
Nanomedicine 264
O(k )  Vector bundle of lines of helicity k .

O(n  2)  Homogeneous vector bundle of lines of helicity n  2. Case of vector bundle used to
electromagnetic fields applied to the diagnostic, modeling and alignment of singularities of the field.
L  Lagrangian Operator
M0,1  Logic reticule to establish the binary codes and binary encoding of the evaluation the path
integrals to along of strings, paths, trajectories and transitive graphs in the corrective and restorative
actions.
A (, , -)  Logic algebra used to digital encoding the Feynman integrals
PB   Two orbital spaces of the body that represent the orbits of solutions to electromagnetic wave
equation. The sign represents polarity.
F  Fermi energy.
TB   Vector Bundle of ionic interchange of the vital field.
C 0 ()  Special graphs space of the symbolic cohomology of strings.
I   Integrals of Field. Integrals of line used in the correspondence with the Feynman diagrams. For
example, between two energy states of one particle,  , and    , exist one infinity of trajectories
that summed give the integral I  .
X (B)  Vital field of the body.
E  Incremental effective energy. This energy is obtained in the spectral analysis realised to all mono-
pharmacists in the Figures 3C), and 11 E).
M  Effectiveness in the space-time of mind-body.
  Path.
H 1 ( A, B)  Space of similarity relations of first duality.
        Virtual edge of photon string graph (string diagrams used to represent binary codes)
   (0)
   (1)
  Quantum wave in the transmitting of cure codes from (binary codes from the energy states).
H 2 ( A, B)  Space of similarity relations of second duality.
E l  Effectiveness/path walked approximate value.
R a  Continuum quantum factor (Neumann factor).
Units and Abbreviations
nm  Nano-meter.
Ge 132  Organic Germanium.
eV  Electron-volts.
E 2005  Epsilon formula.
2006  Gamma formula.
nJ  Nano-joules.
nJ / nm  Effectiveness
Nanomedicine 265
Cycles / sec onds  Frequency
Acknowledgments
We grateful to the international committee of Drug Discovery and Therapy Meetings shaped by the
Nobel laureates and eminent scientists by the many invitations to participate in the nanomedicine
track inside these events, which has been very useful in our research.
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10
Optimization of perfluorocarbon
nanoemulsions for molecular imaging by
19F MRI
1 1 1 1 1*
Christoph Grapentin , Friederike Mayenfels , Sabine Barnert , Regine Süss , Rolf Schubert ,
2 2 2 2*
Sebastian Temme , Christoph Jacoby , Jürgen Schrader , Ulrich Flögel
1
Department of Pharmaceutical Technology and Biopharmacy, Albert Ludwigs University, Hermann-Herder-Strasse 9, 79104
Freiburg, Germany
2
Department of Molecular Cardiology, Heinrich Heine University, Universitätsstraße 1, 40225, Düsseldorf, Germany
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 269
Generation of stable pfc nanoemulsions with defined size …………………………………………..……………. 270
Perfluorocarbons suited for 19F MRI ………………………………………………………………………………………….. 271
Emulsifiers and nanoemulsions …………………………………………………………………………………………………… 272
Manufacturing perfluorocarbon nanoemulsions (PFC-NE) ………………………………………………………….. 272
High pressure homogenization for the generation of PFC-NE with different size ………………………… 272
Removal of liposomes from perflurocarbon nanoemulsions ……………………………………………………….. 273
Stability of PFC nanoemulsions ……………………………………….………………………………………………………….. 274
Size exclusion chromatography (SEC) …………………………………………………………………………………………. 274
Uptake of PFC Nanoemulsions by Macrophages ………………………………………………………………………… 275
Size and concentration dependent uptake ………………………………………………………………………………….. 275
Site-specific delivery of Perfluorocarbon Nanoemulsions …………………………………………………………… 276
Pegylation of PFC-NE to reduce phagocytic uptake …………………………………………………………………….. 277
Antibody-mediated active targeting of PFC-NE …………………………………………………………………………… 279
Perspectives and Conclusions …………………………………………………………………………………………………….. 281
References……………………………………………………………………………………..…………………………………………… 282
Nanomedicine 269
Introduction
Tracking fate and function of specific cell types by molecular imaging methods is of great importance
for basic research and holds the potential to be clinically applied for initial diagnosis as well as the
monitoring of subsequent pharmacological treatment. Key applications include visualization of the
destiny of transplanted stem cells for regenerative therapies [1-6] or dendritic cells for cancer
immunotherapy [7,8]. Furthermore, imaging of immune cells has received increasing attention over the
last decades, since it has become clear that inflammation plays a major role in many diseases. In this
context, macrophages emerged as an attractive target, because these cells are key effectors of both
the progression and resolution of inflammation [9-14]. However, direct monitoring of these cells by
non-invasive imaging methods requires labelling of these cells with contrast agents or genetic
modifications (expression of reporter genes like GFP or luciferase) independent of which modality is
used. Recently, optical techniques, bioluminescence, PET (positron emission tomography), SPECT
(single photon emission computer tomography) or MRI (magnetic resonance imaging) have successfully
been applied to visualize labelled macrophages at inflammatory sites [15]. Nevertheless, optical
techniques and bioluminescence are not suitable for high resolution cell tracking, since these
techniques – although very sensitive and specific – can not resolve the localization of labelled cells
within an heterogeneous inner organ. SPECT/CT and PET/CT display an excellent contrast but require
the administration of radioactive agents and the exposition to high doses of X-ray radiation. On the
other hand, MRI displays high spatial resolution and superior contrast between soft tissues without the
need for radioactivity or X-rays.
Imaging of macrophage immigration by MRI has so far been predominantly performed by use of iron
oxide-based nanoparticles (SPIO = superparamagnetic iron oxide; USPIO ultrasmall superparamagnetic
iron oxide) [16,17]. After intravenous (i.v.) administration, these particles are taken up by monocytes
1
and macrophages and lead to a signal depletion in H MRI upon infiltration of the labelled cells into the
1
inflamed area [18]. However, interpretation of iron-oxide nanoparticle induced H signal erasure is
sometimes difficult, since susceptibility artefacts especially in the boundary zone between different
tissues can also result in signal deprivation and therefore lead to misinterpretation. In addition, the
iron-oxide nanoparticle deposition yields to the loss of precise anatomical information and absolute
quantification is hampered because there is no linear relationship between signal depletion and iron-
oxide content.
19
As an alternative emulsified perfluorocarbons (PFCs) – containing the stable fluorine isotope F –
1 19
recently emerged as promising contrast agents for cell tracking by H/ F MRI combining the
1 19
anatomical information ( H MRI) and the localization of F-labelled target cells Similar to other
nanoparticles, PFC nanoemulsions (PFC-NE) are phagocytosed by monocytes and macrophages after i.v.
injection. PFC-labelled cells subsequently migrate into the inflamed area resulting in a local deposition
19 19
of F nuclei [19,20] (Figure 10.1). Due to the lack of any natural F background in the body, detected
19 1
F signals show a high degree of specificity [21], while its sensitivity is close to that of the H nucleus.
1 19 19
After merging anatomical H and cell-tracking F images the exact anatomical location of the F hot
spot can be precisely determined (Figure 10.1). In particular for inflammation imaging this technique
has been validated so far in a variety of disease models by several groups (Figure 10.1, lower panel)
[19,22-33].
19
Although F MRI is a promising tool for cell tracking and inflammation imaging, sensitivity is a critical
19
concern. F MRI requires several hundreds to thousands of labelled cells per voxel for detection [19-
19
21,34]. On the other hand, increasing the F load per cell by improving the phagocytic uptake of PFC
Nanomedicine 270
particles by monocytes/macrophages (or other cells) in vivo would significantly improve the detection
19
limit of the F MRI technique. However, this is currently hampered by the massive uptake of
“conventional” PFC emulsions by the reticuloendothelial system (RES). After i.v. application, PFC
nanoemulsion particles are coated by serum proteins, which not only enhance their uptake by blood
monocytes, but also cause their fast removal from the circulation by the RES. Thus, the large fraction of
PFC particles absorbed by the RES strongly obstructs an efficient loading of monocytes in the blood and
19
limits the sensitivity of the F MRI approach. Since the major determinants of nanoparticle uptake by
phagocytotic cells are size and surface properties, we aimed at modifying these characteristics to
increase the efficiency and specificity of PFC uptake by distinct cell types.
FIGURE 10.1
19
Principle of F MRI inflammation imaging. Perfluorocarbons are emulsified to generate PFC-NE which can be
injected intravenously. Emulsified particles are taken up by blood monocytes which immigrate into the inflamed
19 1 19
area resulting in a localized F signal. Merging of the anatomical H grey scale image and the corresponding F
signal (red) reveals the exact anatomical position of infiltrated cells and thereby the inflamed area. This technique
has been validated in a variety of clinically relevant disease models. Reprinted with permission from references
[19,24-26,30].
Generation of stable PFC nanoemulsions with defined size

Perfluorocarbons are not mixible with water, therefore PFCs have to be dispersed in water or a
physiological buffer system by the use of an emulsifying agent and energy input. As emulsifying
compounds, lipids or poloxamers can be used to generate the PFC-NE by extrusion, ultrasound or high
pressure homogenization. Nanoemulsions for parenteral application (e.g. labelling of immune cells)
have to withstand heat sterilization or have to be produced under aseptic conditions. The droplet size
of PFC-NE are preferentially of small dimensions (<200 nm), but depending of the nature of PFC,
emulsifier and preparation technique, they are characterized by varying size distribution. To analyze
the properties of PFC-NE photon correlation spectroscopy (PCS) or electron microscopy are routinely
used. PCS yields the average hydrodynamic diameter and the polydispersity index (PI or PDI) that
describes the size distribution. However, this method is limited to dispersions with a rather narrow size
distribution [35].
Nanomedicine 271
19
Perfluorocarbons suited for F MRI
PFCs are synthetic organic molecules where all hydrogen atoms have been replaced by fluorine atoms.
The C-F- and C-C-bonds are exceptionally strong and the electronegativity of fluorine results in weak
intermolecular forces as well as hydrophobic and lipophobic properties [36]. No enzyme is known to
split PFCs and no biological PFC degradation pathway has been reported up to now. PFCs are therefore
regarded as non-toxic, biochemically inert compounds. PFCs usually display a large density of about 2
g/ml [37]. They show a high dissolution capacity for gases and were intensely investigated as oxygen
carriers in the 1960s [38]. In the following decades a multitude of nanoemulsions have been developed
for the use as blood substitutes but also as medical contrast agents, but none has been established on
the market. Among the problems encountered with PFC-NE were insufficient size stability upon
storage, activation of the complement system or other safety and stability concerns [39].
For MRI purposes, the most widely used PFCs are perfluoro-15-crown-5 ether (PFCE), perfluorooctyl
bromide (PFOB) and perfluorodecaline (PFD), whose chemical structures are shown in Figure 10.2. Due
to its 20 chemically and magnetically equivalent fluorine nuclei, PFCE is characterized by a single MR
resonance and therefore has ideal properties for MR imaging techniques, but it exhibits an extremely
long biological half-life and is therefore not suitable for clinical applications [37]. PFD and PFOB have
short biological half-lives but cannot be imaged by conventional methods without artefacts [40]. Of
note, PFOB has a terminal bromine that alters its chemical behavior to slightly lipophilic, thus
simplifying the manufacture of stable nanoemulsions and facilitating its passage over membrane
barriers which contributes to its quick release from the body. Perfluorohexyloctane (F6H8) represents
the class of mixed hydrocarbon-fluorocarbons, or semifluorinated alkanes. Although, it has not yet
been used for MR imaging, it is an interesting candidate, since it is already clinically approved as long-
term tamponade in complicated retinal detachments and as an intraoperative instrument for retinal
surgery. Another important property is it its significantly lower density (1.35 g/ml) compared to the
perfluorocarbons.
FIGURE 10.2
Perfluorocarbons for the generation of PFC-NE. Chemical structures of different PFCs which can be used for the
generation of PFC-NE. PFCE has 20 chemically and magnetically equivalent fluorine atoms which makes it ideally
19
suitable for conventional F MRI but has a very long tissue retention time. PFD and PFOB are characterized by a
short biological half-life but exhibit complex spectra requiring dedicated imaging sequences [40]. F6H8 is a
semifluorinated alkane which has not yet been used for MR imaging.
Nanomedicine 272
Emulsifiers and nanoemulsions
For later pharmaceutical approval, the emulsifiers should already be certificated for i.v. application.
With regard to this, both phospholipids and poloxamers are validated substances. Phospholipids are
naturally occuring reagents and show a good biocompatibility. They can be produced cost efficiently
from well known sources, like egg yolk or soy bean. Phospholipids easily form nanoemulsions with
PFCs, but are prone to hydrolysis and oxidation – factors that limit the stability of NE particles.
However, the main disadvantage of phospholipids is the additional formation of “empty” liposomes.
Liposomes do not contain any PFCs and impair the stability of the nanoemulsion. Poloxamers are block
polymers of an A-B-A type, poly(ethylenglycol)-poly(propylenglycol)-poly(ethylenglycol). Poloxamers
can be produced in large scale, but due to their relative low surface activity they must be used in higher
amounts and tend to form highly viscous dispersions. Another important disadvantage is a possible
activation of the complement system [41]. In our hands, phosholipids are superior to poloxamers for
the generation of PFC-NE.
Manufacturing perfluorocarbon nanoemulsions (PFC-NE)
Because of the quite different physical and chemical properties of the starting ingredients, high energy
inputs are required for the formation of PFC-NE and to reach a fine dispersion of the PFC in aqueous
solutions. This can be achieved by high pressure homogenization, sonication or extrusion. In all cases,
as a first step a crude emulsion (µm size) is produced by means of high shear mixing. Sonication is
limited to small scale production whereas bigger scales of several 100 ml can be produced with
extrusion. Both methods usually lead to a high content of liposomes and NE droplets of a wide size
distribution. The most suitable way to manufacture PFC-NE with a low liposome content and
acceptable size distribution is high pressure homogenization which can be scaled up to large industrial
magnitudes.
High pressure homogenization for the generation of PFC-NE with different size
To generate PFC emulsion particles with different average size, we altered the ratio of the PFC (F6H8,
PFD and PFOB were tested) and phospholipid (here S75; S = soy) – the higher the PFC amount, the
larger the particles. This is because less emulsifier is available to stabilize the inner PFC phase. The
preformed rough emulsions are then subjected to high pressure homogenization (e.g. Avestin,
Emulsiflex C5). By passing a narrow valve, pressures up to 1500 bar are created that disrupt the µm size
droplets of the crude emulsion. Droplet size decreases each time the emulsion passes the valve. By
modifying the ratio of PFC and S75 in the range of 1 to 50, particles with a mean diameter of 100 to 300
nm can be generated (Figure 10.3). However, these preparations show a varying size distribution with a
polydispersity index (PDI) between 0.46 and 0.1 (data not shown). Although differences between
individual PFCs were observed, this technique seems to be suitable for all PFCs investigated and also be
applicable for other PFCs.
Nanomedicine 273
FIGURE 10.3
Dependence of PFC-NE size from PFC/emulsifier ratio. PFC emulsions were prepared by altering the ratio of
emulsifier and perfluorocarbon (PFD, F6H8, PFOB) and subsequent high pressure homogenization (700 bar, 10
cycles).
Removal of liposomes from perflurocarbon nanoemulsions
Liposomes do not contain PFCs but compete with PFC-NE droplets for cellular uptake leading to a
19
decreased F loading of target cells and thereby to a loss of sensitivity for in vivo cell tracking. Since
PFC-NE have a high density, we separated NE droplets from liposomes by repeated centrifugation, e.g.
25 min at 13,000 g for three times. Figure 10.4 shows electron microscopic images of a PFC-NE before
centrifugation (left) as well as the resuspended pellet (right) and the supernatant (middle) after
centrifugation. On the left, empty liposomes can clearly be recognized, which were almost completely
removed upon repeated centrifugation (right). The pictures also show that PFC nanoemulsions are
usually characterized by particles of varying size. The mean diameter and the obtained size distribution
depend on the emulsifier, the homogenization process (pressure and cycles) as well as on the PFC used
and its percentage (see above).
FIGURE 10.4
Removal of liposomes by repeated centrifugation. Electron microscopy of PFC emulsion before and after
centrifugation (3 times 13,000 g) as well as of supernatant after the first centrifugation step. The arrow indicates
liposomes without PFC content. NE droplets appear dark because of their PFC load, while empty liposomes are
bright.
Nanomedicine 274
Stability of PFC nanoemulsions
Stability is a critical issue for the manufacturing of PFC-NE. At higher temperatures, coalescence, i.e.
the irreversible fusion of two or more droplets to a bigger one, is the main mechanism of instability.
Over a longer storage period molecular diffusion, also called Ostwald Ripening, triggers the irreversible
growth of a droplet, at the expense of the smaller ones. Molecular diffusion can be diminished by the
addition of a PFC homologue of lower water solubility, e.g. admixing of small amounts of
perfluorodecyl bromide to perfluorooctyl bromide before homogenization [42]. Another method to
obtain more stable nanoemulsions is to incorporate semifluorinated alkanes as co-surfactants which
generate a stabilizing bridge beween lipids and PFCs [41]. Furthermore, type of PFC and emulsifier as
well as the ratio of both components are factors that critically affect emulsion stability. Of note, the
properties of the aqueous phase also impact on stability, since phospholipids hydrolyze more easily at
elevated pH values and oxidation occurs already at trace amounts of metals. The latter could be
avoided by incorporation of metal chelators like EDTA and antioxidants (i.e. tocopherol), but this would
hamper the biocompatibility of the emulsion. However, PFC-NE should always be prepared with a
degased buffer to prevent oxygen in the formulation. Nevertheless, when stored at 4 °C we observed
properly prepared PFC-NE to be quite stable even without any additives. Figure 10.5 shows excellent
stability for all tested nanoemulsions except for those with a low amount of phospholipid (e.g. PFOB
emulsified with 15.8 mM S75).
FIGURE 10.5 Long-term stability of different PFC-NE. Stability of F6H8-NE (40% PFC), PFD-NE (20% PFC) and PFOB-
NE (20% PFC). ▲= 15.8 mM S75, ■= 31.6 mM S75, ♦ = 47.4 mM S75.
Size exclusion chromatography (SEC)
The uptake of nanoparticles by phagocytic cells is critically dependent on their respective size. Thus,
development of a nanoemulsion should focus on droplets with a well defined size, i.e. with a narrow
size distribution. Otherwise, contaminations with droplets of large size that are taken up more
efficiently and/or carry a higher PFC load may affect both the specificity and quantity of the PFC label.
As can be derived from Figure 10.4, PFC-NE generated by use of phospholipids usually show a broad
size distribution – a problem which cannot be overcome by the conventional manufacturing process.
However, the formation of defined PFC-NE is feasible by combining centrifugation (see above) with size
exclusion chromatography (SEC). Figure 10.6 shows that we were able to successfully separate well
defined fractions of a PFD emulsion via a Toyopearl HW-75S column. Fraction collection in custom
intervals (Figure 10.6A) enabled the isolation of distinct PFC-NE with different sizes and a very narrow
size distribution (PDI of 0.07 after SEC; Figure 10.6B).
Nanomedicine 275
FIGURE 10.6
Isolation of SEC fractions with defined size and very low PDI. A) Fraction collection leads to PFC-NE of defined size.
B) Electron microscopy of PFC emulsion before and after size exclusion chromatography (SEC). PFCs are displayed
with average size of 187 nm and a polydispersity index (PDI) of 0.14 (before SEC) and a size of 180 nm and a PDI of
0.07 (after SEC).
Uptake of PFC Nanoemulsions by Macrophages
Surface characteristics [43], geometry [44] and size [45] are the most important factors which
determine nanoparticle uptake by phagocytes (and other cells). In general, larger particles are taken up
more efficiently by phagocytes than smaller ones, which has been primarily demonstrated by in vitro
uptake investigations with latex beads of distinct sizes. However, almost no systematic studies have
been carried out on the dependence of cellular PFC-NE incorporation from particle size, most likely due
to the difficulties that occur in producing these NE with a narrow size distribution.
Size and concentration dependent uptake
To investigate the cellular uptake of PFC-NE with different size the murine peritoneal macrophage cell
line J774.A1 was used, which is an established model for primary macrophages [45]. For analysis of
uptake, cells were incubated under defined conditions (100,000 cells, 500 µl medium, 2 h, 37 °C) with
rhodamine-labelled PFD nanoemulsions of distinct particle size (generated with SEC; see previous
section) and nanodroplet incorporation was quantifed by flow cytometry via their rhodamine signal. As
shown in Figure 10.7A, the uptake of PFC nanoparticles by this cell type rises with increasing size.
Interestingly, a sigmoid curve was observed for the size-dependent particle uptake by J774.A1
macrophages. Particles smaller than 200 nm were phagocytosed only to a minor amount. However, the
uptake steeply increases for particles larger than 200 nm in diameter and reaches a plateau for PFC
Nanomedicine 276
particles bigger than 300 nm. These findings confirm that uptake of PFC-NE by macrophages can be
governed by the particle size, which thus can be used as a passive targeting strategy.
In a further step, we investigated whether besides size also the concentration of the nanoemulsion
impacts on PFC uptake by J774.A1 macrophages. Again, cells were incubated under strictly defined
conditions (see above) with rhodamine-labelled NE fractions of three different sizes and stepwise
increasing concentrations. Subsequent quantification of the fluorescence signal by flow cytometry
showed that for all individual preparations droplet incorporation followed an exponential increase with
varying slope depending on particle size. As can be seen in Figure 10.7B, 180-nm nanodroplets exhibit a
maximum uptake of about 60% positive cells. The EC 50 (half-maximal effective concentration) value for
this size was determined to be 170 nmol. Particles with a diameter of 239 nm led to about 80% positive
cells and an EC50 value of 80 nmol, whereas particles with a size of 345 nm showed a further increased
uptake to ~ 90% PFC-loaded cells and an EC50 value of 10-20 nmol (Figure 10.7B).
Taken together these experiments demonstrate that the incorporation of PFC-NE by macrophages can
be manipulated by both size and concentration.
FIGURE 10.7
Dependence of PFC nanoparticle uptake from size and concentration: A) Uptake of PFC emulsion particles of
different size by J774.A1 macrophages, 150 nmol droplets per 100,000 cells. B) Concentration-dependent uptake
of PFC emulsion by J774.A1 cells. All data are given as mean values ± SD from n=3-4 independent experiments.
Site-specific delivery of Perfluorocarbon Nanoemulsions
The “passive” uptake of PFC-NE (generated as described above) by cells of the innate immune system
19
can be easily exploited to identify inflamed tissues by F MRI detection of infiltrated, PFC-loaded
immune cells (see introduction). A more specific targeting of PFC-NE is required to visualize cells which
under normal conditions are labelled only to a low extent (e.g. B-cells) or which are not labelled at all
Nanomedicine 277
by PFC-NE (like T-cells or platelets). To this end, the surface of the NE has to be equipped with a site-
specific antigen that directs it to its target [46]. At the same time the normal “passive” phagocytosis of
19
those particles must be prevented to enhance sensitivity and ensure specificity of the detected F
signal.
PEGylation of PFC-NE to reduce phagocytic uptake
After intravenous injection, nanoparticles are coated by serum proteins (so-called opsonisation) which
facilitates the uptake by immune cells. A well-known procedure to impair the attachment of serum
proteins to nanoparticles is the coupling of polyethyleneglycol (PEG) to their surface. PEGylation has
been applied to a variety of nanoparticles to reduce opsonization and to delay their clearance from the
bloodstream [47]. Interestingly, PEGylated nanoparticles have already been used for tumor imaging, in
that the extended persistence in the circulation prolongs the temporal window for passive diffusion
through leaky endothelium which leads to an increased deposition of nanoparticles at the tumor site
[48,49].
For liposomes or nanoemulsions, usually a PEG-modified phospholipid (e.g. distearoylglycero-
phosphoethanolamine-methoxy-PEG) is incorporated during the homogenization process. However, we
used a post-insertion attempt in which the nanoemulsion is modified with a sterol-PEG after its
preparation. Sterol-PEGs can easily be inserted into the preformed phospholipid layer of the
nanoemulsion and have been previously used to modify liposomes [50]. To verify whether sterol-
PEGylation also leads to a stealth effect for our PFC-NE, a PFD-NE with a size of 229 nm (PDI 0.21) was
prepared and modified with different amounts of a sterol-PEG1300. Afterwards, the cellular uptake of
rhodamine-labelled PFC-NE and PFC-PEG-NE particles was compared by flow cytometry. Figure 10.8A
demonstrates that after 2 h of incubation at 37 °C unmodified nanoparticles (“blank”) were internalized
by 85% of the cells, while PEGylation decreased this value to 10% (for 5 mol% PEG) and 2% (for 10
mol% PEG), respectively. Importantly, PEGylation also strongly reduced cellular uptake of PFC
nanoemulsions with larger droplet size: When 5 mol% sterol-PEG1300 was inserted into PFC particles
with a mean diameter of 320 nm and a PDI of 0.13, the cellular uptake of this preparation was
diminished to about 2% (data not shown).
In a next step, we analyzed whether PEGylation of PFC nanoparticles also impacts on the phagocytosis
19
by the reticuloendothelial system in vivo. For this, the biodistribution of the F signal in murine blood,
19
spleen, and liver was monitored by F MRI starting 30 min after PFC application up to 4 h after
injection. A chemical shift imaging (CSI) technique was applied for artefact-free detection of the used
PFOB-NE [40]. Figure 10.8B shows representative images acquired in two separate experiments when
animals where exposed to PEGylated (bottom) and non-PEGylated (top) emulsions, respectively.
Nanomedicine 278
FIGURE 10.8
Impact of PEGylation on PFC uptake in vitro and in vivo
A) PEGylation reduces cellular uptake by J774.A1 cells. J774.A1 cells were incubated with rhodamine-labelled
PEGylated and non-PEGylated PFC emulsion particles for 2 h and analyzed by flow cytometry. B) Representative
19
images displaying the F signal of PEGylated (bottom) and non-PEGylated (top) droplets in liver (right) and spleen
(left) 30 min after injection. Arrows point to location of the spleen; asterisks indicate the position of blood vessels
(i.e. abdominal aorta and vena cava inverior). 100 µl of PFOB emulsion were injected into the tail vein and
1 19
subsequently analyzed by H/ F MRI.
19
Although the F signal in the blood (asterisks) was similar for both preparations 30 min after injection,
the fluorine signal of PEGylated nanoemulsions was hardly detectable in liver and strongly reduced in
the spleen (arrows) as compared to non-PEGylated emulsions. Quantification of the data revealed a
significant retarded uptake of PEGylated droplets by liver and spleen compared to non-PEGylated
nanoemulsions at this time (n=3, P<0.05). Two hours post injection, the signal in the liver was still 3.5-
19
fold higher for non-PEGylated nanoemulsions. Interestingly, the F signal-to-noise ratio (SNR) in the
blood showed the largest difference between PEGylated and non-PEGylated PFCs after 2 h, with a 1.5-
19
fold higher SNR for PEGylated PFCs. However, 4 h after injection the F signal in the blood was similar
for the two PFC emulsions and deposition of PEGylated PFCs was slightly elevated in spleen but much
higher in liver compared to non-PEGylated nanoemulsions. After 24 h both emulsions were fully
19
cleared from blood, and the F signal in liver and spleen was identical between PEGylated and non-
PEGylated emulsions (data not shown).
Altogether, the data show that sterol-PEGylation of PFC-NE almost abolishes the cellular uptake by
19
J774.A1 macrophages in vitro and delays the deposition of F in liver and spleen in vivo. However,
19
several hours after injection the F content in liver and spleen were slightly elevated compared to non-
PEGylated nanoemulsion droplets, which can be explained by the longer circulation of the PEGylated
nanoparticles [51]. The fact that PEGylation delays but does not totally inhibit the PFC uptake by liver
and spleen can be related to the decreased kinetics of serum protein adsorption to the PEGylated PFC
nanoparticles which usually would facilitate their removal from the bloodstream and diminish the
contact time between particles and these organs.
Nanomedicine 279
Antibody-mediated active targeting of PFC-NE
For site-specific targeting of nanocarriers, ligands are coupled to the particle surface, which are mostly
either of peptidic nature or small organic molecules, although other structures are possible as well. For
PFC-NE a targeting via antibodies [52], RGD peptide [53] or folate [54] has been reported. In the
following, we extend the sterol-PEGylation of preformed PFC-NE (described above) to generate cell-
type-specific contrast agents. In a first step, as a proof-of-concept we “rescued” the particle uptake
capability of macrophages for PEGylated PFC-NE by coupling of an antibody directed against the
scavenger receptor A. In another model, we modified PFC-NE with a single chain antibody for the
activated gpIIb/IIIa receptor to direct them specifically to activated platelets which under normal
conditions do not exhibit any affinity towards PFC nanoparticles.
For coupling of ligands to the PFC-NE, we synthesized sterol-PEGs with a reactive N-hydroxysuccinimide
group (NHS) at the distal end of the PEG-chain, yielding a sterol-PEG-NHS-reagent. Anti-CD204 antibody
(raised against scavenger receptor A) can easily be attached to this construct via covalent linking of
amine groups to the NHS-group. The sterol-PEG-antibody is subsequently inserted into the
phospholipid monolayer of the PFC-NE. As untargeted control, neat sterol-PEG-NEs were used. Figure
10.9 summarizes the data which were obtained after incubation of J774.A1 macrophages with the
different rhodamine-labelled PFC-NEs. While the unmodified PEGylated nanemulsion (“PEG”) was again
taken up only by a minority of the cells, the anti-CD204 targeted emulsion (“CD204Ab”) was
internalized by approximately 80% of the J774.A1 macrophages.
In the next step, we aimed at targeting activated platelets as key players in plaque rupture. Early and
non-invasive detection of platelets on micro-atherothromboses would provide a means to identify
unstable plaques and thereby allowing prophylactic treatment towards prevention of stroke or
myocardial infarction. However, platelets are not known to exhibit any phagocytic properties which
might be exploited for internalization of contrast agents enabling their tracking by molecular imaging
methods in vivo.
FIGURE 10. 9
Specific uptake of PFC-NE by coupling of an anti-CD204 antibody. Bars represent mean values ± SD from n=3
independent experiments.
Nanomedicine 280
A promising option for selective direction of PFC-NE to this non-phagocytic target population seemed
to be the coupling of antibodies raised against the activated gpIIb/IIIa receptor which is predominantly
expressed on activated platelets. For this purpose, complete IgGs are not the first choice for in vivo
applications, mainly because of their presence of the Fc-part which might result in recognition by Fc-
receptors on macrophages, B-cells, NK-cells or activation of the complement system as well as
induction of adaptive immune responses. Therefore, we modified PFC-NE with the well-characterized
single chain antibody LB24, which has already been successfully applied in vivo for detection of
thrombii in mouse carotid-arteries using ultrasound [55]. Subsequently, we analyzed the cellular
association of the modified, rhodamine-labelled PFC-NE to clon3 cells, which constitutively express the
activated form of the gpIIb/IIIa receptor. As control, we used the non-specific single chain antibody
LB46 and A5 cells, respectively, which do not express the activated form of the receptor. Figure 10.10
shows that this targeting strategy resulted specifically in clon3 cells in an almost 10-fold increased
uptake of LB24-coupled PFC-NE as compared to LB46-PFC-NE controls.
In summary, these results demonstrate that sterol-PEGs with chemically reactive end groups can serve
as versatile tools to (i) stealth preformed PFC-NE for rapid uptake by the reticuloendothelial system and
(ii) equip them with site-specific ligands against cells or structures which normally could not be
labelled. The described post-insertion technique offers the unique advantage that also antibodies or
other thermodynamically less stable compounds could be coupled to the PFC-NE, which might be
otherwise destroyed or at least modified by either high pressure homogenization or the subsequent
autoclavation process for sterilization of the nanoparticles.
FIGURE 10.10
Specific targeting of PFC-NE against activated platelets. LB24 targeting to the activated gpIIb/IIIa
receptor in clon3 cells (black bars). As control, the non-specific antibody LB46 and A5 cells (striped bars),
which do not express the activated form of the receptor were used. Data represent mean values ± SD
from n=3 independent experiments.
Nanomedicine 281
Perspectives and Conclusions
Since perfluorocarbons are non-toxic and can be emulsified with pharmaceutically approved
19
phospholipids, obtained PFC-NE are promising contrast agents for (immune) cell tracking by F MRI in
19
the clinical setting. F MRI in combination with intravenously administered emulsified PFCs has already
been successfully applied for imaging of macrophage infiltration during myocardial and cerebral
ischemia [19], lipopolysaccharide-induced lung injury [24], bacterial abscess formation [27] and
collagen-induced arthritis [26]. This approach was also applied to monitor anti-inflammatory treatment
during transplant rejection and collagen-induced arthritis [25,26]. A further step towards a clinical
application has recently been made when it could be shown that PFCs with short biological half lives
are suitable for inflammation imaging [40]. Although this approach is highly specific due to the lack of
19
any F background in the body, sensitivity is a key concern. Several hundreds to thousand PFC-loaded
19
cells are required in the area of interest for detection by F MRI [19,34]. Sensitivity is even more
19
relevant for PFCs with short biological half live, since they display complex F spectra resulting in a loss
19
of signal-to-noise during data acquisition and analysis compared to the ideal PFC for F MRI, i.e.
perfluoro-15-crown-5 ether [34,40]. Therefore, an efficient direction of PFC particles to the target cell
type is highly desirable, which is currently hampered by the massive uptake of conventional PFC
emulsions by the reticuloendothelial system.
Passive targeting to macrophages can be achieved by applying NE of distinct sizes. Formation of PFC-NE
with different sizes revealed that particles <200 nm in diameter were less efficiently taken up by
macrophages, while droplets with a diameter of >300 nm resulted in a massive increase in PFC uptake.
Thus, we developed a protocol for the generation of distinct PFC emulsion particles with very narrow
size distribution by modifying the ratio of perfluorocarbon/emusifier and subsequent fractionation by
size exclusion chromatography. The dependency of the particle size from the PFC/emulsifier ratio is
due to the alteration in total surface area to volume. However, there are limits for the smallest and
largest particle sizes. The maximum size is determined by the amount of emulsifier which is sufficient
to stabilize the dispersed phase. Further increases of the ratios will result in leakage of membrane
curvature and instable particle preparations. Lowering the minimum size results in increased
membrane curvature, which results in membrane stress. Therefore, further addition of the emulsifier
will not decrease particle size but lead to the formation of micelles or liposomes.
PEGylation of PFC emulsion particles nearly abolished the cellular uptake, which could be reconstituted
by coupling of a specific antibody recognizing a specific scavenger receptor on macrophages. Thus,
both site specific targeting and prevention of uptake by the reticuloendothelial system can be reached
by modifying the nanoemulsions with a PEG-shield and equipping this with specific ligands. Of note, the
described post-insertion technique can also be applied to antibodies or other thermodynamically
unstable compounds, since the coupling is carried out after high pressure homogenization and
autoclavation of PFC-NE.
19
Although the present study has mainly focused on detecting specifically macrophages by F MRI, the
strategy of optimizing PFC targeting may as well be useful for other cell populations as shown for the
19
direction of PFC-NE to activated platelets. F MRI has been applied to detect stem cell homing [34], T-
cell infiltration [56,57] and migration of dendritic cells [58] after ex vivo labelling of these cells.
Therefore, the presented approach might enable a specific tracking of these cells in vivo, too. In
addition, our targeting technique could also improve ex vivo labelling of cells for subsequent
transplantation, since distinct cells within a mixture of populations could be selected. Finally, the
extended temporal window provided by the addition of PEG to the particles surface can be exploited to
Nanomedicine 282
specifically direct PFCs to cells or structures apart from both the reticuloendothelial system and
19
monocytes/macrophages in vivo, which could strongly expand possible applications for F MRI.
Acknowledgments
The authors thank Bodo Steckel and Nicole Specht for excellent technical assistance and Prof. Karlheinz
Peter (Melbourne, Australia) for kindly providing us with the LB24 and LB46 single chain antibodies.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG), subproject Z2 of the
Sonderforschungsbereich 612 and grant SCHR 154/13-1.
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11
Designing Polymeric Nanoparticles for
Targeted Drug Delivery System
12* 1 1
Ida Idayu Muhamad , Suguna Selvakumaran , Nurul Asmak Md Lazim
1
Bioprocess Engineering Department, Faculty of Chemical Engineering, Universiti Teknologi Malaysia Johor Bahru, Malaysia
2
Cardiovascular Engineering Centre IJN-UTM, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia
Johor Bahru, Malaysia
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 288
Background of study …………………………………………..……………………………………………………………………… 288
Nanostructured-based drug delivery system ……………………………………………………………………………… 290
Types of nanodelivery: natural or synthetic ………………………………………………………………………………… 291
Starch …………………………………………………………………………………………………………………………………………. 292
Chitosan ………………………………………………………………………………………………………………………………………293
Carrageenan ………………………………………………………………………………………………………………………………. 293
Hybrid approach ……………………………………….…………………………………………………………………………………293
Factors affecting nanodelivery system ……………………………………………………………………………………….. 294
Surface modification factor ………………………………………………………………………………………………………… 295
Morphology and shape factor ……………………………………………………………………………………………………..295
Drug release – novel targeted and controlled release …………………………………………………………………. 296
Techniques available ………………………………………………………………………………………………………………….. 297
Nanosuspensions ……………………………………………………………………………………………………………………….. 297
Polymeric nanoparticles ……………………………………………………………………………………………………………… 298
Solid-lipid nanoparticles ……………………………………………………………………………………………………………… 301
Mechanism of nanodelivery inside human body ………………………………………………………………………… 303
Nanodelivery applications ………………………………………………………………………………………………………….. 303
Nanodrug delivery systems for anti-cancer agents ……………………………………………………………………… 304
Tumor-specific targeting with nanocarriers …………………………………………………………………………………307
Physiological system specific nano-delivery ………………………………………………………………………………… 309
Nanoparticle mediated antiretroviral therapy ……………………………………………………………………………. 310
Conclusions ………………………………………………………………………………………………………………………………… 311
References……………………………………………………………………………………..…………………………………………… 312
Nanomedicine 288
Introduction
New opportunities to prevent and to treat diseases are by emerging the understanding of disease
pathways. The intrinsic limitations of therapeutic biomacromolecules, such as proteins and nucleic
acids, can be avoid through rationally-designed delivery vehicles. Drug delivery is becoming an
increasingly important aspect for medicine field, as more potent and specific drugs are being
developed. No longer depending on small-molecule drugs, this field now not only encompasses
prolonging the duration of drug release but also focusing on customized systems that are designed to
achieve specific spatial and temporal control. With the incorporation of nanotechnology, so-called
smart drug-delivery systems integrate biosensing functionalities which support unaided in vivo
feedback control that resulting in part characteristics of the new term Nanomedicine.
Many biomaterials, primarily polymer- or lipid-based, can be used to this end, offering extensive
chemical diversity and the potential for further modification using nanoparticles. The particularly large
surface area on the nanoparticles presents diverse opportunities to place functional groups on the
surface. Particles can be created by expanding or contracting with changes in temperature or pH, or
interact with anti-bodies in special ways to provide rapid ex-vivo medical diagnostic tests.
More practical design extensions have been made in combining inorganic materials with polymers and
in combining different classes of polymers together in nanoparticle form. A whole host of new types of
polymer particles could be designed into reality with the recent advances in chemistry, processing
techniques, and analytical instrumentation. For example now we have particles that are hollow, multi-
lobed, conductive, thermo responsive, magnetic, functionalized with reactive groups on the surface,
and pH responsive. This could be applied into floating carrier, multiparticulate drug delivery, dual core
or multiple layering drugs and more.
Polymeric nanoparticles have been produced for decades for use in a variety of high performance
materials such as high impact resistant polymers and specialty coatings for these purposes. Advanced
analytical techniques and computer simulations of the events occurring during particle formation allow
us to measure structure and develop control strategies to produce structured particles. Our ability to
develop new control process strategies such as modified the of carrier shape, chemical composition,
internal structure, and morphology of the nanoparticles so as to develop new levels of product
performance in the targeted drug delivery system.
Background of study
By year 2050, human population will reach 9,100 million which is about 34% increase in population
from present situation. By increasing in this population, it proportionally increases in global demand for
foods, feed and energy. Despite on the expected demand on food (and water), several technology
should be applied to make a rational use of resources possible. Considering to this situation,
nanotechnology could suppose a great tool in solving that demand [1]. Exploration of nanotechnology
has brought significantly innovations to the pharmacology fields for over past 30 years [2]. Engineered
nanomaterials (ENMs), one of nanotechnology application already became part of human daily life as
food packaging agents, drug delivery systems, therapeutics, biosensors, and many more. By European
Parliament and Council *3+ definition, ‘nanomaterial’ (NM) is any material that is characterized in one
Nanomedicine 289
dimension ≤ 100 nm, or comprises of separate functional parts either internal or on the surface, which
have one or more dimensions ≤ 100 nm, which include structures, in example, agglomerates or
aggregates, which may be larger than 100 nm, but will retain the typical properties of nanoscale [4].
FAO/WHO report [5] that the ENMs have several applications in the agrofood sector include
nanostructured food ingredients, nanodelivery systems, organic and inorganic nanosized additives,
nanocoatings on food contact surfaces, surface functionnalized NMs, nanofiltration, nanosized
agrochemicals, nanosensors, water decontamination, etc. However, in this chapter, authors will
highlight on nanodelivery system from history up to its application.
By definition, drug delivery systems are supramolecular assemblies incorporating agents intended to
treat a disease. They are used to overcome the shortcomings of the conventional drugs, such as
unfavorable pharmacokinetics, poor solubility, instability, high toxicity, drug resistance and low cellular
uptake. Drug targeting is defined as selective drug delivery to specific physiological sites, organs,
tissues, or cells where a drug’s pharmacological activities are required. In fact, a drug distributes
uniformly in the whole body when it is entered the blood stream, and the drug that is distributed at
sites other than the therapeutic sites may cause toxic side effects. By increasing delivery to the
therapeutic sites and reducing delivery to the unwanted sites, an improved therapeutic index can be
obtained with enhanced and reduced drug action at the therapeutic and the unwanted sites,
respectively [6]. The proposed mechanism of drug delivery or drug carriers is represented in figure 11.1
below;
FIGURE 11.1
Drug carriers, drug conjugates and drug-nanosystems can be engineered to control degradation, react to stimuli
and be site-specific [7].
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There are two general methods for drug targeting; active and passive targeting. Active targeting refers
to increasing in the delivery of drugs to a specific target through the use of specific interactions at
target sites where a drug’s pharmacological activities are applied. These interactions for example
include antigen–antibody and ligand–receptor binding. Alternatively, physical signals such as magnetic
fields and temperatures that are externally applied to the target sites may be utilized for active
targeting respectively. Carriers classified into this methodology include antibodies, transferrin, ferrite
containing liposomes, and thermoresponsive carriers.
On the other hand, passive targeting is defined as a method in which the physical and chemical
properties of carrier systems increase the target/nontarget ratio of the quantity of drug delivered by
adjusting these properties to the physiological and the histological characteristics of the target and
nontarget tissues, organs, and cells. Carriers included in this category are synthetic polymers, some
natural polymers such as albumin, liposomes, micro (or nano) particles, and polymeric micelles. This is
because of the challenges with use of large size materials in drug delivery, some of which include poor
bioavailability, in vivo stability, solubility, intestinal absorption, sustained and targeted delivery to site
of action, therapeutic effectiveness, generalized side effects, and plasma fluctuations of drugs. Of
recent, several researches in nanodrug delivery have been designed to overcome these challenges
through the development and fabrication of nanostructures.
It has been reported that, nanostructures have the ability to protect drugs from the degradation in the
gastrointestinal tract (GIT), the technology can allow target delivery of drugs to various areas of the
body. The technology enables the delivery of drugs that are poorly water soluble and can provide
means of bypassing the liver, thereby preventing the first pass metabolism. Nanotechnology increases
oral bioavailability of drugs due to their specialized uptake mechanisms such as absorptive endocytosis
and are able to remain in the blood circulation for a long time, releasing the incorporated drug in a
controlled fashion, leading to less plasma fluctuations and minimized side-effects.
Nanoscale size nanostructures are able to penetrate tissues and are easily taken up by cells, allowing
for efficient delivery of drugs to target sites of action. Uptake of nanostructures has been reported to
be 15–250 times greater than that of microparticles in the 1–10 µm range. Nanotechnology improves
performance and acceptability of dosage forms by increasing their effectiveness, safety, patient
adherence, as well as ultimately reducing health care costs. It may also enhance the performance of
drugs that are unable to pass clinical trial phases. Nanotechnology definitely promises to serve as drug
delivery carrier of choice for the more challenging conventional drugs used for the treatment and
management of chronic diseases such as cancer, asthma, hypertension, HIV and diabetes [7].
Nanostructured-based drug delivery system

Nanostructured- based drug delivery system is one of the rapidly emerging areas currently that has
gained many researchers attention due to the suitable means of both side specific and time controlled
drug delivery. Currently nanostructured- based drug delivery system produce many commercially
available products that is patient compliance and no side effect. Nanostructured- based drug delivery
system offer many advantages, some of which include; (1) they can pass through the smallest and
narrow capillary vessels due to their ultra-tiny volume; (2) they can penetrate cells and tissue gap to
arrive at target organs such as liver, spleen, lungs, spinal cord and lymph; (3) they can provide
Nanomedicine 291
controlled- release for prolong period. These unique properties makes nanostructured- based drug
delivery system better choice to delivery drug compare to convectional drug delivery system.
Recently combination of polymeric system with nanostructured- based drug delivery system provides
sustained drug release. Among different type of polymeric system, hydrogel consider as a suitable drug
carrier for controlled drug release. Hydrogel is cross linked with a three dimensional network that able
to absorb large amount of water due to the presence of hydrophilic group in the network such as
carboxylic, amidic and others. Hydrogel can significantly use in drug delivery system. Hydrogel can be a
suitable carrier for drug delivery system due drug release from its matrix upon swelling, the interaction
of the drug with the polymers, and the solubility of the drug in the release media. Hydrogel have
capability to protect drugs from hostile environment such as presence of enzymes and extreme pH in
the internal organs like stomach. Besides that, hydrogels physical properties make them as good
candidates for drug carrier. For example, hydrogels porosity enable drug loading into gel network and
consequently drug release at desired site. Hydrogel has capable of exhibiting significant volume
changes in response to small changes in pH, temperature and other environmental stimuli.
Types of nanodelivery: natural or synthetic
Two types of polymers can be used in nanodelivery which is natural and synthetic. Natural polymers or
biopolymers may be naturally occurring materials which is formed in nature during the life cycles of
green plants, animals, bacteria and fungi are polymers or polymer matrix composites. Collagen while
synthetic polymers from the ester family. Table 11.1 shows example of natural and synthetic polymers
and Table 11.2 shows adavantages and disadvantages of natural and synthetic polymers
TABLE 11.1
Example of Natural and Synthetic Polymers
Natural Synthetic
cellulose, starch, chitosan, carrageenan, poly(lactic acid) (PLA), poly(cyanoacrylates)
aliginates, xantham gum, gellan gum, (PACA), poly(acrylic acid), poly(anhydrides),
pectins poly(amides), poly (ortho esters),
poly(ethylene glycol), and poly(vinyl
alcohol) (PVA) and other like
poly(isobutylcynoacrylate) (PIBCA),
poly(ethylene oxide) (PEO), poly(å-
caprolactone) (PCL)
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TABLE 11.2
Adavantages and Disadvantages of Natural and Synthetic Polymers
Natural polymers Synthetic polymers

 Less toxic Advantages  Biocompatibility
 Biocompatibility
 Biodegradable
 Easily available
 High degree of Disadvantages  Toxic

variability in natural  Non degrable
materials derived  Synthetic process is
from animal sources very complicated
 Structurally more and high cost
complex
 Extraction process
very complicated
and high cost
Starch
Starch is one example of natural polymers in nanodelivery. Starch is a polysaccharide. It occurs majorly
in plants where they act as storage materials. Chemically, it is composed of recurring units of
glycopyranose in an alpha D-(1, 4) linkage and on hydrolysis yields the monosaccharide, glucose. The
use of starch in pharmaceutics is broad. It is used as co-polymer and excipient in controlled drug
delivery as drug carriers, in tissue engineering scaffolds as hydrogels and as solubility enhancers.
Santander-Ortega et al.[8] investigated the potential of starch nanoparticles as a transdermal drug

delivery system (TDDS). The challenge faced in delivering drug through these systems is that the skin
acts as an effective barrier to drug passage and must therefore be overcome for effective drug delivery.
Nanoparticles were shown to facilitate drug delivery without interference to the skin’s integrity. The
method used to prepare the nanoparticles was emulsification-diffusion due to its reproducibility,
higher yields, ease of scale-up and control over size of particles and degree of polydispersity. Maize
starch modified and un-modified (by the addition of propyl groups) was used as polymeric material to
formulate 2 different types of nano-particles. The modified starch nano-particles were shown to be
non-toxic using LDH (Lactose dehydrogenase) and MTT assay and resulted in particles of uniform size
distribution while the nanoparticles formulated from the native starch was not observable.
Flufenamic acid, caffeine and testosterone were used as model drugs and their delivery across the skin
was analyzed using excised skin from female Caucasian patients who had undergone abdominal plastic
surgery. Permeation data obtained for caffeine and testosterone were similar for nano-encapsulated
and free drugs while the delivery of flufenamic acid using the nanoparticles was enhanced by about
ten-fold.
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Chitosan
Chitosan is another example of natural polymer. This polymer is obtained from the partial N-
deacetylation of chitin found in the shells of crustacean. It is composed of glucosamine and N-acetyl
glucosamine linked by β 1-4 glucosidic bonds and is one of the most widely studied natural polymers
for nano-drug delivery. The deacetylation of chitin is both concentration and temperature dependent
with optimal yields achieved at temperatures between 600C- 800C using 50%w/w alkali. Nano-particles
fabricated with chitosan as co-polymer was used to investigate the controlled release of anti-retroviral
drug, lamivudine.
The nano-particles were prepared by emulsion and solvent evaporation technique and characterised
using dynamic light scattering. The use of this method resulted in monodispersed particles with asize
range of 300-350nm. Two formulations with differences in percentage drug weight (3% and 6%) were
made, of which drug release rate was higher from the nano-particles with higher drug loading, though
both were able to control drug release fairly well. Drug release kinetics showed that the mechanism of
drug release was by diffusion. Conclusions reached suggested that the nano-particles could be applied
for gastrointestinal drug delivery because drug release was relatively slower at neutral pH compared to
acidic pH and also slower in the acidic pH compare to the alkaline pH.
Carrageenan
Kappa carrageenan is also a natural polymers that widely used in biomedical application currently. K-
carrageenan is a sulfated and linear polysaccharide with a repeating d-galactose and 3, 6-anhydro-d-
galactose units and is classically used as an agent for several potential pharmaceutical applications such
as gelling agent in the food and pharmaceutical industries and controlled drug release. Hezaveh
investigated the incorparation of metal nanoparticels such as Ag, MgO and M into kappa carrageenan
hydrogel matrix to sustain drug release and avoid burst release [9].
Hybrid approach
An inorganic-organic composite usually comprises an inorganic phase and a film forming organic phase.
A typical green approach to developing an inorganic-organic composite involves the selection of film
forming organic phase from starches having a degree of polymerization; degree of substitution and
viscosity such that the substituted starches are insoluble in water during mixing but dissolve at a higher
processing temperature during forming, setting or drying of the composite. Thus, excessive migration
of the starch is prevented and the composite is substantially strengthened. There has also been reports
on the lab-on-a-chip approach [13-18], which embodies micron- or nano-sized machines composed of
sophisticated circuits. Small devices have many advantages including portability/disposability, low cost,
high reproducibility, high-throughput screening, and multiple functionalities in a single device.
Recently, combined with other technologies such as optics, single molecular imaging, or cell/protein-
based assay systems, biomedical lab on a chip devices have become an important part of drug
discovery and diagnosis, but its application in drug delivery systems based on are just beginning to
appear. As rightly noted by several authors, to release a drug from a nanodevice is more complicated
than to perform assay or screening drug candidates, this is because, successful drug delivery requires at
least four components namely; drug reservoir, pump, valve, and sensor. Drugs can be placed either in a
fabricated reservoir or in conventional micro- /nanoparticles. Other important organic/inorganic
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composites are metal nanoparticles, such as silver, iron oxide, or gold nanoparticles, coated with
hydrophilic polymers. Their major application has been as theranostics. Only recently, Hirsch et al;
developed gold nanoshell, which provided tunable emission light for bioimaging. Importantly, is the
fact that, gold nanoparticles can be detected by X-ray and emit thermal energy by excitation making it
very useful for medical imaging and thermal therapy (theranostics).
In a related report, Corot et al; developed super paramagnetic iron oxide nanoparticles for magnetic
resonance imaging (MRI) of the whole body. Mechanistically, these nanoparticles are primarily
engulfed by monocyte or macrophage after intravenous administration. However, uptake of super
paramagnetic iron oxide by macrophage does not induce activation of nearby cells making it suitable
for diagnosis of inflammatory or degenerative diseases. Tao and Desai, 2005 had develop
microfabrication as controlled delivery devices. This device provide the capacity to target cells,
promote unidirectional controlled release, and enhance permeation across the intestinal epithelial
barrier. [19-21].
Factors affecting nanodelivery system
FIGURE 11.2
Factors Affecting Nanodelivery System
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Surface modification factor
Nanoparticles can be modified with targeting ligands that selectively recognize and bind to receptors
overexpressed on cancer cells. Examples of common ligands include the native ligand to a receptor
antagonists, peptides, aptamers, and antibodies or their fragments. Targeting ligands may exert their
own therapeutic effects, contributing to treatment efficacy beyond their role in targeting and
specificity.
In selecting an appropriate coupling chemistry, the goal is to achieve high coupling efficiency without
sacrificing binding activity and specificity of the ligand. Especially where chemical modifications are
made on assembled nanoparticles, reactions and processing conditions can disrupt micelle structure or
negatively impact drug activity. The required reagents, potential byproducts, temperature, solvent, and
necessary purification steps must all be given careful consideration. Ideally, the reaction should
proceed under mild conditions, in an aqueous environment, and require minimal post-processing. With
desired chemical functional groups in mind, polymers can be chosen or synthesized to provide
platforms for simple surface modification protocols.
By preserving binding activity, selective nanoparticle uptake by a target cell population is enabled
through receptormediated endocytosis. in vitro, actively targeted formulations have a clear advantage
over unmodified nanoparticles because greater cell uptake transports greater drug doses to their
intracellular targets. However, in vivo, functionalizing nanoparticles with targeting ligands often
reduces the longer-circulation achieved with PEGylation because the targeting ligands may trigger an
immune response. Nevertheless, if cellular uptake can compensate for reduced tumour uptake, overall
anti-cancer efficacy may improve. Nanoparticle internalization rates are likely a function of binding
strength, which depends on both the intrinsic ligand-target affinity and the ligand density. This further
adds to the debate over the optimal ligand conjugation density because increased uptake and
decreased circulation may be linked. One approach is to increase ligand mobility so that they can be
recruited to a common local area on the nanoparticle surface in the presence of target cells. In this
case, multivalent binding would exponentially increase binding strength through avidity without
requiring a high conjugation density.
Morphology and shape factor
Morphology and shape factor, especially particle size are the most important characteristics of
nanoparticle systems. They determine the in vivo distribution, biological fate, toxicity and the targeting
ability of nanoparticle systems. In addition, they can also influence the drug loading, drug release and
stability of nanoparticles. Many studies have demonstrated that nanoparticles of sub-micron size have
a number of advantages over microparticles as a drug delivery system. Generally nanoparticles have
relatively higher intracellular uptake compared to microparticles and available to a wider range of
biological targets due to their small size and relative mobility. Drug release is affected by particle size.
Smaller particles have larger surface area, therefore, most of the drug associated would be at or near
the particle surface, leading to fast drug release. Whereas, larger particles have large cores which allow
more drug to be encapsulated and slowly diffuse out. Smaller particles also have greater risk of
aggregation of particles during storage and transportation of nanoparticle dispersion. It is always a
challenge to formulate nanoparticles with the smallest size possible but maximum stability.
Polymer degradation can also be affected by the particle size. For instance, the rate of PLGA polymer
degradation was found to increase with increasing particle size in vitro. It was thought that in smaller
Nanomedicine 296
particles, degradation products of PLGA formed can diffuse out of the particles easily while in large
particles, degradationproducts are more likely remained within the polymer matrix for a longer period
to cause autocatalytic degradation of the polymer material. Therefore, it was hypothesized that larger
particles will contribute to faster polymer degradation as well as the drug release. Additionally,
nanoparticle geometry impacts transport properties: discs and rod-shaped nanocarriers have shown
improved blood circulation properties over spherical particles [19-21], leading to increased interest in
developing drug carriers that circulate a particular geometry and break into smaller nanocarriers for
improved tumour accumulation, penetration, and cell uptake. Shape also plays a role, where spherical
nanoparticles experience faster uptake than rod-shaped nanoparticles, likely due to changes in local
curvature or due to binding sites being blocked when the longitudinal edge of the rods are oriented
parallel to the cell membrane.
Loading NPs can change the surface morphology of nanocomposite hydrogel. The addition of
nanoparticles has changed the surface morphology of kappa carrageeanan in such a way that the
surface bulge is reduced, resulting in a flatter surface. Moreover, it seems that the porosity of
nanocomposite is increased. It is also clear that the MgO nanoparticles are finely dispersed in the
matrix and a uniform surface is produced. Comparing magnetic nanofillers with Ag nanofillers,
magnetic nanofillers formed more compact surface structure. Also, it is clear that nanofillers create a
uniform structure when synthesized in the blank matrix which was more obvious in magnetic
nanocomposites. Formation of NPs within the hydrogel network results in changing the porosity of
hydrogels which can also affect the MB release from nanocomposite hydrogels [9].
Drug release – novel targeted and controlled release
Drug release and polymer biodegradation are important factors to develop a successful
nanoparticulate system. In general, drug release rate depends on: (1) solubility of drug; (2) desorption
of the surface bound/ adsorbed drug; (3) drug diffusion through the nanoparticle matrix; (4)
nanoparticle matrix erosion/degradation; and (5) combination of erosion/diffusion process. Thus
solubility, diffusion and biodegradation of the matrix materials head the release process.
In the case of nanospheres, where the drug is uniformly distributed, the release occurs by diffusion or
erosion of the matrix under sink conditions. If the diffusion of the drug is faster than matrix erosion, the
mechanism of release is largely controlled by a diffusion process. The rapid initial release or ‘burst’ is
mainly attributed to weakly bound or adsorbed drug to the large surface of nanoparticles. It is evident
that the method of incorporation has an effect on release profile. If the drug is loaded by incorporation
method, the system has a relatively small burst effect and better sustained release characteristics. If
the nanoparticle is coated by polymer, the release is then controlled by diffusion of the drug from the
polymeric membrane. The membrane coating acts as a barrier to release, therefore, the solubility and
diffusivity of drug in polymer membrane becomes determining factor in drug release.
The thickness or geometry does not influence the drug release rate. Furthermore release rate can also
be affected by ionic interaction between the drug and addition of auxillary ingredients. When the drug
is involved in interaction with auxillary ingredients to form a less water soluble complex, then the drug
release can be very slow with almost no burst release effect; whereas if the addition of auxillary
ingredients e.g., addition of ethylene oxide-propylene oxide block copolymer (PEO-PPO) to chitosan,
reduces the interaction of the model drug bovine serum albumin (BSA) with the matrix material
(chitosan) due to competitive electrostatic interaction of PEO-PPO with chitosan, then an increase in
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drug release could be observed. Hezaveh used methylene blue (MB) as model drug to test the drug
release from nanocomposite hydrogel. It can be seen that by increasing the MgO content of
nanocomposites, MB release is significantly increased. By increasing NPs concentration from 0.1 g to
0.2 g, the maximum MB release increases from 0.174 to 0.267 mg/ml. Also, compared to blank
hydrogel, the addition of MgO NPs has increased the cumulative release up to 52%, which means that
more MB release is achieved [9].
Techniques available
Nanosuspensions
Nanosuspension refers to production of sub-micron-sized particles by subjecting the combination of

drug and a suitable emulsifier to the process of milling or high-pressure homogenization. Conventional
milling and precipitation processes generally result in particles with sizes that are much greater than 1
mm. As such, a critical step in the nanosuspension preparation is the choice of the manufacturing
procedure to ensure production of sub-micron particles. Nanosuspension formulations can be used to
improve the solubility of poorly soluble drugs. A large number of new drug candidates emerging from
drug discovery programs are water insoluble, and therefore poorly bioavailable, leading to abandoned
development efforts. These can now be rescued by formulating them into crystalline nanosuspensions.
Techniques such as media milling and high-pressure homogenization have been used commercially for
producing nanosuspensions. The unique features of nanosuspensions have enabled their use in various
dosage forms, including specialized delivery systems such as mucoadhesive hydrogels.
Nanosuspensions can be delivered by parenteral, peroral, ocular, and pulmonary routes. Currently,
efforts are being directed to extending their applications in site-specific drug delivery. Various particle
sizes of spironolactone, a model low solubility drug, have been formulated to yield micro- and
nanosuspensions of the type solid lipid nanoparticles and DissoCubes. The DissoCubes nanosuspension
yielded highly significant improvements in bioavailability. Particle size minimization is not the major
determining factor in the bioavailability improvement. Rather, the type of surfactant used as stabilizer
in the formulations is of greater importance. Improvement in drug solubility in the intestine as well as
in dissolution rate of spironolactone is the most likely mechanisms responsible for the observed effect,
although additional mechanisms such as permeability enhancement may also be involved.
Development of nanoparticle formulations for improved absorption of insoluble compounds and

macromolecules enables improved bioavailability and release. Particle size reduction to sizes below 1
mm is usually difficult due to possible particle aggregation and generation of high surface area
materials. Milling techniques that have been used to generate nano-sized particles are ball milling or
pearl milling that applies milling beads of sizes ranging from 0.4 to 3 mm and these beads may be
composed of glass, ceramics or plastics. The time required for milling depends on the hardness and
brittleness of the drug material in comparison to milling material and inertial forces set up within the
mill. Some of the challenges that milling processes can pose in drug development are
(i) undesirable erosion of the milling equipment components into the drug product;
(ii) the process is usually time consuming, thereby prolonging drug development time;
(iii) milling over a few days may bring the risk of microbiological problems or increases in
the cost of production; also
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(iv) prolonged milling may induce the formation of amorphous domains in crystalline
starting materials or may lead to changes in the polymorphic form of the drug.
The generation of amorphous form of the drug is problematic because these forms may crystallize
during the shelf life of the drug leading to changes in solubility and bioavailability of the drug. An
example of the conversion of crystalline to amorphous form of the drug was observed in jet milling of
albuterol sulfate. Also, the generation of highenergy surfaces that affected wettability was observed
with acetylsalicylic acid. Some examples of nano-sized particles produced by milling are
(i) naproxen nanoparticles approximately 200 nm in diameter and

(ii) danazol particles of a mean size of 169 nm.
There were four approved drug products in the USA that are based on NanoCrystal technology:
a) Rapamune (sirolimus) tablets by Wyeth;

b) Tricor (fenofibrate) tablets by Abbott;
c) Emend (aprepitant) capsules by Merck; and
d) Megace ES (megestrol) oral suspension by Par Pharmaceuticals
High pressure homogenization has also been recognized as an effective method of producing
nanosuspensions. Again, high-pressure homogenization has been applied commercially with the
development of some drug products, such as fenofibrate and paclitaxel. A typical procedure for
preparing nanosuspension involves, preparing an aqueous suspension of drug in surfactant solution,
this is then passed through a high pressure of typically 1500 bar at 3–20 homogenization cycles. The
suspension is then passed through a small gap in the homogenizer of typical width 25 mm at 1500 bar.
Due to built up cavitation forces that are created drug particles are broken down from micro to
nanoparticles. An example is in the micro fluidization of atovaquone to obtain particles in the 100–300
nm size range . It has been reported that, nanosuspension particles in most cases have an average size
ranging from 40 to 500 nm with a small (0.1%) proportion of particles larger than 5 mm. Experts have
recognized that, a major challenge in the use of high-pressure homogenization is the possible changes
in drug crystal structure that may cause batch-to-batch variation in crystallinity level, and have
suggested that, application in drug delivery should include the desired specification by which the
quality of each batch will be evaluated.
Polymeric nanoparticles
The polymeric nanoparticles (PNPs) are prepared from biocompatible and biodegradable polymers in
size between 10- 1000 nm where the drug is dissolved, entrapped, encapsulated or attached to a
nanoparticle matrix. Depending upon the method of preparation nanoparticles, nanospheres or
nanocapsules can be obtained. Nanocapsules are systems in which the drug is confined to a cavity
surrounded by a unique polymer membrane, while nanospheres are matrix systems in which the drug
is physically and uniformly dispersed. The field of polymer nanoparticles (PNPs) is quickly expanding
and playing an important role in a wide spectrum of areas ranging from electronics, photonics,
conducting materials, sensors, medicine, biotechnology, pollution control and environmental
technology. PNPs are promising vehicles for drug delivery by easy manipulation to prepare carriers with
the objective of delivering the drugs to specific target, such an advantage improves the drug safety.
Polymer based nanoparticles effectively carry drugs, proteins, and DNA to target cells and organs. Their
Nanomedicine 299
nanometer size promotes effective permeation through cell membranes and stability in the blood
stream. Polymers are very convenient materials for the manufacture of countless and varied molecular
designs that can be inte grated into unique nanoparticle constructs with many potential medical
applications.
FIGURE 11.3
Schematic representation of various techniques for the preparation of polymer nanoparticles [10]
PNPs can be conveniently prepared either from preformed polymers or by direct polymerization of
monomers using classical polymerization or polyreactions [11]. Methods like solvent evaporation,
salting-out, dialysis and supercritical fluid technology, involving the rapid expansion of a supercritical
solution or rapid expansion of a supercritical solution into liquid solvent, can be utilized for the
preparation of PNP from preformed polymers. On the other hand, PNPs can be directly synthesized by
the polymerization of monomers using various polymerization techniques such as micro-emulsion,
mini-emulsion, surfactant-free emulsion and interfacial polymerization. An illustration of different
preparation techniques for PNP is given in Figure 11.3. The choice of preparation method is made on
the basis of a number of factors such as the type of polymeric system, area of application, size
requirement and others [10].
Advantages of polymeric nanoparticles
 Increases the stability of any volatile pharmaceutical agents, easily and cheaply fabricated in
large quantities by a multitude of methods.
 They offer a significant improvement over traditional oral and intravenous methods of
administration in terms of efficiency and effectiveness.
 Delivers a higher concentration of pharmaceutical agent to a desired location.
 The choice of polymer and the ability to modify drug release from polymeric nanoparticles
have made them ideal candidates for cancer therapy, delivery of vaccines, contraceptives and
delivery of targeted antibiotics.
 Polymeric nanoparticles can be easily incorporated into other activities related to drug
delivery, such as tissue engineering.
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Mechanisms of drug release of polymeric nanoparticles the polymeric drug carriers deliver the drug at
the tissue site by any one of the three general physicochemical mechanisms. (1) By the swelling of the
polymer nanoparticles by hydration followed by release through diffusion. (2) By an enzymatic reaction
resulting in rupture or cleavage or degradation of the polymer at site of delivery, there by releasing the
drug from the entrapped inner core.(3) Dissociation of the drug from the polymer and its
deadsorption/release from the swelled nanoparticles [12].
Biodegradable nanoparticles have been used frequently as drug delivery vehicles due to its grand
bioavailability, better encapsulation, control release and less toxic properties.It also can be used as
carrier for gene delivery. The development of safe and efficient vectors or carriers for in vivo gene
transfer has been one of the key challenges in fulfilling the promises of gene therapy. Viral vectors,
while efficient in many gene transfer applications in vivo, pose safety concerns that are unlikely to
abate in the near future, rendering synthetic carriers attractive alternatives. The synthetic vectors
including cationic liposomes and polycations, while offering better safety profile, continue to suffer
from low gene transfer efficiency. Mechanistic studies are essential to identify the rate-limiting steps in
the non-viral gene transfer process. Controlled and systematic studies are needed in order to reveal the
structure-function relationship.
Hai-Quan Mao have designed and synthesized a series of biodegradable polyphosphoramidate (PPA)
gene carriers with the same backbone but different structural parameters (type and structure of charge
group, charge density, side chain spacer, etc.) in an effort to elucidate the structure-activity
relationship. The aim of this study is to investigate the structure-transfection efficiency relationship of
PPA gene carriers; and to investigate the effect of PPA structure on DNA compaction ability of PPA,
stability of PPA/DNA nanoparticles in physiological medium, cellular uptake efficiency, intracellular
trafficking, DNA unpacking, and nuclear translocation. More importantly, the biodistribution of
PPA/DNA nanoparticles and transport of nanoparticles in the liver will be correlated to the structure
and gene transfer efficiency of the PPA/DNA nanoparticles.
Poly (ethylene glycol) (PEG) has often been used to confer to these drug carriers the desired stability
during the extracellular delivery phase. The incorporation of PEG to lipo- or polyplexes has been proven
effective in reducing undesired effects such as immune response, unspecific interactions, and
degradation. PEGylation can be implemented by using PEGylated components in the initial complex
formation. Alternatively, PEG shielding can be applied to preformed complexes in a secondary
processing step by using either electrostatic self-assembly or chemical grafting. While PEGylation is a
necessity to improve extracellular stability and circulation half-life, it often decreases the transfection
efficiency due to reduced specificity and inhibited cell association and uptake.
Incorporating receptor targeting orusing bioresponsive linkers to release PEG have proven useful to
overcome these intracellular barriers to efficient delivery. Previous work with a copolymerprotected
gene vector (COPROG), consisting of a branched polyethylenimine (bPEI)/ DNA polyplex subsequently
shielded with a copolymer consisting of both PEG and anionic peptides (P6YE5C), showed the presence
of the copolymer, which provides steric stabilization, protection from opsonization, and allows freeze-
drying of the vector with little loss of activity. COPROG particles have proven to be effective gene
delivery vectors with decreased cellular toxicity without impairing gene transfer. The decreased toxicity
of COPROG is likely a result of the removal of unbound polycation by the excess anionic copolymer
emphasizing the potential role of binding stoichiometry in three-component complexes. Likely due to
their stabilizing and opsonization- inhibiting properties, COPROGs have proven advantageous in
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promoting the tranfection capacity of polyplexloaded sponges upon subcutaneous implantation, and
when colyophilized with fibrinogen, are a simple means to achieve an injectable fibrin gene-activated
matrix.
At the level of research, many synthetic DNA particles have been prepared for transfection in cell
cultures and in animal studies. However, several authors are of the opinion that, certain issues must be
addressed in the development of DNA particles with cationic polymers. These are
(i) potential toxicity of cationic polymers especially when administered at high concentrations;
(ii) instability of particles on storage;
(iii) instability of DNA particle size and particle size distribution leading to undesirable particle
aggregation;
(iv) poor transfection efficiency;
(v) poor stability in blood circulation; and
(vi) high cost of scaling up the process to achieve reproducible product quality.
Solid-lipid nanoparticles
Solid lipid nanoparticles (SLN) are particles made from solid lipids with mean diameters ranging
between 50–1000nm and represent an alternative to polymeric particulate carriers. The main
advantage offered by lipid carriers in drug delivery is the use of physiological lipids or lipid molecules
with a history of safe use in human medicine, which can decrease the danger of acute and chronic
toxicity. Sufficient data are available for the use of drug-loaded lipid nano and microparticles for oral
delivery, the main mechanism of lipid particulate materials translocation across the intestine being the
uptake via Peyer’s patches.
Research reported shows that SLN constituted of stearic acid and phosphatidylcholine were evidenced
in lymph and blood after duodenal administration to rats: the small diameters of SLN may facilitate
their uptake by the lymphatics. Up until today, only a few methods are described in the literature for
SLN preparation, including high pressure hot homogenization and cold homogenization techniques
microemulsion-based preparation and solvent emulsification/evaporation method. Particularly, the
emulsification/evaporation method concerns the preparation of nanoparticles dispersions from O/W
emulsions: the lipophilic material is dissolved in a water-immiscible organic solvent that is emulsified in
an aqueous phase.
Upon evaporation of the solvent, a nanoparticle dispersion is formed by precipitation of the lipid in the
aqueous medium. Depending on the composition and the concentration of the lipid in the organic
phase, very low particle sizes can be obtained, ranging from 30–100nm, but a clear disadvantage of this
method is the use of organic solvents, whose toxicity cannot always be neglected. Recently, an
emulsification–diffusion technique was developed using non-toxic and physiologicallycompatible
solvents and monoglycerides or waxes as components of the disperse phase of oil-in-water emulsions
obtained at 50 °C. The solvent-in-water emulsion–diffusion technique was before described in the
literature mostly for the obtainment of polymeric micro- and nanoparticles and only a few authors
proposed its application in the production of SLN.
According to the moderate water solubility of the solvents employed, the dilution of the emulsions
determined the diffusion of the organic solvent from the droplets to the continuous phase with the
Nanomedicine 302
consequent instant solidification of lipophilic material. The emulsion compositions and process
parameters used were the results of a formulative study aimed to develop optimized nanosphere
formulations, whose mean sizes were below 200nm. The possibility of incorporating a peptide drug
such as insulin in the SLN obtained with the developed method was considered, aiming to protect it
from chemical and enzymatic degradation, as it is well-known that the incorporation of peptides in
polymeric or non-polymeric particles should exert a certain protection of the drug against the
proteolytic enzymes present in the gastrointestinal tract. Indeed, the use of lipids as matrix materials
for sustained-release formulations for peptides and proteins has been reported only by few authors,
owing to the hydrophobic nature of the lipid matrix that can be more appropriate to incorporate
lipophilic drugs rather than hydrophilic proteins.
An adequately high solubility of the drug in the lipid melt is therefore the pre-requisite to obtain a
sufficient SLN loading capacity. Considering that the solubility of insulin in most commonly employed
solvents and lipids is quite low, a specific solvent medium of the peptide was required. Isobutyric acid,
a partially water-miscible solvent with low toxicity, revealed a totally unexpected high insulin-
solubilization capacity at 50 °C, further increasing when the solvent was water-saturated. Solid lipid
insulin-loaded microparticles were therefore produced using isobutyric acid as a solvent. Preliminary
analysis of microparticles content after processing showed an insulin-high encapsulation efficiency;
moreover, insulin in SLN did not undergo any chemical modification and its in vitro release from the
microparticles was very low, with an initial burst effect of 20% of the dose.
Of recent, SLN has become a popular drug delivery system for ophthalmic application. It is gaining
prominence as promising approach to improve the poor ocular bioavailability of biomolecules. In
particular, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), regarded as the first
and second generation of lipid nanoparticles are currently being applied. NLC was developed due by
combining the advantages of SLN and avoidance of their limitations such as low drug loading capacity,
poor long term stability and early drug expulsion caused by lipid polymorphism.(107, 108) NLC consist
of a mixture of spacially different solid and liquid lipids molecules, resulting in a structure with more
imperfections in crystal lattice to accommodate drugs. As drug delivery devices, NLC show great
promise for the eye, due to their better biocompatibility, modified drug release kinetics, reduction of
drug leakage during storage, avoidance of organic solvents during production process and feasibility of
large scale production.
To prepare particles using the homogenization method, the drug is dissolved or solubilized in the lipid
that has been melted and heated to a temperature approximately 5–10 °C above its melting point. For
the hot homogenization technique, the drug dissolved in the lipid melt is dispersed under stirring in a
hot aqueous surfactant solution of identical temperature. The obtained pre-emulsion is homogenized
to produce nanoemulsions that are subsequently cooled to room temperature. Solid lipid nanoparticles
are obtained upon lipid recrystallization at room temperature. Some of the process variables that will
affect the particle size of nanoparticles as well as drug loading are
(i) the type of homogenization technique;

(ii) speed of homogenization; and
(iii) rate of cooling in hot homogenization.
Cold homogenization is applied for highly temperature-sensitive drugs and hydrophilic drugs. For the
cold homogenization technique the drug containing lipid melt is cooled and ground to obtain lipid
Nanomedicine 303
particles. The lipid particles are dispersed in a cold surfactant solution that is homogenized at or below
room temperature. The process avoids or minimizes the melting of lipids and therefore minimizing the
loss of hydrophilic drugs to the water surface. Solid lipid nanoparticles can also be prepared by using
microemulsions as precursors.
Mechanism of nanodelivery inside human body

One of example that has been proposed by [9] was about the mechanism of alginate-enclosed
chitosan-calcium phosphate-iron-saturated bovine lactoferrin nanocarrier (AEC-CP-Fe-bLf NCs)
internalization and its action inside human body. From the figure, it shows that the alginate coating of
orally directed AEC-CP-Fe-bLf NCs is degraded in the alkaline environment offered in the small intestine
(A). Then, the alginate coating free C-CP-Fe-bLf NCs enter the blood circulation via endocytosis and/or
transcytosis (B). After that, C-CP-Fe-bLf NCs are released in the tumor site by making use of the
enhanced permeability retention effect(C). Finally, the uptake of C-CP-Fe-bLf NCs in to the cancer cells
is based on oligosaccharide and/or lactoferrin receptor-mediated endocytosis(D).
FIGURE 11.2
Example of mechanism of nanodelivery inside human body
Legend: AEC: Alginate-enclosed chitosan; C: Chitosan; CP: Calcium phosphate; Fe-bLf: Iron-saturated bovine
lactoferrin; NC:Nanocarrier [9]
Nanodelivery applications
Nanodelivery system has been applied previously in many applications for human body [10]. Figure
11.3 below shows a summary of nanodelivery application in the body.
Nanomedicine 304
NANODELIVERY
APPLICATIONS
FIGURE 11.3
A summary of nanodelivery applications in the body
Nanodrug delivery systems for anti-cancer agents
Many researchers have used different approaches and techniques for formulating nanoparticles for
anti-cancer agents. Some of these studies along with their prominent findings are mentioned here.
TABLE 11.3
Different types of anti-cancer agents and their mode of action against cancer cell
Hydrophobic properties Hydrophilic properties

Anti- Techniq Mode of Findings Anti- Techniques Mode of Findings
cancer ues actions cancer actions
agents agents
Paclitax Albumin natural help Pluron Incorporated with interact drastic
el -bound carrier of endothe ics doxorubicin and with multi- sensitizat
paclitaxe endogenous lial other anticancer drug ion of
l (ABI- hydrophobic transcyt agents resitance cancer
007, molecules osis of (MDR) cell
Abraxan such as protein- cancer cells
e®; vitamins, bound
Abraxis hormones and
BioScien and other unboun
ce and water- d
AstraZen insoluble plasma
eca). plasma constitu
substances. ents
stabilize the through inclusion of improve
Nanomedicine 305
drug particle binding paclitaxel in the

and prevents to a cell- liposomal drug’s
any risk of surface formulations (LEP- antitumo
capillary ETU) r efficacy
obstruction
and does not
require any
specific
infusion
systems or
steroid/antih
istamine
premedicatio
n before the
infusion.
Albumin colloidal higher Endost 20 kDa internal inhibit the unstable
-bound suspension penetra atin fragment of the growth of a or
paclitaxe derived from tion into carboxy terminus variety of expensiv
l ABI-007 the tumor of collagen XVIII human e, thus,
lyophilized cells tumors by limits
formulation with an inhibiting their
of paclitaxel increase neovascular clinical
and human d anti- ization applicatio
serum tumor n
albumin activity,
diluted in compar
saline. ed with
an equal
dose of
standar
d
paclitax
el
maximu Endost novel recombinant inhibit approved
m ar human endostatin endothelial by the
tolerate cell Chinese
d dose proliferatio State
of ABI- n, Food and
007 on migration, Drug
patients and vessel Administr
with formation ation for
solid the
tumors treatmen
and t of non-
breast small cell
cancer lung
cancer in
2005 and
has a
broad
spectrum
of activity
Nanomedicine 306
against
solid
tumors
antitum endostar-loaded maintain improvin
or PEG-PLGA adequate g its
activity nanoparticles concentrati antitumo
in ons of reffect
patients endostar in and
with plasma and better
metasta tumor anticance
tic r effect
breast
cancer,
with a
good
overall
respons
e rate
and less
side
effects
micellar Paclitaxel NK105 caused
nanopar was increase slower
ticle incorporated d growth of
formulat into the plasma tumor
ion of inner core of AUC cell
paclitaxe the micelle xenograft
l (NK105 system by s, and
physical prolonge
entrapment d tumor
through doubling
hydrophobic time.
interactions potent CPX-1 novel liposome- prolong in well
between the antitum encapsulated vitro tolerated,
drug and the or formulation of optimized and had
block activity irinotecan and synergistic significan
copolymers against floxuridine molar t
for paclitaxel a ratios of antitumo
human both drugs r activity
colorect following
al infusion
cancer
cell line
HT-29
xenogra
ft
decreas MCC- immunoliposomee positively cytotoxic
ed in 465 ncapsulated reacts to activity
neuroto doxorubicin tagged >90% of against
xicity with polyethylene cancerous several
Osteon bind present in facilitat glycol (PEG) and stomach human
ectin albumin some e intra- the F(ab) fragment tissues but stomach
Nanomedicine 307
(secret neoplasms tumor of human mAb negatively cancer

ed (breast, lung, accumul GAH (goat anti- to all cells
protein and prostate ation of human) normal compare
acid cancer), albumin tissues. d with
rich in leading to -bound doxorubi
cystein the drugs cin or
e accumulatio doxorubi
(SPARC n of albumin cin-
)) in some incorpora
tumors ted PEG
liposome
s
Other than above agents, polymeric micelles also can be utilized to increase the accumulation of drugs
in tumor tissues utilizing the enhanced permeability and retention (EPR) effect and to incorporate
various kinds of drugs into the inner core by chemical conjugation or physical entrapment with
relatively high stability. There are several anticancer drug-incorporated micelle carrier systems under
clinical evaluation, these include;
FIGURE 11.4
Several anticancer drug-incorporated micelle carrier systems
Tumor-specific targeting with nanocarriers
Tumors have different features from normal tissues. They can be either leaky tumor blood vessels or
defective lymphatic drainage, which promotes the delivery and retention of particles called enhancing
permeability and retention (EPR) effect. Nanoformulation, one of technique reported by researchers
can settle this problem by entering and accumulate within tumor cells. This technique can deliver
higher doses of the drug, thus, increasing its anticancer effects besides decreasing the side effects
associated. However, by using this technique, there are many variable factors may affect the delivery of
drug like clearance of nanoparticles by kidneys and uptake by reticuloendothelial cells, which at the
end will retain anticancer nanoparticles in tumor. To overcome these problems, targeted drug delivery
Nanomedicine 308
was introduced. Targeted delivery of therapeutic agents penetrated into cancer cell has important
roles for detection, diagnosis and therapy of cancer. Biomarkers, one of targeted delivery were applied
to differentiate between cancerous tissue and normal tissues. One example of biomarker is ligands.
Ligands were applied by studies for tumor-specific targeting. There are many types of ligands employed
to serve this purposes (see Table 11.4 below);
TABLE 11.4
Ligands employed for tumor-specific targeting and its function
Types of ligands Functions

Folate  nonimmunogenic
Folate nanoparticles  involved in human growth and
development, cell division and
DNA synthesis
Folate-mediated targeting  used to deliver protein toxins,
low-molecular weight
chemotherapeutic agents,
liposomes containing
chemotherapeutic drugs and
immunotherapeutic agents to
cancer cells
Folate-conjugated  used on human cervical
nanoparticles carcinoma cells
Transferrin  essential role in iron
homeostasis and cell growth
Transferrin receptor  initiates receptor mediated
endocytosis and internalization
of transferrin
Transferrin mediated targeting  enhancement of anticancer
activity
Transferrin conjugated  enhance the antitumor activity
nanoparticles and also contributes to the
photo stability and sustain
release of drug
Vasoactive intestinal peptide  angiogenesis
receptors (VIP-R)
Polymer-conjugated  inhibits hyperpermeability of
angiogenesis inhibitor TNP-470 tumor blood vessels
(caplostatin)
Integrin avb3  used targeting moiety on
nanovectors
PLGA nanoparticles  for delivering natural products
like curcumin, that significantly
reported has anti-cancer effects.
Chitosan nanoparticles  inhibition of tumor growth and
induction of tumor necrosis
Nanomedicine 309
One significant benefit of tumor therapy with nanoparticles as a drug carrier is to prolong the duration
of the drug in the body. This will significantly increase the exposure of the tumor to the
chemotherapeutic drug agent, and also prolongs the exposure of the remainder of the body to the
drug. Active targeting of tumor tissues can be achieved by chemically arraying ligands on the surface of
nanoparticles that can recognize and selectively bind to receptors specifically expressed on tumor cells
and vessels. The high surface area to volume ratio of the nanoparticles leads to high local density of
ligands for targeting. Using high-affinity ligands for these transporters along with nanoparticles can
lead to site-directed delivery of drugs.
Physiological system specific nano-delivery
Nano-scale drug-delivery systems are being created in order to regulate the sustained release
especially in pharmacokinetics, pharmacodynamics, solubility, immunocompatibility, cellular uptake,
biodistribution and to minimize toxic side effects, thus enhancing therapeutic impact on traditional
pharmaceuticals. Nanoparticle mediated drug delivery, has potentially contribute to improve the drug
development process which has relied on conventional formulation strategies that are often
inadequate. An underlying concept in drug development process is to establish a link between in vitro
potency, physicochemical properties and absorption, distribution, metabolism, excretion and toxicity
characteristics of a drug candidate which is often cited as a major contributing factor in the failure of
drug functional. Meanwhile, the nanoparticle mediated sustained release of drugs offers an obvious
therapeutic advantage. The targeted delivery of drugs in the body is required to prevent the release of
therapeutics at non-specific sites as well as to protect from any unwanted side effects. Table 11.5
below shows examples of nanodelivery drugs that have been tested by researchers in physiological
systems.
Nanomedicine 310
TABLE 11.5
Physiological system with specific example of nano-delivery drug
Physiological Examples of nanodelivery drugs

systems
Central nervous Amitriptyline, polybutyl-cyanoacrylate nanoparticles coated
system (CNS) with polysorbate-80, dalagrin, kytorphin, neuromuscular
blocking agent tubocurarine, GLUT1 transporter, choline
transporter, insulin, transferrin, beta-endorphin peptides,
OX26, Doxorubicin, Transferrin-liposomes, folates,
Doxorubicin-loaded folic acid-PEG-PLGA micelles, Paclitaxel-
loaded PCL/MPEG micelles
decorated with folic acid, Cationized bovin serum albumin
(CBSA), Polysorbate 80-coated atovaquone-loaded SLN,
dipalmitoylated apoE-derived peptides, camphotericin,
dalargin, diminazene diaceturate, paclitaxel
Pulmonary system Beclomethasone dipropionate loaded polymeric micelles,
liposomes, synthetic lung surfactant Alveofact®, Liposomal
aerosols, non-phospholipid vesicles loaded with
beclomethasone dipropionate, Levonorgestrel encapsulated
liposomes, Liposomes modified with cell-penetrating peptides,
antennapedia, the HIV-1 transcriptional activator, and
octaarginine, Liposomes of EYPC-cholesterol (CHOL)
incorporating dexamethasone palmitate (DEXP), prednisolone,
diazepam, camptotecin, rifampicin, isoniazid, pyrazinamide,
low molecular weight heparin (LMWH)–dendrimer complex,
pegylated dendrimers (mPEG–dendrimer), Pulmospheres™, 9-
nitrocamptothecin (9NC) encapsulated into DLPC liposomes,
Lectins, Mucoadhesive nanoparticles coated with
mucoadhesive polymers, Poly-lactide-co-glycolide (PLGA),
alginate and solid lipid nanoparticles
Cardiovascular Resveratrol-loaded nanoparticles [22], micelles with a clot-
systems (CVS) binding peptide, cysteine-arginineglutamic acid-lysine-alanine
(CREKA), magnetofluorescent nanoparticles, paramagnetic
liquid perfluorocarbon nanoparticles incorporated a
peptidomimetic victronectin antagonist, modified chitosan
nanoparticles with a peptide targeting ligand [23],
Nanoparticle mediated antiretroviral therapy
One of the biggest global threats today is Acquired Immunodeficiency Syndrome, also known as AIDS.
AIDS is the disease presented with lack of treatment. Despite on standard therapy reported, AIDS
couldn’t be treated with any treatment available in the world. The current clinical therapy called ‘highly
active antiretroviral treatment’ (HAART), has made significantly contribute to reducing mortality rate.
However, it should be noted that HAART is not effective method of treatment since it can bring back a
few negative effects to the patient and yet, the drug applied also has a limitation like poor drug
stability under gastric condition. Owing to this, nanodelivery drug was introduced in order to improve
Nanomedicine 311
drug release and prevent drug limitation. Nanoparticles can provide a target specific and sustained
release of these drugs, thus improving their bioavailabilty and protect patient from associated side
effects. Examples of nanoparticle drugs used for AIDS therapy are summarized in the table below;
TABLE 11.6
Examples of nanoparticle drugs used for AIDS therapy and its functions in summary
Examples Functions
Poly (isohexyl cyanate) nanoparticles of for targeting the lymphoid tissue in the
zidovudine gastrointestinal tract
Polyhexylcyanoacrylate nanoparticles for the delivery of zidovudine thus improving its
bioavailability
Zidovudine-loaded poly(isohexyl cyanate) Accumulated in the cells of the reticuloendothelial
nanoparticles system
Poly(epsilon-caprolactone) nanoparticles for targeting the phagocytic mononuclear system
loaded with saquinavir
Stavudine, zidovudine and lamivudine for brain targeting
entrapped in polybutylcyanoacrylate (PBCA)
and
methylmethacrylatesulfopropylmethacrylate
(MMA-SPM) nanoparticles
Dendrimers deliver antiretroviral drugs
Tuftsinconjugated poly(propyleneimine) for targeted delivery to macrophages and
dendrimers loaded with efavirenz enhanced cellular uptake by mononuclear
phagocytic cells
Stavudine loaded into mannosylated and greater cellular uptake by cells of the mononuclear
galactosylated liposomes phagocytic system and greater accumulation in
organs of the reticuloendothelial system
PLGA nanoparticles containing ritonavir, increased uptake of the drugs by macrophages
lopinavir and efavirenz
PHCA nanoparticles containing zidovudine higher drug concentration in the organs of the
reticuloendothelial system
PPI dendrimer-based nanocontainers for targeting of efavirenz macrophages
Conclusions
Nanodelivery systems such as nanosuspensions, polymeric nanoparticles, and solid-lipid nanoparticles,
provide a broad range of techniques and strategies that can optimize the delivery of drug into the
targeted cell. They were very well functioning in releasing the drugs inside human body as well as
manage the time to protect the apopotosis cancer cell, increase the bioavailability and biocompatibility
of potential therapy. Furthermore, the carcinogenicity of the drugs might also be detected in early
stage before releasing to upscale. Moreover, the use of nanodelivery can enhance the physiologically
and specific site of targeted cell. Hence, it may conclude that, nanodelivery drug can be applied at all
stages of drug development, from formulations until therapeutic applications for optimal delivery in
clinical trials.
Nanomedicine 312
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12
Surface modification of metallic implants
with anodic oxide nanotubular arrays via
electrochemical anodization techniques
1* 1
Lu-Ning Wang , Ming Jin
1
School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, China
Outline:
Introduction ……………………………………………….……………………………………………………………………………….314
Fabrication of nanotubular arrays on metals via electrochemical anodization …………………………… 315
Self-Ordering tio2 nanotubular Arrays ………………………………………………………………………………………… 318
Oxide nanotubular arrays on titanium alloys and other metals ………………………………………………….. 320
Biocompatibility approach of metals with nanotubular surfaces ………………………………………………… 324
Hydroxyapatite formation on nanotubular arrays ………………………………………………………………………. 324
In vitro and in vivo studies of nanotubular arrays ………………………………………………………………………..325
Nanotubular arrays for drug delivery and other preloads application ………………………………………… 327
Conclusion and other aspects …………………………………………………………………………………………………….. 327
References……………………………………………………………………………………..…………………………………………… 329
Nanomedicine 314
Introduction
Implantable biomaterials have been extensively applied to treat hard tissue disorders. It has been
reported that 4.5 million Americans are living with artificial knees, including an estimated 500,000
who have had at least two replacement operations on the same knee [1, 2]. In 2011 alone, more
than 600,000 knee replacements were performed in the U.S [1, 3]. The basic property requirements
for materials used in orthopaedic and orthodontic application are mechanically strong and these
materials must be possessing high resistance to corrosion and wear to prevent weakening of the
mechanical properties and the release of toxicity species or debris in physiological culture. In
addition, materials for implant must provide the ability to be machined with complex shapes.
Specifically important, implantable materials must exhibit biocompatibility to avoid adverse
biological response and to minimize allergic immune reactions [4]. Materials are also required to
form a firm and lasting interface with bone in order to survive and function properly. Metals,
ceramics and polymers are three major classes of materials adopted in biomaterials [5-8]. Among
them, biologically compatible metals best satisfy the requirements for implants. The most widely
used ones are surgical stainless steels (such as 316L and 317) [9, 10], Ti and its alloys (such as cp-Ti
and Ti6Al4V) [11, 12], CoCr alloys (such as CoCrMo and CoCrNiMo) [13] and Ta [14]. Surgical
stainless steels are mainly used for temporary implants since the immune system reaction to nickel
is a potential complication [10]. The use of Ti and its alloys is owing to their high specific density
and corrosion resistance. Additionally, Ti alloys with relatively low moduli than other metals have
better match with the modulus of bone [11, 12]. Pure Ti and Ti6Al4V are the prevalent metals used
for orthopedic and orthodontic applications. Because of the concern over the potential toxicity of V
and mutagenicity of Al, new Ti alloys were also developed by substituting Al and V with other less
toxic elements such as Nb, Ta and Zr (Ti-Nb-Ta-Zr) [15, 16]. CoCr alloys are encountered in the
application of artificial knee and hip joint owing to the higher strength, excellent corrosion and
wear resistance. However, their high stiffness causes the adjacent bone to be stress-shielded and
results in disuse atrophy [13]. Ta is considered as the most corrosion resistant metal and is being
exploited to create a highly porous form that favors bone ingrowth and achieve implant fixation
[14].
When exposed to physiological culture after the surgical injury, metallic implants are able to form
stable and compact oxide layers such as Cr2O3 (for stainless steels and CoCr alloys) [9, 13], TiO 2 (for
Ti and its alloys) [11] and Ta2O5 (for tantalum) [14]. These layers insulate the reactive underlying
metal from the surrounding environments and prevent the transmission of undesirable ions.
Moreover, the existing of oxide layers also makes metal materials bioinert, resulting in fibrous
capsules to surround implants *17+. These fibrous tissues forms due to the body’s protection
mechanism against any materials recognized as foreign. On the other hand, bone generation
competes with the rejecting response [18]. Osteoblasts are differentiated from progenitor cells,
migrating to the implant site, and secrete collagen to mineralize into new bone [8, 19]. The
interaction between bone and implant is crucial to determine the performance and life span of the
implant. An immediate bone-implant contact is highly desired to secure the mechanical stability.
On the contrary, the formation of fibrous tissues retards the contact between bone and implants,
resulting in a weak mechanical bonding, which can cause implant to loosen and is susceptible to a
failure implantation [20].
Current implantable metals typically develop a thin layer of fibrous tissue at the interface with
bone. The existing of this thin layer becomes a major challenge to decelerate the process of
osseointegration and to extend the implant fixation time. Fast fixation is critical for the success rate
of implantation and can reduce the micro-motion of implant and minimize the formation of fibrous
Nanomedicine 315
tissue, resulting in early physiologic loading and preventing the bone from disuse atrophy [20]. In
addition, fast fixation of implant reduces the hospitalization time, cost, and improves the quality of
life for patients. As a result, many attempts have been made to improve the interaction between
bone and implants. One of the most actively pursued areas is the development of novel surfaces by
modification techniques to improve the implants’ surface properties and facilitate faster
osseointegration and healing process [20].
Basic bone composition consists of mostly fibrous protein collagen, carbonated apatite [Ca5(PO4,
CO3)3(OH)), CAP] and water [21, 22]. Some previous studies indicated that bone contains many
different structures and is highly porous on the micrometer scale [23-25]. A current strategy is to
consider that natural bone is a nanostructured material [26]. The type I collagen, which is the
organic matrix of bone, has a triple helix structure with 300 nm in length, 0.5 nm in width and
periodicity of 67 nm [26]. CAP (~70 wt% of the bone is CAP) is the inorganic mineral phase of bone
with about 20-40 nm length and is uniquely patterned within the collagen network [27-29].
Considering the geometric factors of collagen and CAP, bony cell may be used to an environment in
nanoscale rather than microscale. Thus proper nano-scale surface modification methods on
metallic implant are highly desired to achieve better and rapid bonding to bone.
An electrochemical technique known as anodization or anodic oxidation is a well-established
surface modification approach for metals to produce protective layers [30]. It has been successfully
applied as a surface treatment for orthopedic implants in the past few decades and it has some
new advances on fabrication of nanostructured surface in recent years [31-35]. Particularly, self-
organized nanotubular oxide structure can be easily formed and controlled by varying the anodic
conditions [33-37]. This type of self-aligned nanotubular structure has attracted more interests
than others over the past 10 years. More than 3,000 papers related to this topic have been
published over the past 5 years [38]. Since Ti-based metals have been paid more attention and
represent an attractive model system for exploring this nanotechnology to create more effective
implantable devices, self-assembled TiO2 nanotubular layer can be easily fabricated on Ti implants
to satisfy requirement for biomedical application [39, 40]. It is further remarkable that the self-
ordering anodization approach is not only limited to Ti and Ti-based alloys but can be applied to a
large range of other transition metals or alloys to form highly ordered nanoporous or nanotubular
oxide layers for potential biomedical application [41-45]. For these reasons, this review focuses on
up-to-date research that describes the synthesis of these nanotubular structures and the factors
that influence the degree of self-ordering, tubular geometry and crystal structure. We will also
focus on the biocompatibility and physiological responses of these nanotubular layers on titanium
and other valve metals, which are pertinent for orthopedic application. The final section
summarizes the main points of this chapter and provides perspectives for future work in this field.
Fabrication of nanotubular arrays on metals via electrochemical

anodization
Electrochemical anodization has been used to fabricate a thick and uniform oxide layers on metals
(normally named valve metals) for almost several decades. Most recently, it has been established
that self-organized nanoporous and nanotubular oxide layer can be grown on suitable metals [31,
32, 34, 46-50]. When most of valve metals (M as a representative symbol) expose to an anodic
voltage in an electrochemical configuration as shown in Figure 12.1 [31, 32, 51], an oxidation
n+ - n+
reaction will be initiated at metal-oxide interface as M → M + ne and the M ions migrate
Nanomedicine 316
2-
outwards under the applied field. At the same time, O ions, provided by H2O in the electrolyte,
n+
migrate towards the metal-oxide interface, react with M and form a compact metal-oxide (MO)
film. The anodization system is normally under a constant applied voltage. As the MO has higher
resistivity than the electrolyte and the substrate, the applied filed within MOs is progressively
reduced by the increasing oxide thickness. Although the oxide film will keep growing as long as the
applied field is strong enough to drive the ion conduction through the oxide, the process is
continuously slowing down resulting in a finite thickness of MO film. Under particular experimental
conditions, a growing of porous MO layer takes place. Furthermore, under even more specific
conditions, self-assembled nanoporous and nanotubular layers can be achieved.
FIGURE 12.1
(a) Mechanism of Oxide formation on valve metals. (b) Various morphologies obtained by electrochemical
anodization of valve metals - a compact oxide film, a disordered porous oxide layer, a self-ordered nanoporous
or a self-ordered nanotube layer (Redraw from Ghicov and Schmuki, Chem. Commun., 2009, 2791–2808).
Copyright © 2009. Reproduced by permission of the Royal Society of Chemistry, from Ghicov A, Schmuki P.
Self-ordering electrochemistry: a review on growth and functionality of TiO2 nanotubes and other self-aligned
MOx structures. Chem Commun (Camb). 2009;(20):2791–2808.
Masuda et al. firstly demonstrated that a self-organized nanoporous oxide layer could be fabricated
on aluminum in oxalic acid under specific voltage conditions [52]. This remarkable work has been
considered a milestone on anodization of metal and triggered hundreds of papers dealing with the
fabrication, modification and application of nanoporous alumina [53-55]. The as-formed
nanoporous alumina was used as photonic crystals and template for nanomaterials synthesis [56-
60]. Accordingly, several models have been put forward to explain the growth mechanism of the
self-organized alumina nanoporous layers [31, 32]. A description can be explained by schematic
steps showing in Figure 12.2 [31].
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FIGURE 12.2
Schematic representation of alumina pore formation by electrochemical anodization: (a) formation of the
anodic oxide on aluminium; (b) local field distribution correlated to the surface morphological fluctuations; (c)
initiation of the pore growth due to the field-enhanced dissolution; (d) pore growth in steady-state conditions;
(e) represents the current transient recorded during anodisation of Al; (f) and (g) show the influence of the
volume expansion and the local acidity on the alumina pore growth, respectively (Redraw from Ghicov and
Schmuki, Chem. Commun., 2009, 2791–2808). Copyright © 2009. Reproduced by permission of the Royal
Society of Chemistry, from Ghicov A, Schmuki P. Self-ordering electrochemistry: a review on growth and
functionality of TiO2 nanotubes and other self-aligned MOx structures. Chem Commun (Camb).
2009;(20):2791–2808. (a) Copyright © 2006 with permission from Elsevier. Reprinted from Bauer S, Kleber S, Schmuki P.
TiO2 nanotubes: Tailoring the geometry in H3PO4/HF electrolytes. Electrochem Commun. 2006;8(8): 1321–1325. (b)
Copyright Wiley-VCH Verlag GmbH & Co. KGaA. © 2005. Reproduced with permission, from Macák JM, Tsuchiya H, Schmuki
P. High-aspect-ratio TiO2 nanotubes by anodization of titanium. Angew Chem Int Ed Engl. 2005; (14): 2100–2102. (c)
Copyright © 2007 Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission, from Albu SP, Ghicov A, Macak JM,
Schmuki P. 250 μm long anodic TiO2 nanotubes with hexagonal self-ordering. Phys Status Solidi Rapid Res Lett.
2007;1(2):R65–R67. (c) Copyright © 2007, American Chemical Society. Reprinted with permission, from Paulose M,
Prakasam HE, Varghese OK, et al. TiO2 Nanotube Arrays of 1000 Length by Anodization of Titanium Foil: Phenol Red
Diffusion. J Phys Chem C. 2007;111(41):14992–14997. (e) Copyright Wiley-VCH Verlag GmbH & Co. KGaA. © 2005. Adapted
with permission, from Macák JM, Tsuchiya H, Schmuki P. High-aspect-ratio TiO2 nanotubes by anodization of titanium.
Angew Chem Int Ed Engl. 2005;44(14): 2100–2102. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. © 2011. Adapted with
permission, from Roy P, Berger S, Schmuki P. TiO2 Nanotubes: Synthesis and Applications. Angew Chem Int Ed Engl. 2011;50
(13):2904–2939. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. © 2005. Adapted with permission, from Macak JM,
Tsuchiya H, Taveira L, Aldabergerova S, Schmuki P. Smooth anodic TiO2 nanotubes. Angew Chem Int Ed. 2005;44:7463–
7465.69 Copyright © 2008, with permission of Elsevier. Adapted from Macak JM, Hildebrand H, Marten-Jahns U, Schmuki P.
Mechanistic aspects and growth of large diameter self-organized TiO2 nanotubes. J Electroanal Chem. 2008;621:254–266.70.
Briefly, as a result of the onset of electrochemical anodization in acidic condition, the surface of
aluminum is covered entirely by a compact, uniform anodic alumina oxide layer (Figure 12.2a).
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Since the surface of oxide layers fluctuates at the microscopic level, the distribution of electric field
in the oxide layer is non-uniform, resulting in focused electric field at some certain place, as shown
in Figure 12.2b. Consequently, field-enhanced dissolution in the anodic oxide takes place and the
nanopores start to form (Figure 12.2c). Successively, the nanopore growth process reaches a
steady-state and uniformly distributed pores are obtained (Figure 12.2d). Additionally, the self-
ordering of nanoporous alumina layers is also contributed by the stress at the metal-oxide interface
owing to volume expansion or electrostriction, repulsion of electric fields, or stabling maximum
current-flow conditions. Many of the mechanisms for self-organized nanoporous alumina layers can
be transferred to the formation of self-ordering nanopores and nanotubular layers on other metals
such as Ti, Zr, Ta, etc [31, 38, 46]. However, for these metals, in contrast to aluminum, an acidic
condition (or a low pH condition) is not sufficient to create self-ordering porous metallic oxide
layers but only to form a compact oxide layer [30-32, 34]. In order to form self-ordering nanopores
-
and nanotubular oxide layer, the existing of fluoride ions (F ) in electrolyte is strictly desired [30, 34,
37]. A key feature of the F- ions is that it is able to form water soluble metal-fluoride complexes.
The complex formation aids the prevention of MO layer formation at the tubular bottom, but this
also leads to mild but permanent chemical dissolution of the formed MO. Another important factor
- 2-
is that F ions are very small and compete with O migration through the oxide layer [31, 32, 34]. It
2-
has been observed that F- ions may migrate at a rate twice as high as O ions through oxide lattices
[31, 32, 42]. As a result, a fluoride rice layer is formed at the metal-oxide interface. This layer is
believed to be the origin of the nanotubular separation and formation. Several excellent reviews
have well explained the formation mechanism of MO nanotubular arrays by means of
electrochemical anodization. This section, therefore, will only give a brief summary of the
formation of some MO nanotubular arrays under various conditions [31, 32, 34].
Self-Ordering TiO2 nanotubular Arrays
The very first paper regarding the formation of porous TiO 2 oxide layer on Ti via electrochemical
anodization in F- containing electrolyte was reported by Kelly in 1979 [61]. However, owing to the
insufficient information of surface morphology by electronic microscopy, it was difficult to observe
the self-ordering TiO2 nanoporous arrays from their work, resulting relative low citation by other
researchers. It is well accepted that the formation of self-ordering TiO2 nanoporous structure by
anodization in fluoride containing chromic acid was reported by Zwilling et al. in 1999 [62]. They
pointed out that a small amount of fluoride ions in the electrolyte is the key form self-ordering TiO2
nanoporous structure. Following this pioneer work, several research groups have carried out
extensive work on optimization of the anodization conditions to develop self-ordering nanotubular
arrays [31, 34].
Anodization to form tube layers is usually carried out by ramping a potential step at a constant
voltage normally between 1-30 V in aqueous electrolytes or 5 - 150 V in non-aqueous electrolytes
containing approximately 0.05 M - 0.5 M fluoride ions [35, 37, 41, 42]. Crucial factors on fabrication
of TiO2 nanotubular arrays are considered to be applied potential, fluoride concentration, pH value
and anodization duration [31, 34]. In general, the nanotubular diameter is reported to be linearly
dependent on the applied anodic potential during growth [31-34]. Yasuda et al. found out that the
diameter of TiO2 nanotubes correlate linearly with the growth factor, f growth, of the Ti, where fgrowth
-1
is growth factor and is 2.5 nm·V for TiO2 (fgrowth being fgrowth = tfilm/U, tfilm being the compact oxide
thickness that grows at a specific potential in Ti) [34]. By assuming that anodic oxide growth begins
from a local oxide breakdown site or a point source on the Ti surface, the oxide growth would take
immediately in all directions leading to a hemispherical oxide structure with a certain radius R =
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fgrowthU. Based on this estimation, TiO2 nanotubular diameters from 5 to about 700 nm can be
achieved in the anodization potential range that has been listed above [31]. Typically at the early
stage of growth, the TiO2 nanotubular length is controlled by the applied electric field and as a
result, the nanotubular thickness is proportional to the applied voltage. Under the constant voltage
U, the electric field is defined as F = U/d, where d is the nanotubular thickness. The electric field
keeps dropping constantly as the d increases, thus lowering the driving force for solid-state ion
4+
(such as Ti ) migration [34]. The result is an exponential drop in the anodic current with time as
shown in Figure 12.2e until the electric field effect is lost. At this point, a practically finite thickness
is reached that mainly depends on the anodization voltage. The presence of fluorides ions strongly
affects the anodization and self-ordering of TiO2 nanotubular arrays. If the fluoride concentration is
very low, normally ≤ 0.05 wt.%, a stable compact TiO2 layer is formed after anodization.
4+
Meanwhile, a high content of fluoride (≥ 1.5 wt.%) results in no oxide formation, as all the Ti
2-
formed immediately reacts with the a drastic amount of fluoride to form soluble [TiF 6] , which is
similar to an electropolishing process [35]. For fluoride content within the intermediate level, 0.05
– 1.5 wt.%, a competition between TiO2 formation and dissolution take place and nanotubular
arrays formation can be observed [31, 32, 34]. Since the dissolution of TiO2 highly depends on
fluoride concentration, the elevation of fluoride content in the anodization electrolyte can lead to a
long tube with large diameter.
When the anodization of titanium is carried out in aqueous electrolyte, most of the composition of
fluoride species is in HF form. In acidic condition such as H 3PO4 and H2SO4, a maximum length of
~500 nm with about 140 nm diameter TiO 2 nanotubular arrays can be obtained under optimum
conditions (Figure 12.3a) [31]. Longer nanotubular arrays (> 1 m) can be formed in buffered
aqueous electrolyte [(NH4)2SO4 + NH4F and Na2SO4 + NaF] (Figure 12.3b). Such neutral or near
neutral electrolyte has less acidity with less dissolution capability on TiO 2, TiO2 nanotubes with
diameter about 200 nm can grow up to 4 m under some optimum conditions. Another strategy to
carry out the anodization of Ti in non-aqueous electrolyte leads to a significant difference in
morphology of as-formed TiO2 nanotubes compared with nanotubes grown in aqueous
electrolytes. Since organic electrolytes, such as ethylene glycol, glycerol, DMSO and ionic liquids,
have a small amount of oxygen, the oxide chemical dissolution in these electrolytes highly depends
on the water concentration. Owing to the low water content, very long (up to 1 mm) TiO2
nanotubes with large diameters (up to 700 nm) can be obtained (Figure 12.3c and 3d) [34]. The fact
that different morphology of TiO2 nanotubes formed in aqueous and non-aqueous electrolytes can
be ascribed to a large extent to the low conductivity of non-aqueous electrolytes and IR-drop
effects, which will decrease the effective voltage of the electrode [31, 32]. The conductivity of the
electrolyte changes as the reaction products are formed with the extension of the anodization
time, resulting in nanotubes with larger diameters with longer thickness.
If the other electrochemical parameters are kept constant, the duration of anodization process,
which can be also converted to the charge passed during the anodization, controls the nanotubular
layer thickness. The thickness of the nanotubes linearly depends on the anodization time (Fig.3e).
However, this only holds for a certain time. Due to etching of TiO 2 by the fluoride species in the
electrolyte, an equilibrium state between the growth of the nanotubes at the bottom and
chemical/electrochemical dissolution of nanotubes at top will be reached, which is commonly
defined as a steady-state condition. At steady-state condition, no further increase in the
nanotubular thickness is observed. If anodization is carried out for extended times, nanotubular
walls are thinned out, perforated and the tube tops become decorated with tube wall remnants.
Since the oxide growth and chemical dissolution of nanotubes highly depend on the water content
in the anodization culture, the amount of water is another factor influencing the nanotube
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formation [31, 32, 34]. A striking effect of the water content is that smooth nanotubular walls are
obtained in low water containing electrolyte while side wall ripples are formed in higher water
contents [34]. The reason for this effect is that for higher water contents, the fluoride rich layer
between the nanotubes shows a faster chemical dissolution rate than the growth rate of the
nanotubes into the underlying substrate; that is, ripples at the walls of the nanotubes can be
ascribed to the continuous etching and passivation of the cell boundary regions.
FIGURE 12.3
Examples of TiO2 nanotubular arrays obtained by electrochemical anodization in different electrolyte: (a)
HF/H2SO4 solution (anodization voltage at 20 V), (b) (NH4)2SO4 + NH4F solution (anodization voltage at 20 V),
(c) and (d) mixed NaF and glycerol solution (anodization voltage at 120 V and 60 V respectively),; (e) TiO2
nanotube-layer thickness with anodization time for different electrolytes (anodization voltage for ethylene
glycol electrolyte held at 60 V, and 40 V for other electrolytes). (a)from Bauer S, Kleber S, Schmuki P. TiO2
nanotubes: Tailoring the geometry in H 3PO4/HF electrolytes. Electrochem Commun. 2006;8(8): 1321–1325.32;
(b) from Macák JM, Tsuchiya H, Schmuki P. High-aspect-ratio TiO2 nanotubes by anodization of titanium.
Angew Chem Int Ed Engl. 2005;44(14): 2100–2102.33; (c) Macak JM, Hildebrand H, Marten-Jahns U, Schmuki
P. Mechanistic aspects and growth of large diameter self-organized TiO2 nanotubes. J Electroanal Chem.
2008;621:254–266.70; (d) From Paulose M, Prakasam HE, Varghese OK, et al. TiO 2 Nanotube Arrays of 1000
μm Length by Anodization of Titanium Foil: Phenol Red Diffusion. J Phys Chem C. 2007;111(41):14992–
14997.35 (E) Data from Macák JM, Tsuchiya H, Schmuki P. High-aspect-ratio TiO2 nanotubes by anodization of
titanium. Angew Chem Int Ed. 2005;44(14): 2100–2102; Roy P, Berger S, Schmuki P. TiO2 Nanotubes: Synthesis
and Applications. Angew Chem Int Ed. 2011; 50(13):2904–2939; Macak JM, Tsuchiya H, Taveira L,
Aldabergerova S, Schmuki P. Smooth anodic TiO2 nanotubes. Angew Chem Int Ed. 2005;44:7463–7465. Macak
JM, Hildebrand H, Marten-Jahns U, Schmuki P. Mechanistic aspects and growth of large diameter self-
organized TiO2 nanotubes. J Electroanal Chem. 2008;621:254–266.
Oxide nanotubular arrays on titanium alloys and other metals
The principle used to grow oxide nanotubular arrays on titanium by using electrochemical
anodization technique in fluoride containing electrolyte can be transferred to biocompatible
titanium alloys (shown in Figure 12.4). Self-organized oxide nanotubular layers have been reported
Nanomedicine 321
on binary alloys, such as Ti-Zr, Ti-Ta, Ti-Nb, and Ti-Mo], tenary alloys, such as Ti-6Al-7Nb, Ti-6Al-
4Vand Ti-35Nb-5Zr, etc, and more complex alloys systems such as Ti-29Nb-13Ta-4.6Zr [34, 41, 45].
The addition of different elements in Ti alloys drastically affects the anodization process and the
ultimate oxide nanotubular morphology and composition. In general, after alloy anodization, the
composition of the oxide layer is consistent with the ratio in the alloy. For instance, the anodic
oxide nanotubular layers on Ti-Al alloys are composed of TiO2 and Al2O3 [41]. The fraction of two
kinds of oxides is the respective fraction of Ti and Al in the base alloys. With the increasing of
titanium content of Ti-Al alloy, TiO2 and Al2O3 nanotubular separation has been observed. Similar
phenomena have also been observed on Ti-Ta and Ti-Nb [45]. In some cases, minor amounts of
mixed oxides may be present in the anodic nanotubular arrays. A mixed oxide nanotubular
structure was reported on Ti-Zr alloys [42]. Zirconium titanate nanotubular arrays were formed on
Ti-50Zr alloys via anodization [42].
Similar to the TiO2 nanotubular arrays formed on pure Ti, the formation of oxide nanotubular
arrays on Ti alloys depends on the anodization parameters, including anodic potential, anodization
time, pH value and fluoride species concentration, that have been discussed in the previous
section. However, owing to the difference in chemistry, including selective dissolution of the oxide
in fluoride and solubility of the respective metal fluorides in different culture in anodization, the
morphology and geometry of oxide nanotubular arrays formed on Ti alloys show some difference
than the TiO2 nanotubular arrays on pure Ti. In the case of Ti-Ta alloys, the anodization process
resulted in the formation of nanoporous oxide layers first and dissolution followed by formation of
nanotubular arrays. For a ternary alloy such as Ti-6Al-4V, both  and  phases of Ti were present
since the addition of other element [41]. Ordered nanotubular arrays were observed on  phase
and a mixture of nanotubular arrays and nanoporous structure was present on a complex  + 
phases. Due to the easily dissolution of V2O5 (mainly in  phase) in fluoride containing culture, the
entire  phase was easily to be attacked and dissolved until the etch reached an underlying 
phase, where a nanotubular structure formed. A very interesting phenomenon was observed on
the anodic nanotubular arrays on Ti-Zr-Nb alloys [43]. Two distinct tube diameters were formed
with one large center tube surrounded by smaller tubes, repeated over the entire anodized area.
The tubes had no difference on the length and showed the same degree of self-ordering, which was
ascribed to availability of current at the different tips. However, the phenomenon is still not well
understood and extensive work is required to explore the formation mechanism.
Depending on the exact electrochemical conditions, self-ordered nanotubular/nanoporous layers
were reported for several other metals, such as Zr [38], Hf [48], Ta [47], Nb [34], W [34], Fe [31] and
Mg [50] (Figure 12.4). For each case, some optimization of the electrochemical conditions specific
to the element is desired to obtain organized high-aspect-ratio nanotubular structures. Lee et al.
and Tsuchiya et al. were the first two groups of researchers to report on the fabrication of self-
ordered ZrO2 nanotubular arrays in fluoride containing species by electrochemical anodization [46].
The formation mechanism of the nanotubular arrays has been described in detail with regard to the
effect of changing the concentration of fluoride ions, pH value, the composition of the electrolyte
and the applied potential. It was shown that by using organic electrolytes, significant thick and
smooth ZrO2 nanotubular arrays up to 200 m was obtained under a 40 V stimulated potential [38].
Irregular ZrO2 nanotubular arrays were obtained by one-step anodization without any
pretreatment owing to the existing of impurities such as carbide in Zr and the inhomogeneity of the
surface. In order to obtain highly ordered nanotubes, pretreatments were applied on Zr to enhance
the self-ordering. Dip-etching, two–step anodizing and electropolishing were applied on Zr
substrate resulting in highly self-ordered ZrO2 nanotubular arrays [38]. The removal of the
impurities and the electropolishing, which reduced the surface roughness, had an influence on the
Nanomedicine 322
homogeneous electric field distribution over the entire metal surface during the anodization, thus
self-ordering nanotubular arrays grew regularly on the entire surface. Similarly, high-aspect ratio
hafnium oxide nanotubular arrays can be achieved under a wide range of anodization parameters
[36, 48]. Tantalum has extreme corrosion resistance in to acidic environments. Thus extreme
conditions are required to obtain nanotubular structures. It was reported that Ta 2O5 nanotubular
arrays formed in a mixed H2SO4 and HF electrolyte with up to 1 wt% H2O under the anodization
voltage from 10-20 V with the anodization time between 5 sec to 120 sec [45]. Extension of
anodization process resulted in the destruction of the nanotubular arrays and dimples on Ta
substrate. The reason of the destruction of nanotubular arrays from surface was shown to be the
formation of thin, fluoride-rich layer built up at the Ta/Ta2O5 interface [47]. Controversially, for
other valve metals such as Nb and W, there were still no highly ordered and only comparably short
nanotubular structures were reported [34]. For some non-valve metals such as Fe [47] and Mg [50],
high-aspect-ratio oxide nanoporous structure and oxide-fluoride nanotubular structure were
recently reported. The reasons for this different behavior may be ascribed to the solubility of a
formed oxide structure in the anodizing electrolyte, the solubility of the fluoride species and the
stress generated when the oxide is formed.
In general, all investigated self-ordering oxide structure fabricated by electrochemical anodization
in fluoride-containing electrolytes on different metals and alloys seem to follow the same growth
principles and key factors that are found out for fabrication of TiO 2 nanotubular arrays: the
diameter of the tubes are determined by the anodization voltage; the tubular length depends on
the chemical resistance of the oxide against fluoride etching, which relates to the anodization
voltage and anodization time and the amount of oxygen, which is provided by water for tube
growth.
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FIGURE 12.4
Cross-sectional and top-view SEM images of ordered oxide nanotube or nanopore layers electrochemically
grown on different valve metals and metal alloys (From Roy, Schmuki et al. Angew. Chem. Int. Ed. 2011, 50,
2904 – 2939). Copyright Wiley-VCH Verlag GmbH & Co. KGaA. © 2011. Reproduced with permission from Roy
P, Berger S, Schmuki P. TiO2 Nanotubes: Synthesis and Applications. Angew Chem Int Ed Engl.
2011;50(13):2904–2939.
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Biocompatibility approach of metals with nanotubular surfaces

Hydroxyapatite formation on nanotubular arrays
In view of a rapid ingrowth of biomedical implants in bone, a key factor is to quickly stimulate
hydroxyapatite (HA) formation from body fluid because HA formation is important for
osseointegration [24]. A number of surface treatments have been explored in order to enhance HA
formation on metal implants, for example using different chemical and physical treatment. It is
therefore of interest to study metallic oxide nanotubular surface in view of HA-induced effects for
biocompatibility approach first.
The formation of HA on biomedical implant is based on heterogeneous nucleation phenomenon.
For a nucleus, assuming spherical shape, to form in a supersaturated solution, the nucleation rate is
[27]
−B −16πγ3 ν2
J = A × exp ( ) = A × exp (1)
KT 3K 3 T 3 ln S 2
Where A is the rate coefficient, B is the activation energy, K is Boltzman’s constant, T is the
temperature, γ is the nucleus-solution interfacical energy, ν is the molecular volume and S is the
degree of supersaturation, defined as the concentration product/solubility product (Ksp). If a
nucleus forms on a foreign substrate (such as implant materials), at a contact angle of θ, the
nucleation rate becomes:
−ϕ ×B
J′ = A × exp (2)
KT
and,
ϕ = 2 + cosθ 1 − cosθ 2 /4 (3)
Since ϕ < 1, J′ in eq. (2) is always higher than in eq. (1). Meanwhile, J′ increases with decreasing θ.
When θ = 0 (spread), the activation energy is also zero and J′ reaches a maximum. Therefore, if the
solution supersaturation (S) and the substrate condition (θ) are properly controlled, nucleation and
crystallization of HA can preferentially occur on the substrate forming the coating. For example,
TiO2 and ZrO2 nanotubular arrays have greater wetting behavior of simulated body fluid than flat Ti
and Zr foils [34, 38]. In addition to surface chemistry, nanotubular arrays change the surface
topography at micro-scale to enhance the nucleation site by large increasing the surface area. Thus
it shows potential that HA formation can strongly accelerated on nanotubular surfaces compared
with flat metal surfaces.
A very thin layer (~25 nm) of nanoscale HA phase was introduced on TiO 2 nanotubular arrays after
immersion in simulated body fluid (SBF) for a week [63]. However, a pretreatment of TiO 2
nanotubular arrays in alkaline solution was required according to the researchers [64]. Thus, the
case might not directly show the benefit of nanotubular arrays for HA formation. Systematic
studies on the formation of HA coating on TiO 2 nanotubular arrays were firstly reported by Schmuki
group [41] (Figure 12.5). In order to obtain a uniform and thick HA coating, it is highly desired to
fabricate nanotubes with larger opening diameters and longer depth for calcium and phosphorous
species nucleation and growth. In order to achieve the HA coating by immersion of TiO 2
nanotubular arrays from SBF, a minimum opening diameter of 15 nm is required [41]. Several
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studies have confirmed that at least 14 days were required to obtain HA coating with over 1
micrometer thick on TiO2 nanotubular arrays, as compared no coating formed on flat Ti [41].
Additionally, the role of TiO2 crystallinity (anatase/rutile) also influenced the HA coating formation.
A mixture of anatase and rutile TiO2 nanotubular arrays showed an enhancement of HA formation
rate at least 2 folds [41]. Moreover, the advantageous 3D structure of nanotubular arrays is optimal
for embedding precursors for HA formation that additionally promote HA nucleation and accelerate
its formation. Several attempts have been carried out to induce amorphous calcium phosphorous
particles or nanocrystalline HA in the nanotubular structure by wet chemical methods before the
formation of HA in SBF [38]. These methods highly relied on the size of the nanotubular arrays
rather than tube crystal structure. By applying these methods, the HA formation rate can be
enhanced about 10 folds. Another attempt to incorporate anion to enhance the HA formation on
nanotubular arrays has been made by Wang et al., who created ZrO 2 nanotubular layers, which was
incorporated with a vast amount of phosphorous anions by anodization in phosphorous species
containing electrolyte [64]. By immersion such nanotubular arrays in SBF, the existing of
2+
phosphorous anions can adsorb Ca to precipitate and form HA coatings only within 4 days. The
incorporation of anions in electrolytes shows a potential to modify the nanotubular arrays with
designated species for a particular application [65].
FIGURE 12.5
SEM images of the as-prepared amorphous TiO2 layers after soaking in SBF for different periods. Compared are
the 2 μm long-nanotubes, the 500 nm nanotubes, and the compact (50 nm thick) TiO 2 layer (From Tsuchiya
and Schumuki, J. Biomed Mater. Res. A 2006, 77A 534-541). Copyright Wiley-VCH Verlag GmbH & Co. KGaA. ©
2006. Reproduced with permission from Tsuchiya H, Macak JM, Taveira L, Ghicov A, Schmuki P.
Hydroxyapatite growth on anodic TiO2 nanotubes. J Biomed Mater Res. 2006;77(3):534–541.
In vitro and in vivo studies of nanotubular arrays
Cytocompatibility leads to promoted bone integration and growth on implant. It is therefore worth
to study the responses of living matter and biological relevant species, such as bone cells, to
nanotubular layers on metallic implants. The most widely used cell types for studies of bioactivity
of nanotubular arrays are osteoblasts (bone cells), fibroblasts (connective tissue cells), bone
marrow cells and stem cells (pluripotent undifferentiated cells) [30]. It is therefore more
spectacular of the interaction of living cells with nanotubular layers. A pioneer work on cell
interactions with TiO2 nanotubular arrays reported by Schmuki group in 2007 showed that
Nanomedicine 326
mesenchymal stem cells react in a very pronounced way to the diameter of nanotubes [66]. The
vitality of the cells was significantly increased for nanotubes as compared with flat metals.
Diameters of ~ 15 nm TiO2 and ZrO2 strongly promote cells adhesion, proliferation and
differentiation, and nanotubes with diameter greater than 50 nm were found to be detrimental on
cells vitality, inducing programmed cell death [67] (shown in Figure 12.6). This effect may be
related to the effective size-scale of the integrin-based focal contact formation between cells and
nanotubular surfaces and the optimum nanotube diameter seems to enhance cellular activities
compared to smooth surfaces. In several conflicting cases, however, osteoblast cells respond to and
proliferate on TiO2 nanotubes greater than 100 nm [67]. Other than the size effect, crystallinity of
nanotubular arrays, remaining fluoride concentration and surface pretreatment were also concerns
of the cell activity. It was shown that anatase/rutile TiO 2 slightly enhanced the proliferation of cell
activities in short term (1 day) in vitro cell culture test. Immersion of as-formed nanotubular arrays
in alkaline solution highly decreases the remaining fluoride species and results in an appropriate
chemical culture for cell proliferation. Additionally, deposition of nanoscale Au particle in TiO 2 and
ZrO2 nanotubular arrays enhanced the mesenchymal stem cells attachment [67]. However, most of
work has clearly demonstrated that the effect of size of nanotubular arrays dominates over tubular
crystal structure, fluoride content and other surface pretreatment. Moreover, some researchers
studied the mechanism of enhancement of bone cell function on TiO 2 nanotubular structure [34].
Two kinds of proteins, fibronectin and vitronectin, are major proteins that involved in osteoblast
adhesion. Results showed significantly increased both fibronectin (15%) and vitronectin (18%)
adsorption on nanotubular structures compared to flat titanium samples. Since the cells adhered to
the metal surface via pre-adsorbed proteins, increased fibronectin and vitronectin adsorpotion on
TiO2 nanotubular structure could explain the observed enhanced osteoblast functions.
FIGURE 12.6
Cell densities of adherent cells on ZrO2 nanotubes with different diameters count under fluorescence
microscope after 24 h adhesion (a) and measured using colorimetric WST-assay after 3 d proliferation (b).
Fluorescence images of GFP-labeled mesenchymal stem cells after 24 h adhesion and 3 d proliferation (c)
(From Bauer et al. Integr. Biol., 2009, 1, 525-532). Copyright © 2009. Reproduced by permission of the Royal
Society of Chemistry, from Bauer S, Park J, Faltenbacher J, Berger S, von der Mark K, Schmuki P. Size selective
behavior of mesenchymal stem cells on ZrO2 and TiO2 nanotube arrays. Integr Biol (Camb). 2009;1(8–9):525–
532.81
Nanomedicine 327
Schmuki et al reported the first and the only work on evaluation of the bioactivity of nanotubular
arrays in vivo [39] (Figure 12.7a and 12.7b). Tests from adult pigs showed that titanium implant
with nanotubular structure surface does influence bone formation and bone development by
enhancing osteoblast function and that higher implant bone contacts can be established if implant
are coated with a nanotubular arrays. Additionally, this nanotubular coatings also resisted shearing
forces that evoked by implant insertion, which were an unexpected advantage for nanotubular
arrays on implant surfaces. However, in terms of a complex culture in vivo, some authors pointed
out that negative effect of nanotubular arrays may also be exploited on surfaces when cell
proliferation is not desired [32].
Nanotubular arrays for drug delivery and other preloads application
The 3-D geometry of the metallic oxide nanotubular arrays indicates that the materials are
appropriate carriers as drug-delivery capsules and drug-eluding coatings on biomedical implant
materials [31]. A potential in vivo capsule in terms of TiO 2 nanotubular arrays has been designed by
Schmuki group [34] (Figure 12.7c). The drug with long molecules can be attached to TiO2 surface by
wet chemical methods. The nanotubes are filled by magnetic Fe 3O4 particles before the attachment
of drugs and these tubes can be magnetically guided to a designated location. Drugs can be
released photocatalytically via ultraviolet reactions [34]. Additionally, drugs can also be released by
electronic stimulated catalysis and more importantly by X-rays, which allows in vivo treatment
through living tissue. Such TiO2 can be used directly for photocatalytic reactions with cells or
tissues, including for the site-selective killing of cancer cells. Metal oxide/aqueous interfaces play
an important role in the adsorption/desorption of organic payloads. Thus the wettability of
nanotubular arrays was adjusted for different payload filling and release. An amphiphilic TiO 2
nanotubular arrays was created composed by hydrophilic drugs and hydrophobic monolayer caps
as an in vivo capsule. The cap does not allow body fluids to enter into the tubes after implantation
in body unless opened by a photocatalytic interaction. Once the hydrophobic layer was removed,
body fluids could enter into the tubes and wash out hydrophilic drugs loaded within the tubes. In
order to achieve an appropriate elution time for loadings, some researchers suggested that capping
of drug-loaded tubular or mesoporous nanotubular layer with biopolymer might represent efficient
and promising drug-release system [34].
Conclusion and other aspects

As a surface modification method, electrochemical anodization can lead to desired chemistry
and/or topography changes and could be used with other treatments (e.g., hydrothermal)
together. First, anodization provides a controlled way to create nano-roughness or even nano-
features. Generally, there are two mechanisms that are responsible for osseointegration of bone:
biomechanical interlocking and biological interactions. For biomechanical interlocking, it depends
on the roughness, and surface irregularity. Current femoral stems made of valve metals or alloys
(e.g. Ti and Ti alloys) are usually macro-textured to provide such surface features for bone to
mechanically interlock. For biological interactions, it involves complex systems. Considering
roughness in different scales, it is reported that increased micro/submicron roughness could
enhance bone cell function, such as ALP activity, while some other studies have revealed the
enhanced cell-implant interactions on nanoporous or nanophase materials. It is thus proposed that
the future titanium implant should possess roughness in a mixed microscale and nanoscale. One
Nanomedicine 328
possible approach to accomplish this is by subjecting implants to techniques like polishing and
mechanical grinding that promote micro-roughness, and then to induce nanotubular structures by
a quick anodization process. Second, HA films produced using nanotubular metallic oxides show
some advantages over conventional ones. Moreover, HA deposited onto the nanotubular metallic
oxides could be nano-scale in dimension. One problem that still needs to be more fully investigated
is how to optimize the bond strength between apatite crystals and the anodic oxide. Furthermore,
electrochemical anodization to form nanotubular structure can be used to incorporate drug
delivery into metallic implants to enhance new bone formation. The nanotubular structures could
serve as reservoirs of chemical mediators, such as bone morphogenetic protein-2 (BMP-2) and
osteogenic protein-1 (OP-1, BMP-7).In a word, electrochemical anodization as a quick and efficient
modification method of metallic implants shows significant potential for enhancing their 10 to 15
year lifetime. This work was prepared in conjunction with project under contract No. YETP0419
with the Beijing Higher Education Young Elite Teacher Project and the start up fund from University
of Science and Technology Beijing.
FIGURE 12.7
SEM pictures of the histological specimen. (a) The interface between the anodic TiO 2 nanotube implant and
the bone can be seen. A partially breakage of the interface is due to the histological preparation. (b)
Magnification reveals that the anodic TiO2 nanotubes keep their structure and do not get damaged by
shearing forces due to the implantation process; (c) TiO2 nanotube for guided drug release: Representation of
magnetically loaded TiO2 nanotubes with attached drug (F). Release is triggered by photocatalytic chain
scission upon UV irradiation. Inset: an example where a blue fluorescent molecule is released from
Nanomedicine 329
magnetically actuated nanotubes [(a) and (b) from Schmuki et al. J. Biomed. Mater. B Appl. Biomater. 2009,
89B, 165-171; (c) from Schmuki et al., Angew. Chem. Int. Ed. 2009, 48, 969]
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13
Unsupervised Medical Image
Segmentation using
Stochastic Optimization Methods
1 1 1
Ivan Cruz-Aceves , Juan Gabriel Avina-Cervantes , Juan Manuel Lopez-Hernandez , Ma. de
1 1 2
Guadalupe Garcia-Hernandez , Miguel Torres-Cisneros , Manuel Ornelas-Rodriguez .
1
University of Guanajuato, Electronics Department (DICIS). Salamanca, Guanajuato, Mexico
2
Leon Institute of Technology, Graduate Department (ITL). Leon, Guanajuato, Mexico
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 334
Image segmentation and optimization methods …………………………………………..……………………………. 335
Active Contour Models (ACM) …………………………………………………………………………………………………….. 335
The ACM Algorithm in General …………………………………………………………………………………………………… 336
Stochastic Optimization Methods ………………………………………………………………………………………………. 336
Particle Swarm Optimization (PSO) …………………………………………………………………………………………….. 336
Differential Evolution …………………………………………………………………………………………………………………. 337
Estimation of Distribution Algorithms (edas) ……………………………………….……………………………………… 338
Application of PSO, DE, and UMDA Algorithms …………………………………………………………………………… 339
Proposed Medical Image Segmentation Methods ………………………………………………………………………. 342
Segmentation through Constrained Polar Sections (CPS) ……………………………………………………………. 342
Pseudocode and Parameter Selection ………………………………………………………………………………………….344
Segmentation by using Maximum Euclidean Distance (MED) ……………………………………………………… 346
Computational Experiments ………………………………………………………………………………………………………. 347
Application to Human Heart on Computed Tomography Images ………………………………………………… 347
Application to White Blood Cells on Microscope Images …………………………………………………………….. 349
Concluding Remarks …………………………………………………………………………………………………………………… 352
Acknowledgements …………………………………………………………………………………………………………………….352
References……………………………………………………………………………………..…………………………………………… 353
Nanomedicine 334
Introduction
In medical image analysis, the automatic segmentation is an important and challenging problem for
the diagnosis and monitoring of different diseases. The objective of image segmentation is to
separate objects of interest from a given image based on different attributes such as shape, colour,
intensity or texture. In recent years, several methods have been proposed for this purpose such as
adaptive local multi-atlas in human heart [1], suppressed fuzzy c-means in brain magnetic
resonance images [2], improved watershed transform in mammograms [3], graph cut in multiple
human organs [4, 5], rule optimization with region growing in pelvic injuries [6] and active contour
models (ACM) in human prostate [7], lungs from magnetic resonance images of the torso [8] and
intravascular ultrasound images [9], to name a few.
The Active Contour Model was introduced by Kass et al. [10] and it is an energy-minimizing spline
curve composed of control points also known as snaxels. This spline evolves through evaluation of
internal and external forces being attracted towards features as edges of a target object. The
classical implementation of ACM presents the shortcomings of sensitivity to initial positioning of
the control points, which must be close to the target object and the propensity to be trapped into
local minima. To solve these drawbacks different improvements have been proposed to adapt
diverse methods working together with ACM such as Finite-element [11], graph cut [12], statistical
methods [13, 14] and population-based methods including Genetic algorithms [15, 16], Differential
Evolution (DE) [17], Estimation of Distribution Algorithms (EDAs) [18] and Particle Swarm
Optimization (PSO) [19, 20, 21]. The use of population-based methods working together with the
classical ACM becomes more stable and efficient in the local minima, which is highly suitable for
medical image segmentation problems.
Population-based methods are an effective way to solve different optimization problems. Three of
the most popular methods are the PSO, DE and EDAs because of their robustness in local minima
and efficiency solving global optimization problems with nondifferentiable functions. Since these
techniques are not computationally demanding and highly efficient, they have been used in many
real-world applications including, tumour classification [22], cancer chemotherapy optimization
[23] and parameter estimation in human immunodeficiency virus (HIV) [24].
This chapter introduces novel unsupervised image segmentation methods based on three different
stochastic optimization techniques. These unsupervised methods use the theory of Active Contour
Model working together with Differential Evolution, Estimation of Distribution Algorithms and
Particle Swarm Optimization to perform the segmentation task. These methods are explored and
applied in the segmentation of white blood cells (leukocytes) from microscope images and for
segmentation of the human heart from Computed Tomography images, where the human heart
results are evaluated using the Jaccard and Dice indexes with respect to regions outlined by
experts.
Nanomedicine 335
Image segmentation and optimization methods

This section introduces the basis of the Active Contour Model for image segmentation, and the
fundamentals of the stochastic optimization methods of Differential Evolution, Estimation of
Distribution Algorithms and Particle Swarm Optimization.
Active Contour Models (ACM)
The classical Active Contour Model, also called snake, is represented by a parametric curve which
can move within the spatial domain of an image where it was assigned [10]. This parametric curve
is defined by p(s, t) = (x(s, t), y(s, t)), s [0, 1], where t is the time parameter whereby the curve
evolves to minimize the total energy function given by Equation (1).
1
𝐸𝑠𝑛𝑎𝑘𝑒 = 𝐸𝑖𝑛𝑡 𝑝 𝑠, 𝑡 + 𝐸𝑒𝑥𝑡 (𝑝 𝑠, 𝑡 ) 𝑑𝑠 (1)
0
This energy function has two components, the internal energy 𝐸𝑖𝑛𝑡 and the external energy 𝐸𝑒𝑥𝑡 .
Internal energy is presented in Equation (2), which is used to maintain the search within the spatial
image domain, and to control the shape modification of the parametric curve using the first and
second derivatives of p(s), the curve tension parameter α(s) and the rigidity parameter β(s).
2 2
1 𝜕𝑝 𝑠 𝜕 2 𝑝(𝑠)
𝐸𝑖𝑛𝑡 𝑝 𝑠, 𝑡 = 𝛼 𝑠 + 𝛽 𝑠 (2)
2 𝜕𝑠 𝜕𝑠 2
The external energy defined by Equation (3) is given by the particular features of the image, where
𝜵𝑰(𝑝(𝑠)) is the surface gradient computed at p(s) and 𝛾 is a weight parameter. The optimal
solution is acquired by solving the Euler equation (Equation (4)), when both external and internal
energies become stable.
2
𝐸𝑒𝑥𝑡 𝑝 𝑠 = −𝛾 ∇𝐼(𝑝 𝑠 )
(3)
𝜕2𝑝 𝑠 𝜕4𝑝 𝑠
∇𝐸𝑒𝑥𝑡 − 𝛼 + 𝛽 =0
𝜕𝑠 2 𝜕𝑠 4 (4)
In the computational implementation of the traditional ACM, the parametric curve is composed by
a set of n control points 𝑝𝑖 | 𝑖 = 1,2, … , 𝑛 , also called snaxels. The internal and external energies
are approximated by Equation (5) and Equation (6) respectively. In both energies 𝑞𝑖,𝑗 represents the
current control point 𝑝𝑖 in the 𝑗 index within its searching window. Accordingly, the local energy
function given by Equation (7) is iteratively evaluated to minimize the 𝑘𝑖 index by using Equation
(8), where 𝑊𝑖 is the predefined searching window for the control point 𝑝𝑖 [21].
1 2 2
𝐸𝑖𝑛𝑡 = 𝛼 𝑠 𝑞𝑖,𝑗 − 𝑝𝑖−1 + 𝛽(𝑠) 𝑝𝑖−1 − 2𝑞𝑖,𝑗 + 𝑝𝑖+1
2 2 2 (5)
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2
𝐸𝑒𝑥𝑡 = −𝛾|𝛻𝐼(𝑞𝑖,𝑗 )|
2 (6)
𝐸𝑖,𝑗 = 𝐸𝑖𝑛𝑡 + 𝐸𝑒𝑥𝑡 (7)
𝐸𝑠𝑛𝑎𝑘𝑒 = 𝐸𝑖,𝑘 𝑖 , 𝑘𝑖 = 𝑎𝑟𝑔 𝑚𝑖𝑛𝑗 𝐸𝑖,𝑗 , 𝑗 ∈ 𝑤𝑖 (8)

𝑖=𝑖
This classical ACM implementation presents two main weaknesses. Firstly, sensitivity to the initial
position of the control points which must be close to target object otherwise failure of convergence
will occur; secondly, these control points are prone to be trapped into local minima problem
deflecting the parametric curve of the optimum edge. A suitable alternative to overcome the
aforementioned drawbacks is to use robust stochastic optimization methods. In our experiments,
three different population-based methods were used, which are described in the following Section
2.2.
The ACM Algorithm in General
According to the previous description in Section 2.1, the classical ACM algorithm can be
implemented by using the procedure in Box 1.
1. Initialize the control points 𝑝𝑖 | 𝑖 = 1,2, … , 𝑛

2. For each control point 𝑝𝑖 :
3. Calculate 𝐸𝑖,𝑗 in searching window 𝑊𝑖 using Equation (7)
4. Find the best index 𝑘𝑖 according to Equation (8)
5. Set 𝑃𝑖 = 𝑞𝑖,𝑘 𝑖
6. Compute 𝐸𝑠𝑛𝑎𝑘𝑒
7. If 𝐸𝑠𝑛𝑎𝑘𝑒 becomes stable, then stop, otherwise repeat steps (2) to (7)
BOX 1
Algorithm of Active Contour Model.
Stochastic Optimization Methods
Stochastic Optimization methods are used to solve numerical optimization problems based on
different strategies. In this work, we focus on Differential Evolution, Estimation of Distribution
Algorithms and Particle Swarm Optimization.
Particle Swarm Optimization (PSO)
Particle Swarm Optimization is a computational intelligence technique proposed by Eberhart et al.

[25] and improved by [26] to solve numerical optimization problems. Similar to evolutionary
techniques, PSO uses a set also called swarm of potential solutions referred to as particles to
perform the optimization task. Each particle represents a solution in an N-dimensional space
𝑿𝑖 = 𝑥𝑖1 , 𝑥𝑖2 , … , 𝑥𝑖𝑁 , which moves through hyperspace to a new position according to the
following velocity equation:
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𝑣𝑖 𝑡 + 1 = 𝜑𝑣𝑖 𝑡 + 𝑘𝑟1 𝑝𝑏𝑒𝑠𝑡 − 𝑥𝑖 𝑡 + 𝑘𝑟2 (𝑝𝑔𝑏𝑒𝑠𝑡 − 𝑥𝑖 𝑡 ) (9)
where 𝑣𝑖 𝑡 is the current velocity of the particle 𝑥𝑖 in the time step 𝑡 , 𝜑 is the inertia weight, 𝑘
represents the learning factor, 𝑟1 , 𝑟2 ~ 𝑈 (0,1) where 𝑈 is a uniform distribution, 𝑝𝑏𝑒𝑠𝑡 is the current
best solution found by 𝑥𝑖 , and 𝑝𝑔𝑏𝑒𝑠𝑡 is the best solution found by the best particle of the whole
swarm. Subsequently, assuming that the new velocity of the particle has been updated, Equation
(10) is used to calculate its new position within the search space.
𝑥𝑖 𝑡 + 1 = 𝑥𝑖 𝑡 + 𝑣𝑖 (𝑡 + 1) (10)
According to the aforementioned description, the PSO algorithm can be implemented by using the
following procedure.
1. Initialize iterations G, and swarm size 𝑁𝑝

2. Initialize each particle 𝑋𝑖 by generating random positions and velocities.
3. For each particle 𝑋𝑖,𝑔 , where 𝑔 = 1, … 𝐺 :
4. Evaluate 𝑋𝑖,𝑔 in fitness function and update its 𝑃𝑏𝑒𝑠𝑡 , if the new fitness is better.
5. Find the best particle in the swarm and update 𝑃𝑔𝑏𝑒𝑠𝑡 , if the fitness value found is
better.
6. Stop if the convergence criterion is satisfied (e.g., stability or number of iterations).
7. Update velocity of all particles using Equation (9).
8. Update position of all particles using Equation (10), then repeat steps 3-8.
BOX 2
Algorithm of Particle Swarm Optimization.
Differential Evolution
Differential evolution (DE) is a stochastic real-parameter heuristic from the family of evolutionary
algorithms proposed by Storn et al. [27, 28] for numerical global optimization problems. DE starts
with a number 𝑁𝑝 of randomly initialized potential solutions, also known as individuals 𝑋 =
{𝑥1 , 𝑥2 , … , 𝑥𝑁𝑝 }. These individuals are iteratively improved through different variation operators
and the solution is chosen to be the individual with the best fitness according to an objective
function.
The main idea of DE method consists of three evolutionary operators: mutation, crossover and
selection on the floating-point encoding.
Mutation: Creates a mutant vector 𝑉𝑖,𝑔+1 at each generation 𝑔 based on the distribution of the
current population {𝑋𝑖,𝑔 |𝑖 = 1,2, … , 𝑁𝑝 } using the following mutation strategy:
𝑉𝑖,𝑔+1 = 𝑋𝑟1,𝑔 + 𝐹 𝑋𝑟2,𝑔 − 𝑋𝑟3,𝑔 , 𝑟1 ≠ 𝑟2 ≠ 𝑟3 ≠ 𝑖 (11)

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where 𝐹 is the differentiation factor also called scaling or mutation factor parameter, and 𝑟1, 𝑟2
and 𝑟3 are the indexes of three individuals mutually different and uniformly randomly selected
from the set {1, … , 𝑁𝑝 }.
Crossover: This operator is used to create the trial vector 𝑈𝑖,𝑔+1 via the following:
𝑉𝑖,𝑔+1 , 𝑖𝑓 𝑟 ≤ 𝐶𝑅 (12)
𝑈𝑖,𝑔+1 =
𝑋𝑖,𝑔 , 𝑖𝑓 𝑟 > 𝐶𝑅
where a uniform random value 𝑟 on the interval (0,1) is generated to be compared with the
crossover rate parameter 𝐶𝑅. If 𝑟 is bigger than 𝐶𝑅, the current information of individual 𝑋𝑖,𝑔 is
preserved, otherwise the values from the mutant vector 𝑉𝑖,𝑔+1 are copied to the trial vector 𝑈𝑖,𝑔+1 .
Selection: This operator is applied using Equation (13) to optimization process. It selects according
to a fitness function, the better one between the current individual 𝑋𝑖,𝑔 and the trial vector 𝑈𝑖,𝑔+1
to be used to replace the current individual in the next generation.
𝑈𝑖,𝑔+1 , 𝑖𝑓 𝑓(𝑈𝑖,𝑔+1 ) < 𝑓(𝑋𝑖,𝑔 ) (13)

𝑋𝑖,𝑔+1 =
𝑋𝑖,𝑔 , 𝑜𝑡𝑕𝑒𝑟𝑤𝑖𝑠𝑒
According to the previous description of the traditional DE algorithm, it is described by using the
following procedure.
1. Initialize generations G, population size 𝑁𝑝 , differentiation 𝐹 and crossover rate 𝐶𝑅

2. Initialize each individual 𝑋𝑖 by generating random candidate solutions
3. For each individual 𝑋𝑖,𝑔 , where 𝑔 = 1, … 𝐺 :
4. Compute 𝑉𝑖,𝑔+1 by using the mutation step in Equation (11)
5. Assign 𝑈𝑖,𝑔+1 according to the crossover operator via Equation (12)
6. Update 𝑋𝑖,𝑔+1 , if 𝑈𝑖,𝑔+1 is better than 𝑋𝑖,𝑔 using Equation (13)
7. If stopping criterion is satisfied (e.g., stability or number of generations), then stop
BOX 3
Algorithm of Differential Evolution.
Estimation of Distribution Algorithms (EDAs)
Estimation of Distribution Algorithms are population-based methods that incorporate statistical

information to solve optimization problems [29, 30, 31]. EDAs are from the family of evolutionary
algorithms since they use a population of individuals, selection operators and binary encoding
replacing the crossover and mutation operators by probabilistic models based on the global
statistical information inferred from the current solutions. One of EDAs that works perfectly for
linear problems or problems with not many significant dependencies is the Univariate Marginal
Distribution Algorithm (UMDA)[32, 33]. This algorithm works on binary strings to infer statistical
dependencies between the variables using a probability vector 𝒑 = (𝑝1 , 𝑝2 , … , 𝑝𝑛 )𝑇 to construct
the probabilistic model, where 𝑝𝑖 represents the probability of obtaining a 1 in position 𝑖. UMDA
approximates the actual probability distribution of the individuals in ℙ𝑡 using the product of the
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univariate frequencies computed from the selected population and assuming that all variables are
independent [34]. The first step of UMDA consists of the selection probability 𝑠, which is computed
using Equation (14) and it is performed after the chromosomes in the search space Ω have been
sorted according to fitness.
ℙ 𝑥 𝑓(𝑥) (14)
ℙ𝑠 𝑥 =
𝑥∈Ω ℙ 𝑥 𝑓(𝑥 )
In the second step a joint probability is calculated through the following:

𝑛
(15)
ℙ 𝑥 = ℙ 𝑋𝑖 = 𝑥𝑖
𝑖=1
where 𝑥 = (𝑥1 , 𝑥2 , … , 𝑥𝑛 )𝑇 is the binary value of the 𝑖th bit in the chromosome, and 𝑋𝑖 is the 𝑖th
component of the random vector 𝑋. Finally, the third step of UMDA is used to generate a new
population of individuals from the probabilistic model, to be evaluated according to a fitness
function in the next generation. These three steps of UMDA are iteratively performed until the
termination criteria are satisfied, and the solution is chosen to be the individual with the best
fitness in the entire population.
Based on the previous description, the UMDA algorithm can be implemented through the following
procedure:
1. Initialize generations T, population size 𝑁𝑝 .

2. Initialize each individual 𝑋𝑖 by generating random candidate solutions
3. For each individual 𝑋𝑖,𝑡 , where t= 1, … 𝑇 :
4. Select a subpopulation S of individuals according to a selection method.
5. Compute the univariate marginal probabilities 𝑝𝑖𝑠 (𝑥𝑖 , 𝑡) of S.
6. Generate 𝑛 new individuals according to 𝑝 𝑥, 𝑡 + 1 = 𝑛𝑖=1 𝑝𝑖𝑠 (𝑥𝑖 , 𝑡) .
7. Stop if convergence criterion is satisfied, otherwise, repeat steps (3)-(7).
BOX 4
Algorithm of the Univariate Marginal Distribution Method.
Application of PSO, DE, and UMDA Algorithms
Since the aforementioned algorithms are optimization techniques, they can be applied to minimize
the following function:
min 𝑓 𝑥 = 𝑥 2 , −64,64 (16)
Figure 13.1(a) presents the graph of the function (16), in which the optimization process will be
explained. The first step in the optimization methods is the initialization of their particular
parameters followed by generating random potential solutions. These solutions referred as
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individuals or particles must be initialized within the search space defined by the function. In our
example, the search space is in the range [-64, 64], and 8 individuals have been randomly
initialized, as shown in Figure 13.1(b) and Table 13.1. The binary representation used for EDAs has
7-bits, since the range of the function ([-64, 64]) can be rearranged to establish a new range in [0,
128].
FIGURE 13.1
(a) Plot of function 𝑓 𝑥 = 𝑥 2 and (b) Initialization of potential solutions.
TABLE 13.1
Initialization of potential solutions according to Figure 13.1(b).
individuals Real encoding Binary encoding (EDAs) Fitness

(PSO/DE)
1 25 1011001 625
2 48 1110000 2304
3 18 1010010 324
4 -50 0001110 2500
5 -42 0010110 1764
6 12 1001100 144
7 35 1100011 1225
8 -26 0100110 676
While EDAs works on the binary encoding, PSO and DE use real encoding to perform their search
strategy. The strategy search of each optimization method is applied through iterations over the
whole set of potential solutions. For instance, PSO uses the velocity and position equations, DE
works with mutation, crossover and selection operator and UMDA uses selection and univariate
marginal probabilities. When the optimization process is finished, in general taking into account the
number of iterations, the optimal solution (Figure 13.2) is chosen to be the individual or particle
with the best fitness in the whole set of potential solutions. These methods are easy to extend for
working with 𝑛-dimensional problems just by modifying their codification, since the search
strategies have been proven to be robust and efficient in different applications [23, 24, 35]. In
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order to adapt the codification presented in Table 13.1 and extending the function (16) to two-
dimensions (function (17)), in the following Table 13.2 and Figure 13.3 the adaptation is illustrated.
FIGURE 13.2
Optimal solution of function (16).
min 𝑓 𝑥, 𝑦 = 𝑥 2 + 𝑦 2 , −64,64 (17)
TABLE 13.2
Adaptation of potential solutions to minimize the function 17 shown in Figure 13.3.
Real encoding Binary encoding

Individual X-axis Y-axis X-axis Y-axis Fitness
1 25 3 1011001 1000011 634
2 48 21 1110000 1010101 2745
3 18 -10 1010010 0110110 424
4 -50 -32 0001110 0100000 3524
5 -42 46 0010110 1101110 3880
6 12 -50 1001100 0001110 2644
7 35 -20 1100011 0101100 1625
8 -26 -2 0100110 0111110 680
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FIGURE 13.3
Surface of spherical function 𝑓 𝑥, 𝑦 = 𝑥 2 + 𝑦 2
In the following Section 3, two medical image segmentation frameworks based on different
strategies working together with the optimization methods described above, are explained in
detail.
Proposed Medical Image Segmentation Methods

The first method uses constrained polar sections to perform the segmentation process. These
constrained sections make it possible to adapt different optimization methods preserving their
original search strategies as it is described in Section 3.1. The second method uses the maximum
Euclidean distance between potential solutions to perform the segmentation task. The search is not
constrained via polar sections; instead, Euclidean distance is used as it is illustrated in Section 3.3.
Segmentation through Constrained Polar Sections (CPS)
In this method, different optimization techniques can be adopted without any modification for
guiding the convergence of multiple active contours on a polar coordinate system to perform the
segmentation task [36, 37]. Due to the local minima disadvantage of the traditional ACM discussed
above, this method uses population-based techniques to solve it. Since the methodology makes it
possible to apply the traditional implementation of the optimization techniques, the advantages of
robustness, low computational time and efficiency are inherently acquired. The procedure of the
segmentation method is illustrated below in Figure 13.4, which consists of the Preprocessing,
Initialization and Segmentation steps.
In the preprocessing stage, we remove noise from the image by using a 2-D median filter (3x3
window size), followed by the Canny edge detector to separate the regions of interest from the
background. The last step in this stage is to produce the Euclidean Distance Map, where low
potential values (ideally zero) are assigned to pixels located close to the target object and high
potential values to pixels far from the object [11].
In the initialization stage, a polar coordinate system on the Distance Map is generated via an
interactively determined seed point composed by the 𝒙 and 𝒚 coordinates. This coordinate system
divides the target object through 𝜃 = 2𝜋 𝑔, where 𝑔 denotes the degrees of each constrained
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polar section 𝑆, in which one edge sectional solution must exist. Subsequently, the object of
interest has to be confined by the spatial domain of the 𝑛 initial contours, which can be generated
in a circular or elliptical shape according to the pattern of the object. After the 𝑛 contours are
produced, 𝑛 equidistant control points (snaxels) are generated and assigned as potential solutions
(individuals or particles) to conform one population 𝑂𝑖 for each polar section 𝑆𝑖 . The third stage
represents the segmentation process, where for section 𝑆𝑖 , the optimization method is applied
individually to minimize the corresponding edge sectional solution. Once the optimization process
is finished, the final segmented object is acquired by connecting the best solution of each polar
section to each other.
The proposed method presents the following advantages on the initialization process. Firstly, the
initial contours can be easily defined in different shapes according to the pattern of the target
object. Secondly, the number of control points (snaxels) per contour, can be modified according to
the number of polar sections in which the target object is divided. The last feature is the seed point,
which is used to generate all the snaxels automatically on the constrained spatial domain of the
object of interest allowing extend the present method to work with sequential images just
reproducing the origin point through the set of images.
Since the segmentation results directly depend from the appropriate parameters selection of the
presented method, they are individually described in the following Section.
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FIGURE 13.4
Workflow of the Constrained Polar Sections method, consisting on the steps of preprocessing of medical
image, initialization of evolutionary methods, and segmentation process through numerical optimization.
Reproduced with permission from I. Cruz-Aceves, J. G. Avina-Cervantes, J. M. Lopez-Hernandez, et al.,
“Multiple Active Contours Guided by Differential Evolution for Medical Image Segmentation”,
Computational and Mathematical Methods in Medicine, Volume 2013, Article ID 190304, 14 pages.
©Hindawi Publishing Corporation.
Pseudocode and Parameter Selection
In this section, the procedure as pseudocode and the parameters description of the image
segmentation method based on constrained polar sections are described as follows:
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1. Initialize coordinates (𝑥, 𝑦) from the origin point, degrees 𝑔 and number of snakes.
2. Initialize parameters of optimization method such as number of iterations and
population size.
3. Generate one population or swarm 𝑂𝑖 for each polar section 𝑆𝑖 .
4. For each population or swarm 𝑂𝑖 :
5. Apply restriction of the search space to ignore improper solutions.
6. Perform the particular search strategy of the optimization method.
BOX 5
Algorithm of the method based on Constrained Polar Sections.
The parameters play an essential role in the success of any optimization algorithm. In our approach,
the parameters have been experimentally tuned taking into account the number of different
potential solutions generated through the iterations, and also by considering the number of
improper solutions to perform local exploitation instead exploration.
Preprocessing step: These parameters have been experimentally tuned to preserve the real edges
in the image, since these can affect the segmentation result. Particularly, the parameters in the
Canny edge detector were used as 𝜎 = 1.3, 𝑇𝑙 = 10.0 and 𝑇𝑕 = 30.0.
Degrees: This parameter 𝑔 represents the degrees of each constrained polar section 𝑆. In our
experiments 𝑔 was statistically tuned in the range [13, 16] degrees, since the relation between
computational time and segmentation results is suitable.
Number of snakes: It has to be defined assuming that the target object is confined within the
region of the initial snakes. According to the human heart segmentation experiments, the number
of snakes was set experimentally between 12 and 15 contours.
Number of snaxels: It depends of the relation 2𝜋 𝑔. The number of snaxels of each snake
determines the number of polar sections in which the target object is divided.
Seed point: This seed point is created interactively by the user, and it is composed by the 𝑥 and 𝑦
coordinates of the pixel where it was assigned.
Iterations: In experiments no more than 10 or 20 iterations are required to become stable, since
the segmentation problem is reduced to minimize constrained polar sections, which are generally
unimodal, computed through the distance map.
The constant parameters in this method are suitable for other medical images, taking into account
that the optimization techniques are directly applied to minimize one edge sectional solution for
each polar area.
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Segmentation by using Maximum Euclidean Distance (MED)
In this method, the Maximum Euclidean Distance is used instead the Constrained Polar Sections to
perform the segmentation task. The key aspect of the method is illustrated below in Figure 13.5,
which consists on the incorporation of the 𝐷𝑚𝑎𝑥 parameter.
FIGURE 13.5
Incorporation of 𝐷𝑚𝑎𝑥 in the Maximum Euclidean Distance method.
This method also makes it possible to apply different meta-heuristics directly in the segmentation
process. The preprocessing step is similar to the process carried out by the method of polar
sections, since we first remove noise from the image followed by an edge detection between the
background and the target object, and finally compute the Euclidean distance map. The
initialization step is different because the polar sections are replaced by the 𝐷𝑚𝑎𝑥 parameter,
which is used to keep the search of the control points within the search space in order to minimize
the closest edge solution. The 𝐷𝑚𝑎𝑥 parameter is initialized by the user in terms of pixel
separation between control points, and it is iteratively evaluated using Equation (18) (Euclidean
distance) as follows:
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𝑛 (18)
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 = (𝑝𝑖 − 𝑞𝑖 )2
𝑖=1
The third step of this method involves the numerical optimization and the segmentation result. The
numerical optimization is performed on the intensity of the Euclidean distance map, which is used
as fitness function. The optimization technique is applied for each population separately in order to
be placed on the closest edge solution keeping 𝐷𝑚𝑎𝑥 between best potential solutions. Finally,
when the optimization process for each set of solutions is finished, the resulting segmented object
is acquired by connecting the best individual of each set to each other.
The procedure of the image segmentation method based on the Maximum Euclidean Distance is
described as follows:
1. Initialize coordinates (𝑥, 𝑦) from the origin point, 𝐷𝑚𝑎𝑥 parameter.

2. Initialize number 𝑛 of active contours and number 𝑚 of control points.
3. Initialize parameters of optimization method such as iterations and population size.
4. Generate one population or swarm 𝑂𝑖 .
5. For each population or swarm 𝑂𝑖 :
6. Apply restriction of the search space to ignore improper solutions.
7. Perform the particular search strategy of the optimization method.
BOX 6
Algorithm of the method based on Maximum Euclidean Distance.
Computational Experiments
In this section, the abovementioned methods are applied in the segmentation of human heart on
Computed Tomography images and white blood cells on microscope images.
Application to Human Heart on Computed Tomography Images
We have applied the Constrained Polar Sections method on a dataset composed of 144 Computed
Tomography (CT) images (size 512 x 512 pixels) from different patients. In Figure 13.6 the human
heart segmentation results on a subset of CT images are illustrated. Figure 13.6(a) presents the
original test images of the subset in order to increase the human perception of the segmentation
task. In Figure 13.6(b) the manual delineations of the human heart made by experts are presented.
Figure 13.6(c) shows the segmentation results obtained through the traditional implementation of
the Active Contour Model, where the fitting problem leads to an inaccurate convergence to heart
boundary. The ACM parameters used in this simulation were set as 45 control points, 𝛼 = 0.017, 𝛽 =
0.86 and 𝛾 = 0.45, according to similar tests reported in [21]. Figures 6(d), 6(e) and 6(f) illustrate the
segmentation results obtained via the CPS method using the UMDA, PSO and DE strategies,
respectively. The general parameters were set as number of contours = 9, number of control points
= 45 and generations = 10. The results obtained with the CPS method are in general more accurate
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than the results obtained with the classical ACM, since it fit to the real human heart boundary
accurately avoiding the local minima problem.
(a)
(b)
(c)
(d)
(e)
(f)
FIGURE 13.6
Human heart segmentation on CT images. (a) Test images, (b) Delineation by experts, (c) Segmentation
results using the classical ACM implementation, (d) Segmentation results using UMDA, (e) Segmentation
results using PSO, and (f) Segmentation results using DE.
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From the abovementioned dataset of CT images, in Table 13.3 a comparative analysis using the
average of the segmentation results obtained from computational methods and the regions
outlined by experts is introduced. The similarity measures used for this analysis are the Jaccard and
Dice indexes, which evaluate the segmentation result in terms of overlapping regions and they are
situated in the range [0, 1], according to [3]. This comparative analysis suggests that the CPS
method using different optimization strategies can lead to more efficiency in human heart
segmentation regarding the traditional Active Contour Model, which can be significantly help
cardiologists in clinical practice.
TABLE 13.3
Average similarity measure among the regions segmented by ACM, and the constrained polar sections
framework via UMDA, PSO and DE compared to those regions outlined by expert from the set of CT images.
Comparative Similarity Measure

Studies Jaccard index (J) Dice index (D)
ACM vs Expert 0.5272 0.6904
UMDA vs Expert 0.7142 0.8333
PSO vs Expert 0.8260 0.9047
DE vs Expert 0.8666 0.9285
The use of optimization strategies in the CPS method provides robustness, accuracy and stability in
the local minima problem, improving the segmentation accuracy. As shown in similarity analysis,
the CPS method is able to detect the human heart with a high accuracy and effectiveness, which
can help cardiologists to better analyze the medical images and increase their monitoring abilities.
Application to White Blood Cells on Microscope Images
The Maximum Euclidean Distance method is applied in the segmentation of white blood cells (also
called leukocytes) on microscope images. The experimental data set includes images with one and
two leukocytes to be detected, which are collected from the ALL-IDB database [38, 39]
(http://homes.di.unimi.it/scotti/all/).
We can apply the MED method in different ways; for instance, by using the preprocessing
procedure for the CPS method, or adopting techniques such as the Generalized Hough Transform
[40] to detect circles avoiding the interactive seed point just applying a slight adaptation as in [41,
42]. This preprocessing step uses a binarization of the test image to separate the target objects
from the background image, followed by applying the Sobel edge detector to introduce just the
boundary pixels to the Generalized Hough Transform in order to detect the most promising circles
close to the leukocytes. Finally, when the circle of the object of interest is detected, we can use the
MED method to generate the potential solutions around this circle to adapt it to the shape of the
target leukocyte.
In Figure 13.7, the preprocessing steps for the segmentation of white blood cells are illustrated.
Figure 13.7(a) presents the test microscope images; Figure 13.7(b) shows the edge detection results
using the Sobel operator. From the edge detection procedure, the Generalized Hough Transform is
able to detect circular shapes (target leukocyte) in the image as it is shown in Figure 13.7(c). This
Nanomedicine 350
circle represents the input for the MED method generating multiple concentric circles as potential
solutions. The best contour solution found by the method represents the final segmentation result.
This MED method shows a high accuracy segmentation locating the edge of the white blood cells as
it is illustrated in Figure 13.7(d).
(a)
(b)
(c)
(d)
FIGURE 13.7
White blood cells segmentation. (a) Test images, (b) Edge detection, (c) Circle detection using Hough
transform and (d) Segmentation results using the MED method.
The aforementioned procedure to detect one leukocyte can be applied to detect multiple
leukocytes in an image as it is shown in Figure 13.8(a) and (b), respectively. The procedure only
depends on the number of circles which are detected by the Generalized Hough Transform.
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(a)
(b)
FIGURE 13.8
(a) and (b) Multiple white blood cells segmentation using the procedure illustrated in Figure 13.7.
The segmentation results acquired by the CPS and MED methods show their flexibility to be applied
in different problems in medical image segmentation only by using different preprocessing steps.
Also, these methods are easy to adapt for working with different population-based methods, since
the methodologies allow preserving the original implementation of the optimization techniques.
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Concluding Remarks
In this chapter, we have presented two different frameworks based on the theory of active contour
models for medical image segmentation. The first framework through an interactive initialization
divides the target object in polar sections, in order to avoid the local minima and the sensitivity to
initial positioning problems regarding the classical implementation of the Active Contour Model.
This framework was applied in the human heart segmentation on Computed Tomography images
using different optimization strategies such as Estimation of Distribution Algorithms, Particle
Swarm Optimization and Differential Evolution. Moreover, the second framework involves a
distance parameter between potential solutions instead the constrained polar sections, and it also
introduces the Generalized Hough Transform to avoid the interactive seed point to perform the
segmentation task. This second framework was applied in the white blood cells segmentation
problem on microscope images. The experimental results revealed that by using optimization
techniques as strategy search in both frameworks, it is possible to attain high accuracy, robustness
and efficiency in the human heart and leukocytes segmentation. Finally, the experimental results
have also shown that these frameworks are appropriate and efficient to be used in different
medical segmentation problems, which is very useful for clinical decision support applications.
Acknowledgements
This work has been supported by the National Council of Science and Technology of México
(CONACYT) under Grant 241224-218157. The authors wish to thank to the cardiology department
of the Mexican Social Security Institute, UMAE T1 León for the precious clinical advice and for
kindly providing us the sources of cardiac CT images.
Nanomedicine 353
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14
Biological applications of Semiconductor
Nanoparticles
Salam Massadeh, Manal Alaamery
Brain Genome Laboratory, King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz
University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard - Health Affairs (MNGHA), Riyadh,
Kingdom of Saudi Arabia.
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 357
Properties of semiconductor nanoparticles …………………………………………..…………………………………… 358
Optical properties ………………………………………………………………………………………………………………………. 358
Quantum confinement and quantum size effect …………………………………………………………………………. 360
Biological Applications of semiconductor nanoparticles …………………………………………………………….. 361
Nanosensors ………………………………………………………………………………………………………………………………. 361
Immunochromatographic assays ……………………………………………………………………………………………….. 361
Glucose nano-sensing ……………………………………….………………………………………………………………………… 362
Live cell labeling ………………………………………………………………………………………………………………………… 364
In Vivo applications of Quantum Dots ………………………………………………………………………………………… 364
Gold nanoparticles for intracellular imaging ………………………………………………………………………………. 366
Encoding …………………………………………………………………………………………………………………………………….. 366
Flouroimmunoassays using antibody-conjugated Quantum dots ……………………………………………….. 366
Conclusions ………………………………………………………………………………………………………………………………… 367
References……………………………………………………………………………………..…………………………………………… 368
Nanomedicine 357
Introduction
Bionanotechnology is one of the key technologies of the 21st century. It has attracted the attention of
many scientists, due to the vast applications that can be produced by merging material science and
biotechnology. This field involves the utilisation of biological systems such as cells, cellular components,
and proteins, to manufacture efficient nanostructures (1–5). Nanotechnology is the new utensil that
explores biomolecular structures, functions and properties. Bionanotechnology have already been used
to measure structural elements of cells, molecular recognition and also have been applied in drug
delivery (6–12). Biomolecules and biological complexes are naturally existing building blocks that have
a great importance in the direction of molecular recognition and self-assembly. More complex
structures such as viruses and bacteriophages could be assembled at the nanoscale. Such bio-
nanostructures can direct the "bottom up" assembly (13–17).
In the same vein, the unique electronic, optical, and catalytic properties of nanoparticles associate in
the generation of new devices and material with new functions and properties (18–21). Additionally,
the conjugation of nanoparticles with biomaterials may render new magnitudes in nano-biotechnology.
Biomolecules have a size range between 2-100 nm. These proportions are similar to those of the
nanoparticles which increase the possibility of their applications into living cells (22–26). Figure 14.1
below shows the dimensions of nanoparticles compared with other species.
Nanomaterials have at least one spatial dimension in the range of 1-1000 nm, and show unique
mesoscopic properties that will be discussed later in this chapter. Additionally, semiconductor
nanoparticles (NPs) or also called nanocrystals (NCs) are characterised as small inorganic solids that
have a size range of 1-100 nm. Figure 14.1 below displays the dimension of different species showing
the position of the nanoparticles in between (18,27–29).
Atoms Proteins Viruses Bacteria
Volume
1 nm 10 nm 100 nm 1 µm
H3C CH3
Molecules Nanocrystals Visible light
FIGURE 14.1
dimensions of different species showing the position of nanomaterials.
In this chapter we will be discussing a specific type of semiconductor nanoparticles called Quantum
dots (QDs). QDs are highly luminescent, colloidal semiconductor nanocrystals. QDs have unique size-
dependant properties, which make them highly attractive for applications in catalysis, phosphors,
Nanomedicine 358
photovoltics, light emitting diodes (LEDs) and biological labeling. The main appealing feature of
semiconductor NCs, are their mesoscopic properties that differentiates them from bulk crystals. The
mesoscopic properties will be discussed in details later in this chapter (4,29–37).
When quantum dots (QDs) were first explored 20 years ago, they were applied in electronics and
optics. At that time, it was still unrealized that QDs could be suitable for applications in biology and
medicine. However, their use as research devices has extended prominently in the last decade, and
currently QDs are being used as probes for high resolution molecular imaging of cellular components
and for tracking cell activities and movements.
Besides, it is possible to bind quantum dots to proteins and receptors to check with which molecules
they interact and to explore their location in the cell. Hence, QDs are used in biomedical applications
because of their unique tuneable optical properties, (38–40). The semiconductor QDs are usually
synthesized in organic media, which will produce hydrophobic QDs, in order to use these QDs in
biological applications they should be rendered biocompatible; by masking their surface with a
hydrophilic coating. The key approaches to make QDs biocompatible include silanisation and surface
exchange with bifunctional molecules (38–41). Consequently, forming nanobioconjugates should be
synthesised in a way that keeps the bioconjugate in the nano size regime. (42).Typically, the
nanoparticles are coupled to the biomaterials through two main approaches; direct covalent linkage
and non-covalent interactions between the particle and biomolecules (43). QDs of different emission
wavelengths could be excited with a single light source, which gives these particles an advantage to be
used for multiplex imaging of molecular targets coating layer of wider band gap semiconductor plays an
important role to passivate its surface, and reduce non-radiative carrier recombination and thus
increases the optical characteristics of the semiconducting nancrystal (20,27,44).
Properties of semiconductor nanoparticles

Optical properties
QDs are spherical semiconductor nanocrystals made of elements from the periodic groups II-VI (CdSe)
or III-V (InP)(34,45). QDs are extremely fluorescent, due to their spatially confined band gaps resulting
in physical, and optical, properties in-between compounds and single molecules. Quantum
confinement allow QDs to emit light at different wavelengths directly related to their core
diameter(46). Also, the relatively small size of QDs allows the crystal to behave as a single molecule
with all its atoms being excited and emitting light together, resulting in a high signal intensity. In the
same vein, one of the main optical properties of QDs is their resistance to photobleaching, and their
long fluorescence lifetime when compared with organic flourophores. Organic flourophores or organic
dyes tend to decay within nanoseconds and exhibit photobleaching (24,31,47–49) figure 14.2.
FIGURE 14.2
Florescent core/Shell quantum dot, with hydrophobic ligands on the surface.
Nanomedicine 359
Additionally, quantum dots are made up of 100–2000 atoms. This unique type of nanocrystals has
physical and chemical properties that are determined by two factors. The first factor is the high
surface/ volume ratio of the nanoparticles. The second factor is the size of the particle, which
determines the electronic and physical properties of the material. Yet, the optical spectra of
nanocrystalline semiconductors (figure 14.3) exhibit a blue shift in their photoluminescence spectra as
the particle size decreases. This size dependent optical property is an example of the size quantisation
effect which occurs when the size of the nanoparticle is smaller than the bulk-exciton Bohr radius, aB,
of the semiconductor (equation 1)(36).
2   1 1 
aB   *  * 
Equation 1
e2  e
m mh 

Where, aB is the bulk-exciton Bohr radius, є is the bulk optical dielectric coefficient, e the elementary
charge, and me* and mh* the effective mass of the electron and hole respectively.
2 nm 8 nm
FIGURE 14.3
Emission spectra of differently colored InP/ZnS quantum dots (excited at 400 nm), displaying the increase in size
near the IR region.
Moreover, nanoparticles have very little scattering of visible light due to their small size. Mie's solution
to maxwell's equations describes all aspects of light scattering. For spherical particles much smaller
than the wavelength of light (0.1xλ) it can be simplified to the Rayleigh approximation. For Rayleigh
scattering the intensity I of light scattered by a single small particle from a beam of nonpolarised light
6
of wavelenghth λ and intensity I0 is proportional to (d/2) (equation 2).
Nanomedicine 360
I  I0
1  cos 2
    2 
 n 2  1   d 6

4
  
2
Equation 2
    
n 2  2 
2 2
2R
Where R is the distance to the particle, θ is the scattering angle, n is the refractive index of the particle,
and d is the diameter of the particle [].
Quantum confinement and quantum size effect
The optical absorption spectrum of semiconductor nanocrystals is a direct method for the assessment
of quantum size effects. The absorption of a photon excites an electron from the valence band to the
conduction band, which is associated with the band gap energy (Eg). The absorption of photons with
energy similar to that of the band gap, hv ≥ Eg, activates an optical transition that creates an electron in
the conduction band of the semiconductor alongside with a hole in the valence band.
Absorption of photons with energy superior to Eg leads to excitations exceeding the conduction band
edge. The absorption (A) of light by a semiconductor material with thickness l can be expressed by an
expression similar to the Beer law, where α represents the absorption coefficient of the solid and is a
function of the radiation frequency (equation 3)
A  I Equation 3
The electronic band structure of semiconducting nanoparticles is different from the bulk
-
semiconductors, in an intrinsic semiconductor an electron (e ) is excited into the conduction band by
+
photon absorption, leaving a positively charged hole (h ) in the valence band. When these two charges
communicate, by their Coulumb potential developing a quasiparticle called an exciton, they are bound
in a way that resembles the electron-proton binding in the hydrogen atom, these charges interact as
the exciton recombines, excess energy is released either as photoluminescence (radiative decay, Kr) or
heat (non-radiative decay) the equation 1-4 presents the exciton's Bohr radius (45,50–52).
Semiconductors have band gap energy (Eg) wich separates the valence and conduction bands. The
density of bonding orbitals (σ) and antibonding orbitals (σ*) decreases as the size of the semiconductor
decreases, which directs to a discrete energy level within the valence and conduction bands which are
comparable to the discrete energy levels of individual atoms (Figure 14.4).
Antibonding
orbitals
Band gap
Bonding
Energy orbitals
FIGURE 14.4
the fluorescence properties of quantum dots arise from the energy gap of the confined QD.
Nanomedicine 361
The nanocrystal is in the weak confinement regime when the semiconductor radius r is larger than the
exciton Bohr radius aB, in this case the quantisation of the exciton centre-of-mass motion take place
and the exciton is competent to shift as a net uncharged particle. It is assumed that the nanocrystal is
strongly confined when r<<aB (53). Furthermore, the strongly confined semiconductor nanocrystals
have a size dependent photolomuniscence wavelength, the energy levels in a QD become quantised
E
due to the confinement of electrons (figure 14.4 above), the band gap energy g , is determined by
the orbitals inside the particle. The Brus equation below shows these changes as a function of
nanocrystal size(Equation 4) (46,53,54).
h 2 2 1.8e 2
E g (r ) E g (r  )   Equation 4
2r 2  4 r  0 r
Where r=radius of the nanocrystal, εr=the relative dielectric constant of the semiconductor and ε 0=
vacuum permittivity.
Biological Applications of semiconductor nanoparticles

Nanosensors
Quantum dots (QDs) have attracted the attention of many scientists working in the field of biosensors.
Their long-term photostability, makes real-time and continuous monitoring possible. Lately, scientists
have focused on creating sensors for different target analytes, where QDs are utilised as electron
donors for fluorescence resonance energy transfer (FRET) between a donor (the QDs) and an
acceptor(49,55,56). In addition to FRET, QDs have many valuable properties that have been exploited
for the development of nanosensors that depend on the alteration in the emission wavelength, voltage
2+ 2+ 2+ 2+
or fluorescence intensity. QDs have been used as optical sensors of ions (Cu , Mn , CO , Hg ), drugs,
organic pollutants such as polycyclic aromatic hydrocarbons, and small biological molecules (glucose,
folic acid)(57–61).
Immunochromatographic assays
A promising model of nanosensors is the development of immunochromatographic test strips, to

construct easy-handling nano-biosensors. Gold nanoparticles (GNPs) have formed the basis of an
accessible tool already marketed; which is the home pregnancy test. The GNPs are used in such test
strips because of their high stability compared with other systems that rely on the use of fluorescence
or enzymatic labels. Also, the surface of GNPs makes them good candidates for rapid antibody–antigen
recognitions. In the presence of the human chorionic gonadotropin hormone (HCG), a sandwich-type
assay is formed between the secondary antibody–immobilized GNPs immunocomplex and the primary
antibody immobilized on the membrane. When the antigen–antibody reaction takes place, the viewing
window of the home pregnancy test, shows a red line caused by the aggregation of gold nanoparticle at
that location. The red line is a visual indicator of the presence of the HCG hormone in the urine sample.
Figure 14.5 below demonstrates the principel of the home pregnancy test based on the use of GNPs
(62).
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GNPs-AB1
GNPs-AB2
GNPs reservoir
Filter
Result window
FIGURE 14.5
the use of gold nanoparticles (GNPs) in the home pregnancy test. The GNPs are conjugated to Antibodies that
recognises the human chorionic gonadotropin hormone (HCG); Urine passes from the flow stick to a central
reservoir containing gold nanoparticles. If pregnancy hormone is present, the GNPs form a complex with the HCG,
which results in the aggregation of the nanoparticles, this aggregation is diplayed as a red signal in the viewing
window of the test.
Glucose nano-sensing
The detection of glucose is an important application of nanotechnology. Scientists have widely

investigated this specific type of nanosensor. The function of the glucose nanosensor is based on the
presence of highly fluorescent nanoparticles, QDs. The quantification of glucose concentrations is of
high importance in controlling different biotechnological processes, and in diagnosing several
metabolic disorders, like diabetes. Previously, other glucose detection methods have been applied, like
amperometric, spectrophotometric, fluorometric methods.
In the case of the QD-glucose sensor, the enzyme glucoseoxidase (GOX) is coupled to the nanoparticles
to create an optical detection tool. In the presence of glucose the enzyme GOX releases Hydrogen
Peroxide (H2O2) as a result of the enzymatic reaction; Hydrogen Peroxide initiates the formation of free
radicals, which reduces the luminescence of the nanoparticles. The quenching of the fluorescence of
QDs is a direct indicator of the presence of glucose in the system (63). Figure……below shows an
example of fluorescent detection of glucose. Hydrophilic CdSe/ZnS QDs have been used to sense
glucose. The fluorescence quenching of the QDs has been applied to measure the concentrations of
glucose in aqueous solution. The quenching process was based on the transfer of electrons from the
QDs to enzymes (glucose oxidase (GOD), peroxidase (HRP)), which catalyze the oxidation/reduction
reactions of glucose and all of the components involved in the process. The QDs have been able to
detect glucose by introducing them directly into the glucose solution after their conjugation with the
GOX enzyme(58,64–67) (figures 14.6 and 14.7).
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FIGURE 14.6
Optical sensing of Glucose. In the presence of Glucose, Hydrogen Peroxide is released and causes a rapid decrease
in the photoluminescence of quantum dots from reference (64).
FIGURE 14.7
Change in fluorescence intensities of the QD-FRET-based probes at different glucose concentrations and various
volume ratios of QDs/GOD/HRP added to glucose solution (64).
Nanomedicine 364
Live cell labeling
Quantum Dots have been projected as alternatives for traditional organic dyes and fluorescent proteins
in imaging applications due to their exceptional photophysical properties. QDs are about 10–100 times
brighter and show narrower and more symmetric emission spectra than organic fluorophores.
Additionally, QDs present large absorption cross-sections, which make them 100–1000 times resistant
against photobleaching. QDs with different emission wavelengths could be excited with a single light
source, producing spectrum that ranges from the ultraviolet to the NIR (68–71).
Mainly, live cells studies with QDs have shed the light on the presence of cellular membrane markers.
As they ease the access of QDs and do not require passage of the probes through the cell membrane.
The next step is to target the cytoplasmic molecules. It has been really challenging to determine the
best technique for cytoplasmic translocation of QDs. A variety of techniques have been extensively
used for intracellular labelling with QDs. For instance: passive uptake, receptor-mediated and non-
specific endocytosis, cell penetration, liposome mediated intracellular delivery, electroporation and
microinjection (72–75). Electroporation of QDs into cells involves the application of electric pulses,
which temporarily disturb the phospholipid bilayer, consequently, increasing the permeability of
cellular membranes. Nevertheless, it may cause the aggregation of QDs inside the cells causing cell
death. However, direct injection (micro-injection) is one of the most commonly applied methods of
QDs translocation into living cells. Microinjection of QDs into cells, is a direct method that introduces
QDs into the cytoplasm or the nucleus of the cell by applying pressure or electrical impulse. This
method have made intracellular organelles labeling feasible and more specific, however, it is a time
consuming and might cause the aggregation of the QDs due to the high pressure applied when applying
this technique. Hence, scientists working in this field are highly encouraged to develop new techniques
that could be less time-consuming and less aggressive on the QDs samples.
Jaiswal et al. have applied the QD cell labeling method to investigate the effect of starvation on
Dictyosteliumdiscoideum cells. These cells were starved for different periods of time, and then they
were labeled with differently colored QDs. Consequently the labeled cells were imaged; it has been
observed that the QDs have the tendency to aggregate depending on the degree of starvation of cells.
The experiments executed by Jaiswal et al. could not be achieved using the conventional organic dyes
due to their susceptibility to photobleaching(76).
Moreover, Dubertret et al. has reported a very important study about the use of CdSe/ZnS Qds in
bioimaging. PEG functionalized QDs were synthesized to study the development in Xenopus
embryos(41) Figure 14.8. The QDs were internalized by microinjection into individual cells of the
growing embryo; the embryonic development has been studied for individual cells, since the
fluorescence of QDs was confined to the offspring of the studied cells. This study has revealed that QDs
used were highly stable and had low toxicity. Additionally, QDs have been utilized to measure cell
motility via imaging of phagokinetic tracks. It has been shown that cells have been able to engulf the
QDs, through a mechanism that was undefined(13,77,78).
In Vivo applications of Quantum Dots
Many research groups have focused their research on the use of QDs in vivo, replacing the fluorescent
polymers and the organic dyes. QDs have superior qualities over the conventional fluorescent dyes, as
QDs are photostable when used during long-term experiments. Akerman et al. have applied specific
targeting of peptide-QDs bioconjugates in mice. QDs have been conjugated to peptides that specifically
target lung blood vessel endothelial cells, tumor cell blood vessels, and tumor cell lymphatic vessels
were conjugated to QDs, the QD-bioconjugate were introduced intravenously into mice. QD
Nanomedicine 365
bioconjugates have specifically targeted the lung and tumor vasculature, with no observed toxicity (74).
Also, it has been noticed that the QDs have accumulated in the liver, Spleen and the targeted tissues.
On the other hand, when the quantum dot conjugates were specifically targetting tumor, the QD-
bioconjugates have only accumulated in the tumor cells, which indicates that the QD-bioconjugates
were able to specifically target the tumor cells.
Moreover, QDs have displayed great promise when imaging the vascular networks of mammals such as
lymphatic and cardiovascular systems.It have been demonstrated by Kim et al. that the NIR
fluorescence of QDs could be used to locate the position of sentinel lymph nodes in mice and pigs(79).
Various studies have focused on the movement of QDs through the lymph system to nodes, this
application have been of tremendous importance in assisting surgeons to locate and remove lymph
nodes in special medical cases (80,81).
FIGURE 14.8
QDs labeling of Xenopus embryos at different stages and specific QD intracellular localizations. (A) Schematic
showing the experimental strategy. QD-micelles, were injected
into an individual blastomere during very early cleavage stages (B) to (E), transmission and fluorescence images
have been superposed. (B) Injection of one cell out of an eight-cell-stage embryo resulted in labeling of individual
blastomeres. (C) Same embryo shown 1 hour later. The daughter cells of the injected blastomere are labeled (D)
and at a later stage (E) show two neurula embryos, which were injected into a single cell at the eight-cell-stage in
the animal pole. The QDs can be visualized through the pigmented layer of the epidermis. (F) Intracellular labeling
of an axon (arrow) and somites at tadpole stage 40. The QD-micelles migrate into axons all the way to growth
cones. In the somites, the QD-micelle seems to localize in subcellular structures. (G) QDs localized in the nucleus
during mid-blastula stages. This localization is reduced in later stages of the development. (H) Labeled neural crest
cells migrating into the branchial arches. (I) QD fluorescence observed in the gut of an injected embryo. Printed
from (41) with permission.
Nanomedicine 366
Gold nanoparticles for intracellular imaging
Gold nanoparticles (GNPs) have been used in several biological applications; like drug delivery of
hydrophobic drugs, photothermal agents, radiotherapy dose enhancer and in nanosensing. Moreover,
recent studies have shown that gold nanoparticles are promising candidates for cancer stem cell
therapy. Ai et al. have utilized gold nanoparticles in cancer cell imaging and photodynamic therapy
(PDT). The GNPs have been functionalized with AS1411 aptamer and with one prphyrin derivative.
Cancer cells (HeLa cells) have been targeted due to their overexpressed nucleolin. The functionalized
GNPs were able to specifically bind to the cell surface through specific interaction between the AS1411-
nucleolin and the AS1411 aptamer conjugated to the GNP. Hence the conjugated GNPs were able to
differentiate between the cancer cells and the normal cells. The cancer cells were distinguished by the
increase in their fluorescence intensity, caused by the specific binding of the GNPs(82).
Also, Kim et al. have applied GNPs for gene delivery(83). The GNPs were covalentely bound to small
interfering RNA to its surface. siRNA-gold nanoparticles were introduced onto PBMCs or 293T cells.
This study has found that the covalentely conjugated siRNA-gold nanoparticles were highly stable. The
amount of GNPs uptake into the cells, have increased with increasing the concentration of the
functionalized GNPs, in a linear manner. The GNPs internalized by the cell have been located in
membrane-bound organelles with electron-dense cores, and they were not free in the cytosol. Also,
the oligonucleootides bound to the GNPs have activated the immune-related genes and pathways in
human peripheral blood mononuclear cells.
Encoding
Single coulour QDs could be applied to color-code or identify objects. Also, differently couloured QDs
can produce clear spectral codes that can be used effectively in multiplexed assays. QDs are highly
advantageous when used to produce spectral codes, because different colours of QDs can be excited at
a single excitation wavelength and they are photo-resistant. The color -coded units can be successfully
decoded using imaging techniques to determine their unique fluorescence spectra(84,85).
In the same vein, Han et al. have applied the multicolor coding in biological assays. Various sizes of
CdSe/ZnS QDs have been embedded into polymer microbeads(85). The unique optical properties of
QDs made them suitable for wavelength-and-intensity multiplexing. In this study, six colors of QDs
having ten different intensity levels. It have been found that the QD-embedded beads were highly
stable and uniform, also it has been shown that DNA hybridization studies show that the coding and
target signals could be read at the single bead level.
Flouroimmunoassays using antibody-conjugated Quantum dots
Quantum dots are highly stable, with excellent quantum yields that can reach up to 90%. The relatively
intense luminescence of QDs could be detected at concentrations comparable to standard fluorescent
organic dyes (86). Many conjugation protocols have been developed to conjugate quantum dots to
antibodies. One of the main applications of Antibody-conjugated QDs is their use in
fluoroimmunoassays for the detection of proteins or small molecules. Goldman et al. have developed a
conjugation strategy based on electrostatic self-assembly between negatively charged dihydrolipoic
acid (DHLA)-capped CdSe/ZnS QDs and positively charged proteins, that will form a bridge between the
quantum dot and the antibody(87).
Nanomedicine 367
Wang et al. have synthesized CdTe QD- bioconjugates based on antibody−antigen interactions. In this
case bovine serum albumin (BSA) was the antigen conjugated to red fluorescent CdTe QDs. On the
other hand, Anti-BSA antibody (IgG) has been coupled to green fluorescent CdTe QDs. The
bioconjugates have been characterized by SDS−PAGE electrophoresis, gel-permeation HPLC, and
circular dichroism. The Antigen−antibody binding affinity was assessed by enzyme-linked
immunosorbent assay (ELISA). Additionally, FRET analysis have shown that the luminescence of green-
emitting NPs have been quenched whereas the emission of the red-emitting NPs have been enhanced.
However, the luminescence of the two colored CdTE QDs has been recovered as the immunocomplex
was exposed to an unlabeled antigen(88).
Conclusions
This chapter has demonstrated the tremendous applications of nanotechnology in biological
applications. More specifically, we have shed the light on the use of semiconductor nanoparticles in
biology. The superior set of properties that quantum dots offer makes them suitable alternatives of the
traditional organic dyes. Also, we have shown many examples on promising biological applications of
QDs. QDs have made the single-molecule detection feasible. Plus, the use of QDs in encoding has great
potential to reform high-throughput biology. The spectral coding technology is anticipated to open new
eras in gene expression studies, high-throughput screening, and medical diagnostics. Future research
will explore the use of the multiple emission colors of quantum dots for tracking of intracellular
compartement.
Additionally, the toxicity of nanomaterials is a major factor that scientists should focus on during their
research. Many research groups have already paid so much attention on the toxicity of QDs, some of
them have discontinued the use of Cd containing QDs, and replaced them with less toxic alternatives
such as; Indium, Silicon…etc. When toxicity is observed, it has been found to occur from controllable
parameters, such as NP stability or dose. Hence, it is doubtful that toxicity issues should hinder the
development of the exciting bio-applications offered in this chapter.
In conclusion, the surface functionalization of nanocrystals adds more functions and uses for the
nanoparticles, opening doors to new biological applications. Advances involving the surface
modification of QDs have enabled many novel biological experiments. Albeit, quantum dots could be
challenging to deal with in every application, it is very likely that they will become a leading fluorescent
probe in biology over the next several years.
Nanomedicine 368
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15
Biosensors and Nanobiosensors: Design
and Applications
Ahmed Touhami
Physics & Astronomy Department, University of Texas at Brownsville

One west university boulevard, Brownsville, Texas, 78520, USA
Outline:
Introduction: What is a Biosensor? ……………………………………………….……………………………………………. 374
Micro-Biosensors …………………………………………..…………………………………………………………………………… 375
Biosensing Techniques ……………………………………………………………………………………………………………….. 375
Electrochemical Biosensors ………………………………………………………………………………………………………… 376
Potentiometric Biosensors ………………………………………………………………………………………………………….. 376
Amperometric Biosensors ……………………………………………………………………………………………………………377
Voltammetric Biosensors ……………………………………………………………………………………………………………. 378
Optical Biosensors ……………………………………….…………………………………………………………………………….. 379
Fluorescence-based Biosensors …………………………………………………………………………………………………… 380
Surface Plasmon Resonance Biosensors ……………………………………………………………………………………… 380
Optical fiber based biosensors ……………………………………………………………………………………………………. 382
Acoustic Biosensors ……………………………………………………………………………………………………………………. 384
Quartz Crystal Microbalance ………………………………………………………………………………………………………. 386
Application and Examples of Micro-Biosensors ………………………………………………………………………….. 386
In Vivo Biosensor ………………………………………………………………………………………………………………………… 386
The Glucose Biosensor ………………………………………………………………………………………………………………… 387
Array-type Biosensors ………………………………………………………………………………………………………………… 388
Microbial Biosensor ……………………………………………………………………………………………………………………. 389
Nanobiosensors: Basic concepts & Applications …………………………………………………………………………. 391
Nanomaterials for new biosensing principles ……………………………………………………………………………… 391
Immobilization Strategies at the Nanoscale ……………………………………………………………………………….. 392
Examples of nanobiosensors ………………………………………………………………………………………………………. 393
Nanowire Biosensors ………………………………………………………………………………………………………………….. 393
Cantilever Biosensors …………………………………………………………………………………………………………………. 395
Ion-channel based sensing …………………………………………………………………………………………………………. 396
Nanobiosensors in Nanomedicine ……………………………………………………………………………………………….398
Conclusion …………………………………………………………………………………………………………………………………. 398
References……………………………………………………………………………………..…………………………………………… 399
Nanomedicine 374
Introduction: What is a Biosensor?

The most widely accepted definition of a biosensor is: “an analytical device which incorporates a
biologically active element with an appropriate physical transducer to generate a measurable
signal proportional to the concentration of chemical species in any type of sample” *1-5].
Type of Biosensors: Biosensors can be categorized according to the basic principles of signal
transduction and biorecognition elements. In the general scheme of a biosensor (figure 15.1), the
biorecognition element responds to the target compound and the transducer converts the
biological response to a detectable signal, which can be measured electrochemically, optically,
acoustically, mechanically, calorimetrically, or electronically, and then correlated with the analyte
concentration. Biological elements include enzymes, antibodies, micro-organisms, biological tissue,
and organelles. When the binding of the sensing element and the analyte is the detected event, the
instrument is described as an affinity sensor. When the interaction between the biological element
and the analyte is accompanied or followed by a chemical change in which the concentration of
one of the substrates or products is measured the instrument is described as a metabolism sensor.
Finally, when the signal is produced after binding the analyte without chemically changing it but by
converting an auxiliary substrate, the biosensor is called a catalytic sensor [5, 6]. The method of
transduction depends on the type of physicochemical change resulting from the sensing event [5].
Often, an important ancillary part of a biosensor is a membrane that covers the biological sensing
element and has the main functions of selective permeation and diffusion control of analyte,
protection against mechanical stresses, and support for the biological element.
The research field of biosensors started with the introduction of the first generation glucose
oxidase (GOx) biosensor in 1962 by Clark and Lyons [7]. Since then, biosensors have been
intensively studied and extensively utilized in various applications, ranging from public health and
environmental monitoring to homeland security and food safety [5, 8-11]. According to the Web of
Knowledge, over 4000 research papers about biosensors have been published each year for the
past three years (2011-2013). Though a lot of research activity has been involved in developing
biosensors for various purposes the time has come to bring this technology to the forefront and
make it commercially available [12-14]. Efforts and funds need to be mobilized to manufacture
biosensors on a large scale so as to benefit and be of use to the general public. With exposure to
the commercial market the applications of this technology would be greatly enhanced. A few such
applications could be detection of virulence of a vaccine just before it is injected so as to prevent
accidental acquisition of a disease, bandages detecting a septic wound, deadly viruses in the
environment or from the patient sample (rapid and early detection) and so forth in the medical
field. Real time monitoring of dairy products and breweries might help foster a cleaner and hygienic
environment and experiment with different tastes imparted by specific microorganisms in specific
concentrations giving rise to new products. A farfetched and plausible use of this technology could
be in space exploration where if present the concentration of the living organisms would be very
low and might lead to answering many of the long standing questions regarding the presence of life
in space.
The goal of this chapter is to cover the full scope of biosensors. It offers a survey of the principles,
design, operation, and biomedical applications of the most popular types of biosensing devices in
use today. By discussing recent research and future trends based on many excellent books and
reviews, it is hoped to give the readers a comprehensive view on this fast growing field.
Nanomedicine 375
FIGURE 15.1
Schematic of a typical biosensor: a) biorecognition element that specifically bind to the analyte; b) an interface
architecture where a specific biological event takes place and gives rise to a signal picked up by c) the
transducer element; the transducer signal (which could be anything from the in-coupling angle of a laser beam
to the current produced at an electrode) is converted to an electronic signal and amplified by a detector circuit
using the appropriate reference and sent for processing by d) computer software to be converted to a
meaningful physical parameter describing the process being investigated; finally, the resulting quantity has to
be presented through e) an interface to the human operator. (Modified and adopted from reference 11).
Micro-Biosensors
The major progress in microsystem technologies for creating small, integrated and reliable
microtransducers devices in combination with biological sensing elements has revolutionized the
field of biosensors during the last decade. Such micro-biosensor systems raised the expectation to
get a comprehensive insight into dynamic cellular metabolic events and subsequently a complete
understanding of the metabolism of human biology. Currently, cancer can be detected by
monitoring the concentration of certain antigens present in the bloodstream or other bodily fluids,
or through tissue examinations [13]. Correspondingly, diabetes is monitored by determining the
glucose concentrations in the blood over time [14]. However, despite their widespread clinical use,
these techniques have a number of potential limitations. For example, a number of diagnostic
devices have slow response times and are burdensome to patients. Furthermore, these assays are
expensive and cost the health care industry billions of dollars every year. Therefore, there is a need
to develop more efficient and reliable sensing and detection technologies.
Optical transduction will be the focus of this article
Biosensing Techniques
Biosensors can be classified either by the type of biological signaling mechanism they utilize or by
the type of signal transduction they employ. Transduction can be accomplished via a great variety
Nanomedicine 376
of methods. Most forms of transduction can be categorized in one of three main classes. These
classes are: 1) electrochemical detection methods, 2) optical detection methods and 3) mass
detection methods. However, new types of transducers are constantly being developed for use in
biosensors. Each of these three main classes contains many different subclasses, creating a nearly
infinite number of possible transduction methods or combination of methods. Here, we will discuss
each of these three detection mechanisms.
Electrochemical Biosensors
The first scientifically proposed as well as successfully commercialized biosensors were those based
on electrochemical sensors for multiple analytes [7, 14-17]. At present, there are many proposed
and already commercialized devices based on the electrochemical principle including those for
pathogens and toxins [18]. This stems from a number of attributes of electrochemistry including
the high sensitivity of electrochemical transducers, their compatibility with modern
miniaturization/microfabrication technologies, minimal power requirements, economical cost, and
independence of sample turbidity and color [16]. The basic principle for this class of biosensors is
that chemical reactions between immobilized biomolecule and target analyte produce or consume
ions or electrons, which affects measurable electrical properties of the solution, such an electric
current or potential [15, 16]. The electrochemical signal produced is then used to relate
quantitatively to the amount of analyte present in a sample solution. Potentiometry, amperometry,
voltammetry, and, more recently, electrochemical impedance spectroscopic measurements are
among the electrochemical detection techniques often used in conjunction with immunoassay
systems and immunosensors, leading to their respective categories according to the type of signal
measured. The fundamental principles of each of these techniques are presented below, followed
by discussions based on some recent work that have specifically addressed problems encountered
in these areas.
Potentiometric Biosensors
These biosensors are based on ion-selective electrodes (ISE) and ion-sensitive field effect
transistors (ISFET) [14-18]. The primary outputting signal is possibly due to ions accumulated at the
ion-selective membrane interface. Current flowing through the electrode is equal to or near zero.
The electrode follows the presence of the monitored ion resulting from the enzyme reaction. For
example, glucose oxidase can be immobilized on a surface of the pH electrode. Glucose has only
minimal influence on pH in the working medium; however, the enzymatically formed gluconate
causes acidification. A biorecognition element is immobilized on the outer surface or captured
inside the membrane. In the past the pH glass electrode was used as a physicochemical transducer
[19]. Nowadays, semiconductor based physico-chemical transducers are more common. ISFETs and
LAPS (light addressable potentiometric sensor) based systems especially are convenient for
biosensor construction. The ISFET principle is based on a local potential generated by surface ions
from a solution [16, 20]. This potential modulates the current flow across a silicon semiconductor.
The transistor gate surface in ISFET is covered by a selective membrane; for pH detection this could
be made from compounds such as silicon nitride (Si 3N4), alumina (Al2O3), zirconium oxide (ZrO2) and
tantalum oxide (Ta2O5). The LAPS principle is based on semiconductor activation by a light-emitting
diode (LED). The sensor is made from an n-type silicon typically coated with 30 nm of silicon oxide,
100 nm of silicon nitride, and indium-tin oxide. The LAPS measures a voltage change as a function
of medium pH in the LED activated zone 9figure 15.2). This opens the way for multiposition sensing
Nanomedicine 377
and construction of an array of biorecognition zones. A good example of a potentiometric

immunosensor involves the detection of enzyme-labeled immunocomplexes formed at the surface
of a polypyrrole coated screen-printed electrode [20].
In potentiometric measurements, the relationship between the concentration and the potential is
governed by the Nernst equation, where 𝐸𝑐𝑒𝑙𝑙 represents the observed cell potential at zero
0
current. This is sometimes also referred to as the electromotive force or EMF. 𝐸𝑐𝑒𝑙𝑙 is a constant
potential contribution to the cell, R the universal gas constant, T the absolute temperature in
degrees Kelvin, n is the charge number of the electrode reaction, F is the Faraday constant and Q is
the ratio of ion concentration at the anode to ion concentration at the cathode [21].
0
𝑅𝑇
𝐸𝑀𝐹 𝑜𝑟 𝐸𝑐𝑒𝑙𝑙 = 𝐸𝑐𝑒𝑙𝑙 − 𝑙𝑛𝑄
𝑛𝐹
The direct determination of the analyte ion concentration with the Nernst equation is referred to as
direct potentiometry. The lowest detection limits for potentiometric devices are currently often
achieved with ion-selective electrodes (ISE). Therefore, by definition the detection limit is analyte
-8 -11
specific and current devices have limits of detection in ranges between 10 to 10 M.
Potentiometric sensors prove suitable for measuring low concentrations in tiny sample volumes,
since they ideally offer the benefit of not chemically influencing a sample. The variety of ions, for
which low detection limits are possible, is currently quite limited and missing such important
analytes as: nickel, manganese, mercury and arsenate ions. Detailed information about
potentiometry and their limit of detection (LOD) is provided in the review by Bakker et al. [22].
FIGURE15.2
Block diagram of the light addressable potentiometric sensor with biorecognition component bound into
membrane and with buffered reaction cell.
Amperometric Biosensors
In amperometry, the current produced by the oxidation or reduction of an electroactive analyte

species at an electrode surface is monitored under controlled potential conditions. The magnitude
of the current is then related to the quantity of analyte present [16]. Clark oxygen electrodes
perhaps represent the basis for the simplest forms of amperometric biosensors, where a current is
produced in proportion to the oxygen concentration. This is measured by the reduction of oxygen
Nanomedicine 378
at a platinum working electrode in reference to a Ag/AgCl reference electrode at a given potential

[23]. Typically, the current is measured at a constant potential and this is referred to as
amperometry. If a current is measured during controlled variations of the potential, this is referred
to as voltammetry. Furthermore, the peak value of the current measured over a linear potential
range is directly proportional to the bulk concentration of the analyte, i.e. the electroactive species
[15]. However, not all protein analytes are intrinsically capable to serve as redox partners in
electrochemical reactions, a suitable label must be introduced to promote the electrochemical
reaction of the analyte at the working electrode [23] (figure 15.3). Despite the disadvantage of this
often indirect sensing system, it is claimed that amperometric devices maintain a sensitivity
superior to potentiometric devices [24]. An example of an amperometric device is the
aforementioned glucose biosensor, which is based on the amperometric detection of hydrogen
peroxide. A very tangible application of amperometry is used in combination with immunosensing
techniques to measure levels of the human chorionic gonadotropin -subunit (-HCG) in advanced
pregnancy testing.
FIGURE15.3
(A) Example of the three-electrode screen-printed sensor produced by BVT (Brno, Czech Rep.). The sensor
body is made from ceramics. A gold working electrode (a) is surrounded by an Ag/AgCl reference electrode (b)
and gold auxiliary electrode (c). Letter d means silver output contacts. The ruler in the bottom is in millimeter
scale (from refer [25]). (B) A sample amperometric measurement: This is a typical hydrodynamic response of
their biosensor to glucose followed by several injections of ATP measured in phosphate buffer at 650 mV in
reference to Ag/AgCl. The change in current response is proportional to the ATP concentration as glucose is
consumed at the glucose oxidase (GOD) and hexokinase (HEX) modified electrode surface (from refer [26]).
Voltammetric Biosensors
Voltammetry belongs to a category of electro-analytical methods, through which information about

an analyte is obtained by varying a potential and then measuring the resulting current. It is,
therefore, an amperometric technique. Since there are many ways to vary a potential, there are
also many forms of voltammetry, such as: polarography (DC Voltage) [25], linear sweep, differential
staircase, normal pulse, reverse pulse, differential pulse and more. Cyclic voltammetry is one of the
most widely used forms and it is useful to obtain information about the redox potential and
electrochemical reaction rates (e.g. the chemical rate constant) of analyte solutions. More recently,
interdigitated array (IDA) microelectrodes have gained popularity as an alternative transducer in
electrochemical immunoassays. In general, a simple design of an IDA consists of a pair of
interdigitated microelectrode “fingers”. When an IDA is used as a sensing electrode in a
Nanomedicine 379
voltammetric experiment, the two interdigitated electrodes are usually held at different potentials
to achieve “redox” cycling of the electroactive species to be detected. A major advantage of this
redox cycling is that it improves the signal-to-noise ratio by enhancing the Faradaic current relative
to the background current, resulting in lower detection limits and improved sensitivity. These
features opened up many opportunities in which IDA electrodes were applied as electrochemical
detectors in analytical chemistry and biosensor systems [26].
FIGURE15.4
An example of cyclic voltammogram. The cholesterol/PB sol-gel modified glassy carbon electrode in a
phosphate buffer (pH 6.8) at varying concentrations of the analytic solution: a) blank solution; b-f) blank
-5 -5 -5 -5 -5
solution + cholesterol 10 , 2.10 , 3.10 , 4.10 , 5.10 mol/L. (from reference [27])
Optical Biosensors
Optical detection biosensors are the most diverse class of biosensors because they can be used for
many different types of spectroscopy, such as absorption, fluorescence, phosphorescence, Raman,
SERS, refraction, and dispersion spectrometry. In addition, these spectroscopic methods can all
measure different properties, such as energy, polarization, amplitude, decay time, and/or phase.
Amplitude is the most commonly measured as it can easily be correlated to the concentration of
the analyte of interest [28]. In optical biosensors, the optical fibers allow detection of analytes on
the basis of absorption, fluorescence or light scattering. Since they are non-electrical, optical
biosensors have the advantages of lending themselves to in vivo applications and allowing multiple
analytes to be detected by using different monitoring wavelengths. The versatility of fiber optics
Nanomedicine 380
probes is due to their capacity to transmit signals that reports on changes in wavelength, wave
propagation, time, intensity, distribution of the spectrum, or polarity of the light. In general,
acquisition of the signal from these devices is accomplished through flexible cables, which can
transmit light to the biological component. Optical methods are readily multiplexed; samples can
be interrogated with many wavelengths simultaneously without interfering with one another. A
large variety of optical methods have been used in biosensors, however, those devices based on
fluorescence spectroscopy, surface plasmon resonance, interferometry and spectroscopy of guided
modes in optical waveguide structures (grating coupler and resonant mirror) are the most
common. However, other emerging optical sensing technologies have been under investigation,
such as optical ring resonators and photonic crystals.
Fluorescence-based Biosensors
Fluorescence is a widely used optical method for biosensing due to its selectivity and sensitivity. A
fluorescence-based device monitors the frequency change of electromagnetic radiation emission
stimulated by previous absorption of radiation and subsequent generation of an excited state that
only exits for a very short time. Single molecules could be repeatedly excited and detected to
produce a bright signal easily measured even at single-cell level. There are three types of
fluorescence biosensing. The first is direct sensing when a specific molecule is detected before and
after a change or reaction takes place. The second form is indirect biosensing when a dye is added
that will optically transduce the presence of a specific target molecule. The use of green fluorescent
protein (GFP) is a powerful fluorescent tag that has enabled investigators to study the location,
structure and dynamics of molecular events within living cells. However, binding interactions
between an activated signalling molecule and its target could be difficult to detect due to the
difficulty of seeing this localized interaction over background fluorescence. A third type of
fluorescence biosensing, called fluorescence energy transfer (FRET), can be used and it generates a
unique fluorescence signal. In a typical fluorescence measurement, the fluorophore is excited by a
specific wavelength of light and emits light at a different wavelength. However, when two
fluorophores are paired in such a way that the emission wavelength of one overlaps with the
excitation wavelength of the other, the excitation of one of them will stimulate fluorescence of the
complementary pairing one (if they reside within about few Angstroms from eachother). FRET has
tremendous utility because the unique fluorescence signal generated under these circumstances
can be used to visualize and quantify the position and concentration of interacting fluorophores.
Two major strategies have been used to develop FRET biosensors: (i) two chain probes in which the
fluorophores are on two different molecules resulting in intermolecular FRET when the two
molecules come into proximity, or (ii) single chain probes in which different regions of a single
molecule are tagged and undergo FRET due to intramolecular, conformational changes [29].
Surface Plasmon Resonance Biosensors
Surface plasmon resonance (SPR) biosensor was first demonstrated for biosensing in 1983 by
Liedberg et al. [30]. Since then it has been extensively explored and has gradually become a very
powerful label-free tool to study the interactions between the target and biorecognition molecules.
The principle, development, and applications of SPR biosensors have been well described in several
excellent review papers [31, 32]. A quick survey of the literature points to the success of surface
plasmon resonance (SPR) biosensors in a wide range of fields from fundamental biological studies
to clinical diagnosis applications. In SPR biosensing, the adsorption of a targeted analyte by a
Nanomedicine 381
surface bioreceptor is measured by tracking the change in the conditions of the resonance coupling
of incident light to the propagating surface plasmon wave (SPW). The SPW is a charge density
oscillation that occurs at the interface of two media with dielectric constants of opposite signs,
such as a metal and a dielectric. The existence of this surface plasmon wave is dictated by the
electromagnetic (EM) properties of the metal, typically gold or silver, and the dielectric interface
(sample medium). The resonance coupling appears as a dip in the reflectivity of the light spectrum,
which is traditionally tracked by measuring the wavelength, the incident angle or the intensity of
the reflected light (figure 15.5). The coupling of the light to the SPW requires, for electromagnetic
reasons, a high-index prism or a periodic grating surface. The sensitivity of the SPR lies in the strong
electromagnetic enhancement of the SPW. Commercial SPR biosensors are generally capable of
2
detecting 1 pg/mm of absorbed analytes. This sensitivity is strongly dependent on many
parameters, but is particularly dependent on surface functionalization. Sensor detection limit (DL)
is another important parameter to characterize the sensor performance. The DL can be deduced by
taking into account the noise in the transduction signal,, i.e., the minimum resolvable signal: DL =
/S, where S is the sensitivity. Improvement in the DL can be accomplished by increasing the
sensitivity or reducing the noise level. Sensitivity can be enhanced by increasing the light-matter
interaction. Today, the key challenge in the SPR biosensor development lies not primarily in the
integration of the various components of the biosensor (sampling handling, control electronics,
2
etc.) but on providing robust integrated SPR biosensors that are as or more sensitive (<pg/mm )
than their current counterparts, such as interferometer, optical ring resonator, and optical fiber
based biosensors.
There are four basic methods to excite the SPR, as shown in figure 15.5: prism coupling, waveguide
coupling, fiber optic coupling, and grating coupling. In the prism coupling configuration (Fig. 5A),
the incident light is totally reflected at the prism–metal interface and generates an evanescent field
penetrating into the metal layer. At the resonant angle or resonant wavelength, the propagation
constant of the evanescent field matches that of the SPW as described in Eq. (2), and as a result,
the photon will be coupled into the SPW.
2𝜋
𝑛𝑝 𝑠𝑖𝑛𝜃 = 𝛽𝑠𝑝

Where  is the incident wavelength, np the prism refractive index, the incident angel and sp is the
propagation constant of the SPW. Prism coupling is the most convenient SPR configuration and
generally has the best sensing DL; however, the prism is bulky and it is difficult to integrate.
Waveguide coupling offers a good alternative to the prism; it is robust and easy to integrate with
other optical and electrical components. The light propagates in a waveguide through total internal
reflection and generates an evanescent field at the waveguide–metal interface, which excites the
SPW in the same way as in the prism configuration (Fig. 5B).Although these basic SPR structures
have good DL that can satisfy most research requirements, there still exist two potential problems
that will limit their applications in some fields. First, the evanescent field in those basic SPR
structures only penetrates into the surrounding medium for about 100 nm, and thus it is very
difficult to detect the large target molecules like cells and bacteria. Second, there is only one SPW
to detect the RI change. It is impossible to differentiate the surface RI change and the bulk solution
RI change. As a result, the sensing performance is deteriorated when detecting the target
molecules in a complex solution, such as blood samples. To overcome those two problems, a new
optical structure has been developed, as illustrated in figure 15.5F, where the metal layer is
sandwiched by two dielectric layers with a similar RI [33–34]. Two new surface plasmon modes
Nanomedicine 382
called long range surface plasmon (LRSP) and short range surface Plasmon (SRSP) form, which are
bound to both metal–dielectric interfaces. Using this dual mode of the LRSP and SRSP, the sensor is
able to differentiate the background RI change and surface bound RI change [35]. Furthermore, the
LRSP has a longer penetration length in the surrounding medium, making it suitable for cell and
−7 −8
bacterium detection. The LRSP also exhibits an excellent DL as low as 2.10 to 2.10 RIU [33, 36].
FIGURE 15.5
Various SPR sensor configurations. (A) Prism coupling, (B) waveguide coupling, (C) optical fiber coupling, (D)
side polished fiber coupling, (E) grating coupling and (F) long-range and short-range surface plasmon (LRSP and
SRSP). (from reference [39])
Optical fiber based biosensors
Among optical-based biosensors, optical fibers are new, tiny, flexible platforms that are being used
with increasing frequency as biosensor transducers. Optical fibers are able to make quick and
sensitive responses, and can be employed as an intrinsic or extrinsic biosensor [37]. Optical fibers
are a convenient material for optical sensor design because they can be inexpensive and provide
easy and efficient signal delivery. Fiber Bragg gratings (FBGs), while developed as a tool for the
telecommunications industry, have flourished as a versatile sensor with a wide breadth of
applications. They are currently among the most popular of all fiber-based optical sensors for
analyzing load, strain, temperature, vibration, and RI [38].
Optical fibers transmit light on the basis of the principle of total internal reflection (TIR). Fiber optic
biosensors are based on the transmission of light along silica glass fiber, or plastic optical fiber to
the site of analysis. Optical fiber biosensors can be used in combination with different types of
spectroscopic technique, e.g. absorption, fluorescence, phosphorescence, surface plasmon
resonance (SPR), etc. Optical biosensors based on the use of fiber optics can be classified into two
different categories: intrinsic sensors, where interaction with the analyte occurs within an element
of the optical fiber; and extrinsic sensors, in which the optical fiber is used to couple light, usually to
and from the region where the light beam is influenced by the measurand. In practice, fiber optics
can be coupled with all optical techniques, thus increasing their versatility.
Nanomedicine 383
The simplest optical biosensors use absorbance measurements to determine any changes in the
concentration of analytes that absorb a given wavelength of light. The system works by
transmitting light through an optical fiber to the sample; the amount of light absorbed by the
analyte is detected through the same fiber or a second fiber. The biological material is immobilized
at the distal end of the optical fibers and either produces or extracts the analyte that absorbs the
light. Illuminating the fiber with two focused intersecting laser beams, RI perturbations can be
written into the fiber core that have periodicities  on the order of the wavelength. The resulting
structure functions as a band rejection filter, reflecting a narrow band of light at the Bragg
wavelength (B) according to the following relationship:
B = 2𝑛𝑒𝑓𝑓 
Where neff is the effective RI encountered by the fiber core mode. By monitoring B, the system
functions as a RI sensor which serves as the foundation for biochemical sensing functionality.
As described in figure 15.6, in order to expose the evanescent field from the FBG, several strategies
have been pursued. One approach is to create a surface grating on the side of the fiber. This usually
requires bending and polishing until the fiber’s core is exposed, and then a grating is physically
patterned on that surface. Another way of exposing the evanescent field involves chemically
etching the optical fiber down to its core (see Fig. 6B)
Long-period gratings (LPGs), have attracted a great deal of attention in recent years for biochemical
sensing applications although the advantages of LPGs have been known since the early 1990’s.
These devices are manufactured with periodicity on the order of 100 µm to 1mm, three or four
orders of magnitude larger than traditional FBGs. They therefore have the advantage of being able
to sense the RI changes around the cladding without etching or complex structural design. Due to
the large grating pitch, they are also far easier to manufacture. LPGs can also be customized by
chemically etching the cladding material down in order to enhance the sensitivity.
Nanomedicine 384
FIGURE 15.6
Overall, FBG and LPG sensors are very attractive due to their simple design, low manufacturing costs, and high
compatibility with standard optical fibers. Most applications, however, focus on physical and basic RI sensing,
because they lend themselves so readily to these purposes. For biochemical detection, these sensors cannot
compete with other types of optical sensors in terms of their DLs even though their sensitivities can exceed
−1
100 nmRIU . (from reference [39]).
Acoustic Biosensors
Electroacoustic devices used in biosensors are based on the detection of a change of mass density,
elastic, viscoelastic, electric, or dielectric properties of a membrane made of chemically interactive
materials in contact with a piezoelectric material. Bulk acoustic wave (BAW) and surface acoustic
wave (SAW) propagation transducers are commonly used. In the first, a crystal resonator, usually
quartz, is connected to an amplifier to form an oscillator whose resonant frequency is a function of
the properties of two membranes attached to it. The latter is based on the propagation of SAWs
along a layer of a substrate covered by the membrane whose properties affect the propagation loss
and phase velocity of the wave. SAWs are produced and measured by metal interdigital transducers
Nanomedicine 385
deposited on the piezoelectric substrate as shown in figure 15.7 [40]. Even though SAW-based
biosensor systems have been the focus of academic and industrial research for a number of years,
most of these approaches only feature laboratory setups that are suitable for proof-of-principle
evaluation and first experimental tests. For real commercial success, two crucial issues need to be
solved: an appropriate production process is required, as is an applicable handling process for
future SAW based biosensors. Most contributions to the scientific community relating to SAW-
based sensor technology do not suggest overall system designs but rather basic approaches limited
to the sensor element itself. Apart from the sensor, there are a number of additional issues which
must be addressed when considering a market-compatible overall system [40]. However, surface
acoustic wave biosensors are inexpensive devices to manufacture, and require inexpensive
electronics to run and to disseminate data. They offer a flexible approach to point-of-care, real-
time diagnostics and their small size allows flexibility in the samples to be analyzed; the devices can
be incorporated into airway tubing to capture proteins and other molecules in breath condensate.
Therefore SAW-based biosensor technology is a promising approach which may eventually be able
to compete against established but much more complex optical- based biosensing techniques, like
surface plasmon resonance (SPR).
FIGURE 15.7
Basic SAW biosensor setup exemplified by a SAW immunosensor. The arrows at the top indicate the flow of
the liquid sample (1) in which the sensor is immersed. The elements of the SAW biosensor are a piezoelectric
crystal (2), IDTs (3), the surface acoustic wave (4), and immobilized antibodies (5) corresponding to the analyte
molecules (6) in the sample. The driving electronics (7) operate the SAW biosensor and generate changes in
the output signal (8) as the analyte binds to the sensor surface. (From reference [40])
Nanomedicine 386
Quartz Crystal Microbalance
The Quartz Crystal Microbalance (QCM) has been the most used acoustic device for sensor
applications since 1959, when Sauerbrey established the relation between the change in the
resonance frequency and the surface mass density deposited on the sensor face [41]. The classical
QCM sensor typically consists of an oscillator circuit containing a thin AT-cut quartz disc with
circular electrodes on both sides of the quartz (figure 15.8). Due to the piezoelectric properties of
the quartz material, an alternating voltage between these electrodes leads to a mechanical
oscillations of the crystal. These oscillations are generally very stable due to the high quality of the
quartz. If a mass is adsorbed or placed onto the quartz crystal surface, the frequency of oscillation
changes in proportion to the amount of mass. Therefore, these devices can be used as high
sensitivity microbalances intended to measure mass changes in the nanogram range by coating the
crystal with a material which is selective towards the species of interest. The quartz crystal acts as a
signal transducer, converting mass changes due to the hybridization process into frequency
changes. One of the main advantages of this device is the ability to control a QCM’s selectivity by
applying different coatings, which makes this sensor type extremely versatile. Despite of the
extensive use of QCM technology, some challenges such as the improvement of the sensitivity and
the limit of detection in high fundamental frequency QCM, remain unsolved; recently, an
-2
electrodeless QCM biosensor for 170MHz fundamental frequency, with a sensitivity of 67 Hz cm
-1
ng , has been reported [42]; this shows that the classical QCM technique still remains as a
promising technique.
1 to 5 mm
50 to
250 µm
Electrodes
Piezoelectric Material
FIGURE 15.8
Schematic of a classical Quartz Crystal Microbalance
Application and Examples of Micro-Biosensors

In Vivo Biosensor
The field of biosensors may be viewed as comprising essentially two broad categories of
instrumentation: i) sophisticated laboratory machines capable of rapid, accurate and convenient
measurement of complex biological interactions; ii) easy-to-use, portable devices for use by non-
specialists for in situ or home analysis. Although biosensor development made a huge progress in
recent years, their application in clinical diagnosis is not very common, except for glucose
biosensors which represent about 90 % of the global biosensor market. Interferences with
undesired molecules during measurements with real samples and also high selectivity and accuracy
are still serious issue. This is very important, since treatment is often dependent on individual levels
of clinical markers. The emergence of semi-synthetic and synthetic receptors is yielding more
Nanomedicine 387
robust, versatile and widely applicable sensors, while nanomaterials are facilitating highly sensitive
and convenient transduction of the resulting binding and catalytic events. Escalating healthcare
costs together with consumer demand is likely to generate a new generation of inexpensive
wearable, integrated and less-invasive sensors amenable to mass production to support the
maintenance of wellbeing, care of the elderly, pharmaceutical development and testing, and
distributed diagnostics.
Electrochemical biosensors currently dominate the field, but are focused mainly on metabolite
monitoring, while bioaffinity monitoring is carried out principally using optical techniques.
However, both transducers find utility across the whole field, along with piezoelectric,
thermometric, and micromechanical transducers.
The Glucose Biosensor
Blood glucose measurement for the management of diabetes comprises approximately 90% of the
world market for biosensors. Millions of diabetics test their blood glucose levels daily, making
glucose the most commonly tested analyte. Such huge market size makes diabetes a model disease
for developing new biosensing concepts. Glucose concentration is also one of the most monitored
indicators in many endocrine metabolic disorders. The glucose biosensor is the most widely used
example of an electrochemical biosensor which is based on a screen-printed amperometric
disposable electrode.
The first developed glucose enzyme electrode relied on a thin layer of glucose oxidase (GOx)
entrapped over an oxygen electrode via a semi-permeable dialysis membrane. Measurements were
made based on the monitoring of the oxygen consumed by the enzyme-catalyzed reaction [14]:
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 → 𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐻2 𝑂2 (1)
The entire field of biosensors can trace its origin to this original glucose enzyme electrode. A wide
range of amperometric enzyme electrodes, differing in electrode design or material, immobilization
approach, or membrane composition, has since been described. In 1973, Guilbault and Lubrano
described an enzyme electrode for the measurement of blood glucose based on amperometric
(anodic) monitoring of the hydrogen peroxide product [43]:
𝐻2 𝑂2 → 𝑂2 + 2𝐻 + + 2𝑒 − (2)
The resulting biosensor offered good accuracy and precision in connection with 100 µl blood
samples. The most suitable concept for glucose determination rely on the use of the natural oxygen
cosubstrate and generation and detection of hydrogen peroxide (equations 1 and 2). The
biocatalytic reaction involves reduction of the flavin group (FAD) in the enzyme by reaction with
glucose to give the reduced form of the enzyme (FADH2) followed by re-oxidation of the flavin by
molecular oxygen to regenerate the oxidized form of the enzyme GOx(FAD). The resulting electric
current from the re-oxidation on the working electrode at applied constant potential is
proportional to the glucose concentration:
𝐺𝑂𝑥 𝐹𝐴𝐷 + 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 → 𝐺𝑂𝑥 𝐹𝐴𝐷𝐻2 + 𝐺𝑙𝑢𝑐𝑜𝑛𝑜𝑙𝑎𝑐𝑡𝑜𝑛𝑒

𝐺𝑂𝑥 𝐹𝐴𝐷𝐻2 + 𝑂2 → 𝐺𝑂𝑥 𝐹𝐴𝐷 + 𝐻2 𝑂2
This model is now utilized in the most commercially successful glucose biosensors utilizing glucose
oxidase or glucose dehydrogenase. Varieties of enzymes were used for biosensor construction, for
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example oxidoreductase enzymes were used for lactate, malate, and ascorbate. Extensive review of
commercially available biosensors for glucose, cholesterol, lactate, triglycerides and creatinine
determination can be found in the review by Monošík et al. *44+. This type of biosensor has been
used widely throughout the world for glucose testing in the home bringing diagnosis to on site
analysis.
Array-type Biosensors
The rapid detection and monitoring of toxins in clinical fluids require new approaches in order to
expedite appropriate countermeasures. For example, many toxins are secreted by bacteria during
–1
the course of infection and should be detected in low quantities (ngmL ) in urine or blood products
following intoxication. In recent years, the fabrication of biosensors able to distinguish multiple
analytes in a single sample has become an increasingly well recognized research goal. In such
bioassays, it is imperative to implement the sensor in an array format. Instruments for reading
microarray systems and microplate readers are a few examples (figure 9). The detectors for such
systems need to measure the analyte quantity at different locations, which is typically carried out
sequentially by a single detector across the array (scanning) or to dedicating an individual detector
to each site. Most array-biosensors require a single measurement (usually when the assay reaches
its biochemical equilibrium) per detection site (pixel) [45]. Others require the capturing of the
reaction kinetics, necessitating multiple measurements per pixel. For instance, fluorescence
detection in microarray systems is extensively used for a variety of applications. These include but
are not limited to nucleic acid/protein detection and quantification, DNA sequencing, blotting, and
real-time PCR analysis. The single frequency excitation induces photon emission (at a different
frequency) from the fluorescent label in the sample material. Filtration of the emission spectrum
and detection follow to form the fluorescence image of the sample. There are two types of
fluorescence detection systems for array-based platforms. In one system, the excitation source is
scanned across the array and a single-pixel very high-performance detector such as a
photomultiplier tube is used for detection. The other approach is to use a homogeneous excitation
light source for the entire assay at once and measure fluorescence emission at different pixels
through a 2D array of detectors e.g. a CCD camera. In these implementations, the resolution of the
CCD camera and the number of photosensitive pixels determine the possible array size [46].
Array-type biosensors offer many advantages over the conventional analytical methods, the most
significant of which are: i) a variety of analytes can be investigated simultaneously in the same
sample, ii) the required sample quantities are minimal, iii) low consumption of scarce reagents, iv)
high miniaturization and v) high sample throughput. These advantages become very evident if we
consider the workflow during a typical drug screening process. At the beginning there is the need
for a selection of few eligible compounds out of a large variety of molecules for a given purpose.
Since the most limiting factor is the ratio between the number of data points per day and the cost
per data point, this first mass-selection is best done at a molecular level where DNA-chips and
protein microarrays find their most common application.
Microarrays are not limited to DNA analysis; protein microarrays, antibody microarray, chemical
compound microarray can also be produced using biochips. Randox Laboratories Ltd., the first
protein Biochip Array Technology analyzer in 2003. In protein Biochip Array Technology, the biochip
replaces the ELISA plate or cuvette as the reaction platform.
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FIGURE 15.9
An example of a microfluidic-based biosensor that can be incorporated onto a wristwatch. The lab-on-a-chip
system relies on manipulation of small volumes of fluid in microchannels using microvalves (from reference
[46]).
The biochip is used to simultaneously analyze a panel of related tests in a single sample, producing
a patient profile. The patient profile can be used in disease screening, diagnosis, monitoring disease
progression or monitoring treatment. Performing multiple analyses simultaneously, described as
multiplexing, allows a significant reduction in processing time and the amount of patient sample
required. Biochip Array Technology is a novel application of a familiar methodology, using
sandwich, competitive and antibody-capture immunoassays. The difference from conventional
immunoassays is that the capture ligands are covalently attached to the surface of the biochip in an
ordered array rather than in solution.
Microbial Biosensor
Microorganisms have been integrated with a variety of transducers such as amperometric,

potentiometric, conductimetric, luminescence and fluorescence to construct biosensor devices.
Since microbial biosensor response, operational stability and long-term use are, to some extent, a
function of the immobilization strategy used, immobilization technology plays a very important role
and the choice of immobilization technique is critical. Microorganisms can be immobilized on
transducer or support matrices by chemical or physical methods. Several reviews papers and book
chapters addressing microbial biosensor development have been published [5, 15, 23, 28, 47].
Based on the sensing technique, recent reported microbial biosensors can be classified into two
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major groups: electrochemical microbial biosensors and optical microbial biosensors.

Amperometric microbial biosensors have been extensively exploited for the determination of
biochemical oxygen demand (BOD) for the measurement of biodegradable organic pollutants in
aqueous samples [47]. Amperometric microbial biosensors also provide rapid and sensitive tools in
health and fermentation applications. The detection of glucose, which is of great interest in the
diagnosis of diabetes and quality control of fermentation and food, accounts for about 90% of the
entire biosensor market. Several microbial biosensors for glucose detection have been fabricated
based on the oxygen consumption of the respiratory activity in the microbes. Generally, the
bacteria, which can uptake glucose, also possess the enzyme activity to metabolize other
carbohydrates, such as galactose, catechol, mannose, and xylose. Selective biosensors can still be
developed for different sugars as long as the bacteria are adapted to the specific analyte in advance
through the selective cultivation. The qualitative and quantitative detection of alcohols with high
sensitivity, selectivity, and accuracy is required in many fields. Several microorganisms which can
metabolize ethanol with the consumption of oxygen, such as G. oxydans, Pichia angusta, and
Candida tropicalis have been applied to the construction of ethanol whole-cell biosensors.
Potentiometric microbial biosensors detect the amount of analytes by measuring the potential
difference between the working electrode and the reference electrode separated by a selective
membrane. Recently, a potentiometric biosensor based on the pH electrode modified by
permeabilized P. aeruginosa was developed for selective and rapid detection of cephalosporin
group of antibiotics [48]. The hydrolysis of cephalosporin, due to the enzyme activity of the
microbial layer, was accompanied by the production of protons near the pH electrode. The
response came from the change of electric potential difference between the working electrode and
the reference electrode. Another potentiometric biosensor for the identification of -lactam
residues in milk was also reported [49].
Microbial biosensors have been under extensive investigation over decades. Particularly, some
electrochemical and optical microbial biosensors developed for environmental applications have
been commercialized. For example, commercial on-line BOD microbial biosensors were available
from Biosensores SL Moncofar, Spain and Isco GmbH, Gross Umstadt, Germany. The Green Screen
Environmental Monitoring (EM) with a yeast cellular sensing element was designed for the
simultaneous detection of genotoxicity and cytotoxicity by Gentronix Ltd., Manchester, UK. In
addition, amperometric and bioluminescent whole-cell toxicity biosensors have been developed by
Euroclone Ltd., West Yorkshire, UK and Remedios Ltd., Aberdeen, UK [50]. Nevertheless,
commercial microbial biosensors are just tips of the iceberg compared to the great amount of
academic research on them. The intrinsic disadvantages (slow response, low sensitivity, and poor
selectivity) using microorganisms as the biosensing element limit the widespread interest of
microbial biosensors on the market. Microbial biosensors typically suffer from the poor selectivity
because of the non-specific cellular response to substrates. With the development of biotechnology
and the availability of genome sequence for more microorganisms, we can genetically engineer
microbes with specific metabolic pathways up-regulated or downregulated, thus providing
enhanced selectivity to specific targets. Another way to improve the selectivity of microbial
biosensors is to develop microbial sensor arrays. The introduction of analyte to the microbial
sensor arrays will generate a finger-printed response pattern. By combining with artificial neural
network analysis, the target compound can be identified [47].
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NanoBiosensors: Basic concepts & Applications

Advances in nanotechnology have led to the development of nanoscale biosensors that have
exquisite sensitivity and versatility. The ultimate goal of nanobiosensors is to detect any
biochemical and biophysical signal associated with a specific disease at the level of a single
molecule or cell. They can be integrated into other technologies such as lab-on-a-chip to facilitate
molecular diagnostics. Their applications include detection of microorganisms in various samples,
monitoring of metabolites in body fluids and detection of tissue pathology such as cancer. Their
portability makes them ideal for pathogenesis of cancer applications but they can be used in the
laboratory setting as well.
The ability to detect disease-associated biomolecules, such as disease-specific metabolites, nucleic
acids, proteins, pathogens, and cells such as circulating tumor cells, is essential not only for disease
diagnosis in the clinical setting but also for biomedical research involving drug discovery and
development. Nanotechnology, with its enhanced sensitivity and reduced instrumentation size, will
rapidly improve our current biodiagnostic capacity with respect to specificity, speed, and cost.
Reduction in sensor size provides great versatility for incorporation into multiplexed, transportable,
portable, wearable, and even implantable medical devices. The integration of nanoscale
ultrasensitive biosensors with other medical instruments will open the door to emerging medical
fields, including point-of-care diagnostics and ubiquitous healthcare systems. The biomedical
application of nanobiosensors is wide; moreover, the future impact of nanobiosensor systems for
point-of-care diagnostics will be unmatched. This technology will revolutionize conventional
medical practices by enabling early diagnosis of chronic debilitating diseases, ultrasensitive
detection of pathogens, and long-term monitoring of patients using biocompatible integrated
medical instrumentation.
There are different strategies for creating next generations of nanobiosensor devices: i) the use of a
completely new class of nanomaterial for sensing purposes, ii) new immobilization strategies, and
iii) the new nanotechnological approaches. In the second part of this chapter, current state-of-the
art principles of nanobiosensor systems are discussed along with future perspectives.
Nanomaterials for new biosensing principles
One of the first new nanomaterials to impact on amperometric biosensors was the carbon
nanotube (CNT), which was blended into a number of formulations to improve current densities
and overall performance of enzyme electrodes and enzyme-labelled immunosensors [51].
Amperometric enzyme electrodes benefited from enhanced reactivity of NADH and hydrogen
peroxide at CNT-modified electrodes and aligned CNT ‘‘forests’’ appeared to facilitate direct
electron transfer with the redox centers of enzymes. The most widely used nanomaterial in
industry overall to date, however, is the silver nanoparticle. These have also been harnessed as a
simple electrochemical label in a highly sensitive amperometric immunoassay intended for
distributed diagnostics and as an inexpensive solution for immunoassays performed in developing
countries. In this electrochemical sandwich immunoassay, silver nanoparticles are used as a robust
label, which can be solubilised after the binding reaction has occurred, using thiocyanate, to form a
silver chelate. This benign chemistry replaces earlier versions using aggressive chemical oxidants
such as nitric acid. Once solubilised, the silver concentration can be very sensitively determined
using stripping voltammetry on a single-use screen-printed carbon electrode. The silver colloid
aggregates due to the presence of thiocyanate and the negatively charged aggregates are attracted
to the positive potential of the carbon electrode during the pre-treatment. Once in direct contact
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with the electrode surface, the silver is oxidised at 0.6 V to form soluble silver ions, which are
immediately complexed by the thiocyanate and detected by the ensuing anodic stripping
voltammetry. Hence, the analyte concentration yields a signal which is directly proportional to the
anodic stripping voltammetry peak of silver. In one example, the cardiac marker myoglobin, was
-1
measured down to 3 ng mL , which was comparable with the conventional enzyme-linked
immunosorbent assay (ELISA). Samples volumes of less than 50 mL could be handled and the assay
worked in turbid solutions without the need for sample clean up [52].
A variety of other nanoparticle-based strategies have been described in the literature for
electrochemical affinity assays [53]. Most recently, nanostructured materials have been used to
deliver label-free electrochemical immunoassays. Gooding’s group in Australia described a direct
electrochemical immunosensor for detection of veterinary drug residues in undiluted milk. They
used a displacement assay for with a mixed layer of oligo(phenylethynylene) molecular wire, to
facilitate electrochemical communication, and oligo(ethylenelycol) to control the interaction of
proteins and electroactive interferences with the electrode surface [54]. More recently, Turner et
al. reported on the use of a highly conductive N-doped graphene sheet-modified electrode, which
exhibited significantly increased electron transfer and sensitivity towards the breast cancer marker
-1
CA 15-3. This label-free immunosensor delivered a low detection limit of 0.012 U mL and worked
-1
well over a broad linear range of 0.1–20 U mL [55].
Despite using new nanotechnologies for biosensors the application of nanomaterials to bioanalytics
in array-type assays or in vivo monitoring is currently a replacement of organic dyes, radioactive or
metal labels and contrast agents by metal, oxide or luminescent nanocrystals. Such methods have
to be used to investigate metabolic pathways on cellular levels where conventional device-based
nanobiosensors have no chance to measure. Using such new labelling nanomaterials the
bioanalytical and imaging methods remain mostly unchanged, whereas the tagged or labelled
biomolecule is replaced by a bionano-system. The conjugation between biomolecule and
nanocrystal is crucial for every bionano-system as it determines the overall biological properties of
the conjugate.
Immobilization Strategies at the Nanoscale
Since the development of the first biosensor, biosensors technology has experienced a
considerable growth in terms of applicability and complexity of devices. In the last decade this
growth has been accelerated due the utilization of electrodes -modified nanostructured materials
in order to increase the power detection of specific molecules. Other important feature can be
associated with the development of new methodologies for biomolecules immobilization. This
includes the utilization of several biological molecules such as enzymes, nucleotides, antigens, DNA,
amino acids and many others for biosensing. Moreover, the utilization of these biological molecules
in conjunction with nanostructured materials opens the possibility to develop several types of
biosensors such as nanostructured and miniaturized devices and implantable biosensors for real
time monitoring. The interface between the nanostructure and the biomolecule requires significant
attention as it dictates the biosensor performance and sensitivity. Based on the physical and
chemical properties of both the nanostructure and the biolmolecule, a number of immobilization
methods have been proposed. The key problem during the immobilization is how to fully maintain
the biomolecule's conformation and activity. Non-specific biomolecule adsorption onto the
nanostructure is the initial stage of the degradation mechanisms that will ultimately compromise
the functionality of the biosensor. The different methods of conjugation between nanostructures
and biomolecules can be divided into three categories.
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The first category includes methods where biomolecules are bound non-covalently to
nanoparticles. Therefore, nanoparticles are first derivatized with a chemisorbed monolayer or the
capping agent from synthesis to have hydrophobic surfaces. In a second step, these hydrophobic
nanoparticles are precipitated and redissolved in water within tensidic micelles. In principle, this
method works with all common micelle building agents such as phospholipids and sodium
dodecylsulfate. In a final step, biomolecules are coupled covalently to functional groups at the
outer sphere of the micelles [56]. A major advantage of this method is that the whole process, from
non-polar/polar solvent transfer to the coupling, is relatively easy to perform. The bond between
nanoparticles and biomolecules is based on hydrophobic interactions within the micelles.
Therefore, the conjugate disintegrates relatively easily.
The second category contains methods in which biomolecules are chemisorbed onto nanoparticles
by means of a ‘linker’. This can be realized in two variations: first, the biomolecules contain surface-
active groups such as, e.g., thiols, and are directly chemisorbed onto the nanoparticles. Second, a
bifunctional molecule is chemisorbed onto the nanoparticles and biomolecules are coupled to
these molecules in a second step [57], similar to the micelles from the first category.
Chemisorption of thiols onto gold surfaces is well known and as long as the adsorption energy is
–1
less than −40 kJ mol , this bond has mostly covalent character. However, from a practical point of
view, on a longer time-scale these conjugates can disintegrate by desorption, what could become
critical for long-term experiments in the range of days.
The third category of coupling methods includes methods in which biomolecules are bound
covalently to modified nanoparticles. Therefore, the nanoparticles have to be derivatized with a
cross-linked surface shell, which contains binding sites for biomolecules. This cross-linked surface
shell could consist of functionalized polymers or inorganic networks like silica. Second, the
biomolecules have to be coupled to these surface shells [58]. Such conjugates are very stable due
to covalent bonds. The major disadvantage of these methods is the sometimes difficult and costly
preparation. Compared to the other categories these methods are recommended when long-term
stability of the conjugate is necessary.
In conclusion, it can be stated that different methods of producing bionanosystems with different
advantages and disadvantages are available. The major problem with all of them is that the
biomolecule is turned into a colloid by attaching it to a nanocrystal. Because colloids have very
different ‘solubility’ from biomolecules, there is always a tendency for coagulation within biological
media.
Examples of NanoBiosensors
Nanowire Biosensors
Nanowire biosensors are a class of nanobiosensors, of which the major sensing components are
made of nanowires coated by biological molecules such as DNA molecules, polypeptides, fibrin
proteins, and filamentous bacteriophages (figure 15.10). A bionanowire is a one-dimensional fibril-
like nanostructure, with the diameter constrained to tens of nanometers or less and unconstrained
length. Since their surface properties are easily modified, nanowires can be decorated with virtually
any potential chemical or biological molecular recognition unit, making the wires themselves
analyte independent. The nanomaterials transduce the chemical binding event on their surface into
a change in conductance of the nanowire in an extremely sensitive, real time and quantitative
fashion. One dimensional nanowires, nanotubes, nanobelts and nanosprings have become the
Nanomedicine 394
focus of intensive research in biosensing due to their unique properties and their potential for
fabrication into high density nanoscale devices. The nanowires can be used for both efficient
transport of electrons and optical excitation, and these two factors make them critical to the
function and integration of nanoscale devices. In fact, they are the smallest dimension structures
that can be used for efficient transport of electrons and are thus critical to the function and
integration of these nanoscale devices. Because of their high surface-to-volume ratio and tunable
electron transport properties due to quantum confinement effect, their electrical properties are
strongly influenced by minor perturbations. One of the excellent candidates for development of
enzyme/protein-based biosensors is the CNT, due to its unique electric, electrocatalytic, and
mechanical properties. Wang and co-workers employed CNT/Nafion-based electrodes for
immobilizing glucoseoxidase (GOx) enzyme for sensitive detection of glucose [59]. This CNT/Nafion
composite was prepared by dispersing solubilized CNTs in Nafion solution onto an electrode
surface. The CNT-based biosensor offers substantially greater signals, especially at low potential,
reflecting the electrocatalytic activity of CNTs. Such low potential operation of the CNT-based
biosensor results in a wide linear range and a fast response time. Boron-doped silicon nanowires
(SiNWs) have been used to create highly sensitive, real-time electrically based sensors for biological
and chemical species [60]. Biotin-modified SiNWs were used to detect streptavidin down to at least
a picomolar concentration range. The small size and capability of these semiconductor nanowires
for sensitive, label-free, real-time detection of a wide range of chemical and biological species
could be exploited in array-based screening and in vivo diagnostics. Furthermore, the highly-
ordered nanowires array combined with multiple biorecognition holds the promise of developing
multiplexed nanobiosensors. They will be very useful for high-throughput diagnosis and screening.
Due to their small size and robustness, the nanowires are a good candidate material for fabricating
nanoscale biosensors for in-body biosensing and for making remote-controlled nanbiosensors for
environmental monitoring. In general, nanobiosensors based on nanowires such as CNTs show
great promise for future applications in health-care testing, disease diagnostics and environmental
monitoring.
FIGURE 15.10
The image on the left illustrates the sensing principle of nanowire-based biosensing with an FET configuration.
The image on the right demonstrates the concept of multi-analyte biosensing with nanowire FET
configurations (from reference 61)
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Cantilever Biosensors
The best example of the use of nanotransducers is cantilever based biosensors which utilize a
micromechanically produced cantilever in a similar manner as for the production of (atomic force
microscopy) AFM probes. A several 100 nm thick cantilever is bent due to biosensing interaction on
the surface (figure 15.11), which can be optically sensed by a laser. The sensitivity can be tuned
down to single molecule interaction analysis [60]. The high sensitivity of microcantilever sensors
has proven to be a powerful platform for detecting molecular interactions in a label-free, time
resolved manner [63]. By an asymmetrical chemisorption of molecules (i.e., on one side of the
microcantilever), the sensors can detect processes in “static” mode by measuring the bending of a
microcantilever due to stress formation during the adsorption process; or in “dynamic” mode
where the resonant frequency of an oscillating microcantilever shifts due to mass adsorption on its
surface. The versatility of the microcantilever technique as a chemical/biological sensor has been
demonstrated for vapors, ions, DNA, proteins, antibiotics and pathogenic microorganisms. The
mechanical sensitivity of the static mode technique stems from changes in surface stress caused by
molecular interactions with the surface (change in the electronic charge distribution of the
substrate’s surface atoms) and by lateral interactions within the molecular layer (electrostatic
forces, structural changes and steric competition) [64]. This sensitivity to structural changes in
static mode operation has shown to be particularly suited for measuring binding processes based
on conformational changes of molecules attached to the microcantilever’s surface such as proteins,
DNA or lipid bilayers. Recently, Bumbu et al. [65] applied the static mode technique to study the
behavior of poly(methyl methacrylate) brushes that had been polymerized from the silicon surface
of a microcantilever sensor, i.e., using a “grafting from” approach. While this allowed the authors
to study the in situ swelling and collapse of poly(methyl methacrylate) brushes, the kinetics of
brush formation could not be monitored in real-time. The driving impetus behind this work is to
apply microcantilever sensors operated in static mode to study in real-time i) the kinetic aspects of
surface PEGylation, and ii) conformational changes in the PEG layer over a timescale of tens of
minutes in situ.
In the last decade, several research groups observed that microcantilevers can transduce a number
of different signal domains, e.g. mass, temperature, heat, electromagnetic field, stress, into a
mechanical deformation: either a bending or a change in the resonance frequency, with a
resolution which is orders of magnitude higher than that achievable with macroscopic structures.
Over the last years, the number of applications of these sensors has shown a fast growth in diverse
fields, such as genomic or proteomic, because of the biosensor flexibility, the low sample
consumption, and the non pretreated samples required.
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FIGURE 15.11
(A) Scanning electron microscope image of a silicon microcantilever array consisting of eight cantilevers and
two sidebars. (B) Schematic drawing of the sensor instrument: 1) the measurement cell with a mounted
microcantilever array, 2) optical-read out system comprising vertical cavity surface emitting lasers (VCSELs)
and a position sensitive detector (PSD), 3) data processing and acquisition, 4)valve selector connected to liquid
samples (from reference 63).
Ion-channel based sensing
Biological ion channels are water-filled subnanosized pores formed by protein molecules in the
membranes of all living cells. Ion channels play a crucial role in living organisms by selectively
regulating the bflow of ions into and out of a cell thereby controlling the cell’s electrical and
biochemical activities. New generations of nanobisensors have been developed in which the
conductance of a population of molecular ion channels is switched by the recognition event. The
approach mimics biological sensory functions and can be used with most types of receptor,
including antibodies and nucleotides. The technique is very flexible and even in its simplest form it
is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance
element whose dimensions can readily be reduced to become an integral component of a
microelectronic circuit. It may be used in a wide range of applications and in complex media,
including blood. These uses might include cell typing, the detection of large proteins, viruses,
antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds [66,
67].
The active elements of the ion-channel switch comprise a gold electrode to which is tethered a lipid
membrane containing gramicidin ion channels linked to antibodies (figure 15.12). The molecular
structure of the tethered membrane results in an ionic reservoir being formed between the gold
electrode and the membrane. The ionic reservoir can be accessed electrically through connection
to the gold electrode. In the presence of an applied potential, ions flow between the reservoir and
the external solution when the channels are conductive. The ion current is switched off when
mobile channels diffusing within the outer half of the membrane become crosslinked to antibodies
immobilized at the membrane surface. This prevents them forming dimers with channels
immobilized within the inner half of the membrane. The number of dimers is measured from the
electrical conduction of the membrane. The switch has a high gain; a single channel facilitates the
flux of up to a million ions per second. A quantitative model of the biosensor has been verified
experimentally. The detection of analytes possessing multiple recognition sites is performed using
the structure shown schematically in figure 15.12. This structure is assembled using a combination
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of sulphur–gold chemistry and physisorption. The membrane consists of lipids and channels, some
immobilized on the gold surface and some diffusing laterally within the plane of the membrane.
The antibodies on the mobile channels scan an area of the order of 1 m in less than 5 minutes.
2
Thus with a low density of channels and a high density of immobilized antibodies, each channel can
3
access up to 10 more capture antibodies than if the gating mechanism were triggered by a
directing binding of analyte to the channels. The speed and sensitivity of the biosensor response
may be adjusted in direct proportion to the number of binding sites accessible to each mobile
channel. This allows for quantitative detection of analyte from sub-picomolar to micromolar
concentrations in less than 10 minutes.
FIGURE 15.12
Large analyte transduction mechanism. The binding of analyte (green) to the antibody fragments (Fab) (red)
causes the conformation of gramicidin A to shift from conductive dimers to nonconductive monomers. This
causes a loss of conduction of ions across the membrane. The scale can be visualized by the fact that the
tethered lipid bilayer is 4 nm thick (from reference 67).
Several companies/research groups have developed biosensors based on synthetic lipid

monolayers and bilayers. For example, OhmX Corporation is currently developing a reagentless
biosensor system using self-assembled monolayers tethered to a gold surface for the electronic
detection of biomarkers in clinical samples. Stochastic signal analysis has been employed by
Bayley’s group at Oxford and has made substantial contributions in advancement of ion channel
biosensors.
The fabrication of the ion-channel switch (ICS) biosensor has several interesting properties that
make it an appealing case
study. The ICS biosensor incorporates a self-assembled monolayer providing enhanced stability.
The tethered bilayer permits 2-D diffusion of gramicidin channels that provides a remarkable gating
mechanism. Since gramicidin has a terminal ethanolamine group that permits a range of
chemistries, the biosensor may be prepared for use with a wide range of receptors to detect many
different analytes. The ICS sensing mechanism does not require washing (unlike an ELISA assay),
provides large transduction amplification (millions of ions for every channel dimerization), and a
high detection sensitivity since a single channel can diffuse and identify analyte molecules bound to
many capture sites. The ICS biosensor also provides an objective electrical readout that is
intrinsically digital. The digital output permits the use of sophisticated statistical signal processing
algorithms to estimate the type and concentration of analyte [67].
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NanoBiosensors in Nanomedicine
Nanomedicine involves cell-by-cell regenerative medicine, either repairing cells one at a time or
triggering apoptotic pathways in cells that are not repairable. Multilayered nanoparticle systems
are being constructed for the targeted delivery of gene therapy to single cells. Cleavable shells
containing targeting, biosensing, and gene therapeutic molecules are being constructed to direct
nanoparticles to desired intracellular targets. Therapeutic gene sequences are controlled by
biosensor-activated control switches to provide the proper amount of gene therapy on a single cell
basis. The central idea is to set up gene therapy "nanofactories" inside single living cells. Molecular
biosensors linked to these genes control their expression. Gene delivery is started in response to a
biosensor detected problem; gene delivery is halted when the cell response indicates that more
gene therapy is not needed. Cell targeting of nanoparticles, both nanocrystals and nanocapsules,
has been tested by a combination of fluorescent tracking dyes, fluorescence microscopy and flow
cytometry. Intracellular targeting has been tested by confocal microscopy. Successful gene delivery
has been visualized by use of GFP reporter sequences. DNA tethering techniques were used to
increase the level of expression of these genes. Integrated nanomedical systems are being
designed, constructed, and tested in-vitro, ex-vivo, and in small animals. While still in its infancy,
nanomedicine represents a paradigm shift in thinking – from destruction of injured cells by surgery,
radiation, chemotherapy to cell-by-cell repair within an organ and destruction of non-repairable
cells by natural apoptosis.
Conclusion
Simple, easy-to-use measurement devices for a diverse range of biologically relevant analytes have
an intuitive appeal as portable or pocket-sized analysers, and this has driven the diverse range of
applications reported in the literature. However, both historical precedent and a critical analysis of
potential markets leads to an indisputable conclusion that healthcare is and will continue to be the
most important area for the application of biosensors. The maintenance of health is one of the
most laudable technological objectives challenging science and technology and diagnosis is an
essential prerequisite for treatment and prevention of disease. Moreover, related applications of
biosensors, such as the maintenance of food safety and environmental monitoring can be aligned
with this central objective. The developing world has a desperate need for robust diagnostics that
can be deployed in the field by both healthcare professionals and volunteers. Infectious diseases
account for around a quarter of worldwide deaths, although they are projected to decline as a
percentage of total deaths over the coming 20 years, as other cause become more prevalent. In
developing countries we are faced with diseases of poverty such as HIV/AIDS and tuberculosis,
where the former kills 1.8 million people each year and the latter still affects around a third of the
world’s population and accounts for an estimated 1.4 million deaths, according to the WHO (2012),
although the incidence has been falling globally at a rate of 2.2% in recent years. In addition there
are 2.5 million deaths from diarrheal infections and almost 800 000 from malaria. Of the estimated
57 million global deaths in 2008, 36 million (63%) were due to non-communicable diseases.
Technology needs to offer more economic solutions and distributed diagnostics enabled by
biosensors and enhanced by consumer products available over-the-counter are a key part of the
solution. This is also commercially attractive, with in vitro diagnostics already worth an estimated
US$40 billion per year. While glucose biosensors for diabetes have had the most profound effect on
Nanomedicine 399
disease management to date, biosensors for other metabolites promise utility for other non-
communicable diseases such as kidney disease, which is increasingly being recognised as an
emerging problem in a rapidly ageing population. Multifarious affinity biosensors have been
described to detect cardiac disease markers such as creatine kinase and troponin, while cancer
markers and single cell cancer detection have attracted considerable recent literature.
We hope that this brief overview has illustrated that biosensors have achieved considerable
success both in the commercial and academic arenas and that the need for new, easy-to-use, home
and decentralized diagnostics is greater than ever. The enormous success of the glucose sensor
serves as a model for future possibilities and should not overshadow the multifarious other
applications that this versatile technology can address. Emerging science, driving new sensors to
deliver the molecular information that underpins all this, includes the development of semi-
synthetic ligands that can deliver the exquisite sensitivity and specificity of biological systems
without the inherent instability and redundancy associated with natural molecules. Currently
aptamers, affibodies, peptide arrays and molecularly imprinted polymers are particularly promising
research directions in this respect. Chances of success are enhanced by the potential utility of some
of these materials for novel therapeutic, antimicrobial and drug release strategies, since these
complimentary areas will drive investment in these approaches.
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16
Titanate Nanotubes as a Versatile
Platform for Nanomedicine
Julien Boudon, Anne-Laure Papa, Jérémy Paris and Nadine Millot
Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), UMR 6303 CNRS-Université de Bourgogne, BP 47870, F-21078,
Dijon Cedex, France.
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 404
The preparation of titanate nanotubes and their characterization …………………………………………….. 404
Titanate nanotubes obtained by anodization ……………………………………………………………………………… 404
Hydrothermal synthesis of titanate nanotubes …………………………………………………………………………… 405
Titanate nanotubes features and characterizations ……………………………………………………………………. 407
The surface modification of titanate nanotubes …………………………………………………………………………. 408
Tiont modification by silane derivatives ……………………………………………………………………………………… 409
Tiont modification by phosphonate derivatives ……………………………………….…………………………………. 410
Characterizations of surface-modified tionts ………………………………………………………………………………. 410
The grafting on titanate nanotubes with a view to in vivo imaging …………………………………………….. 416
Optical imaging ………………………………………………………………………………………………………………………….. 416
Nuclear imaging …………………………………………………………………………………………………………………………. 417
Magnetic resonance imaging ……………………………………………………………………………………………………… 418
Therapeutic applications of titanate nanotubes …………………………………………………………………………. 419
Tionts as radiosensitizers ……………………………………………………………………………………………………………. 419
Chemotherapy by taxane-grafted tionts …………………………………………………………………………………….. 421
Photodynamic therapy by phthalocyanine-grafted tionts …………………………………………………………… 423
Conclusion …………………………………………………………………………………………………………………………………. 424
References……………………………………………………………………………………..…………………………………………… 425
Nanomedicine 404
Introduction
Titanate nanotubes (TiONts) have received more and more attention [1] since their pioneering
hydrothermal synthesis [2]. Recently they have been considered for biomedical applications [3, 4] but
not yet as carriers of therapeutic molecules. The synthesis of TiONts with a controlled morphology has
been reported [5, 6] and the studies have demonstrated that TiONts were internalized by cells without
inducing cytotoxicity [7, 8]. Nevertheless, prior to their use as carriers, it was necessary to determine
TiONt biodistribution which has not been reported till date.
In this chapter, the preparation will be first described then the surface modification of TiONts will be
exposed; once the TiONt surface is prepared molecules of interest can be grafted such as probes for
optical imaging (OI) or macrocyclic chelating agents for the loading of radionuclides to allow nuclear
imaging (SPECT, PET), the latter will be developed in the third part along with magnetic properties that
can be implemented on TiONts by the grafting of superparamagnetic iron oxide (SPIO)
nanoparticles [9]. The fourth and last part will present TiONts themselves or the combination of TiONts
with therapeutic molecules. Thereby this chapter covers the development of a novel versatile
theranostic (for both therapeutic and diagnostic applications) TiONt-based nanomaterial for
nanomedicine.
The preparation of titanate nanotubes and their characterization

Titanate nanotubes can be obtained via two methods: first by electrochemical treatment of titanium
foil, where tubes grow from the surface by anodization [10] and which leads to nanotubes with a
“large” diameter (typically > 25 nm, often 100 nm) [10]; and secondly by hydrothermal treatment [2]
which leads to a smaller diameter (around 10 nm). In both cases their length can reach a few hundred
nanometers, even the micrometer size in the second case. The TiONts described in this chapter have all
been synthesized by hydrothermal treatment of TiO2 powder.
Titanate nanotubes obtained by anodization
Titanate nanotubes can be obtained from electrochemical treatments (Figure 16.1): an oxidation
reaction of a metallic titanium substrate under a specific set of environmental conditions leading to
self-organized TiO2 nanotube layers [11]. Afterwards, TiO2 nanotubes can be converted into perovskite
oxide such as PbTiO3, BaTiO3 or SrTiO3 by hydrothermal treatment in the presence of the corresponding
precursor solution. This yields interesting materials for piezoelectric or ferroelectric applications.
In this case, the TiO2 nanotube growth is controlled by the applied voltage: for example Hashishin et al.
showed that for a 5h experiment, the growth rate was 321 nm/V in length and 2.33 nm/V in inner
diameter resulting in 12-µm-long and 110-nm-inner-diameter nanotubes [12].
Nanomedicine 405
FIGURE 16.1
The electrochemical anodization process and possible anodic morphologies: a) the different phases of anodization
process, b) highly ordered nanoporous alumina, c) highly ordered TiO2 nanotubes, d) disordered TiO2 nanotubes
obtained by rapid breakdown anodization (RBA). Reprinted with permission from Ref. [11]. Copyright 2011 Wiley.
However, only a few studies involving anodized nanotubular titanium mentioned their use for
biomedical applications. For example, Yao and Webster had an interesting approach by loading
penicillin-based antibiotics for prolonged delivery [13]. Still, if these micrometer-sized anodized
nanotubes can be used at the surface of titanium implant, they cannot be considered for in vivo
injection. Indeed large microsized particles are filtrated mechanically by sinusoids and cleared by the
reticuloendothelial system (RES) of liver and spleen [14]. In addition, smaller titanate nanotubes could
be synthetized by hydrothermal synthesis.
Hydrothermal synthesis of titanate nanotubes

Titanate nanotubes are obtained by the hydrothermal treatment of TiO 2 precursor (anatase [15],
-1
rutile [16], both [17] or amorphous [18]) in strongly basic conditions (from 5 to 10 mol.L NaOH).
Temperature range typically between 100 and 180°C and reaction time ranging from 20 to 72 h have
been used in their preparation (lower and higher temperatures are possible depending on the NaOH
Nanomedicine 406
concentration used). The time of reaction can be reduced to a few hours with presonication of the
reaction mixture [19], however, a 100% yield is difficult to reach [5, 15, 20]. Nanosheets are produced
as byproducts of the reaction and they represent about 10% of the batch with optimal conditions of
synthesis [5]. It has been shown that the amount of byproducts produced can be quantified from the
XRD (X-Ray Diffraction) patterns of the samples [5]. Complex schemes and morphology diagrams have
been proposed, trying to emphasize the sensitive nature of the synthesis (Figure 16.2) [6, 21]. The main
parameters that must be controlled to yield a majority of tubular-shaped titanate nanomaterials have
been extensively investigated [5, 22, 23]. Titanate nanotubes’ synthesis is highly sensitive to reaction
conditions and their yield relies on various parameters such as precursor phase, NaOH concentration,
reaction time, mixing mode and speed. Indeed, a slight variation of these parameters can preferentially
lead to the formation of a majority of titanate nanoribbons or nanosheets rather than nanotubes. Our
-1
studies have shown that the optimal parameters are the following ones: rutile precursor, 10 mol.L
NaOH, 36 hours of hydrothermal treatment, magnetic stirring at 120 rpm [5]. An acidic post treatment
(to the hydrothermal synthesis) was initially described by Kasuga et al. as a key parameter of the
nanotubes formation. Later in literature, it has been shown that acid washings were not required to
obtain nanotubes [24], however this treatment has been described for increasing the porosity
+ +
(consequently the specific surface) and to modulate the composition (Na substitution by H ) of the
already formed nanotubes [25, 26].
The formation mechanism of titanate nanotubes is still a matter of debate. Several phenomena are
discussed in literature: the dissolution of the precursor crystallites in bulk followed by their
precipitation into nanosheets which then curl into nanotubes [27], and an energy-dependent curling
process of nanosheets first exfoliated from the particle block [28, 29]. The kinetics of their formation
has also been studied and demonstrated to be precursor size dependent [30]. TiONt formation
mechanism has been extensively reviewed by Bavykin, Walsh and colleagues [31, 32].
FIGURE 16.2
Morphological phase diagram ([NaOH],T) proposed by Morgan et al. [6, 15, 33] Reprinted with permission from
Ref. [6]. Copyright 2008 American Chemical Society.
Nanomedicine 407
Titanate nanotubes features and characterizations
Titanate nanotubes, whose phase composition is NaxH2-xTinO2n+1 · yH2O, are rolled up like a spiral
(contrary to carbon nanotubes the walls of which are concentric). They have an inner cavity (around
4nm, Figure 16.3a) and they are defined by two crystal lattices: 0.7 nm perpendicular to their
longitudinal axis (and also the distance between two consecutive walls) and 0.36 nm at 76 from the
main axis [33, 34]. The size characteristics (such as length, inner diameter and outer diameter
distributions) may vary depending on the synthesis parameters. It has been shown that titanate
nanotubes can display asymmetry in the number of walls that they have (Figure 16.3b). Beyond their
morphology, the interesting feature developed by these tubes is their highly increased specific surface
area (Figure 16.3c) in comparison to their spherical TiO2 precursors. In the first published synthesis of
2 -1
nanotubes [2], Kasuga et al. have reported a 400 m .g surface area. The surface of these tubes is also
interestingly covered with hydroxyl groups, which allow anchoring for further functionalization
(described in part 2). The ζ-potential of TiONts is negative after synthesis followed by washing till pH 6
and this is due to these hydroxyl groups. It has been shown that their isoelectric point (IEP) is 3.6 [9].
After synthesis, nanotubes appear aggregated (micrometer size) but a post-functionalization by
polymers (PolyEthylenImine (PEI) [7, 35], PolyEthylene Glycol (PEG) [35], Poly(-CaproLactone
(PCL) [36]) is able to disperse and stabilize their suspension. This step is also essential to improve the
circulation properties of the particles in the blood stream for nanovectorization purposes and to
improve their biocompatibility in the case of PEGylation.
Nanomedicine 408
FIGURE 16.3
TiONts: (A) TEM image, (B) HRTEM image depicting wall asymmetry and (C) specific surface area of titanate
-1
nanoparticles synthesized by hydrothermal treatment from anatase TiO2 and P25 (Degussa) in 10 mol.L NaOH at
various temperature during 24 h and products obtained from P25 with various NaOH concentrations at 110C.
Figure 16.3c is reprinted from Ref. [37], Copyright 2004, with permission from Elsevier.
The surface modification of titanate nanotubes

The physicochemical properties of TiONts and more generally speaking nanoparticles (NPs), such as
size (i.e. hydrodynamic diameter dH), hydrophobicity, surface charge, will affect the in vivo
biodistribution and clearance [38]. NPs with dH below 6 nm can be excreted through the renal route,
whereas larger uncoated NPs are more easily recognized and cleared by the liver and phagocytic cells
of the reticuloendothelial system (RES) [39] and thus excreted through the hepatobiliary route. Rapid
clearance can be partially alleviated by adding polymer coatings, like poly(ethylene glycol) (PEG).
Nanomedicine 409
Surface charge also plays a crucial role in the rapid elimination of NPs from the blood by the RES uptake
because surface charge can increase dH of NPs by non-specific adsorption of plasma protein, which
increases opsonization, i.e. removal of NPs by RES macrophages. In the meantime, NPs have to be in
the size range of a few hundreds of nm in order to target and stay into tumors in case of NP-mediated
targeting therapy: the enhanced permeability and retention (EPR) effect is harnessed by an acute
control of the size and surface chemical groups of NPs [39].
After synthesis, TiONts – and more generally speaking metal oxides – undergo agglomeration because
of the hydroxyl functions present at their surface that create neither sufficient electrostatic nor enough
steric repulsion for stable colloidal suspensions. In order to circumvent the relatively low reactivity
and/or the lack of mild coupling conditions involving hydroxyl functions, surface modifications have
been developed to adapt to a wide majority of molecules of interest for imaging and/or therapy. The
aim is to tune the TiONt surface by converting initial OH groups into amines, carboxylic acids or thiol
functions capable to react with imaging probes or biological molecules bearing activated ester, amine
or maleimide moieties. These modifications can not only enhance the reactivity towards molecules of
interest but also increase the electrostatic repulsion via ammonium (positive charges) or carboxylates
(negative charges). Surface modification by silane and phosphonate derivatives are described in the
following paragraphs.
TiONt modification by silane derivatives
Amino- and mercapto-alkoxysilane derivatives are used to modify TiONt surfaces to introduce amine
(Figure 16.4) or thiol groups (not shown). It is a convenient organic approach to the surface
modification of TiONts [40, 41] as numerous derivatives are commercially available including various
chemical groups borne on different chain-length alkanes.
FIGURE 16.4
Example of surface modification of TiONts by an ω-aminosilane derivative to yield amino-functionalized TiONts.
Short carbon chains are preferred so that the implemented functions do not imbed into the organic
layer. For example, 3-aminopropyltriethoxysilane (three-carbon chain) was used to bring amine
functions to the surface of iron oxides nanoparticles [42, 43]. The reaction of silane on metal oxides
occurs in three consecutive steps under mild temperature and reduced pressure conditions [44]: i)
silanes are first hydrolyzed into silanols; ii) silanols form polysiloxanol films; iii) polysiloxanol films are
condensed on the metal oxide surface (briefly illustrated on Figure 16.4). It is a convenient way to yield
stable surface-modified titanate nanotubes exhibiting readily available chemical functions (NH2, COOH,
SH) that are conventionally encountered in biochemistry (e.g. amines and carboxylic acids forming
amides, thiols and maleimides forming thioethers).
The titanate nanohybrids formed are characterized by a series of techniques to assert the presence of
the grafted silane derivatives: thermogravimetric analysis (TGA), X-ray Photoelectron Spectroscopy
(XPS), Fourier-transformed Infrared Spectroscopy (FTIR), elemental analysis (EA, also referred to as
Nanomedicine 410
CHNX analyses), Energy-dispersive X-ray spectroscopy (EDS) associated with transmission electron
microscopy (TEM) observations, ζ-potential measurements according to pH variations.
TiONt modification by phosphonate derivatives
Another suitable approach to the surface modification of TiONts implies phosphonate derivatives [45]
to introduce e.g. carboxylic acids groups. A recent review clarifies the interactions of phosphonate
derivatives with metal oxide surfaces [45]: contrary to the silane homocondensation before
condensation on the metal oxide surface, phosphonates individually condensate on metal oxide
surfaces potentially conferring a better stability of the grafted organic layer. Additionally the
interaction of phosphonate derivatives, e.g. ω-functionalized alkylphosphonate derivatives X–
RPO(OH)2, which could involve two (cf. Figure 16.5 as illustration) or three oxygen atoms to coordinate
the metal oxide surface depending on the deprotonated phosphonate anion intermediate: mono-
– 2-
deprotonated RPO2(OH) or fully deprotonated RPO3 .
FIGURE 16.5
Example of surface modification of TiONts by an ω-carboxysilane derivative to yield carboxyl-functionalized TiONts.
6-phosphonohexanoic acid (PHA) was successfully used to modify TiONt surface. As a consequence
TiONt suspensions are more stable after grafting than before (Figure 16.6 left) and even more stable
than aminosilane-functionalized TiONts (Figure 16.6 right).
FIGURE 16.6
On the left: result of surface modification of TiONts by 6-phosphonohexanoic (PHA) acid ligands on the suspension
stability. On the right: difference between stabilizers (PHA lead to TiONts–COOH and APTES to TiONts–NH2) on the
suspension stability.
As an illustration of successful graftings by aminosilanes or phosphonohexanoic acids on TiONts, a

selection of characterizations is provided in the following paragraph.
Characterizations of surface-modified TiONts
Despite a low isoelectric point (pH 3.3) and a high value of ζ-potential (approximately -30 mV) in
physiological conditions at pH 7.4 (Figure 16.7), bare nanotubes suspensions are not stable.
Unfortunately the same phenomenon is observed for APTES-modified TiONts (Figure 16.6), but it is not
Nanomedicine 411
the case of PHA-modified TiONt suspensions which are stable during at least 24 h in the same
conditions. As shown in Figure 16.7, TiONt surface modification by APTES shifts the isoelectric point
towards higher pH values (ca. 6.3). However, the surface modification by PHA barely changes the
isoelectric point (ca. 3.2) as well as ζ-potential at pH 7.4 but increases stabilization (Figure 16.6).
FIGURE 16.7
-2
TiONts, TiONts–NH2 and TiONts–COOH ζ-potentials according to pH variations measured in 10 mol.L-1 NaCl
solution.
Significant differences in IsoElectric Points (IEP) and ζ-potentials at pH 7.4 are noticed when bare
TiONts and aminosilane-modified TiONts or carboxyphosphonate-modified TiONts are compared (Table
16.1).
TABLE 16.1
TiONts, TiONts–NH2 and TiONts–COOH isoelectric points (IEP) and ζ-potentials at physiological pH
ζ-potential (mV)
IEP
at pH 7.4
TiONts 3.3 ± 0,2 -32 ± 3
TiONts–NH2 6.3 ± 0,8 -13 ± 4
TiONts–COOH 3.2 ± 0,3 -35 ± 3
As could be expected from ζ-potential values at pH = 7.4 presented in Table 16.1, TiONts–NH2 are not
stable into physiological medium (pH 7.4 and 0.9 weight% NaCl) or into PBS buffer while TiONts–COOH
are stable during at least 24 hours.
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FIGURE 16.8
TiONts–NH2 (top) and TiONts–COOH (bottom) TEM observations showing small bundles of functionalized TiONts
According to TEM observations (Figure 16.8), the surface modification leads to nm-sized agglomerates
of TiONts contrary to the µm-sized agglomerates showed by bare TiONts (not shown in this chapter,
see [7] for example).
FIGURE 16.9
TiONts, TiONts–NH2 and TiONts–COOH FTIR spectra
-1
FTIR spectroscopy (Figure 16.9) was also used to characterize TiONts (note that the 2400 cm band
-1
represents CO2 pollution): metal oxide hydroxyl groups exhibit vibration bands at 1630 cm and 3400
Nanomedicine 413
-1 -1 -1 -1
cm . TiONt specific vibration modes are located from 470 cm to 917 cm of which the 700-750 cm
partial band is still present after grafting.
After grafting, the presence of the APTES ligand carbon chains is confirmed by the appearance of bands
-1
at 2915 and 2960 cm corresponding to the C–H vibrational modes, N–H vibration bands are located at
-1 -1
3250 cm and 1625 cm but are combined with that of the adsorbed water. Vibration modes around
-1
1050 cm may correspond to the vibrations of C–C bonds but also to the characteristic ones of APTES
ligands (Si–O–Si) or PHA ligands (P–O). This difference is not readily observable in the FTIR spectra but
suggests the presence of the ligands on the surface of TiONts.
FIGURE 16.10
TiONts, TiONts–NH2 and TiONts–COOH thermogravimetric analyses
Relative weight losses between 150 °C and 800 °C of surface-modified TiONts are greater than that of
bare TiONts suggesting the presence of organic ligands at the nanoparticles surface (Figure 16.10).
TiONts–COOH exhibit a greater relative weight loss when compared to that of TiONts–NH2, which is not
surprising since the carbon chain of PHA (6-carbon chain) is heavier than the APTES one (3-carbon
chain). Thus the ligand grafting rate of surface-modified TiONts is estimated using the BET specific
2 -1
surface area of the bare TiONts which is (163 ± 25) m .g determined on ten different TiONt syntheses.
TABLE 16.2
Grafting rates of surface-modified TiONts in the cases of APTES (NH2) or PHA (COOH) molecules modifications
(standard deviations on grafting rates are obtained from repeated experiments).
Molecular weight
Relative weight loss Grafting rate
of leaving species -2
(%) -1 (molecules.nm )
(g.mol )
TiONts 3.8 18 (6.0 ± 4.0) OH
TiONts–NH2 8.0 58 (5.7 ± 0.8) NH2
TiONts–COOH 10.9 115 (3.1 ± 0.5) COOH
When TiONt samples are analyzed by XPS (Figure 16.11), carbon and oxygen 1s energy levels are
decomposed: the C1s level highlights the C–NH2 bond of APTES-modified TiONts. On the other hand,
Nanomedicine 414
the components related to the PHA carbon skeleton on TiONts–COOH are identified as the components
corresponding to the C–P and (C=O)–OH bonds. The proportion increase of the C–C/C–H component
also reveals the presence of carbon ligands at the surface-modified TiONts.
Decomposition of the modified TiONts O1s energy level emphasizes the emergence of new
components. Concerning TiONts–COOH, the component at 529.7 eV corresponds to the oxygen
network and the one at 532.2 eV to the (C=O)–OH double bond. The component at 531.0 eV can be
attributed to oxygen bonds within phosphonates (P=O/P–O–), but also to the remaining surface
hydroxyl. In addition to the presence of the ligands at the surface of TiONts–COOH, sodium Auger
electrons are no longer present at 535.0 eV (when compared to bare TiONts). Because the PHA grafting
occurs at acidic pH, the sodium ions are thus exchanged with hydrogen ions from the H2O/EtOH
environment.
The XPS analysis of APTES-modified TiONts shows no difference in the decomposition of the O1s energy
2–
level. The same components as bare TiONts are observed: (i) the O oxygen network component at
529.7 eV, (ii) the OH surface hydroxyls component at 531.5 eV, and (iii) the sodium Auger electrons at
535 eV. The presence of the surface hydroxyls component at 531.5 eV suggests that all surface
hydroxyls were not involved in the APTES grafting. The atypical morphology of TiONts as well as an
incomplete silane condensation could explain the existence of the surface hydroxyls component after
APTES grafting.
The characterizations of TiONt nanohybrids are in correlation with TiONt suspensions observation
because surface-modified TiONts appeared more stable when compared to their parent unmodified
TiONts (Figure 16.6).
Depending on the post-functionalizing steps reaction conditions, silane-modified TiONts generally lead
to unstable suspensions because of isoelectric points (IEP) around pH 7 too close to the pH of
physiological conditions (pH 7.4). In that particular case further stabilizing molecules must be grafted to
sterically stabilize them. In other cases another approach involving phosphonate derivatives is
preferred and will be detailed in the following paragraph.
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FIGURE 16.11
TiONts, TiONts–NH2 and TiONts–COOH XPS analyses at C 1s and O 1s energy levels
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The grafting on titanate nanotubes with a view to in vivo imaging

One of the major challenges with the use of TiONts for molecular imaging is a covalent grafting of
imaging probes to assert the presence of TiONts. This part presents the successful synthetic strategies
developed to realize TiONt-based imaging.
Optical imaging
FIGURE 16.12
A zinc phthalocyanine derivative bearing an alkyne ready for coupling by click chemistry on TiONts.
The introduction of fluorescence-based optical probes is of great interest for optical imaging. However
in the case of in vivo optical imaging the absorption of the various tissue and blood components [46]
rather limits the use of conventional fluorophores (e.g. fluorescein or rhodamine derivatives). Other
approaches are developed for in vivo optical imaging based on phthalocyanine derivatives [43] (Figure
16.12) the absorption band of which lies in the 650–800 nm region where light penetration in tissues is
the deepest.
Phthalocyanine compounds are synthetized by the condensation of four isoindole groups leading to a
5
fully conjugated system capable of high molar extinction coefficients (e.g. ε(ZnPc) = 1.85 10 L.mol-1.cm-1),
very low photobleaching – when compared to conventional fluorophores – and high quantum yields. In
addition some of the isoindole can be substituted in order to add anchoring possibilities to the
macrocycles which forms complexes with a wide variety of metal ions (e.g. Zn, Al, Si). Indeed, even if
phthalocyanines are analogues of porphyrin macrocycles, they do not exhibit hypsochromic shifts when
grafted on nanoparticles [47]. For example, a recent communication used an alkyne moiety on zinc
phthalocyanines to run click chemistry on azide-functionalized iron oxide nanoparticles and no
wavelength shift was observed (Figure 16.13) [43].
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FIGURE 16.13
On the right: fluorescence emission and excitation of phthalocyanine-grafted iron oxide nanoparticles in ethanol.
Upon excitation of zinc phthalocyanine-grafted metal oxide nanoparticles at 600 nm in ethanol

solution, a fluorescence emission (Figure 16.13) at 673 nm was observed, which is comparable to the
fluorescence emission of the parent free zinc phthalocyanine. The results seem to indicate that there is
no Aggregation Caused Quenching (ACQ) due to π-stacking and Förster Resonance Energy Transfer
(FRET) quenching in the organic solvent. As a consequence phthalocyanine-based nanohybrids are
promising candidates for in vivo optical imaging.
Nuclear imaging
FIGURE 16.14
111
In radiolabeling of DOTA-grafted TiONts for SPECT imaging.
The grafting of macrocyclic DOTA-based chelators is an elegant way to ensure that the radionuclide
chosen for Single Photon Emission Tomography (SPECT) or Positron Emission Tomography (PET) is
actually tethered to TiONt carrier. DOTA derivatives grafting, radiolabeling (Figure 16.14) and imaging
capabilities is of particular interest to follow TiONt biodistribution.
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FIGURE 16.15
Reconstructed images of in vivo SPECT/CT dual imaging of Swiss nude mice intravenously injected with 15 MBq
111 111
DOTA[ In] (left panel), TiONts-DOTA[ In] (right panel), at 1 h, 4 h and 24 h post-injection.
111
Just after DOTA-grafted TiONts were radiolabeled by In, Swiss nude mice were injected. 1 h after
injection the difference between the two compounds is obvious highlighting the success of the
111
functionalization and radiolabeling TiONts-DOTA. Indeed, the free DOTA[ In] compound is rapidly
eliminated because the radioactivity is exclusively in the bladder after an hour and it is then removed
continuously until total elimination by excretion.
111
Instead, TiONts–DOTA[ In] flow through the lungs, liver and spleen, but they are also gradually
111
eliminated by excretion through the bladder. At 24 hours, the TiONts–DOTA[ In] are no longer
present in the bladder and are most likely be subsequently removed by natural means (no imaging was
performed after 24h).
Magnetic resonance imaging
Figure 16.16
T2-weighted MRI phantoms for six different echo times TE and for six increasing concentrations (from left to right)
of two different samples (in triplicates) of TiONts–SPIO nanocomposites.
The combination of TiONts to superparamagnetic iron oxide (SPIO) nanoparticles is an interesting

approach for magnetic resonance imaging (MRI) based on inorganic nanomaterials. Indeed SPIO
nanoparticles are effective T2 contrast agents (negative contrast), the synthesis of which is also well-
known [9, 48, 49].
Three different pathways were used to obtain TiONts-SPIO nanocomposites: TiONt hydrothermal
synthesis in the presence of SPIO nanoparticles, [9] SPIO soft chemistry synthesis in the presence of
TiONts and finally co-synthesis of both SPIO and TiONts by hydrothermal synthesis (Figure 16.17).
Nanomedicine 419
FIGURE 16.17
TEM observations of TiONts–SPIO nanocomposites (Fe/Ti mass ratio is 1:2) obtained by co-synthesis.
SuperParamagnetic Iron Oxide (SPIO) nanoparticles grafted onto TiONts allow the resulting
nanocomposite to be itself superparamagnetic because of the SPIO crystallite size which is less than 20
nanometers. MRI measurements on acrylamide gels (Figure 16.16), lead to relaxivities comparable to
that found in literature [50]. Thus TiONts–SPIO nanocomposites are capable to act as nanocarriers that
can be followed by magnetic resonance imaging.
Therapeutic applications of titanate nanotubes

In the biomedical field, titanate nanotubes have been investigated for their use in bone
regeneration [51, 52], dopamine detection [53] and molecule vectorization [7, 54]. In this last part of
the chapter are presented three examples of TiONts use for therapeutic applications: the first one is
the use of TiONts themselves to increase the efficacy of a radiotherapy treatment; the second one is
the grafting of docetaxel, a taxane derivative for antitumor activity and the third one is the use of a
phthalocyanine derivative for photodynamic therapy (PDT).
TiONts as radiosensitizers
The major challenge in radiation oncology is to exert therapeutic effects on tumor while preventing
unwanted effects on normal surrounding tissue. To remedy this limitation, one interesting approach in
research so far has been to use nanoparticles able to enhance radiation effects (due to their intrinsic
properties [55] or by carrying a radioelement [56]), while focusing the dose on a minimal area of
treatment. Indeed, nanoparticles containing a high atomic number (Z) element, such as gold (Z =
79), [55] platinium (Z = 78), [57] gadolinium (Z = 64) [58] and silver (Z = 47), [59] have been attractive
for their radiosensitizing effect on tumor cells. Particles under radiation stabilize their energy by both
re-radiation photons (photoluminescence phenomenon) and emission of secondary and Auger
electrons, thus leading to the generation of Reactive Oxygen Species (ROS). These oxygen species
generate DNA double strand breaks and consequently, cause nuclear damage and have deleterious
effects on cell survival [60]. Nanoparticles offer a dual advantage as their tunable size and surface-
grafting permit the generation of suitable size distributions at which EPR (Enhanced Permeability and
Retention) effect can be achieved [61, 62]. The vasculature surrounding the tumor is disorganized and
highly permeable while the lymphatic system is defective. These factors taken together lead to the
Nanomedicine 420
passive targeting (so called EPR) of the nanoparticle to the tumor site. If nanoparticles are able to
increase cell sensitivity to radiation, a minimal dose can be administered and be effective on tumor
burden, while remaining safe for surrounding healthy tissues.
Nanoparticle-cell interaction is reported in many reviews [63-73] but the main observation regarding
the effect of nanoparticles on cells is the production of reactive oxygen species, leading to oxidative
stress. Functionalized and naked nanoparticles have been shown to increase p53 (a tumor suppressor
protein) and phospho-p53 expression, as well as leading to Rad51 up-regulation and an increase in
H2AX [8, 65]. All these proteins are involved in the DNA repair pathway. Nanoparticles have also been
shown to induce cell cycle arrest [8, 74]. Based on their cytotoxic effects, targeted nanoparticles could
then offer new avenues in cancer research [75].
Though titanium is not a heavy metal (Z = 22), its potential as radiosensitizer has been considered as
other low atomic number elements like calcium (Z = 20) and phosphorus (Z = 15) have demonstrated
DNA damage incidences after X-ray exposure [76, 77]. Also, titanate nanotubes were a prime candidate
for this application as their internalization ability in cells has been demonstrated to be higher than their
spherical counterparts [7]. Similar enhancement of internalization as a function of physical shape has
been observed for organic particles as well [78]. Titanate nanotubes have shown no cytotoxicity when
incubated with glioblastoma cell lines (SNB-19 and U87MG) but exert radiosensitization on both cell
lines with a significant decrease of the SF 2 parameter (survival fraction of cells at 2 Gy) [8] (Figure
16.18). This effect of TiONts has been validated at low and high doses as confirmed by an increase and
decrease respectively of  and  parameters. Biological consequences have been investigated by
detection of apoptosis, autophagy and ROS production without concluding that any of these factors
could explain the radiosensitization effect of TiONts (in the experimental conditions tested). However,
a clear implication of cell cycle impairment has been highlighted. Indeed, DNA damage and repair
assessed by H2AX quantification 1 h and 24 h after irradiation at 2 Gy, with and without TiONts, has
demonstrated that TiONts induce a cell accumulation at the G2/M checkpoint [8]. Prior literature has
shown that cancer cells are more sensitive to radiation when they are in G2 phase. It has been
hypothesized that the observed accumulation of cells at the G2/M checkpoint could be due to TiONt-
induced DNA damage, and that this G2/M arrest would subsequently promote the radiation induced
therapeutic effect [8]. However further investigation on the effect of TiONts on DNA need to be
pursued to clarify the process.
In perspective, if improving the tumor targeting and lowering the radiation dose can be achieved by
using selected nanoparticles, a gap still remain in mitigating the bystander effect [60] produced by
radiation therapy. The radiation-induced bystander effect are collateral damages produced in
surrounding cells due to signaling molecules traveling from irradiated cells to neighboring normal ones.
This effect induces genome instability and may contribute to secondary cancers [79]. Small signaling
inhibitors delivered with nanoparticles could potentially address this complex issue and offer an
additional level of protection to healthy surrounding tissues. To date, the potential of a combined
radiosensitizer and a signaling pathway inhibitor has not yet been reported and could offer a
synergistic response.
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FIGURE 16.18
Survival fraction curves obtained from SNB-19 and U87MG showing the effect of X-ray exposure (different doses)
without TiONts (control: black curve) or with 1 µg/mL TiONts incubation (grey curve). Radiosensitization
parameters (: initial slope, : degree of downward curvature and SF2: survival fraction at 2 Gy) were extracted
from these curves with the Linear Quadratic model (LQ-model). Reprinted from reference [8], copyright 2013 with
permission from Elsevier.
Chemotherapy by taxane-grafted TiONts
Docetaxel (DTX) is a clinically well-established anti-mitotic chemotherapy taxane-type drug. Its

combination with TiONts is of great interest for tumor treatment, TiONts acting as a drug carrier in case
of systemic injection. The grafting strategy of a docetaxel derivative is presented in Figure 16.20 and
the results of MTS biological tests on a PC3 human prostate cancer cell line in Figure 16.19.
Preliminary biological results did not show any cytotoxicity for surface-modified TiONts before
cytotoxic docetaxel drug molecules were grafted on them (Figure 16.19, green dashed-dotted line) as
opposed to free docetaxel having a 4 nM IC50 (Figure 16.19, orange dashed line). In order to have DTX
drug covalently bound on TiONts, DTX had to be modified by a crosslinker: modified DTX molecules are
4-fold less toxic (IC50 is increased to ca. 20 nM, see Figure 16.19, dark red line) than unmodified
docetaxel. The activity of docetaxel grafted directly at the surface of TiONts did not lead to satisfactory
IC50 cytotoxicity (not shown) but the introduction of a PEG spacer – between TiONts and docetaxel –
decreases the steric bulkiness brought by the proximity of docetaxel molecules on TiONts. Thus
PEGylated docetaxel molecules more freely interact with tubulin subunits in microtubules. That is the
reason why TiONts-PEG-DTX nanohybrids are considered for further studies on mice xenografted by
prostate tumors. The next elaboration steps will notably consist in optimizing the docetaxel grafting
rate on TiONts in order to increase treatment efficacy without increasing the nanotube load.
Nanomedicine 422
FIGURE 16.19
MTS cytotoxicity test results on PC3 cell lines showed 4 nM IC50 for docetaxel drug (orange dashed line), the
absence of cytotoxicity for surface-modified TiONts (green dashed-dotted line) and intermediate toxicity for
modified-docetaxel (dark red line).
To the best of our knowledge, no TiONt-based chemotherapy treatment has been yet developed and as
a consequence it represents a novel and innovative way to deliver such a chemotherapy drug by
increasing its load on TiONts and potentially reducing undesired side-effects. In the current study
TiONts-docetaxel nanohybrids were engineered for intratumoral injections so that a maximum
docetaxel payload could be delivered into the tumor but a systemic injection could be considered after
the nanohybrids are modified by a targeting molecule. Detailed results are to be published in a
dedicated communication [88].
Nanomedicine 423
FIGURE 16.20
Grafting strategy for docetaxel-modified TiONts: once PEGs are tethered on TiONts–NH2 via a peptide coupling,
docetaxel molecules are then grafted on PEG function X via a linker R.
Photodynamic therapy by phthalocyanine-grafted TiONts
The phthalocyanine molecule described in paragraph 3.1 is not only capable of optical imaging but also
to undergo intersystem crossing with oxygen to produce highly cytotoxic singlet oxygen upon
absorption of light.
The grafting of zinc phthalocyanines is almost quantitatively realized via a click chemistry reaction (cf.
1,4-triazole cycloaddition on Figure 16.21) as was already achieved on iron oxide nanoparticules [43]. In
this manner TiONts could act as bimodal therapeutic composites but studies are still in progress on this
promising aspect.
From the photodynamic therapy (PDT) point of view, zinc phthalocyanines (ZnPc) are of great
interest [80]. PDT implies light, oxygen and the presence of a photosensitizing compound. This
combination forms reactive species causing oxidation reaction within cell leading to necrosis.
Phthalocyanines can be used as photosensitizers as well as porphyrin macrocycles already used in vivo
for PDT applications because they have a high singlet oxygen quantum yield (e.g. ΦΔ(ZnPc) = 0.50 in
DMSO as a comparison to unchelated phthalocyanines ΦΔ(H2Pc) = 0.14 in the same solvent or
tetraphenylporphyrin ΦΔ = 0.55 in chloroform).
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FIGURE 16.21
Grafting way of phthalocyanine-functionalized TiONts: carbonyl-functionalized phosphonate derivatives are
condensed on TiONts, amino-PEG undergo peptide coupling on the latters and click chemistry is finally realized
between alkyne-phthalocyanines and azide-PEG-TiONts.
In addition the wavelength of the light source must be correctly selected to the photosensitizer
excitation wavelength to produce oxygen reactive species. From this standpoint, phthalocyanines
appear to have a more appropriate absorption wavelength than the porphyrins of the same metal
chelate λemission(max, ZnPc) (cf. Figure 16.13) to be compared to λemission(max, ZnPorphyrin)) [81] with
respect to the optimum in vivo optical window (650-1450 nm) [46].
Phthalocyanine-grafted TiONts present two major interests: on the one hand zinc phthalocyanine is
capable of in vivo photodynamic therapy and on the other hand TiONts are themselves prone to
increase the radiosensitizing effect. Eventually they can be used for bimodal therapeutic applications.
Conclusion
As a conclusion, the development of a TiONt-based nanomedicine is of great interest: the parameters
of their synthesis are from now on known and set [5]. They are needle-shaped, ca. 150 nm long and
have about a 10 nm diameter. More importantly, TiONt surface can be tuned by surface modification
(silanization or phosphonatation for example) so that they become more stable in suspension (a
mandatory prerequisite to any biomedical application) and ready for the grafting of molecules of
interest one after the others. This step-by-step scaffold allows the development of a thoroughly
characterized versatile platform.
Moreover surface-modified TiONts belong to the theranostic nanomaterial class [82-85] and appear as
a potent tool, as recently shown in this chapter, that combine therapeutic aspects (enhanced
radiotherapeutic treatment by radiosensitizing TiONts [8], nanocarrier for DNA transfection by PEI
Nanomedicine 425
coating [7] chemotherapy by grafting of taxane derivatives [86], or photodynamic therapy thanks to
phthalocyanines [87]) as well as imaging ones (MRI probe [9], optical imaging probe [87], nuclear
imaging probe [88]).
Future prospects will underscore efficacy and safety: TiONts can affect a wide range of diseases by
improving both parameters, prolonging circulation time, and enhancing delivery specificity. Indeed
safety is of paramount importance in any drug’s translational roadmap [39]. Preliminary toxicity tests
have been performed on TiONt nanohybrids at the different stages of elaboration but careful and
exhaustive cytotoxicity, genotoxicity and other nanotoxicological tests would be an essential step [89]
to evaluate the possibility of future clinical translation of these titanate nanotubes. According to the
studies conducted in recent years, the authors are confident in the future relevance of a TiONt-based
nanomedicine.
Acknowledgements
All the people who contributed in the results presented in this chapter are greatly acknowledged:
Richard Decréau, Yann Bernhard, Céline Mirjolet, Bertrand Collin, Alexandra Oudot, Laure Dumond,
David Vandroux, Mathieu Moreau, Claire Bernhard, Paul Walker, Thomas Gautier, Renée Mayap Talom,
Olivier Heintz and Rémi Chassagnon as well as all the people from the IMAPPI program, the
PharmImage® consortium and the 3MIM agreement who are involved but not mentioned in the
presented results.
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17
Bismuth nanoparticles: antimicrobials of
broad-spectrum, low cost and safety
1 2
Cabral-Romero Claudio , Shankararaman Chellam
1
Facultad de Odontologia, Universidad Autonoma de Nuevo Leon,UANL. Monterrey, N.L. Mexico
2
Departments of Civil and Environmental Engineering and Chemical and Biomolecular Engineering, University of Houston,
Houston, TX USA 77204-4003
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 430
Antimicrobial resistance among pathogen microorganisms …………………………………………..…………… 430
Nanotherapeutic; a way to control multiresistant microorganisms ……………………………………………. 432
Bismuth nanoparticles as antimicrobial agents …………………………………………………………………………… 433
References……………………………………………………………………………………..…………………………………………… 435
Nanomedicine 430
Introduction
In spite of the great succes of penicillin in the 1950s, nowadays we are dealing with pathogenic bateria
resistant to many common antibiotics. Staphylococcus aureus, Klebsiella pneumoniae, and
Pseudomonas aeruginosa constitute the most frequent multiresistant bacteria associated with
nosocomial infections. However, this phenomena is not exclusive of bacteria, resistant strains of
Candida albicans were detected since 1980s. In the field of virology, there is a lot of viral infections
whithout any treatment option. There is an urgent need to develop new alternatives to combat the
multiresistant pathogens and this include antimcirobials of broad-spectrum, low cost and safety.
Over the last twenty years, the field of nanotechnology have had an amazing growth and advances. The
area of nanoparticle-based medicine receives particular attention as it holds the promise to
revolutionize medical treatment with more potent, less toxic, and smart therapeutics. Although
questions still exist about their safty, with substantial efforts by both academia and the
biopharmaceutical industry, a few nanomedicines have been successfully developed and approved for
clinical use such as DOXIL nanoencapsulated by liposomal pegylation and liposomal formulations
containing irinotecan, a derivative of camptothecin[1, 2] .
Although several choices of nanoparticles exhibit medicinal properties, this chapter is focused in the
usefulness of bismuth nanoparticles as an alternative broad-spectrum antimicrobial agent. We describe
their efectivness as bactericidal, antibiofilm, fungicidal and antiviral agent. Finally, we comment the
possible toxicity of bismuth-based nanoparticles base on experiments with cell cultures and genotoxic
assays.
Antimicrobial resistance among pathogen microorganisms

The increasing prevalence of resistance pathogenic bacteria to common antibiotics is one of the most
important problems in modern medicine [3]. The incidence of infections by methicillin-resistant S.
aureus increased dramatically in the past two years [4]. Pseudomonsa aeruginosa, Escherichia coli,
Mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis and Klebsiella
neumoniae cause 60% of intrahospital infections and are the pathogenic bacteria that most commonly
exhibit antibiotic resistance [5]. Several reports indicate the emerging multiresistant strains of Candida
since 1980s, demonstrating that antimicrobial resistance is not exclusive to bacteria [6, 7].
Microorganisms have several mechanisms to acquire resistance against antimicrobials. They include;
intrinsic resistance, excretion of bombs efflux, adaptive resistance, indifference to drug, persistence
and formation of biofilms. The term “biofilm” refers to growth of microbes, forming highly organized
communities attached to a surface through a matrix of exopolysaccharides (EPS) (Figure 17.1). Amongst
other things, EPS matrix surrounding the microbial cells protects them from very high concentrations of
many different antibiotic agents, often leading to chronic infections despite antibiotic treatment [8].
Cells growing within biofilms are able to evade the host immune system [9] and require frequently
1000 times more antimicrobial agent to be eliminated than planktonic cells [10].
Nanomedicine 431
FIGURE 17.1
Scanning electron micrograph of bioaggregates from wastewater plant treatment. Adapted from Badireddy and
co-workers [11] and [12].
In 2010, the American Society for Infectious Diseases, call to develop 10 new antibacterial drugs by the
year 2020, showing that it is urgent to invent new drugs to fight multiresistant microorganisms. Several
research groups are focusing on alternatives in plant extracts, synthetic molecules, and modifications
to known antimicrobials like methicillin, first semi-synthetic penicillin. The presence of the ortho-
dimethoxyphenyl group directly attached to the side-chain carbonyl group of the penicillin nucleus
facilitates the β-lactamase resistance. One of the most promising strategies for overcoming microbial
resistance is the use of nanoparticles.
Nanomedicine 432
Nanotherapeutic; a way to control multiresistant microorganisms

There are many reazons why nanoparticles inhibit the development of multiresistance by microbes.
Nanoparticles prevent drug resistance because they use several mechanisms act simultaneously to
inhibit the microbial growth like oxide nitric release [13], packaging several antimicrobial and anti-
inflamatory drugs within same nanoparticle and controlling their realise (Figure 17.2) [14],
nanoparticles have been used to target antimicrobial drugs to site of infection, letting the use of lower
quantities of drugs to control the infection [15]. Metal nanoparticles with bactericidal, fungidal and
antiviral activities could be employed as surface disinfectant and mixed with other materials like paint,
clothes, etc.
FIGURE 17.2
Diagram that ilustrate packaging of active ingredients into nanoparticles.
For every plausible application, is basic the nature of nanoparticle surface which determines their
interaction with the environment. These interactions could modify both nanoparticle and target; if it is
a cell, it could alter cell psysiology, structure (e.g. membrane disruption including its permeability), or
fundamental process like DNA division or protein synthesis [16, 17]. One kind of nanoparticle could
present one or more of these action mechanisms to inactivate microbes fundamentally explaining the
reasons why nanoparticles can prevent the development of drug resistance.
Different types of metals have been used to synthesize nanoparticles and explore their possible
antimicrobial effect. Each metal can employ different mechanisms to inhibit the microbial growth
(Figure 17.3). The metals most commonly use to synthesize nanoparticles for biomedical applications
include silver (Ag), zinc (Zn), copper (Cu), titanium (Ti), magnesium (Mg), gold (Au) and bismuth.
Inorganic colloidal nanoparticles are very small, nanostructured materials dispersed in a solvent. Base
on the material of origin, nanoparticles can have several different properties such as hardness, active
surface, chemical reactivity and biological activity [18]. The nanoscale materials are more
andvantageous than their bulks elements, they can be applied to many fields like magnetism,
electronic and medicine [13, 19, 20].
Nanomedicine 433
FIGURE 17.3
Main mechanisms to inhibit the microbial growth by nanoparticles.
Bismuth nanoparticles as antimicrobial agents

Bismuth is a crystalline, brittle metal and constitutes the most naturally diamagnetic metal. Typically,
bismuth is found as bismuthinite (bismuth sulfide), bismite (bismuth oxide) and bismuthite (bismuth
carbonate) [21]. Bismuth has the property that it expands as it freezes and also has unusually high
electrical resistance for a metal. Its thermal conductivity is lower than any metal except mercury [22].
Bismuth oxide is a derivative of great technological importance, and it is used in the manufacture of
glass and ceramic products and also, as catalyst in the oxidation of hydrocarbons. It is widely used in
applications such as microelectronics, sensor technology and optical [23, 24].
Colloidal chemistry provides opportunities to generate straightforward synthetic routes to obtain

bismuth nanoparticles with well-controlled size distributions and high crystallinity. In general,
commercial bismuth salts are used as precursors; also, surface modifier species and a reducing agent
are added to produce the nanoparticles.
Structural characterization of the NPs is obtained by X-ray diffraction analyses of the bismuth colloids
and transmission electron microscopy of high resolution (HR-TEM) [25]. This method of synthesis is the
most common used to obtain metal nanoparticles; it is not expensive and scalable to industrial
production. This is an important characteristic for their use in humans.
Bismuth compounds are most commonly used for treating gastrointestinal disorders. Although
elemental bismuth exhibits antimicrobial activity, it does so only at relatively high (order of millimolar)
concentrations due to its limited water solubility. However, solubility is increased upon chelation and
Nanomedicine 434
bismuth´s antimicrobial properties are manifested at much lower (order of micromolar) concentrations
with bismuth dimercaptopropanol (BisBAL) being highly effective against several bacteria [26].
However, BisBAL’s long-term efficacy might be limited as it is readily consumed upon contact with
microorganisms. For this reason, we have been exploring BisBAL in its nanoparticulate form with the
idea that its slow dissolution would enable it to act as an antimicrobial agent for extended time period
[27]. Bismuth compounds remain important components of stomach remedies, such as Pepto-Bismol
(bismuth subsalicylate, BSS)[28] and De-Nol (colloidal bismuth subcitrate, CBS), and derivatives of CBS,
such as ranitidine bismuth citrate (RBC), are currently under development [22]. The diversity of
bismuth compounds in medicine extends to the treatment of syphilis[29] and tumors[30], and in
radioisotope therapies[31]. Recently, bismuth nanoparticles had been employed in diagnosis of
biomolecules as well as antimicrobial agents of broad-spectrum [32-38]. It has been reported that
bismuth nanoparticles (Bis-NPs) can inhibit bacterial growth at concentrations lower than 1 mM [32].
Bismuth oxide nanoparticles exhibited antifungal activity since 2 mM, showing better results than
commercial antifungals [33]. The most interesting is that Bis-NPs interfere with biofilm formation of S.
mutans, main etiological agent of caries. Early results indicate that Bis-NPs inhibit rotavirus replication,
probably due to inactivation of capsid proteins (unpublished data). BisBAL significantly reduced
extracellular polymeric substances (EPS) expression by Brevundimonas diminuta in suspended cultures
at levels just below the minimum inhibitory concentration [39]. Bismuth-containing nanoparticles in
combination with X-ray treatment also have potential in treating drug-resistant bacteria [40]. Since X-
rays can easily penetrate human tissues, this bactericidal strategy has the potential to be used in
effectively killing deeply buried MDR bacteria in vivo. Recently, it was reported that Bis-NPs inhibited
Helicobacter pylori growth altering their Krebs cycling, nucleotide and amino acid metabolism [41].
In summary, Bis-NPs present bactericidal, fungicidal and antiviral activity. Base on bismuth subsalicylate
is used to treat stomach ailments; we hypothesize that Bis-NPs will be not toxic towards human cells,
until the moment there are not reports indicating secondary effects of bismuth nanoparticles. When
monkey kidney cells were expose for 24 hours at a final concentration of 2 mM of Bis-NPs, no cytotoxic
effect was detected [33]. Bismuth nanoparticles constitute a promising approach to fight infectious
diseases, but more testing is necessary to ensure their safe use in humans. Silver nanoparticles show an
important antimicrobial activity, but several reports indicate that they may present important toxic
effects [16, 17, 42]. Genotoxic effects of Bismuth (III) oxide nanoparticles (BONPs) were investigated on
the root cells of Allium cepa by Allium and Comet assay. Results indicate that BONPs exhibit genotoxic
activity in A. cepa root meristematic cells [43]. More studies about the possible cytotoxity of bismuth
nanoparticles are needed to detect any secondary effect in humans.
These results permit us to be excited about a new kind of antimicrobial of broad-spectrum, effective
and low cost. Bismuth nanoparticles could be used as surface disinfectant; hospitals, pediatric and
geriatric clinics, food and pharmaceutical industries, laboratories and in general all locations were
microbial contamination needs to be counteracted. One interesting alternative is to adhere as a film
within valves, catheter, prosthesis, and dental implants. Silver nanoparticles have been incorporated to
several items; however Bis-NPs would have the advantage of being more effective and lower toxic side-
effects.
Nanomedicine 435
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Nanomedicine 438
18
Surface Modification of Porous Anodic
Alumina for Medical and Biological
Applications
*
Morteza Aramesh and Jiri Cervenka
School of Physics, The University of Melbourne, VIC, Australia
Outline:
Introduction ……………………………………………….………………………………………………………………………………. 439
Fabrication and Properties of Uncoated Anodic Alumina …………………………………………..………………. 440
Surface Modification Methods …………………………………………………………………………………………………… 442
Grafting methods ……………………………………………………………………………………………………………………….. 443
Sol-gel methods ………………………………………………………………………………………………………………………….. 444
Atomic Layer Deposition …………………………………………………………………………………………………………….. 446
Plasma modification and Chemical Vapor Deposition Methods ………………………………………………….. 448
Other Deposition Methods ……………………………………….………………………………………………………………… 450
Applications of Different Surface-Modified AAO Materials ………………………………………………………… 450
Template ……………………………………………………………………………………………………………………………………. 450
Separation ………………………………………………………………………………………………………………………………….. 452
Sensing ………………………………………………………………………………………………………………………………………. 454
Drug Delivery ……………………………………………………………………………………………………………………………… 456
Future of the Modified Membranes …………………………………………………………………………………………… 459
Conclusions ………………………………………………………………………………………………………………………………… 460
References……………………………………………………………………………………..…………………………………………… 461
Nanomedicine 439
Introduction
Membranes are one of the key elements of medical, biological, chemical and environmental industries
[1]. In the last decades, the emergence of nanotechnology has led to the fabrication of high-
performance nanoporous membranes with well-defined properties and pores sizes. Nanoporous
membranes have been used for numerous medical and biological applications [2], including biosensors
[3, 4], protein separation and DNA sequencing [5, 6], cell growth [7, 8], tissue engineering [8] and drug
delivery [9]. The potential applications of membranes are strongly determined and influenced by the
physical and chemical properties of a membrane material. The other important parameters of effective
membranes are pores size, uniformity, porosity, thickness, aspect ratio, surface chemistry and
morphology [10]. Membranes used in biological and medical environments should also have very good
biocompatibility as well as excellent physical and chemical stability. Solid state membranes made of
inorganic materials, such as oxides (Al, Si, Ti, Nb, Zr, W, Ta oxides), are well-known for their mechanical
stability and robustness. They can be prepared by different methods to achieve nanopores with well-
engineered pore size, shape and aspect ratio. Anodization is one of the methods which is most
commonly used for their production because it is a simple and scalable method that offers a unique
control over the membrane pore shape, size, thickness and chemical composition.
Nanoporous anodic aluminum oxide (AAO) membranes have been the center of the attention since
1995 [11], due to their fast, facile and cheap fabrication. AAO can be easily fabricated by simple
anodization of aluminum in an acidic electrolyte. Different anodization regimes can be applied in the
fabrication process, leading to different pore diameters in the range of 10-450 nm [12, 13] . The two-
step anodization of aluminum gives rise to a well-ordered closed pack hexagonal porous oxide
structure, with vertical pores that have almost the same pore sizes and pore distances. All these
properties have made AAO a very popular materials platform for different membrane applications,
such as separation, filtration, catalysis and chemical reactors [14, 15]. However, the use of AAO in bio-
medical applications has been limited owing to its low chemical stability in acidic or basic conditions
[16]. Biocompatibility of AAO has also not been well established yet [17, 18]. For this reason
researchers have been trying to modify and improve the surface of AAO by different biocompatible
materials [19-23]. The advantage of this surface modification approach is that it largely preserves the
nanoporous structure of AAO templates, while opening up a range of new possibilities to improve its
chemical, physical and biological properties. Depending on the purpose and the applications, different
types of fabrication methods and materials can be used to modify the surface of AAO structures.
In this chapter, we review a range of different surface modification methods and materials that have
been applied to AAO membranes over the past years. In particular we focus on the surface
modifications that are suitable for biomedical applications and discuss their advantages and
disadvantages. We summarize different available surface modifications techniques, including chemical
immobilization (grafting) [24], sol-gel [25], thermal evaporation [26], atomic layer deposition [27] and
chemical vapor deposition [28]. The use of these techniques for the fabrication of surface modified
AAO membranes using different types of materials is discussed. We describe how these techniques can
modify the surface of AAO templates to improve its surface properties, such as surface charge, surface
chemistry, porosity, hydrophobicity, selectivity, surface activity, permeability, protein and cell
adhesion. Under the surface modification methods, we also review different types of materials used for
surface modifications, including metals, silica, polymers, diamond and diamond-like carbon. We
present an overview of how surface modified AAO membranes have been used for drug separation,
microbe detection, protein adsorption, purification and sensing [19, 29-32]. Finally, we discuss possible
future directions and challenges of surface modified membranes.
Nanomedicine 440
Fabrication and Properties of Uncoated Anodic Alumina

Anodic porous alumina (AAO) membranes have been intensively studied by many different groups all
over the world [11, 33-39]. AAO is a relatively robust porous ceramic material which has a great
potential in drug delivery [40], bio-sensing [31, 32, 41], bio-patterning [7, 29], bio-separation [6, 42],
catalysis [10] and many other applications [14, 33]. AAO is obtained by electrochemical etching of
aluminum in an acidic electrolyte. Figure 18.1.a shows a simple setup for anodization of aluminum. A
piece of aluminum foil is set in front of another electrode (e.g. graphite or platinum) in an electrolyte
solution, and an electric field is applied over the two electrodes. The acidic electrolyte contains both
+ - 2-
positive (H ) and negative (e.g. X-O & X-O ) ions and they accelerate as a result of the applied electric
field. Positive hydrogen ions travel to the cathode and turn to hydrogen gas by receiving an electron.
Negative ions, on the other hand, move toward the anode (aluminum), giving rise to the following
chemical process:
3+ 2-
(1) 2Al + 3O  Al2O3 ,
+ 3+
(2) Al2O3 + 6H  2Al (aq) + 3H2O.
Reaction (1) & (2) leads to the formation and dissolution of aluminum oxide, respectively. The
anodization conditions are crucial because they determine the rate of the formation and dissolution of
the oxide layer. Higher formation rates lead to the growth of the oxide layer. This process is called
anodization. Whereas higher dissolution rates produce a polished surface of the metal and therefore
this process is called electropolish. In anodization process, the formed oxide layer is usually porous as a
result of the penetration of the ions into the formed oxide layer [43].
In 1995, Masuda and Fokuda reported the formation of the ordered porous structure of AAO by
controlling the anodization process using selected acid solutions, applied voltages and temperatures
[11]. The formed structure was a close packed array of uniform cylindrical pores arranged in a
hexagonal form (Figure 18.1.b). Their method is known as “two-step anodization”, and is schematically
described in Figure 18.1.c. In the first step of anodization, random pores nucleate at different positions
of the aluminum surface, but as they grow more towards the inside of the material, they become more
regular in a self-organized manner. The formed oxide is chemically removed at the end of the first step,
leaving the ordered pattern on the aluminum surface. By anodizing the patterned aluminum in the
second step (with the same anodization conditions as the first step), an ordered porous oxide structure
is formed as shown in Figure 18.1.b. Finally, the aluminum substrate can be removed to form a free-
standing AAO membrane. There have been reported different chemical and electrochemical methods
to detach the oxide layer from the aluminum substrate to achieve a free standing AAO membrane [44,
45].
Nanomedicine 441
FIGURE 18.1
a) A simplified setup for anodization, b) an SEM image of a nanoporous AAO structure (Adapted from [34],
Copyright (2007), with permission from Elsevier), c) schematics of a “two-step” anodization method followed by
detachment from the alumina substrate and pore opening to achieve a free-standing AAO membrane.
The thickness of the formed oxide layer depends on the anodization time, and it can range from
-1
100 nm up to few hundred microns [33, 34]. The growth rate is usually very low (~1-2 µm h ), but by
-1
using different anodization conditions in a so called “hard anodization” it can be as high as 70 µm h
[35]. The size and the distance of the pores can also be precisely controlled by using anodization
parameters such as applied voltage, temperature, electrolyte type and concentration [34, 39]. Pore size
can be tuned from 10 to 650 nm, and the porosity can reach up to 50% [12, 13]. Although the resulting
pore size strongly depends on the applied voltage it can also be tuned by the type of electrolyte and
widened by post chemical etching [45]. Figure 18.2. shows a graph of commonly used electrolytes and
a range of voltages to achieve different interpore distances. Fabrication of different pore shapes of
AAO has also been reported by pre-texturing the aluminum surface with triangle or square shaped
holes before the anodization process [36]. This pre-texturing allows even a change in the arrangement
of the pores in AAO. For more details on the anodization process and how to obtain controllably
different pore structures in AAO membranes we refer readers to recent reviews [34, 46].
FIGURE 18.2
The range of achieved pore diameters and pore distances of AAO as a function of different common acids and
anodization voltage used in the anodization process.
Nanomedicine 442
Various interesting AAO nanoarchitectures can be obtained with anodization when cleverly engineered.
For example Li et al. [47] reported the fabrication of Y-branched carbon nanotubes inside the branched
pores of AAO. A fast formation of three-dimensional pore structures with variable pore sizes inside the
channels has been reported by Mayamai et al. [38]. Zhao et al. [37] fabricated different pores in the
shape of nanocontainers. Anodic alumina tubular membranes have also received a lot of interests and
there is a great potential to use them for drug delivery or as filters [48, 49]. These examples show that
the control over the nanostructure shape and size in the anodization process is quiet unique.
Nanopores in AAO membranes can be made in an array of uniform, cylindrical, straight and various
other pore shapes. Additionally, this fabrication process is cheap, straight forward and very consistent
and therefore it is suitable for large scale production [34].
As a result of the anodization process, the surface chemistry of AAO is complicated. The AAO material
in addition to aluminum oxide contains also some other anion contaminations from the anodization
process, which diffused from the electrolyte into the formed oxide during the fabrication process [45].
For this reason, the surface of AAO contains a combination of different atomic and molecular bonding
such as oxides, hydroxides, carboxyl and etc. [45]. It is very important to note that anodic aluminum
oxide is very different from pure aluminum oxide (e.g. sapphire) in terms of the atomic structure,
chemistry and chemical stability. AAO is amorphous and dissolves in most of acidic or basic conditions
[16]. Consequently, the biocompatibility of AAO varies in the literature [17, 18]. All these drawbacks
strictly limit the direct application of AAO in different biomedical applications. However, these
limitations can be overcome by modifying the surface and surface chemistry of AAO by different
materials and coatings. These coatings can be made of other materials with better bioresistivity and
biocompatibility than AAO. Additionally, the surface of AAO can also be functionalized with different
organic and inorganic molecules to be used for other advanced applications that would not be possible
with uncoated AAO. In the next section we review different methods and materials for surface
modification and functionalization of AAO for various biomedical applications.
Surface Modification Methods

As mentioned in the previous section, despite of the great aspects of AAO, its surface chemistry is not
suitable for biomedical applications. The best approach to make the structure more biocompatible and
chemically stable is to modify the surface with other materials. This approach provides an attractive
and versatile strategy for fabrication of nanoporous AAO-based materials with different and improved
physical and chemical properties. Both organic and inorganic materials can be used for the surface
modification and functionalization of AAO. Over the last years, the surface of AAO have been coated
with different materials, such as polymers [20, 24, 40, 50-52], proteins [53, 54], DNAs [29], metals [26],
oxides [19, 25, 55-58], diamond [59, 60] and diamond-like carbon [26, 59, 61]. Figure 18.3. shows a
schematic illustration of an ideal surface modification of AAO, where all the surface of nanoporous AAO
is uniformly and homogenously coated with a more resistive, robust and biocompatible material, while
maintaining the original nanoporous structure. The most common methods to fabricate these coatings
are as follows: physical evaporation [26], grafting [52], sol-gel [19, 25], (electro-)chemical deposition,
chemical vapor deposition (CVD) [28, 59, 61], plasma deposition [22], and atomic layer deposition [27,
57]. Each of these methods has got its advantages and disadvantages. In the following text, we describe
these methods in more detail, overview different materials that they can produce and discuss how they
are suitable for surface modification of nanoporous AAO and biomedical applications.
Nanomedicine 443
FIGURE 18.3
Schematic illustration of an ideal surface modification of an AAO membrane, where the entire inner and outher
surface is coated by a more resistive, robust and compatible material, while maintaining the original structure of
nanoporous AAO.
Grafting methods
Grafting is a technique to covalently immobilize a molecule (usually polymer) to a surface. The process
happens by immersing a membrane into a grafting solution. A grafting solution usually contains
polymer chains, a linking molecule and a catalyst. The linking molecule is attached at the end of the
polymer and covalently bonds to the surface of a membrane in the presence of the catalyst.
Temperature, polymer type and concentration are effective parameters in this method [20].
Popat et al. [20] have applied this grafting method by attaching poly(ethylene glycol) (PEG) to the
surface of AAO. The reaction is shown in Figure 18.4. A PEG-OSiCl3 couple forms by reacting PEG with
silicon tetrachloride in the presence of triethylamine as a catalyst. Then the PEG-OSiCl3 reacts with the -
OH groups on the surface of the membrane, by forming a covalent bonding network of Si-O-Si bonds on
the surface interface, resulting in a PEG-immobilized on the alumina surface. The thickness of the PEG
layer on AAO was ~2.5 nm and it covered both the pore walls and the top surface of the membrane
[20]. PEG coatings have been used in this study for their good biocompatibility and low protein fouling.
As a result the PEG-modified membranes have shown 70% less protein adsorption compared to the
non-modified membranes [20].
FIGURE 18.4
Reaction mechanism of PEG immobilization on alumina surfaces using a covalent grafting technique. (Adapted with
permission from [20]. Copyright (2004) American Chemical Society.)
Nanomedicine 444
In another study, a grafting method has been used to cover the top surface of AAO by hyperbranched
poly(acrylic acid) (PAA) without filling the pores (Figure 18.5) [52]. This approach is suitable for
encapsulation of the pores and might find its application in drug delivery. First a 5 nm thick gold layer
was evaporated on the top surface of AAO membrane, as a grafting layer. A monolayer mixed
anhydrides were formed on the gold layer which then had been used as an attachment point for the
grafting of a PAA layer. The advantage of this process is that the molecular grafting can be repeated
several times resulting in a hyperbranched PAA film. Although a hyperbranched 6-layer PAA film
covered the whole top surface of AAO, it did not block the inside of 20 nm pores of the AAO template
as schematically shown in Figure 18.5. PAA is a biocompatible material which is suitable for biological
studies. Moreover it can be further modified to show fluorescent and/or hydrophobic characteristics
[62].
FIGURE 18.5
PAA grafting to the top surface of an AAO membrane: a) idealized formation and b) schematics of the mechanism.
(Adapted with permission from [52]. Copyright (2000) American Chemical Society.).
Generally polymer grafting is one of the easiest and cheapest methods to attach polymers to the
surface, but there are some problematic issues associated with it [63]. First of all the surface chemistry
of AAO is usually not homogenous because it contains different hydroxyl, oxide and carboxyl groups on
the surface [33, 43]. This might result in incomplete coverage of the grafted molecules on the surface.
This can be overcome by using other techniques, such as an hydrogen peroxide treatment [64], which
can maximize the -OH bonding groups on the surface of AAO. Secondly, polymers are usually suffering
from some drawbacks such as short life time, inhomogeneous structure, limited chemical and biological
stability and functionality [65]. Therefore these coatings on AAO will be applicable only for applications
which require a short term usage in biomedical environments.
Sol-gel methods
Generally, the term “sol-gel” denotes the formation of a solid material remained after hydrolysis of the
adsorbed molecules and subsequent evaporation of the solvent part of the precursor. Sol-gel is a
widely used technique to synthesize silicon and titanium oxide structures, and there have appeared
different alternatives of this process in the literature [3, 19, 25, 42, 56]. One of the main disadvantages
of the sol-gel methods is that they are limited to a certain type of materials and they contain traces of
the used precursors, which hinders their wider usage.
Nanomedicine 445
Figure 18.6 shows an example of a sol-gel process, where the inside of the pores of an AAO membrane
is coated with a silica film [56]. It uses SiCl4 that adsorbs on the surface of AAO, which is then
hydrolyzed to SiO2. CCl4 is used to wash the solvent after each cycle. The chemical composition of the
resulting surface coating is (SiO2)x(SiOH)y [56]. The advantage of this process is that the thickness of the
formed layer can be varied by the number of the cycles, where an each cycle adds approximately a 1
nm layer on the surface [56]. In another sol-gel approach, TIP (titanium isopropoxide) or TEOS
(tetraethoxysilane) has been used to synthesize TiO2 and SiO2 on AAO, respectively [25]. TiO2 and SiO2
layers have been formed by a quick immersion of the membrane into a TIP or TEOS solution,
respectively.
FIGURE 18.6
An example of a sol-gel process inside the channels of AAO producing free-standing SiO2 nanotubes by subsequent
etching of an AAO membrane. (Adapted with permission from ref [56].)
Sol-gel approach can also be used for reducing the pore size of nanoporous AAO membranes, which
might be useful for applications that require pore size < 10 nm. Figure 18.7. shows a formation of well-
ordered silica-surfactant nanocomposite channels inside the nanopores of AAO, which has been
demonstrated by Yamaguchi et al. [42]. The formed surfactant channels in the nanopores have a
parallel arrangement to the channels of AAO with a very narrow pore size diameter of the order of 3.4
nm. This has been achieved by passing the precursor solution through AAO membranes with 25 nm and
47 nm pore sizes [42]. The precursor contained a mixture of TEOS and cetyltrimethylammonium
(CTAB). The exact concentration of each precursor in this mixture has shown to play a crucial role on
the size of the formed surfactant nanochannels [42]. These nanoscopic AAO-based nanocomposite
channels show a great potential for size-exclusive separation of molecules.
Nanomedicine 446
FIGURE 18.7
TEM images of an alumina membrane with nanoporous silica–surfactant nanocomposites inside the columnar
alumina pores: a) low-magnification top view, b) higher magnification top view, and c) side view. Scale bars
correspond to 100 nm (a) and 50 nm (b,c). (Reprinted by permission from Nature Publication, ref [42] , copyright
(2004))
Atomic Layer Deposition
Atomic layer deposition (ALD) is conformal deposition using sequential, self-limiting surface reactions
on the sample surface [27]. In this process, one or more chemical interaction(s) take place on a surface,
giving rise to deposition of a thin film, usually a monolayer. ALD is commonly used for deposition of a
whole range of oxides (Al2O3 HfO2, HfSiO, La2O3, SiO2, STO, Ta2O5, TiO2, ZnO), nitrides (AlN, HfN, SiNx,
TaN, TiN) and metals (Au, Cu, Pt, Ru, W). This method has been successfully applied to deposit oxides in
AAO, such as aluminum oxide [28], zinc oxide [55], and titanium and silicon oxides [57].
ALD is a well-controlled method that can be used to reduce the pore size of AAO membranes below 10
nm, as demonstrated by Velleman et al. [23]. In this study, a SiO2 layer has been deposited on AAO with
20, 100 and 200 nm pore sizes using ALD to reduce the pore size to 1-2 nm. The growth rate of SiO2 on
AAO was estimated to be about 2.5-3 nm/cycle (at 250°C) when using tri (tert-butoxy) silanol and
trimethylaluminum (TMA) as precursors and nitrogen to purge the reactor at the end of each cycle.
Although ALD is considered as a conformal coating technique, the deposition of the added layer can be
different on the surface than inside the nanopores of AAO. Figure 18.8. shows a SEM image of a SiO 2
coated AAO by ALD [23]. Due to the space confinement in the nanometer-sized pores, the deposition
inside the pores has a lower rate, and even it can be totally different, than on the top surface. Even
though it is not possible to see any changes in the pore size from the profile of AAO in Figure 18.8 the
size of the pores is shrunken at the top surface due to the formation of multilayers. The thickness of
the deposited layer in ALD decreases as a function of depth from the surface towards the channels
inside the material [23]. In a short cycle, the precursors do not have enough time to fill the pores and
they are purged by the gas before they completely cover the surface of the pores. This is the reason
why ALD methods need to be carefully optimized to achieve conformal deposition inside of confined
nanospaces. By adjusting different ALD conditions, it has been possible to fabricate nanotubes and
nanowires inside AAO’s channels using ALD (Figure 18.9) *66+. This method employed a combination of
a delayed reaction that happened after one (or more) cycle and a partial purge of the chamber, which
was used to let some time for the residues to reach and saturate the bottom of the pore walls.
Nanomedicine 447
FIGURE 18.8
SEM images of AAO membranes with 200 nm pore size after 20 cycles of ALD deposition of silica. (Adapted from
ref [23], Copyright (2009), with permission from Elsevier.)
FIGURE 18.9
Schematic of an ALD process to create titania nanotube arrays on a substrate. 1) Nanoporous-alumina template on
a substrate created by anodization of an Al film, 2) TiO2 deposited onto the surface of the template by ALD. 3) Top
layer of TiO2 on alumina removed by gentle mechanical polish. 4) Alumina template chemically etched away to
reveal array of titania nanotubes on the substrate. (Adapted with permission from ref [66].)
Nanomedicine 448
Overall the advantage of ALD methods lies in excellent conformal coverage of the surface, in a self-
limiting manner. These methods have a high control over the thickness of the grown layers and
therefore are very suitable for deposition of different inorganic materials in AAO. Additionally, ALD can
be combined with plasma to deposit nanostructures even at lower temperatures than in the usual ALD
[27]. Current limitations of ALD, however, include a typically small size of the chamber, slow and
expensive procedure, and a limited choice of the deposited elements.
Plasma modification and Chemical Vapor Deposition Methods
Plasma modifications and chemical vapor deposition (CVD) methods are becoming increasingly popular
due to several advantages compared to other conventional surface modification and deposition
techniques. CVD is a method for depositing solid materials onto a substrate as a result of chemical
reactions between precursors and a substrate. The precursor needs first to become reactive by using a
source of energy, such as heat or plasma. Then the reactive high energy precursor molecules can
chemically react with the substrate, giving rise to the formation of a new layer on the surface. Plasma
can be used to assist the CVD process by accelerating the process due to the higher activity of ions
compared to neutral molecules. The detailed CVD process of the deposition is quite complicated and
out of the scope of this chapter. To get a more advanced understanding of CVD using plasma and its
applications, interested readers are encouraged to read a recent review on versatile nanofabrication
using reactive plasmas [67].
Thermal (hot filament) CVD and plasma-enhanced CVD (PECVD) methods are currently the most
popular CVD techniques used for deposition of different materials [67]. PECVD has the additional
advantage in comparison to thermally driven CVD because it offers extended capability for changing
the deposited structure morphology and surface properties by using different plasmas [68-75]. CVD
methods are relatively cheap, energy efficient and capable of producing conformal coatings on
different substrates. For all these reasons, CVD has been widely used for a broad range of biological
and medical purposes [9, 22] . One of the most promising ways is to use plasmas at room temperatures
*70+ and atmospheric pressures *72+, as recently developed by Ostrikov’s group. CVD is an especially
useful method for deposition of different carbon materials, such as diamond [68], diamond-like carbon
(DLC) [61], graphene [71], carbon nanotubes and nanofibers [73]. Carbon-based materials possess a
wide range of physical and chemical properties which are suitable for biomedical applications [74]. In
3
particular, the biocompatibility of sp -bonded carbon materials - such as DLC and ultra nanocrystalline
nanodiamond (UNCD) - is well established and it has been already successfully used in many
commercial bionic devices, such as bionic eye [75].
PECVD deposition of polymer based materials on AAO have been recently demonstrated by Losic et al.
[22]. In this work, a plasma polymerization of n-heptylamine plasma polymer (HAPP) was employed to
functionalize and reduce the pore size of AAO membranes. A 13.56 MHz RF generator was used to
polymerize HAPP on AAO using a very low power (40W) and high pressure (0.2 Torr) for a very short
time (20 s - 200 s). Longer deposition times have caused further polymer growth but blocked the pores
as schematically shown in Figure 18.10. The advantage of HAPP coatings on AAO is that their surface
can be subsequently functionalized with amino groups to attach other chemical functionalities, which
offers a broad range of possibilities to use these coatings in various separation and drug delivery
applications [23].
Nanomedicine 449
FIGURE 18.10
Schematic representation of the different stages of surface coating of AAO membranes (the top part of pore
structure).
CVD methods have also been used for template growth of nanostructures in AAO nanopores [37, 69].
Carbon nanotube-AAO composites [76] and free-standing carbon nanotube arrays [77] have been
obtained using CVD growth of carbon nanotubes inside of porous anodic alumina templates. These
composite materials have shown improved mechanical and electrical properties, which make them
useful for sensing, catalysis and battery applications.
Deposition of diamond-like carbon (DLC) on AAO has also been recently demonstrated [59, 61]. Karan
et al. [61] has fabricated subnanometer-sized nanopores in 10-40 nm thick free-standing DLC coatings
on AAO membranes by plasma polymerization of organic compounds. These highly cross-linked
3
networks of sp carbons have demonstrated ultrafast permeation of organic solvents through the
membranes. In our recent study, the whole surface of AAO has been coated with a homogeneous
ultrathin DLC using chemical vapor deposition [59]. A very thin layer of DLC (~2 nm) was grown
homogenously all over the internal and external surface of AAO in a self-limiting manner. The
advantage of this CVD process is that it covers the whole surface of the membrane (Figure 18.11.a,b) in
a very short time (~ 5 min). Membranes with variable pore sizes (10-200 nm) have been produced using
this method. DLC is a very strong, robust and biocompatible material which has been shown very
suitable for different biomedical applications.
Ultra-nanocrystalline diamond (UNCD) films can also be grown on the top surface of AAO using CVD
[78]. For this purpose, however, the membrane needs to be first pre-seeded with detonation
nanodiamond before putting the membranes in a CVD diamond chamber. The seeding process is done
in an ultrasonic condition where the membrane is merged in a solution containing nanodiamond
nanoparticles. The nanodiamonds stick to the surface and act as the nucleation sites in the CVD
diamond growth [68]. Figure 18.11.c shows a nanocrystalline diamond-coated AAO membrane. The size
and the thickness of the nanocrystalline diamond layer depend on the time of the deposition. By
controlling the deposition time nanometer pore size is achievable. Another advantage of the diamond
coatings - in addition to their biocompatibility and chemical resistivity - is that they can be
functionalized with different chemical functional groups to be used for further applications [74].
Nanomedicine 450
FIGURE 18.11
a) A SEM image of the top surface of DLC coated AAO with 40 nm pore size, b) a TEM image of a pore wall of a DLC
coated AAO, and c) a SEM image of the top surface of nanocrystalline diamond coated AAO with reduced pore size.
Other Deposition Methods
In addition to the techniques discussed so far, there are also other techniques that have been used for
modifying nanoporous AAO structures in the literature. These methods include electron beam
evaporation [26], laser pulse deposition [26], and electrochemical and electroless deposition methods
[79]. Thermal, electron-beam and other evaporation methods are commonly used for deposition of
thin films made of almost any material. However, evaporation in vacuum usually leads to deposition of
a material only on the top surface of porous AAO structures similarly to Figure 18.10. and as
demonstrated in references [26, 41, 80]. Laser pulse deposition is also a directional deposition method
that can effectively coat only the top surface of nanoporous AAO, for example by DLC layer [26]. On the
other hand, electrochemical and electroless deposition methods offer the possibility to coat the whole
surface of AAO with different materials, such as metals [79]. Electrochemically grown gold nanotubes
and nanowires inside the channels of AAO have been recently reported [79]. These materials hold a
great promise for bioseparation applications.
Additionally it is important to note that the coatings on AAO membranes should be very well bonded to
the AAO substrate for biomedical applications in order to possess a good stability in different chemical
and biological solutions. Therefore methods using physical bonding between the coated material and
AAO, such as evaporation, do not always produce highly stable layers. Therefore it is better to use
methods that utilize chemical bonding [52].
Applications of Different Surface-Modified AAO Materials

Template
AAO has been used widely as a template for the growth of a broad range of nanostructures and
nanomaterials, including nanodots, nanotubes, nanowires, nanocones and nanocontainers [37, 73, 79,
81, 82]. Nanostructured AAO templates have a great potential to be used in different biological and
medical applications due to their easy and cheap way of fabrication [2, 8, 15, 63, 83, 84]. Formation of
a highly ordered array of biological molecules can improve the performance of different functional
devices [8, 29, 30]. The high surface to volume ratio of AAO templates can also be utilized to produce
Nanomedicine 451
high density and three-dimensional (3D) arrays of enzymes, proteins and DNAs. In this section we
present a few examples how AAO templates have been used in different biomedical experiments and
applications.
AAO is a very suitable template for immobilization of the biological molecules, due to the adjustable
pore size and interpore distance. Matsumoto et al. [29] have studied the fabrication of flow-through
type 3D DNA arrays inside the channels of AAO. This is an engineered type of sensor to optically detect
target DNAs which flow through the channels of a membrane and hybridize with the immobilized probe
DNA array. This sensor takes the advantage of high density DNAs arrays. Figure 18.12.a shows the
immobilization of probe DNAs on the pore walls of AAO [29]. By passing a complementary fluorescent
DNA through the membrane, some of the DNAs hybridized and captured with immobilized DNAs. Other
non- complementary DNAs would pass through the membrane without any hybridization. Later on, it is
possible to detect the hybridized DNA under a fluorescence microscope, due to the fluorescent
emission of the target DNAs. Figure 18.12.b shows fluorescent microscopy images of the DNA-array
inside AAO, where the emitted light from DNAs is guided by the AAO channels. Different pore sizes can
be used for this aim. However, the used interpore distance should be at least half of the wavelength of
the fluorescent light due to the diffraction limit. In this example the wavelength of the emitted
fluorescent light (Cy3 dye) was 562 nm, and a good optical resolution of the DNA array was achieved by
a membrane with 400 nm interpore distance [29].
FIGURE 18.12
a) Schematic of a DNA capture concept in an AAO nanopore and b) fluorescent images of complementary DNA’s
captured by the probe DNA. Adapted with permission from ref [29].
Another example of a template use of AAO is the fabrication of bio-imprinted polymers. It has been
shown that bio-molecules attach to a bio-imprinted polymer selectively [30]. As made bio-imprinted
polymers can be used as a sensor or as carrier for drug delivery. Ince et al. [30] have demonstrated
fabrication of Immunoglobulin G (IgG)-imprinted nanotube polymers using AAO templates. The
resulted nanotubes showed a great selectivity to post protein bonding. Briefly the fabrication
technique includes the immobilization of IgG inside the channels of AAO, and subsequent growth of
polymer nanotubes; resulting in the formation of IgG-imprinted polymeric nanotubes. The nanotubes
then can be released by washing away the AAO membrane with 1 M HCl for 24h. Freed nanotubes
were soaked in 1 M HCl for another 24 hours followed by several washes with DI water. The imprinted
nanotubes can be further loaded with some drugs for drug delivery purposes. The advantage of AAO
templates is that they can be easily fabricated in different pore sizes, so that the dimensions of the
filled nanotubes can be varied. In this way it has been possible to control the release and diffusion
parameters of the targeted biomolecules [30]. As made patterned nanotubes were tested for
selectivity in fluorescent BSA, IgG and Lysozyme solutions and shown dominantly higher adsorbed
amount of IgG than the non-imprinted nanotubes.
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In addition to the above shown template applications, AAO have been used as a template for cell
cultures [17]. AAO with its large surface area and nanoporous structure is a suitable material for
orthopedic implants which allows the formation of bone inside the nanochannels [85]. Porous
materials are widely used for implants due to their ability to mimic the nanoporous dimensions and
structure of the bone components, whilst hosting specific genes or drugs within the pores for
therapeutic treatments [83, 85]. Swan et al. [7] have reported the growth of the osteoblast cells (bone
forming cells) inside the channels of AAO with normal phenotype and morphology. However, more
studies are needed for optimization of the cell growth inside the channels.
Although the membrane preparation and functionalization methods often use strong acidic/alkali
solutions this might not be a problem for further biomedical applications. The biocompatibility of the
resulting membrane will be mainly determined by the final surface treatment and impermeability of
the top protecting surface coating on AAO membranes. So far there have not been observed any strong
effects of these treatments on the biological activity of the membranes. However a more work should
be undertaken in this direction as well as proper testing of the biocompatibility of the surface modified
membranes and suitability of the used chemical methods and materials.
Separation
Nanoporous structures and especially AAO can be used for different kind of molecular separation and
filtration applications. Uniform and narrow pore size of the whole-through AAO membrane is playing a
crucial role in most of these applications. Selectivity and flux rate are one of the most important factors
in the membrane separation applications. Separation of biomolecules has been the subject of long
research [5, 6, 19, 22, 79] and many studies have dealt with this important topic. Nanoporous
membranes with uniform, aligned and straight pores with a high chemical and mechanical stability
offer one of the most suitable methods for separation of biomolecules [2, 6, 79]. There are two main
mechanisms of biofiltrations which are based on (1) size limitation and (2) chemical affiliation. In the
first mechanism, molecules which are smaller than the pore size of the membrane can pass through,
while the bigger molecules are blocked [22, 42]. The second mechanism is related to the bonding of the
molecules with the membrane, which would let transfer faster the selected molecules with lower (or
higher) chemical affinity through the membrane than the other types of the molecules [5, 19]. Here we
give an example of each of these two mechanisms.
Transport of nitrogen, water and ovalbumin molecules through AAO and functionalized AAO has been
investigated by Lee et al. [24]. In this study, poly(ethyleneimine) (PEI) was first physically attached to
the surface of AAO, and then poly(ethylene glycol) (PEG) were grafted by PEI-AAO samples. The PEG
modified membranes have shown a reduction in the transportation rate of different molecules, due to
the reduction of the membrane permeability. Because PEG has a neutral chemical structure with a
hydrophilic character and its brush-like chains sterically exclude other macromolecules, it has a
repulsive behavior to proteins. In another protein adsorption test (FITC-BSA), PEG modified samples
have shown 20% less bonding to proteins than unmodified AAO [24]. In these experiments it is
-
important to perform a protein concentration test of the membranes at low concentrations (< 1 mg ml
1
) to make sure that the adsorption is caused just by the surface interactions and not due to trapping of
the proteins inside the pores.
The transport behavior of fluids in a nanometer size membrane has been experimentally and
theoretically well studied [19, 24, 86]. In an experimental study by Lee et al. [24], it has been shown
that the pore size of AAO membranes can effectively change the transport rates of fluids such as
nitrogen and water. As mentioned in the previous section, pore size of an AAO membrane can be
reduced to molecular size by using different methods, and then molecules bigger than the pore size will
Nanomedicine 453
be excluded from transport. As it has been shown in the study of Lee et al. [24], a PEI-PEG coating have
been able to reduce the size of the pores until the transport of a small molecules such as ovalbumin
was totally excluded.
A modified AAO can also be used for separation of two hydrophobic and hydrophilic dyes. As we
mentioned in the previous section, ALD is a common method used to coat AAO with SiO 2. The SiO2-
modified AAO can be further modified with perfluorodecyldimethylchlorosilane (PFDS) using other
chemistry methods to reduce the pore size to 10 nm [23]. SiO 2-modified AAO is hydrophilic, while
whole surface PFDS-SiO2-modified AAO is highly hydrophobic, showing 12° and 109° contact angles
using a water drop measurement [23]. The rate of the diffusion of the two different dye molecules
(Rubpy (hydrophobic) and rose bengal (RB) (hydrophilic)) with a size smaller than the pore diameters
has been measured by Velleman et al. [23]. This study suggested that the flow rate of the hydrophilic
dye can be reduced by a PFDS functionalized membrane, while not changing significantly the flow rate
of the hydrophobic dye. The flux (Rubpy:RB) of the modified membrane was two times larger than for
the unmodified membranes. The lowest flux has been found for RB in PFDS-modified membranes. The
proposed experiment demonstrated the selectivity of the membrane based on their hydrophobic
properties, which might be very useful for many applications such as protein separation and
purification.
FIGURE 18.13
Schematic illustration of the modification process to fabricate a PFDS-SiO2-AAO membrane used for separation of
hydrophilic and hydrophobic dyes. (Reprinted from ref [23], Copyright (2009), with permission from Elsevier)
Nanomedicine 454
Chemical affinity separation by nanofilters can be applied for chiral separation. Chiral separation of
drugs is one of the most important issues in pharmaceutical industry [19]. (4-[3-(4-fluorophenyl)-2-
hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzonitrile) is a chiral drug with RS and SR enantiomers [19]. It is
desired to selectively separate these two enantiomers. Since they have the same size and same
chemical bonding, it is not possible to separate them via size and charge exclusion. The separation of
these enantiomeric drugs has been demonstrated in Charles R. Martin’s group *19+ using silane
modified AAO membranes. Transport of the RS enantiomer measured by a chromatography method
has been found twice faster than of SR enantiomer in the silane modified AAO membranes. The
principle of this transport difference has been based on the affinity bonding of drug to an immobilized
antibody-loaded-silica nanotube [19]. The binding character has been adjusted by addition of dimethyl
sulfoxide (DMSO). For the membrane with 35 nm pore diameter, the ratio of RS/SR flux behave been as
high as 2.6 at optimal concentration of DMSO [19]. By reducing the pore size to 20 nm the RS/SR flux
increases to 4.5 [19]. The stability of these modified AAO membranes confirmed that these flux ratios
remained constant even after 1 week.
Sensing
AAO materials with specifically modified surfaces show a great promise for sensing of different
chemical and biological molecules [4, 31, 41, 64, 80, 87-90]. The main advantage of porous AAO in
these sensing applications lies in improved sensitivity of the devices due to a large surface area of AAO
in comparison to other materials with flat surfaces. Selectivity is usually provided by different
specialized surface coatings on AAO. The sensing principle of AAO-based sensors can be based either
on optical or electrical detection mechanism. Electrical sensing usually monitors the change of sensors’
current, resistance, capacitance, impedance and etc. [41, 91]. Optical detection can have different
mechanisms, such as fluorescent emission [29, 32], interference [4, 87, 88], surface plasmon excitations
[80], surface enhanced Raman spectroscopy (SERS) and optical waveguide coupling. There are also
many other integrated methods for biosensing applications, for example AAO grown on quart crystal
microbalance (QCM) can be used for mass adsorption detection. Here we focus on two examples of
electrical and optical sensing methods that will demonstrate the benefit of using AAO-based materials
in sensing applications. For more information on sensing using AAO we refer interested readers to a
recent review [15].
There are many pollutants in the environment that require detection in parts per billion. Achieving such
a high sensitivity is often difficult with currently available sensors [32]. Poly chlorobiphenyls (PCBs) are
among the toxic organic pollutant which can lead to serious liver damage [32]. To sense these
pollutants, Wang et al. [32] has immobilized fluorophore phenyl isothiocyanate (PITC) on AAO
membrane (PITC@AAO) to optically detect a presence of PCB. Since it is very hard to covalently bond
PITC to AAO, the immobilization took place via simple immersion of the membrane in a PITC-contained
solution. The as-made PITC@AAO membrane was used to detect PCBs using a UV-Vis spectrometer. By
increasing concentration of PCB from 1 ppb to 6 ppb, the fluorescent intensity of the membrane
increased by a factor of 18 (Figure 18.14.a). Furthermore, the selectivity of the membrane to a certain
type of PCB (PCB101) among 4 other more PCBs has been tested, and a very good selectivity has been
obtained (Figure 18.14.b) [32]. The achieved minimum amount of PCB detected using PITC@AAO
membrane has been around 1 ppb [32]. The experiment with the same concentration of the same
fluorescent molecules placed on a flat quartz substrate lead to a 25% less intensity than on PITC@AAO
[32]. This results show that the high surface area of AAO-based sensors increases the sensitivity. The
confinement of the fluorescent molecules inside the channels might have some effects on the
enhancement of the fluorescent emission as well. More studies are needed to get a better
Nanomedicine 455
understanding of the optical enhancement mechanism in AAO nanochannels. Despite of this the study
of Wang et al. has shown the potential of optical detection of pollutants using surface modified AAO
sensors which promise high sensitivity and selectivity to the targeted biomolecules.
FIGURE 18.14
a) UV-vis spectra of the AAO membrane immobilized with (red) and without (black) PITC. b) Fluorescence
intensity of PITC@AAO after exposing it to different types of PCBs. (Adapted from Ref. [32] with permission from
The Royal Society of Chemistry)
In another example [41], the electrical properties (impedance) of a modified AAO have been measured
to detect label-free DNAs based on charge effects (Figure 18.15). For this purpose AAO membrane was
first silanizied with different proportions of APTS/ETS, and then the two opposite surfaces of the
membranes were coated with a thin layer of gold as electrodes. The impedance measurement was
done by applying an AC mode over the electrodes. In this case, the impedance of the membrane is
given by [41]:
k BT L
Z mem 
e  D C  4C A
2 2 2
bulk
ΔC is a measure of the changes in density of surface charge for a certain pore diameter. e, α, D, T, L and
A are electron’s charge, porosity, diffusion constant, temperature, channel length and cross section
area, respectively. Any changes in the surface charge would have a direct effect on conductance of the
nanochannel and the total impedance of the membrane. By comparing this formula to a flat capacitor
plate based sensor [41], it is obvious that the impedance change is much larger than in the case of the
porous AAO structure. This sensing principle has been utilized by Wang et al. to detect label-free DNAs
[41]. In this study, the surface of the modified AAO was positively charged, and since DNA is a highly
charged molecule, adsorption of the oppositely charged DNAs by membrane has caused a drop in
impedance (Figure 18.18). This sensing mechanism has been suggested as one of the option to detect
cystic fibrosis.
In conclusion, we have shown that AAO provide an interesting material platform for different sensing
schemes. The main advantage of AAO in these applications lies in its large surface area, porous
structure with high density of pores and narrow pore size and distribution. Large surface area of AAO
provides a unique platform to capture and sense a very low amount of molecules. Electrical sensing is
possible also because anodic alumina is a robust insulator with a relatively high break-down limit. Its
adjustable pore size is well suited for optical visualization. High transparency of AAO structure can also
be helpful for optical sensing applications and interferometric measurements.
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FIGURE 18.15
a) A schematic image of an AAO-based electrical sensor. b) Impedance measurement of the AAO-based
membranes as a function of solute concentration, with and without DNA. (Adapted with permission from ref [41].
Copyright (2009) American Chemical Society.)
Drug Delivery
One of the promising applications of nanoporous membranes is controlled drug delivery [8, 14, 40, 50,
65, 83, 85, 91]. In this application, the volume of the nanopores in AAO membranes is utilized for drug
storage and subsequent release. An application of a suitable surface modification to AAO membranes is
important mainly because it can improve its biocompatibility as well as other important properties for
drug capture and release. Biocompatibility of the membranes used in drug delivery is the first condition
that should be fulfilled by a device. So far all the illustrated experiments were done in vitro. When it
comes to in vivo applications, the stability and biocompatibility of a membrane and its behavior inside a
living tissue becomes critically important [18]. Any failure of the device can be dramatic. In vitro tests
can be also used for prediction of relative biocompatibility of a material [17, 18]. Cytotoxicity
determination is a standard method to test biocompatibility of a material and to get some information
about the interaction of the body cells and implanted device [17]. However, in vivo studies give much
clearer and reliable information about the body response to the implanted device. This has been
illustrated by La Flamme et al. [18], who have investigated the biocompatibility of PEG modified and
bare nanoporous AAO membranes both in vitro and in vivo. Although in vitro data showed no toxicity
of the samples the in vivo study has shown clear signatures of inflammatory response of the body to
the implanted membranes [18]. In the in vivo study, two membranes (PEG-modified and unmodified
AAO) were planted in the lower abdominal region of a male Lewis rats for up to 4 weeks. Then
membranes and surrounding tissues were removed to observe the fibrous growth and inflammations.
There was no fibrous growth in any of the samples after 4 weeks, but there were some moderate
inflammations observed after one week in both membranes, with more indications in unmodified
membrane (Figure 18.16.). Although it has been argued that this inflammations might be caused by the
surgical process, this and other studies clearly suggested that AAO membranes need to be made more
bioresistive and biocompatible [18, 20, 22, 24, 26, 92]. The interaction of AAO membranes with
different cell interfaces have been studied in great detail by Brüggemann [17].
Anti-biofouling of a device is very important to resist the adhesion of the proteins of blood or immune
system to its surface. PEG coatings on AAO have shown a great improvement in its anti-biofouling
characteristics [18]. Other suitable materials for anti-biofouling are diamond [60, 75], diamond-like
carbon [26, 60, 93], noble metals and high quality metal oxides such as sapphire [92].
Nanomedicine 457
FIGURE 18.16
Histological examination of tissue exposed to no material (A) and (D), unmodified capsules (B) and (E), and PEG-
modified capsules (C) and (F) after 1 and 4 weeks. Arrows indicate the portion of the tissue that was exposed to
the capsule (Adapted with permission from ref [18]. Copyright (2007) American Chemical Society)
Another important factor in drug delivery is the controlled delivery. Figure 18.17.b shows the release of
drug from a normal tablet inside the body, showing a safe profile and a dangerous profile of release
[14]. The idealized concentration release is shown in Figure 18.17.b, where the drug releases with
almost constant rate after an initial stabilization. Any drug release from nanoporous membranes is
required to be in the safe region and therefore it is better to have a release profile similar to the
idealized profile. Figure 18.17.a shows a different models of drug release from a nanoporous
membrane [14]. Pore size and drug particle size play the most important role in these drug release
mechanisms. For a membrane with pores much bigger than drug particles, Fick’s mechanism is found
as the dominant mechanism of release profile. As the pore size decreases, the single file diffusion
profile dominates because the release profile gets closer to the concentration diffusion profile.
FIGURE 18.17
a) Release mechanism of drugs from porous structures and b) the therapeutic range of a release profile (top) and
the idealized release profile (bottom). (Reprinted from ref [14], Copyright (2012), with permission from Elsevier)
Nanomedicine 458
Tubular AAO membranes (Figure 18.18) with different pores sizes have also been used for controlled
molecular transport [94]. In this study, fluorescein isothiocyanate (FITC)-dextran molecules with
different molecular weight (4 kDa, 20 kDa, 70 kDa and 150 kDa) were encapsulated inside the tube and
the diffusion rates were measured. As it is expected the release rate of fluorescein (400 Da) is higher
for the bigger pore size (55 nm) [94]. Heavier (bigger) molecules release much slower than lighter
(smaller) molecules. It has been observed that even after 24 h there has not been a considerable
release of big molecules. This study has clearly shown that the controlled idealized release of the drug
from AAO capsules is possible.
FIGURE 18.18
An example of a tubular AAO capsule for drug release from the capsules with pore diameters (Reprinted from ref
[95], Copyright (2005), with permission from Elsevier).
Usually the size of the drugs is much smaller than the pore sizes of AAO, which limits the drug release.
To improve the release characteristics of drugs from AAO membranes, Losic et al. [50] suggested
coating the top surface of the membrane with a plasma polymer after drug loading. Coatings on AAO
will decrease the pore size, which will consequently lead to a better release characteristic. This
principle has been demonstrated in an innovative integrated method using carriers (polymeric micelles)
loaded in the pores of AAO to carry the hydrophobic drugs (Figure 18.19.a) [50]. The top surface of
AAO was coated with a plasma polymer to reduce the pore size. Figure 18.19.c shows the release
profile of the micelles from the membrane. After the burst release (~ 35%) in first 6 hours, the drug
release slowed down for the next couple of days (6-14 days). It can be seen that the profile
characteristics is very close to the ideal drug release, which represents the single file diffusion
mechanism [14, 50].
Another innovative method is a switchable drug release from AAO membranes which has been
demonstrated by Jeon et al. [91]. For this purpose, AAO was first coated with gold and then a layer of
polypyrrole was formed on the gold layer by an electropolymerization method.
Nanomedicine 459
FIGURE 18.19
a,b)schematics of the concept, and c) release profile. (Reproduced from Ref. [50] with permission from The Royal
Society of Chemistry.)
The principle is based on a special characteristic of polypyrrole, which is able to change its volume in
different electrochemical conditions. When the polymer gets oxidized, the hydrated ions expel each
other and the chains get shrunken. As a result the pore diameter will increase. On the other hand,
when the polymer gets back to the reduced state, chains are expanded and the pore size will decrease
(Figure 18.20). This is a reversible process which can be used for on-demand drug release.
FIGURE 18.20
Schematics of reversible change of pore size and the drug release rate between oxidation and reduction states.
(Adapted) with permission from ref [91]. Copyright (2011) American Chemical Society)
Future of the Modified Membranes

Currently AAO membranes are among the best potential membranes for biomedical use due to their
robustness, uniform and narrow porous structure, and also because of their easy and cheap way of the
fabrication. We have shown in the previous sections that there are many research groups around the
world who are investigating the potential applications of this great material, while other are trying to
modify this material for different desired applications. Surface modification of AAO membranes is used
Nanomedicine 460
to improve various properties of the AAO material, opening up new opportunities for other advanced
applications, especially for biomedical application that have a big demand for functional membranes.
Functional nanoporous membranes can be used in catalysis or as separators and filters which are
commonly used in biology and chemistry. A better and more accurate filter is always demanded for
pharmaceutical industries or for blood purification applications. Excellent bio filters should separate
maximum amount of targeted molecules with a high speed and without any impurities. The separation
of the biomolecules by their size is very difficult so the functional membrane should be chemically able
to select the targeted molecules to pass. The speed of the permeate is shown to be related to the pore
diameter, however some other forces, such as electrostatic or hydrophobic, can be used to fasten the
permeation through membranes. More studies on chemical selectivity of the molecules can push the
current membranes toward the idealized membrane.
For sensing application, there is a need for a more conformal and uniform membranes that can
improve both sensitivity and selectivity of AAO based sensors. Ideally ordered nanoporous structure
with a very conformal coating is required for a highly sensitive and reliable membrane. For template
applications, again the uniform structure of the pores and more control over the architecture of the
pores is needed. If the architecture of the membrane can be controlled in both nanoscale and
microscale, there will be many great opportunities for designing implants with specific geometry and
functionalization. At the moment the design of the AAO membranes is limited to the flat and tubular
membranes, with almost the same pore diameter along the pore length in the membrane. The
development of improved anodization or modification techniques promise fabrication of more complex
AAO-based membranes with various geometries and desired surface properties and functionalities.
For in vivo applications (e.g. drug delivery) the first important and crucial factor is biocompatibility of
the used membranes. AAO is not sufficiently biocompatible and chemically stable in the biological
conditions [18, 22, 23, 25, 28]. Release of the aluminium ions from the membrane would have some
side effects on the long use of uncoated AAO membranes in the body. To prevent infections, the
membrane should be coated with a more resistive and biocompatible material. The ideal coating
should be homogenous and all over the membrane including inside the pores of the membrane. Any
uncoated or agglomerated area might lead to total failure of the device. Engineering of such a coated
layer is not easy, especially in the nanopores, but it should be possible with the use of one of the
coating methods discussed in section 3. In choosing the best material for the coating the specific
function of the device should be considered. However, generally the coated material should lead to an
improvement of the chemical and mechanical properties of AAO. Carbon based materials such as
diamond and diamond-like carbon are considered among the best candidates for this purpose. Their
biocompatibility has been proven and they are very strong materials in terms of physical and chemical
properties.
Conclusions
Since the discovery of nanoporous anodic aluminium oxide (AAO), this material has been used for
various applications. The main advantage of AAO is that its nanopores are well-ordered, uniform, and
organized in a parallel columnar way. Also adjustable and narrow pore size distribution of AAO
membranes is very unique. Anodization is a cheap and straight forward method of fabrication of AAO
which has made it a very popular membrane and template for different purposes. Despite of all the
great advantages, the chemical instability and slight toxicity of these membranes have limited their
applications in biomedical devices. In this chapter, we have shown that to take advantages of the
unique properties of AAO in biomedical applications, it is important to provide the membranes with
Nanomedicine 461
different surface modifications of functionalizations. Very interesting and break-through advances have
occurred in this path. We have presented different chemical and physical methods that have been used
to modify the surface of AAO in respect to their efficiency, film properties and type of coated materials.
Additionally, we have discussed various applications of the coating methods and differently surface
modified AAO membranes. Polymer modifications seem very straight forward and cheap, but they are
suffering from the polymer drawbacks; such as short-time stability and limited chemical and physical
stability. Oxide materials are physically strong and biologically suitable, but usually their fabrication
methods require very expensive and slow deposition methods, such as ALD. There have been
developed other cheap methods for the deposition of oxide films by using chemical methods such as
sol-gel. The other materials such as bioresistive and biocompatible carbon-based materials are very
good for biomedical purposes but the current methods are not developed for depositing them on
nanoporous AAO nanoarchitectures.
The surface modified AAO membranes can be functionalized with different biological molecules for
different purposes. Some of the main application of these membranes are: (1) templates for fabrication
of the ordered biomolecules or patterned growth of the cells, (2) biosorting and filtering biomolecules
by selectively passing the targeted molecules through the membrane, (3) sensing of the targeted
molecules, such as DNAs, proteins or even cancer cells and (4) capsules for drug delivery by releasing
the previously-loaded drugs. There are many researches active in these areas, who are passing through
the cutting-edge technology by introducing new and creative ideas and possibilities.
The future of the biology and medicine will be different from now by developing and designing better
and improved membranes, sensors and drug delivery methods. More sensible devices, more accurate
measurements, more purified drugs, smarter functions and more targeted delivery of the drugs are
some of the possible outcomes of better control to fabricate functional nanoporous membranes. There
is lots of work needed to be done to enhance the current limits of the surface modified AAO
membranes. More control over the fabrication and modification in nanoscale and microscale is needed.
More understanding of the chemical selectivity of the materials and their functionality is needed. Also
the modification techniques should be improved for a cheap and reliable method to coat or modify the
whole surface of the three-dimensional nanoporous materials.
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19
Tissue visualization mediated by
nanoparticles: from tissue staining to
mass spectrometry tissue profiling and
imaging
1# 2# 3 2,4 1,2,5,6*
Lenka Kolářová , Petr Vaňhara , Eladia María Peña-Méndez , Aleš Hampl and Josef Havel
1
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5/A14, 625 00 Brno, Czech Republic
2
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 3/A1, 625 00 Brno, Czech
Republic
3
Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, University of La Laguna, Campus de
Anchieta, 38071 La Laguna, Tenerife, Spain
4
International Clinical Research Center, St. Anne´s University Hospital, Brno, Czech Republic, Pekařská 53, 656 91 Brno, Czech
Republic
5
Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
6
R&D Centre for Low-Cost Plasma and Nanotechnology Surface Modifications, CEPLANT, Masaryk University, Kotlářská 2, 611 37
Brno, Czech Republic
#
These authors contributed equally.
Introduction ……………………………………………….………………………………………………………………………………. 468

From tissue staining to tissue imaging …………………………………………..…………………………………………… 468
Nanoparticles as visualization agents …………………………………………………………………………………………. 470
Tissue imaging by MS …………………………………………………………………………………………………………………. 474
Ionization methods for IMS ………………………………………………………………………………………………………… 476
MALDI MS ………………………………………………………………………………………………………………………………….. 476
SIMS …………………………………………………………………………………………………………………………………………… 477
DESI MS ……………………………………….…………………………………………………………………………………………….. 478
Application of MALDI-TOF MS in tissue analysis …………………………………………………………………………. 478
Histological staining and IMS ……………………………………………………………………………………………………… 479
NP-Mediated IMS ………………………………………………………………………………………………………………………. 479
Conclusions ………………………………………………………………………………………………………………………………… 481
References……………………………………………………………………………………..…………………………………………… 482
Nanomedicine 468
Introduction
For decades, visualization of cell and tissue structures by chemical staining developed for optical
microscopy was the only option for a clinical histopathological diagnosis or biomedical research in a
tissue context. A cardinal breakthrough was later achieved by the introduction of antibody-based
techniques that provided spatial resolution and antigen specificity at the cellular and sub-cellular levels.
Presently, classical methods of tissue analysis are complemented with various instrumental methods to
provide global information on tissue architecture, reflecting the chemical composition of the tissue,
including spatial distribution of metabolites, regional structural differences of the extracellular matrix
or secreted proteins and other large molecules. These techniques include Raman spectroscopy (RS),
magnetic resonance imaging (MRI), ultrasonography (US), computed tomography (CT) or positron
emission tomography (PET), single-photon emission CT (SPECT) or mass spectrometry (MS). In this
review, we provide a brief overview of the application of nanotechnology in histology methods,
particularly in bioanalytical tissue MS imaging.
From tissue staining to tissue imaging

Tissue staining is an established histological technique that reveals structural patterns that are not
clear or sufficiently visible to be observed directly. The first dyes used for staining of tissue structures
were colored substances isolated from natural resources—e.g., indigo, saffron, hematoxylin,
azocarmine, and orcein. These dyes were commonly used for dyeing textile fibers and later found wide
application in histology. The onset of synthetic dyes further enhanced histopathological analyses since
the invention and application of aniline dyes in 1856 [1]. However, various tissues, including neural or
lymphatic tissue, resisted staining using large molecular dyes. When the impregnation by silver salts
and other non-classical-dyes was introduced to histological analysis [2], fine molecular structures of
complex organs were revealed—e.g., reticulin networks in the spleen or the architecture of the brain
isocortex (Fig. 19.1A). Decades later, the application of fluorescent dyes or enzymes linked to specific
antibodies, together with confocal microscopy, shifted histological analysis from subjective
morphological evaluation to the analysis of particular molecular patterns or epitopes even in an
automated manner [3]. Advances in analytical chemistry, particularly in MS, and its orientation in
complex biological problems, contributed to the development of techniques for the direct analysis of
chemical tissue composition, thereby enhancing the potential of current diagnostics in cancer [4] or
pharmaceutical research [5]. Mass spectra with individual peaks corresponding to molecules of a
particular molecular weight and charge (i.e., the m/z ratio) provide individual tissues with their unique
spectral fingerprint (Fig. 19.1B) and an input for “bottom-up” proteomics. The progress of
micromanipulation techniques, such as laser-captured microdissection allowed topologically highly
correlated MS-based proteomic analysis on very small but precisely defined cell populations [6].
Nanomedicine 469
A
Hematoxylin & Eosin Azocarmine Silver impregnation
10
20
40
FIGURE 19.1
(A) Architecture of the mouse isocortex and adjacent cerebral white matter, visualized by classical histological
techniques. Brain tissue was fixed in formalin and embedded in paraffin (FFPE). Sections of the isocortex were
stained with hematoxylin and eosin, azocarmine and silver impregnation according to standard protocols [7].
Roman numerals correspond to individual layers in the isocortex (I: Lamina zonalis; II: L. granulairs externa; III: L.
pyramidalis; IV: L. granularis interna; V: L. ganglionaris; VI: L. multiformis). Deposition of silver nanocrystals within
neural structures and neurons reveals structural patterns that are poorly distinguished by standard staining. (B) A
unique molecular fingerprint of mouse brain tissue. The spectral profile of the digested surface proteins was
generated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI TOF) MS (blue). Brain tissue in the
form of FFPE sections was deparaffinized and trypsinized (red) prior to MS.
Nanomedicine 470
Presently, the term “tissue imaging” is dedicated to advanced methods of visualizing tissue structures
ranging from in vivo imaging of whole organs to high-resolution cell tracking or revealing the chemical
composition and metabolomic profiles. For decades, classical histological methods have been based on
specific chemical staining of tissue sections, selective for proteins, peptides, lipids, metabolites or other
biomolecules or drugs. Despite precise positional information within a tissue, standard topological dyes
have provided only limited information concerning chemical composition or structural specificity.
However, antibody-based techniques developed for visualization of fixed structures are generally not
compatible with imaging in vivo due to protein degradation or limited penetration into tissues.
Nanoparticles containing a metallic core and a functionalized shell that were recently introduced to
bioimaging technology showed surprisingly effective capability to migrate to the desired site, interact
with the target tissue and deposit in specific sites.
Nanoparticles as visualization agents

A nanoparticle (NP) is defined as a single unit with a size varying between ~1 and 100 nm and showing
uniform parameters and properties that are not present on a larger scale [8]. Recent advances in
nanotechnology brought NPs to the broad interest of the scientific community due to their unique
chemical, optical, electronic, magnetic and structural properties. NPs have found wide applications in
biomedicine, ranging from research to diagnostics, prediction or therapeutics [9]. Because the size of
NPs reaches cellular or sub-cellular levels while maintaining their capability to directly interact with
molecular structures, NPs are greatly attractive in fluorescent microscopy as quantum dots [10, 11] or
contrasting agents for tissue in vivo imaging (MRI), particularly regarding the labeling and tracking of
migrating cancer or stem cells [12] or enhancing other types of analysis—e.g., MS. NPs can be
synthesized from various materials ranging from carbon to metals and functionalized according to the
desired function or visualization techniques. Functionalization of NPs covers a broad range of
modifications of the NP core or engineering of an NP shell. Properties of NPs can be further tuned to a
particular task by the composition of different biocompatible compounds that allow use in living cells.
This is particularly true for many cancers that preferentially uptake various metabolites or biomolecules
compared with normal tissue. Iron oxide NPs coated with the heavy chain of human ferritin (M-HFn)
were found to interact with the transferrin receptor that is predominantly expressed on cancer cells.
The iron core possesses catalytic activity and allows the oxidation of standard peroxidase substrates—
e.g., tetramethylbenzidine or diazoaminobenzene. The deposition of M-HFn NPs in cultured cancer
cells or within a fixed tissue allowed for precise identification of malignant cells in histological sections
[13]. Recently, fluorescent nanodiamonds were applied to track implanted lung progenitor cells during
engraftment from intravenous administration to final homing into terminal bronchioles [14]. Therefore,
simple administration of such biospecific NPs provides an analytical output without any further
targeting ligand of contrasting substrate. Specific interaction of engineered NPs with cellular or tissue
structures opens a possibility to detect bound para- or supermagnetic NPs in vivo—e.g., using MRI or
other multimodal imaging techniques such as CT, SPECT or fluorescence-based techniques. In a breast
cancer cell line study, Tietz and colleagues encapsulated an iron oxide core in an epichlorohydrin cross-
linked dextran polymer, conjugated with a cyclopentapeptide with high affinity to a CXCR4 chemokine
receptor. Breast cancer MDA-MB-231 cells overexpressing CXCR4 preferentially bound CXCR4-specific
NPs and provided a negative contrast MRI image [15]. Iron oxide NPs were used for MRI tracking of
implanted mouse embryonic stem cells into the site of the lesion where the labeled cells provide a
hypointense MRI signal [16]. The superparamagnetic iron oxide NPs are generally safe and well-
tolerated compounds [17] currently approved for administration in patients using various modalities.
Nanomedicine 471
Lanthanide core NPs specifically influence the relaxation times of protons in their close proximity and,
compared with iron oxide NPs, provide a positive MRI contrast. Contrasting agents based on
3+
gadolinium (Gd ) chelates, however, may induce adverse reaction in some patients—e.g., nephrogenic
systemic fibrosis [18]. To overcome this obstacle, modification of the structures coating the Gd core has
been described—e.g., polyethylenglycol/polyethylenimine [19], silica coating [20], various metal-
organic frameworks (MOFs) [21] or nanocarbon (nanodiamonds) [22]. Due to their molecular structure,
MOFs are highly attractive as drug-delivery vehicles or imaging agents (Fig. 19.2). Targeted complex Gd
NPs then show high specificity to the investigated tissue—e.g., cancer-lesioned liver [23] or melanin-
containing melanoma cells [24]. Replacement of the coated core metal with an inert element—e.g.,
gold—and building up a scaffold with similar structural properties to the extracellular matrix or a cell
membrane have decreased the toxicity of MRI-contrasting NPs, enhanced the imaging properties [25]
and also allowed for multiple analysis using, for example, fluorescence sorting, MS profiling, antibody-
based techniques, magnetic labeling or electron microscopy [22, 26, 27]. The mesoporous silica-coated
hollow manganese oxide demonstrated low cellular and systemic toxicity when electroporated into the
mesenchymal stem cells (MSCs) and tracked by MRI after stereotactic injection into the putamen of the
mouse. Moreover, MSCs containing silica-coated manganese NPs retained their viability, and their
differentiation potential was uncompromised. Importantly, the architecture of mesoporous silica shell
NPs provided access of water molecules to the manganese core and enhanced the positive contrast of
the MR image. Deposition of labeled MSCs in hyperintense MRI loci allowed stable tracking with high
spatial resolution over prolonged time periods [28].
Recently, nanoscale metal-organic frameworks (NMOFs) emerged as promising biomedical or
bioengineering tools for imaging and drug delivery [29-31]. NMOFs are a new class of hybrid materials
in nanometer size consisting of metal ions and organic bridging ligands (Fig. 19.2). The chemical
properties of these materials, such as their structural and molecular diversity, type of metallic core
(e.g., Yb, Gd, Mn, or Fe), high loading capacity, and intrinsic biodegradability make them suitable for
direct in vivo imaging using near-infrared microscopy [32] or MRI [33].
FIGURE 19.2
Molecular structure of an MOF NP.
Nanomedicine 472
Gold NPs (AuNPs) are a promising class of NPs with a lower frequency of adverse effects of lanthanide
NPs in vivo and a longer life-time than iron NPs. Individual AuNPs (Fig. 19.3) or their clusters (Fig. 19.4)
can be directly modified by various structures that either improve a detection method, enhance
specificity or reduce toxicity and improve clearance. For example, nanoflares are highly functionalized
NPs that bring extreme detection specificity to the level of single molecules of nucleic acids. Thus, the
conjugation of specific oligonucleotides to AuNPs combines very high specificity with a potent
fluorescence activity and low toxicity to the cell. Standard techniques for mRNA detection are based on
the detection of an interaction event between a fluorophore-labeled oligonucleotide with its target
sequence. However, major obstacles lie in the delivery method into cells, a high background
compromising the specificity of the assay and enzymatic degradation of the reporters. AuNP-based
nanoflares possess the unique capability of detecting individual mRNAs in living cells without enzymatic
degradation of the vehicles or the need for transfection reagents for delivery [34, 35].
Stabilization
Derivatization
Targeting
FIGURE 19.3
Example of direct modifications of AuNPs. AuNPs can be stabilized using citric acid or gallic acid. Derivatization by
introducing various functional groups or even biomolecules (e.g., thiol, amino, antibody, and oligonucleotides) can
enhance the binding specificity to particular tissue structures, antigens or even individual sequences of DNA or
RNA. Dashed or full lines indicate non-covalent or covalent bonds, respectively.
Nanomedicine 473
FIGURE 19.4
AuNPs form clusters of various sizes and complexity. Gold clusters up to Au10 are known to be planar, higher Au
clusters possess three-dimensional structure, and some of them (Aum, where m=16,17,18) might be empty,
representing gold fullerenes, which are hollow gold cages that can form endohedral complexes—e.g., M@Au16,
where M is a foreign metal inside the cage.
Therefore, NP engineering defines physical parameters that are critical for the particular imaging
method but also determines biological properties, such as target specificity, migration through the
tissue environment, retention in the vascular system, renal excretion or the efficacy of the cellular
uptake, which can dramatically influence the information value of the diagnostic or analytical output
(Fig. 19.5).
Nanomedicine 474
FIGURE 19.5
Engineering of the core and shell determines performance in various imaging techniques and targeting specificity,
and prevents degradation or adverse effects in vivo.
Tissue imaging by MS
MS allows the rapid detection, localization and identification of many molecules from the very simple
to the most complex (e.g., biomolecules). Tissue MS is a label-free technique that can provide detailed
understanding of biological processes in a broad cellular context and whole biological systems. A
principle of MS techniques is demonstrated in the MALDI-TOF MS example summarized in Fig. 19.6.
The development of imaging MS (IMS) provided the unique ability to analyze hundreds of analytes in a
one experiment without prior knowledge of the tissue composition or the use of antibodies or staining
reagents. Another advantage of this technique is the maintenance of spatial molecular patterns
because tissue samples are analyzed without prior homogenization (mechanical disruption of tissue,
creating a homogenous mixture) or fractionation (division of homogeneous mixture into individual
fractions) [36, 37]. Homogenization and fractionation belong to classical approaches of sample
preparation; however, these techniques affect the distribution of particular molecules (spatial
information) and destroy morphological structures. Moreover, the IMS approach can visualize the
global biochemical heterogeneity of individual cell populations and tissue structures at the single-cell
level [38]. IMS can be applied in a wide area of “omics” research such as proteomics, lipidomics,
Nanomedicine 475
metabolomics, metallomics, and drug discovery, and is a topic that was reviewed in detail recently [39].
FIGURE 19.6
Principle of MALDI-TOF MS. Tissue molecules co-crystallized with a matrix (defined below) are desorbed by a laser
beam from a sample surface. Ions enter into the flight tube towards a detector. The time of flight and intensity of
the signal are recorded; next the ratio of the molecular weight (m/z) to charge is determined.
Depending on the experimental design, the output is either a single mass profile of a nonspecific tissue
region or a homogeneous cell population, or a panel of mass spectra recorded in high resolution from
defined coordinates and containing spatial information [40]. The IMS approach efficiently combines the
analysis of molecular species and their distribution together with morphological and histopathological
information. Moreover, fixed tissues prepared for routine histology are compatible with IMS or other
various modifications of MS, enabling analysis of various samples stored at medical facilities [41].
However, IMS requires modifications of virtually all steps of the standard “profiling” MS protocol. To
achieve a desired resolution, the sample preparation and application retrieving the surface molecules
for ionization, instrumental setup, data acquisition and mass spectra analysis in parallel with
histological assessment need to be optimized [42-45]. A scheme depicting the difference between MS
profiling and MS imaging is shown in Fig. 19.7.
Nanomedicine 476
FIGURE 19.6
Principle of mass spectrometry profiling and imaging on intact tissue sections.
Ionization methods for IMS
Ionization is a critical step in the entire process of IMS, determining the quality of recorded spectra,
spatial resolution and content of biologically relevant information. Presently, there is a broad range of
surface ionization methods adopted for IMS from bioanalytical MS; however, MALDI MS, secondary ion
MS ﴾SIMS﴿ and desorption electrospray ionization ﴾DESI﴿ are the most common.
MALDI MS
MALDI is a soft ionization technique based on mixing a sample with an excessive amount of a matrix
and subsequent desorption and ionization of the sample molecules by a short (~ns) laser pulse usually
Nanomedicine 477
-4
in a high vacuum (about 10 Pa) [46]. The matrix plays a key role in the absorption of laser energy of a
certain wavelength, which is then passed to the analyte, causing a transition of the analyte from the
solid phase into the gas phase and preventing fragmentation molecules of the sample by using a direct
laser beam. The matrix is at the same time a donor or acceptor of protons to the sample molecules in
the positive or negative mode. The mechanism of proton transfer from the matrix to the analyte
±
remains unclear. This technique preferentially provides pseudomolecular [M ± H] ions of the analyte
2+ 3+
but sometimes can be observed in the spectra of other ions, such as [M + 2H] , [M + 3H] ions or
+
dimers [2M + H] , and others. MALDI enables the ionization of biomolecules over a wide mass range,
including DNA, peptides, proteins, lipids and many other substances. MALDI MS is often the chosen
bioanalytical method because of its sensitivity, its tolerance to impurities and simple preparation of the
sample [38].
The correct choice of matrix is one of the most important steps in MALDI IMS. As the matrix, organic
aromatic acids are usually used. Each matrix is suitable for different analytes. For example, the most
commonly used matrix in MALDI-TOF of cellular or tissue samples is sinapinic acid suitable for the
detection of higher molecular weight proteins, α-cyano-4-hydroxycinnamic acid for the detection of
lipids and lower molecular weight peptides and 2,5-dihydrobenzoic acid or 2,6-dihydroxyacetophenone
for the ionization of phospholipids and drugs [47]. Various matrices differently affect desorption and,
thus, ionization of the analyte. The interaction of the analyte with the matrix might also improve the
ionization efficiency. The application of a MALDI matrix on the sample of tissue section affects
substantially the assay output. In MALDI IMS of a tissue section, the matrix solution needs to be
homogeneously deposited over the sample—e.g., by spraying the matrix over the entire surface of the
tissue. However, the regions desired for single profiling can be defined by simple deposition of matrix
droplets to particular regions of the tissue section [48].
Manual methods of matrix deposition such as airbrush spraying or dipping the tissue sections into a
matrix-containing solution usually suffer from poor reproducibility [49]. Automated deposition allows
the deployment of a very thin layer of matrix with an IMS quality suitable also for high-throughput
preparations [50]. The most common mass analyzer in combination with the MALDI technique is the
TOF, which provides high mass resolution, excellent mass accuracy, and a rapid acquisition rate over a
large mass range [51].
SIMS
SIMS was the first ionization technique in MS used for chemical imaging [52]. The main advantage of
SIMS is the spatial resolution level at the submicron level as low as 50 nm, making this approach
suitable for investigating the chemical composition of single cells or even subcellular structures [38].
SIMS is based on irradiation of the sample surface by a primary beam of high energy containing metal
+ + +
ions (e.g. Ar , Ge , or In ). These primary ions generate the emission of secondary ions from the sample
surface. The energy of the primary ion is much higher (range 5–25 keV) than the energy of the laser
beam in MALDI experiments; thus, SIMS often leads to extensive fragmentation of surface molecules
and is classified as a hard ionization technique. SIMS typically desorbs and ionizes elements and small
molecules, such as lipids, metabolites, and small drugs, with an upper mass limit about 2 kDa. However,
ion beams can be focused with a much higher spatial precision than laser beams. SIMS represents a
unique tool for high-resolution IMS of single cells, their organelles and structures or to accurately
define tissue regions [53]. This technique does not require any special sample preparation or a matrix.
-7
SIMS analyses are carried out in an ultrahigh vacuum (<10 Pa) to facilitate the transfer of ions after
desorption from the sample surface.
Nanomedicine 478
SIMS is commonly combined with TOF, magnetic sector or Orbitrap analyzers [54]. According to the
experimental design, SIMS works in either the static or dynamic mode. In the static mode, a primary ion
12 2
beam is used at a dose less than 10 primary ions/cm for desorption of the molecule from the surface
and provides information about the composition of the surface. Static SIMS is mostly used for
12
qualitative imaging. Dynamic SIMS is more destructive because a much larger primary ion dose (>10
2
primary ions/cm ) is applied on the sample, uncovering deep structures. Dynamic SIMS is mainly used
for quantitative elemental imaging [55].
DESI MS
The DESI ionization technique was developed by the Cooks group in 2004 [56] and was first used for MS
imaging of biological tissues in 2005 [57]. The DESI principle is based on the concept of the combination
of two MS ionization methods—electrospray ionization (ESI) and desorption ionization (DI). DESI occurs
by the interaction of charged droplets of solvent (often a mixture of water and methanol at a ratio of
1:1) produced by electrospray with the sample surface. By the collisions of drops with the sample
surface, secondary charged droplets containing dissolved surface molecules of sample are generated.
These secondary droplets are immediately converted on gaseous ions, which are directed at an
appropriate angle into the inlet of the mass analyzer [58]. DESI provides several advantages compared
with MALDI and SIMS. DESI is performed at atmospheric pressure and requires no additional sample
preparation or addition of the organic matrix. DESI also induces very little fragmentation of the sample,
making it suitable for analysis of complex molecules. DESI provides multiply charged ions in the form of
n+ n-
[M + nH] and [M – nH] but at a lower spatial resolution than MALDI or SIMS [53]. The rapid analysis
time and its ability to be combined with various MS analyzers make this technique attractive for
imaging all types of tissue [51] or particular structures, including polymers, drugs, and lipids [59].
Application of MALDI-TOF MS in tissue analysis
MALDI-TOF MS for tissue analysis has already demonstrated its applicability in clinical and biological
problems [60-62]. The generation of tissue-specific molecular weight or m/z maps or images with high
resolution and sensitivity provides a tool for pathology, chemotherapeutics, and discovery of disease
biomarkers. MALDI-TOF MS allows the rapid detection of more than a thousand peptides and proteins
from various tissues covering diverse fields of medicine, ranging from oncology, regenerative medicine,
and neurology to pathology, tissue architecture or biomedical research [42, 45, 62-68]. MALDI MS or
IMS, with laser capture microdissection, generates expression profiles from individual cells or
clinicopathologically relevant regions of tissue. For example, the molecular profiles of tumor tissues can
correctly predict tumor behavior, diagnosis, prognosis or response to therapy. Bottom-up proteomics
based on complex patterns can lead to the discovery of novel biomarkers [42, 65].
MALDI-TOF MS/IMS has the potential to identify patient subpopulations that are not evident based on
the cellular phenotype determined microscopically [58]. In surgical pathology, IMS allows the highly
sensitive and rapid evaluation of surgical intraoperative margins [69]. The IMS spectra generated in
high resolution from tumor lesions surrounded by healthy tissue requires sophisticated computational
analysis to correctly discriminate and classify the samples. For this purpose, a new method was
developed involving reconstructing the image from the raw mass-spectral data, preprocessing of IMS
data and subsequent classification and identification of IMS data based on artificial neural networks
(ANNs) [70]. ANNs are computational simulations of human neural networks for modeling highly
nonlinear systems in which the relationship between the variables is unknown or very complex [71].
Nanomedicine 479
Histological staining and IMS
Histological staining remains a cornerstone in the routine diagnosis of cellular and tissue
pathomorphology. Classical histological techniques require skilled clinical pathologists to diagnose
tissue abnormalities. Spectral profiles or images can complement structural information and facilitate
or specify a diagnosis [72]. Correct interpretation of MALDI MS/IMS information requires the
correlation of specific ion images to histological information. The latter can be accomplished either
using the same tissue section for both MALDI MS/IMS and histopathology or by analysis of two
consecutive sections. The first approach is complicated by interference of the dye used for tissue
staining with ionization during MS. Covalent binding of a dye—e.g., hematoxylin/eosin (H&E)—to the
sample surface, reduces the quality of a mass spectrum. However, chemically distinct dyes, such as
cresyl violet or methylene blue, are compatible [72]. Nevertheless, the staining pattern provided by
these dyes is different than that provided by H&E, and pathological information does not need to be
exhaustive. Recently, a staining protocol for tissue sections already analyzed by MS was introduced
that allowed a clear correlation to MALDI IMS results [73]. Analysis of consecutive sections requires a
precise alignment of neighboring structures. This is often complicated in complex samples, such as
neural tissue samples, due to the different microarchitecture and molecular profiles of adjacent tissue
sections [74].
The data outlined above apply only to fresh-frozen tissue samples. As the commonly used stains,
including H&E, are compatible with formalin-fixed paraffin-embedded tissues [36], the presence of a
cross-linked surface due to formalin bridges introduced by fixation of FFPE tissues prevents analysis by
MS. The peptide cross-links can be released using enzymatic treatment to make the surface available
for MS analysis [75]. The availability of FFPE samples archived throughout the clinical facilities
represents an enormously rich material for detailed MS/IMS studies with clear links to clinical practice
[76].
NP-Mediated IMS
Nanoscale materials have been widely introduced into bioanalytical MS and IMS research rather
recently to overcome obstacles in MS analysis of complex or unstable biological samples. Although it
has been demonstrated that classical organic chemical matrices in MALDI-TOF MS enable the detection
of peptides, proteins and nucleic acids and other biomolecules [77], there are still unresolved problems
in the adaptation of MALDI-TOF MS protocols to IMS:
1) The co-crystallization of analytes with matrices does not produce homogeneous mixtures,
which cause hot spots, thereby requiring sweet-spot searching [78].
2) MALDI-TOF MS can hardly detect small molecules because of the high background signals
coming from small organic matrices, which are present in the low-mass region (500 Da) [79].
3) The presence of salts in a sample solution increases the intensities of salt-adducted forms
[80].
4) Neutral molecules such as carbohydrates are poorly ionized by MALDI because of the absence
of either a basic or an acidic group in their structures [81].
To resolve these problems, the use of inorganic compounds with NP properties as matrices has been
introduced for the determination of analytes ranging from small organic molecules to biopolymers [82-
85].
Nanomedicine 480
MALDI requires photon-absorbing matrix compounds to enhance desorption [86]. UV-absorbing

materials can be used as energy mediators to transfer the photon energy from the laser source to the
surrounding analytes for effective desorption and ionization with minimum fragmentation of analyte
molecules. Cobalt NPs (30 nm in diameter) as matrices have been used by the pioneers in the MALDI
field for the analysis of lysozyme [87]. The cobalt NPs possess a large surface area, show high
photoabsorption, and a low heat capacity compared with those of microparticles. Inspired by Tanaka’s
results, a series of inorganic micro- and nanomaterials have been investigated as potential inorganic
matrices—e.g., graphite particles [88], AuNPs [89], silver NPs [90, 91], titanium dioxide NPs [82, 92],
silicon NPs and nanorods [93, 94], Au nanorods [95], and carbon nanotubes [96].
Nanomaterial matrices compared with organic matrices offer better sample homogeneity [82, 89, 90,
92-99] and elimination of matrix ion interference. Flocculated and trapped mixtures can be detected
using nanomaterials in various matrices [100-102]. AuNPs are suitable matrices for the determination
of biomolecules in high-salt solutions, such as biological buffers, by MALDI-TOF MS [102]. AuNP-
assisted laser desorption/ionization (LDI) was used for the determination of neutral carbohydrates [81],
where the ionization efficiency of neutral carbohydrates can be greatly amplified without derivatization
steps. Bare, capped, and functionalized AuNPs are good candidates as new generation matrices for
high-resolution imaging and profiling analysis [87]. As described elsewhere, AuNPs are particularly
interesting because they do not (or seldom) ionize by laser irradiation; however, a local temperature
increase occurs on the particle surface [103]. Heat produced by the laser beam is then rapidly
transferred to the analyzed sample. As a result, molecules of the analyte are desorbed/ionized with
minimal background signal coming from the matrix. Compared with a commonly used 2,5-
dihydrobenzoic acid matrix, the AuNPs showed the successful detection of small carbohydrates up to
m/z 500 [101].
AuNPs were demonstrated to mediate the ionization of several peptide systems and small proteins [89,
97, 100-102, 104-108]. Additionally, the complexity and size of AuNPs influence the ionization of
biomolecules in MALDI experiments. Russell and co-workers [106] published the detection of peptides
with molecular weights of 500–2500 using 2- to 10-nm AuNPs. Moreover, coating AuNPs
(functionalization) allows for selective extraction of desired substances from the complex solutions [89,
109]. For example, polyethylene-modified magnetic NPs (Fe3O4) to extract phosphoproteins from
complex protein digests for MS analysis were used (Chen et al. 2011). Interestingly, the deposition
method—e.g., the order of the layout of reagents and sample—influences either the interaction of the
AuNP matrix with the sample or exposition of the sample to the laser beam. Samples covered with
AuNPs provided better detection sensitivity and sample homogeneity compared with either the
deposition of a mixture of matrix and sample onto the target plate or deposition of AuNPs onto the
target before the sample [110].
NPs is now widely applied in various fields of tissue visualization—e.g., immunohistochemistry [13], MS
profiling and imaging [38, 42, 60], detection of individual biomolecules in living cells, delivery as vectors
[34, 35, 111] or systemic tracking of defined cell populations in whole organisms [11, 22]. Modification
and functionalization of their surface, selection of their physical properties and biological targeting
make NPs highly attractive for diagnosis, targeted therapy and biomedical research. A brief overview of
the use of NPs in tissue visualization is provided in Fig. 19.8.
Nanomedicine 481
FIGURE 19.8
Brief overview of NP use in tissue visualization.
Conclusions
Nanomaterials have wide applications in visualization strategies in cell and tissue biology. In particular,
specifically engineered NPs can complement classical techniques of analysis on fixed tissues and
enhance the performance of in vivo MRI. Biocompatible NPs provide a tool for in vivo cell tracking in
regenerative medicine or cancer research and provide deep insight into tissue ultrastructure and
chemical composition. IMS combines efficient analysis of chemical composition, spatial distribution and
structural information, reflecting the complexity of the biological systems. Classical histological staining
techniques are still indispensable particularly for daily clinical routine; however, the use of advanced
methods such as NP-mediated IMS complement greatly the palette of available diagnostic approaches
in the clinic and research.
Bringing the bioanalytical MS analysis to the cellular and subcellular levels allows the identification of
molecular composition related to precise spatial localization. The combination of structural
visualization and bioanalytical analysis complements greatly and contributes to the entanglement of
complex interactions and mechanistic phenomena in tissues. Therefore, nanomaterial-mediated
visualization is an important tool in standard clinicopathological techniques and tissue engineering.
Nanomedicine 482
Acknowledgements
We thank Mr. Filippo Amato and Ms. Mireia Puig-Romero for their contribution to Fig. 19.1. Work in
our laboratories is supported by the Ministry of Education, Youth and Sports of the Czech Republic
(Projects MSM0021622411, MSM0021627501, MSM0021622430, the Czech Science Foundation
(Projects No. 104/08/0229, 202/07/1669) and the Grant Agency of Masaryk University
(MUNI/M/0041/2013). We also thankful for the support of CEPLANT, the project R&D Center for Low-
Cost Plasma and Nanotechnology Surface Modifications (CZ.1.05/2.1.00/03.0086 funding from the
European Regional Development Fund).
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20
Recent trends in tissue engineering
applications of atom transfer radical
polymerization
a, § a, § a, § b,c
Masoud Mozafari , Vahid Shabafrooz , Mostafa Yazdimamaghani , Daryoosh Vashaee Lobat
ad
Tayebi
a
School of Materials Science and Engineering, Helmerich Advanced Technology Research Center, Oklahoma State University,
OK 74106, USA
b
Electrical & Computer Engineering Department, North Carolina State University, Raleigh, NC 27606, USA
c
School of Electrical and Computer Engineering, Helmerich Advanced Technology Research Center, Oklahoma State
University, Tulsa, OK 74106, USA
d
Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI 53201, USA
Outline:
Tissue Engineering approach ……………………………………………….…………………………………………………….. 491
Controlled radical polymerization …………………………………………..………………………………………………….. 492
Tissue engineered surfaces via atom transfer radical polymerization …………………………………………. 493
Functional polymeric brushes ……………………………………………………………………………………………………… 493
Antifouling surfaces ……………………………………………………………………………………………………………………. 497
PEG-modified surfaces ……………………………………………………………………………………………………………….. 498
PHEMA- and paam-modified surfaces ………………………………………………………………………………………… 500
Antibacterial surfaces ……………………………………….………………………………………………………………………… 500
Stimuli-responsive surfaces ………………………………………………………………………………………………………… 502
Hydrogel scaffolds via ATRP ……………………………………………………………………………………………………….. 504
Injectable thermo-responsive scaffolds via ATRP ……………………………………………………………………….. 510
Conclusion …………………………………………………………………………………………………………………………………. 515
References……………………………………………………………………………………..…………………………………………… 515
All authors have contributed equally to this work.

Nanomedicine 490
Abbreviations:
Description Abbreviation
Atom transfer radical polymerization ATRP

Reversible-addition fragmentation chain transfer RAFT
Surface-initiated atom transfer radical polymerization SI-ATRP
Activators generated electron transfer atom transfer radical AGET ATRP
polymerization
Deactivation enhanced atom transfer radical polymerization DE-ATRP
Poly (ethylene glycol) PEG
oligo (ethylene glycol) OEG
oligo (ethylene glycol), methacrylate OEGMA
Poly (poly (ethylene glycol) methacrylate) PPEGMA
Poly (ethylene glycol) methacrylate PEGMA
Poly (glycidyl methacrylate) PGMA
Poly (propylene glycol) methacrylate PPGMA
Poly (ethylene glycol) methyl ether methacrylate PEGMEMA
Ethylene glycol dimethacrylate EGDMA
Poly (glycidyl methacrylate)-co- (methylmethacrylate) PGMAMMA
Poly (methyl methacrylate) PMMA
Poly (N, N-diethyl aminoethyl methacrylate) PDMAEMA
Poly (2-(tert-butylamino)ethyl methacrylate) PTBAEMA
Poly (N-isopropylacrylamide) PNIPAAm
Poly (2-hydroxyethyl methacrylate) PHEMA
Polypropylene hollow fiber PPHF
Polycaprolactone PCL
Poly (ethylene glycol)methyl ether methacrylate-co-2-(2-methoxyethoxy) PEGMEMA475–
ethylmethacrylate-co-poly(ethylene glycol) diacrylate MEO2MA–PEGDA258
Poly (N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane) poly(NIPAAm-co-
BMDO)
Bovine serum albumin BSA
Magnetic nanoparticles MNPs
Magnetic resonance MR
Thermoset polyester TPE
AAm polymer PAAm
2- dimethylaminoethyl methacrylate DMAEMA
3-sulfopropylmethacrylate SPMA
glycidylmethacrylate GMA
4 vinylbenzyl chloride VBC
3-chloropropionic acid CPA
Extra cellular matrix ECM
Hyaluronic acid-glycidyl methacrylate HAGM
Hyaluronic acid HA
Free radical photopolymerization FRP
Hydroxyapatite HAP
HAP–poly (l-lactide) PLLA
Poly (methyl methacrylate) PMMA
Nanomedicine 491
Methyl methacrylate MMA

Lower critical solution temperature LCST
Lactate dehydrogenase LDH
Radical ring-opening polymerization RROP
Dulbecco’s modified Eagle medium DMEM
Controlled radical polymerization CRP
Free-radical polymerizations FRP
Polypropylene PP
Gel permeation chromatography GPC
2-hydroxyethyl methacrylate HEMA
Acrylamide AAm
2-methacryloyloxyethyl, phosphorylcholine MPC
sulfobetaine methacrylate SBMA
carboxybetaine methacrylate CBMA
Tissue Engineering approach

There are numerous biomaterials, including different kinds of metals, ceramics, glasses, polymers,
nanocomposites, and soft matters, introduced during the last couple of decades that has been
scientifically investigated or engineered for biological and biomedical applications [1,2,3,4,5].
In order to choose a material to act as the foundation for a tissue engineering scaffold, the material
should be nontoxic, mechanically similar to the native tissue, safe, capable of attachment with
molecules of normal tissue, and not cause excessive immune responses [6,7,8,9,10]. Moreover, a
suitable scaffold should be biocompatible and starting to degrade as cells develop and lay down the
extracellular matrix [11,12]. The materials of scaffolds and their coating and surface modifications have
a decisive role in the rate of degradation that affects the macroscopic shape and the appropriate
development of new tissues [13,14,15,16,17]. In addition, the molecular weight of scaffold degradation
products should be less than 50 kDa in order to be excreted from the body [18]. Materials with these
specifications can be classified into three main categories:
 Natural polymers, which often easily fulfill these expectations such as gelatin [1922],
hyaluronic acid (HA) [23], alginate [24], chitosan [25,27] and collagen [28,29],
 Synthetic polymers, mainly aliphatic polyesters[30,31], and
 Inorganic biomaterials [32,33], including hydroxyapatite [34,35].
One of the key challenges in tissue engineering techniques is creating biodegradable polymeric
materials with appropriate properties that can be modified to incorporate specific proteins, growth
factors or functional groups. The discovery of controlled radical polymerization (CRP) methods has
recently shown the high ability of these methods for creation of well-defined materials with
incorporated functional groups such as the ones required for tissue engineering constructs [36-41].
Nanomedicine 492
Controlled radical polymerization

Recently, there has been considerable interest in synthesizing functional polymeric surfaces with
precise control over molecular weight, architecture, composition, and end groups for biomedical
applications. Due to the slow initiation, fast propagation and subsequent transfer or termination in
conventional free-radical polymerizations (FRP), these methods are not compatible to provide
polymers with narrow molecular weight distributions and defined chain ends. Modern CRP methods
display synthesis of polymers with low polydispersities and tailored molecular weights, allowing
tolerance to functional groups and monomer types with direct polymerization [42,43]. Mechanistically,
in CRP methods an adequately large number of activation–deactivation cycles make the total number
of dead chains sufficiently smaller than that of living chains (dormant plus active chains) to attain low
polydispersity [44]. This includes reversible-addition fragmentation chain transfer (RAFT)
polymerization [4548], nitroxide mediated living free radical polymerization (NMP) [4952], and atom
transfer radical polymerization (ATRP) [5364], which have all been used to prepare polymers with
reactive side chains and applied in the surface modifications [66,67]. For example, ATRP has been
employed for polymerization of a wide range of monomers such as styrenes, acrylates, methacrylates
and zwitterionic [68,69], including a variety of functional monomers and varying the topology of the
polymer (linear, branched, hyperbranched, stars, etc.) and block copolymers since the end groups
remain active at the end of the polymerization [43,66,67,70,71]. ATRP was first reported in 1995, and
showed a precise control over polymerization by using readily accessible and inexpensive catalyst
components and easy experimental setups. After that it has attracted great commercial interest in
many different aspects. Figure 20.1 shows a typical schematic of ATRP equilibrium. We can see the
reciprocating nature of the activation and deactivation steps in order to form a high mole fraction of
dormant species that still preserve the ability to grow. In fact, the control over radical polymerization
can be obtained by keeping the concentration of active species or propagating radicals adequately low
in the polymerization. This method can be conducted in bulk, solution or a variety of homogeneous and
heterogeneous media in different ranges of emulsion, suspension, and dispersion. Furthermore,
polymers can be grown from surfaces, proteins, nanoparticles etc. Overall, this method has had
remarkable growth in the past 15 years, and will continue growing and entering into many other areas
of science and applications [72,73,74].
FIGURE 20.1
Normal ATRP equilibrium [55].
Nanomedicine 493
Tissue engineered surfaces via atom transfer radical polymerization

Functional polymeric brushes
The interactions between a biomaterial and its biological environment are overseen by its surface
properties as they dominate the interactions between the material and the biological environments. In
the field of tissue engineering, polymeric materials provide surfaces for the immobilization of
biologically active molecules and living cells. Therefore, the ability to control the surface properties of
biomaterials is of fundamental importance in the design of biomedical materials [75,76]. Polymer
brushes refer to an assembly of polymer chains which are chained by one end to a surface or an
interface. In comparison with other surface modification methods (e.g. self-assembled monolayers);
polymer brushes can increase the spatial density of functional groups on a surface, as they extend the
two-dimensional distribution of the functional compounds to a three-dimensional one. Polymer
brushes are robust either in mechanical or chemical properties and exhibit a high degree of synthetic
flexibility to introduce a variety of dense functional groups. Polymer brushes are considered as a
central model for many polymer systems including polymer micelles, grafted polymers, adsorbed
diblock copolymers and also block copolymers at fluid–fluid interfaces. All of these systems with their
deformed configurations as a common feature are illustrated in Figure 20.2.
FIGURE 20.2
Examples of functional polymeric brushes [204].
Nanomedicine 494
The preparation methods of polymer brushes are physisorption or covalent attachment. In the first
method, the sticky parts of polymer chains are adsorbed onto a suitable substrate. Non-covalent
adsorption of polymers to surfaces is capable of being revoked and such polymer brushes are often
unstable. Covalent chaining of polymer brushes on solid substrates is an effective method for
modifying surface properties, such as antifouling ability, biocompatibility, and bimolecular recognition.
It can be accomplished by either the grafting to or grafting from approaches. For the grafting to
approach, polymer chains are attached directly on a suitable surface via reaction between end-
functionalized polymers and appropriate reactive groups on the substrate surface. The method is
experimentally simple, but has its limitations. For instance, it is difficult to achieve high grafting
densities because of the steric gathering of the already adsorbed polymer chains on the surface
reactive sites. Furthermore, the thickness of the graft layer can be determined by the molecular weight
of the polymer in solution. The grafting from approach via polymerization from the initiators bound on
substrate surfaces can be discussed as a convenient alternative to control various parameters such as
functionality, density and thickness of the brushes. The substrate surface is first modified with an
initiator monolayer followed by the growth of polymer chains directly from the reactive sites of the
immobilized initiator layer. The screening of grafting sites is effectively reduced, because the grafted
chains on the surface prevent the addition of monomers to growing chain ends or to primary radicals.
This method has been attractive in recent years because of its effects in producing controllable
functional polymer brushes of large thickness and high density. With the purpose of achieving
maximum control over brush length, density, and composition on the surface, several grafting from
methods have been developed, including surface-initiated cationic or anionic polymerization, ring-
opening polymerization, and CRP techniques [204,77,78]. Among all above techniques, cationic, anionic
and ring-opening polymerization techniques require accurate experimental conditions and
sophisticated catalysts which are often moisture-sensitive. These requirements make their common
application quite difficult in surface functionalization. Recently, the development of CRP techniques,
including ATRP, has opened up new routes to the preparation of precise polymer brushes of controlled
structures. Going through the surface-initiated CRP techniques, surface-initiated ATRP has been
established to be the most versatile technique for surface functionalization [75,79]. It is easy to prepare
the ATRP initiator layers on substrates using commercially available α-haloesters or benzyl halides,
circumventing the multistep synthesis necessary for the introduction of functional alkoxyamine
initiators of polymerization. Significantly, surface-initiated ATRP can also be carried out in the absence
of sacrificial initiators to produce thick and dense polymer brushes. ATRP has been achieved from
various surfaces, including surfaces of inorganic particles, planar surfaces, polymer networks, and even
from dendrimers [75, 79]. In order to prepare the functional polymer brushes via surface-initiated
ATRP, the presence of a uniform monolayer of initiators on the target substrate surfaces plays a vital
role (Figure 20.3). Versatile immobilization methods of ATRP initiators have been developed for an
extensive range of biomedical substrates, including inorganic surfaces of silicon, silica, titanium, gold
and Fe3O4, and surfaces of films such as polypropylene (PP), aromatic ring-containing polymers,
cellulose, and nylon [80-86].
As a new approach for implantable titanium substrates, bromomethyl-terminated biomimetic catechol
and chloromethyl-terminated silanes can be immobilized on the oxidized titanium surfaces to serve as
ATRP initiators. Bromomethyl-terminated thiol agents as ATRP initiators can be directly immobilized on
gold surfaces [87,88]. The attachment of ATRP initiators on magnetic Fe3O4 nanoparticles can be
accomplished via treatment with silanes (containing chloropropyl, chloromethyl, or chlorosulfonyl
groups) and organic acids containing bromomethyl groups [Figure 20.3 (b)] [89,90]. For the common
biomedical polymer films, the ATRP initiators can be introduced onto PP films via UV- or ozone-induced
coupling of 2-bromoisobutyrate. Aromatic ring-containing polymer films can be functionalized via
Nanomedicine 495
chloromethylation, cellulose via direct coupling of 2-bromoisobutyrate, and nylon via

formaldehydeinduced coupling of 2-bromoisobutyrate [91,92]. After the immobilization of ATRP
initiators on the target substrates, surface-initiated ATRP can be carried out in the presence of a copper
halide/nitrogen-based ligand catalyst system. The main difference between ATRP from a surface and
ATRP in bulk or solution is related to the extremely low concentration of initiators immobilized on the
surface. After the halogen atom transfer to the transition metal catalyst, the concentration of
persistent radical (deactivator) may be too low to reversibly trap the propagating radicals, leading to
uncontrolled chain growth [93]. Therefore, a sufficiently high concentration of the deactivating Cu (II)
complex (CuCl2 or CuBr2) is required at the beginning of ATRP to rapidly establish equilibrium between
the active and inactive (dormant) chains. The Cu (II) complex can be obtained by either the reaction of
Cu (I) complex with the initiator or addition of the complex at the initial stage of ATRP.
Accordingly, the addition of free initiators or extra deactivating Cu (II) complex is usually chosen to
guarantee the presence of an enough amount of deactivator to control the equilibrium between the
inactive and the active chains during surface-initiated ATRP. In the first approach where free initiators
are added, the molecular weight of free polymers formed by the free initiator in solution is used to
serve as a measure of the molecular weight and polydispersity of the grafted polymers on the surface,
due to the usual difficulty in obtaining the molecular weight of the grafted polymer on the solid
surface. Consequently, the free initiator serves not only as a mediator for ATRP on the surface, but also
as an indicator of surface graft polymerization. However, this approach enforces an intrinsic limitation
to the maximum thickness of the obtained polymer brushes, because most of the monomers are
utilized by homopolymerization in solution. The second method in which additional deactivating Cu(II)
complex is added allows the growth of thicker polymer brushes, as the brush growth can proceed in a
faster rate [94]. For the second method, the molecular weight and polydispersity of the surface-grafted
polymer can be determined only with the division of grafted chains without any degradation. The
amount of grafted polymer on the planar surface is minute. Only about 0.01mg of the polymer can be
2
obtained from a 100-nm thick film grown on a 1cm flat surface. Thus, the quantity of polymers cleaved
from the surface does not allow an accurate analysis of molecular weight and polydispersity by gel
permeation chromatography (GPC). Compared to ATRP in bulk or in solution, in surface-initiated ATRP
the problem of purifying the final products by removal of the metal catalyst has been minimized.
Interestingly, the catalyst complex can be readily removed from the well-defined polymer brushes by
suitable solvent extraction. Surface-initiated ATRP has been utilized widely for biomedical applications.
Table I summarizes the types of bioactive surfaces prepared via surface-initiated ATRP. Details on the
preparation and characteristics of these bioactive surfaces are given below [93,95].
TABLE I
Bioactive surfaces prepared via surface-initiated ATRP
Nature of Surface Functional Monomers Application

Antifouling Surfaces PEG-containing methacrylate, Biomedical devices, Tissue
HEMA, AAm, MPC, SBMA,CBMA Engineering, and Filtration
Membrane
Antibacterial Surfaces DMAEMA, 4-VP, SPMA, PTBAEMA Medical devices, Tissue
Engineering, Filtration, and
Fibers
Stimuli-responsive bioactive NIPAAm, DMAEMA, NaMA Cell culture, drug delivery,
Surfaces and Tissue Engineering
Nanomedicine 496
FIGURE 20.3
Methods of immobilizing ATRP initiators on various substrate surfaces for the preparation of functional polymer
brushes by surface-initiated ATRP (Si–H: hydrogen-terminated Si wafer; UME: 10-undecylenic methyl ester [80];
BIBB: 2-bromoisobutyrate bromide [90]; VAn: 4-vinylaniline [89]; VBC: 4 vinylbenzyl chloride [205]; NBS: N-
bromosuccinimide [81]; APTS: 3-aminopropyltriethoxysilane [76]; CPA: 3-chloropropionic acid [76]; CTS: 4-
(chloromethyl)phenyl trichlorosilane [101]; CTCS: 2-(4-chlorosulfonylphenyl)ethyl trichlorosilane [84,206]; BMPA:
2-bromo-2-methylpropionic acid [83]).
Antifouling surfaces
Non-specific adsorption refers to the tendency of proteins or cells of being physically adsorbed on a
substrate without specific receptors. This occurs at the surface after the exposure of a foreign device to
a biological environment. The protein adsorption processes are complicated. The adsorption of plasma
proteins plays an important role in incurring subsequent undesirable events, including platelet
adhesion, thrombus formation, foreign body reaction, bacterial infection, and adhesion of
macrophages through which the tissue destruction has been hidden [9698]. In addition, non-specific
adsorption can typically reduce the efficiency of biosensors, single molecule detection and single cell
Nanomedicine 497
analysis, giving rise to undesirable features, such as high background noise and false positives. The
factors governing protein surface interactions include the physical state of the material, protein
properties, and solution environment. It is without question that the surface first comes into contact
with the biological environment. However, the substrate surface must be modified to provide it with
resistance to protein adsorption and cell adhesion. Accordingly, the functionalization of the substrate
surface with antifouling coating to improve the performances of biomedical devices and biosensors is
of great importance. ATRP has been widely utilized to impart various substrate surfaces with
antifouling properties. The monomers used in ATRP synthesis of antifouling surfaces are: oligo(ethylene
glycol), methacrylate (OEGMA), 2-hydroxyethyl methacrylate (HEMA), acrylamide (AAm), zwitterionic
monomers of 2-methacryloyloxyethyl, phosphorylcholine (MPC), sulfobetaine methacrylate (SBMA),
and carboxybetaine methacrylate (CBMA) [99-113].
PEG-modified surfaces
Poly (ethylene glycol) (PEG) and oligo (ethylene glycol) (OEG) have been the most commonly used
antifouling materials. PEG has many remarkable features such as physical and biochemical properties,
including non-toxicity, non-immunogenesis, non-antigenicity, excellent biocompatibility, and miscibility
with many solvents [114,115]. PEG and its components demonstrate good antifouling effects on a wide
diversity of proteins, suppress platelet adhesion, and reduce cell attachment and growth [116,121]. It is
clear that the protein molecules or cells have been inhibited from approaching the substrate surface by
excluded volume of PEG units and the mobility or flexibility of highly hydrated chains in water. In fact,
water molecules in a range of two or three per EG unit and up to a maximum of 10 required for
hydration within the PEG layers play a vital role for protein resistance. Conventional methods to
immobilize the PEG coatings on substrates include direct attachment of self-assembled PEG monolayer
to surfaces, graft polymerization of PEG monomers to a polymer backbone, and adsorption of PEG
block copolymers at multiple sites on the surface [118]. The efficiency of each strategy for constructing
a protein and cell-resistant surface not only depends on the unique antifouling properties of PEG units,
but the molecular structure resulting from the surface coverage has also been important [119].
Recently, dense non-fouling polymer brushes have been synthesized via ATRP of various OEGMA macro
monomers from planar substrates of gold, silica, Ti, stainless steel and hydrogel [122, 120,121,123].
The thickness of the precise OEGMA polymer, poly(poly(ethylene glycol) methacrylate) (PPEGMA)
brushes was tunable, and the surfaces exhibited first-rate antifouling effects to many proteins,
including fibrinogen, bovine serum albumin (BSA), globulin, lysozyme, peptide, and lactamase [124]. In
general, they are resistant to platelet and cell adhesion [122,123]. The non-fouling properties of the
PPEGMA brushes are stable under long-term cell culture conditions. PPEGMA brushes grafted from
gold substrates have been shown to prevent nonspecific cell adhesion for up to 30 days while PEG
brushes of approximately 100 nm in thickness on Ti substrates coated with catechol-anchor exhibited
excellent resistance to cell fouling for up to 3 weeks independent of the EG side chain length, after
which the long-term antifouling performance depended on the EG chain length [125,126].
The stability of trialkoxysilane-anchored PPEGMA brushes from silica substrates was shown to be
dependent on chain density [127]. Increasing the chain densities makes the brushes separate rapidly.
On the other hand, the stability of the PPEGMA brushes in cell culture medium could be improved by
decreasing the grafting density from less than 1 day to more than 7 days, without cooperating the
antifouling properties. Precise antifouling PPEGMA brushes from magnetic nanoparticles (MNPs) were
also grafted via surface-initiated ATRP [128,130]. Although, the MNPs that were taken up by
macrophage cells were less than those in the pristine MNPs, after characterization of the macrophage
cells cultured with MNPs, the similar morphology and viability to those without the nanoparticles was
Nanomedicine 498
achieved (Figure 20.4) [128, 129]. The PEGylated MNPs demonstrated long-term colloidal stability in
the physiological buffer and the nanoparticles tolerated longer circulation in the bloodstream in
comparison with conventional magnetic resonance (MR) image contrasting agents [131].
FIGURE 20.4
(a) RAW 264.7 cells in control culture (without any nanoparticles) after 1 day, (b) cells after culturing in medium
containing pristine MNPs (0.2 mg/mL) for 1 day and (c) for 4 days, and (d) cells after culturing in medium
containing P(PEGMA)-immobilized nanoparticles (0.2 mg/mL) for 1 day. The P(PEGMA)-immobilized nanoparticles
were obtained after polymerization time of 2 h. Scale bar ) 40 µm [129].
With the purpose of improving the performance of membranes in biomedical applications, ATRP of
OEGMA is widely carried out to alter the membrane surfaces with antifouling properties. The
membranes studied include cellulose, nylon, poly (vinylidene fluoride), and poly (phthalazinone ether
sulfone ketone) membranes [132-135]. Increasing the length of the poly (ethylene glycol) methacrylate
(PEGMA) brushes leads to decrease of the pore size, considering the ability to change the membrane
pore size with ATRP processing time. The membranes with grafted PPEGMA brushes demonstrate good
resistance to protein adsorption and fouling under continuous-flow conditions, thus prolonging the
useful lifetime of the filtration membranes. Surface-initiated ATRP of OEGMA is also carried out to
introduce a PPEGMA graft layer on the surfaces of poly (methyl methacrylate) (PMMA), thermoset
polyester (TPE), and poly (glycidyl methacrylate)-co- (methylmethacrylate) (PGMAMMA) microfluidic
devices. The PMMA microcapillary electrophoresis (iCE) devices with grafted PPEGMA brushes exhibit
significantly reduced electroosmotic flow and non-specific adsorption of proteins on microchannel
surfaces [136,139]. The reproducibility of column efficiency and migration time of the PPEGMA-
modified PMMA microchips was improved by one order of magnitude over the untreated PMMA iCE
chips. The PPEGMA-grafted TPE microchannel showed low and pH-stable electroosmotic flow and low
non-specific protein adsorption. Capillary electrophoresis separation of amino acid and peptide
mixtures in these PPEGMA-modified TPE microchips also exhibited good reproducibility. For the PEG-
modified PGMAMMA microdevices, fast and efficient separations of amino acids, peptides and proteins
were obtained due to denser and more uniform antifouling PEG brushes on the PGMAMMA surface
[136,137,138].
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PHEMA- and PAAm-modified surfaces
Dense hydrophilic poly (2-hydroxyethyl methacrylate) (PHEMA) brushes indicating remarkable

biocompatibility and physical properties also exhibit excellent protein repellency [140]. It has been
presented that PHEMA brushes are grafted via ATRP from silicon wafer, silica particles, and nylon
membranes [141-143]. The size-exclusion effect of the dense PHEMA brushes plays an important role
in suppressing protein adsorption [144]. In fact, the chains can become elongated and oriented to
physically exclude the protein molecules from the entire brush layer. In addition, the PHEMA brushes
can also effectively prevent cell adhesion [143]. Cell adhesion can be tuned by controlling the grafting
density of PHEMA brushes. Decreasing the graft densities of PHEMA brushes leads to the adhesion
followed by proliferation of cells. AAm polymer (PAAm, Figure 20.4(c)) is a biocompatible, water
soluble, polar, electrically neutral and stable polymer. Highly hydrophilic PAAm brushes can also avoid
the adsorption of proteins and prevent cell growth [145]. Well-defined PAAm brushes have been
prepared via surface-initiated ATRP from silica microfluidic chips [146], poly (dimethylsiloxane) [147],
and silicon wafer [148]. Antifouling PAAm brushes are attached on electrophoretic microfluidic chips
via surface initiated ATRP for improving protein separation [146]. In fact, the higher the density the
much less forces between the surface and microorganisms [148].
Antibacterial surfaces
Infections caused by microorganisms still remain as a major concern, especially in the healthcare sector
where bacterial infections arising from implants and medical devices result in increased suffering,
lengthy hospital visits, regular operations, and sometimes even death. In spite of the high success rate
in dental and orthopedic implant surgery, many studies have reported bacterial infections associated
with implants. To reduce the risk of infection, much research has been applied in the production of
antibacterial surfaces [149,150]. Antimicrobial surfaces are broadly used to avoid microbial infection in
a wide range of industrial, medical and private settings. Different strategies have been developed to
realize the necessity for antibacterial surfaces. One of the most useful approaches for active
antimicrobial agents is being permanently attached on the surface through covalent interactions. The
antibacterial action results from the contact of the microorganisms with the biocidal surface without
releasing the biocide into the environment. This reduces the possibility of generating drug resistance to
the active agent through the microbial segment. In general, antimicrobial surfaces have been prepared
via covalent immobilization of antimicrobial polymers onto different substrates [151,153]. The
antimicrobial polymers generally contain cationic groups, such as alkyl pyridinium or quaternary
ammonium moieties. The interaction of the cationic sites of quaternized groups with the negatively
charged membrane of bacteria has an adverse effect on the integrity of the bacterial cell, as the
quaternary ammonium groups can disrupt the plasma membrane to cause the release of intracellular
substances. Hence, cationic antimicrobials play a vital role in the development of permanent and non-
leaching antibacterial surfaces. The conventional antibacterial surfaces have been synthesized by either
classical free-radical polymerization or by simple coupling reactions [86]. These methods possessed less
control over the polydispersity, molecular weight, and density of functional groups.
In order to fabricate a successful antibacterial surface on a tissue scaffold, the facilitation of tissue
repair and regeneration by enabling the localized production of therapeutic drugs is of crucial
importance. Although polycaprolactone (PCL) has been extensively employed as a scaffold biomaterial,
its undesirable cell-adhesion property still needs to be improved. ATRP has been used to impart
antibacterial surfaces to filter paper, titanium, gold, glass, silicon, polyolefin, fibers, polymer
microspheres and poly (vinylidene fluoride) [154,161].
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Recent research has shown that some vinyl monomers containing tertiary amino groups, such as 2-
dimethylaminoethyl methacrylate (DMAEMA) and 4-vinyl pyridine, can be polymerized or
copolymerized via ATRP, followed by quaternization, to induce the antimicrobial activity. Control over
both the polymer length and the effective number of quaternary ammonium groups could result in a
highly effective biocidal polymer [155, 156,162]. According to a recent study [162] the PCL film surface
was conjugated with poly ((2-dimethyl amino) ethyl methacrylate) (P (DMAEMA))/gelatin complexes
via surface-initiated atom transfer radical polymerization (SI-ATRP) for improving cell immobilization
and subsequent gene transfection. Matyjaszewski et al. recently proposed the original idea of
preparing permanent, non-leaching antibacterial surfaces via ATRP [155]. Polymerization of tertiary
amine-including DMAEMA via ATRP is illustrated in Figure 20.5. The process is performed directly from
the filter paper to produce polymer chains of controlled molecular weight and low polydispersity. The
biocidal functionality on the surfaces has been produced by subsequent quaternization of the amino
groups of poly (N,N-diethyl aminoethyl methacrylate) (PDMAEMA) brushes. The modified surfaces
exhibited considerable antimicrobial capacity over E-coli and Bacillus subtilis. It has been demonstrated
that the available surface of PDMAEMA brushes synthesized by ATRP was the critical element in
designing a surface for maximum efficiency [163,156]. Most biocidal surfaces had charge densities
15
greater than 10 accessible quaternary groups/ [156]. The antimicrobial surfaces of
PDMAEMA/poly (3-(trimethoxysilyl) propyl methacrylate) can also be prepared by ATRP by the grafting
onto technique. [157]. The higher concentrations of quaternary groups achieve a higher biocidal
functionality. Considering the same density of quaternary groups, the biocidal activity of surfaces
prepared by the grafting onto technique has been higher than those of surfaces prepared by the
grafting from technique [108]. Hence, biocidal activity was not strongly affected by polymer
architecture. This phenomenon could be described by the non-uniform coverage of the polymer on the
former surfaces, and the biocidal activity was affected by localized patches of high concentration of
quaternary ammonium groups.
FIGURE 20.5
Surface-initiated ATRP of DMAEMA, and subsequent quaternization of PDMAEMA, from cellulose surfaces [155].
Viologen has been used to quaternize the tertiary amino groups of the monodispersed PDMAEMA
brushes from ATRP. Antimicrobial capability and inhibition of biofilm formation were enhanced by the
substantially increased polycation concentration on the surface. For example, compared to quaternized
PDMAEMA brushes exhibited by an alkyl halide, the viologen-quaternized PDMAEMA brushes exhibited
enhanced antimicrobial functionality. Recently, novel surfaces with grafted block copolymer brushes of
Nanomedicine 501
PEGMA and DMAMEA (PPEGMA-b-PDMAEMA) were prepared via consecutive surface-initiated ATRPs
from PP hollow fiber (PPHF) membranes [164].
The ATRP time can affect the length of the PPEGMA-b-PDMAEMA brushes resulting in the regulation of
the pore size of PPHF. Quaternization of the PDMAEMA block by alkyl bromides demonstrated both
antibacterial and antifouling effects due to the hydrophilic antifouling nature of the PPEGMA blocks.
Moreover, 3-sulfopropylmethacrylate (SPMA) incorporation with silver ions inside the polyelectrolyte
film was synthesized via ATRP followed by fabrication of polyelectrolyte brushes of poly (3-
sulfopropylmethacrylate) [165]. The silver-incorporated brushes successfully hinder the growth of
bacteria. Furthermore, during leaching, the brushes were able to keep the silver ions on the surface.
Consequently, the silver-incorporated brushes exhibited highly desirable properties of an antibacterial
surface. In addition to PDMAEMA, antibacterial surfaces of the neutral polymeric biocide, poly (2-(tert-
butylamino)ethyl methacrylate) (PTBAEMA), were also prepared via ATRP [166,167]. Thus, ATRP is a
versatile tool for creating antibacterial surfaces, with the antibacterial properties derived from cationic
or neutral polymeric biocides.
Stimuli-responsive surfaces
The design and synthesis of materials so that their physico-chemical properties respond to any external
stimuli highlights the potential of these materials in biomedical areas. Grafting the stimuli-responsive
materials containing polymers may yield surfaces that control biological interactions such as
bioadhesions. The applications include surface adhesion modifiers, cell culture, and tissue engineering
[168]. Different stimuli-responsive polymer brushes such as pH-responsive [169,170], and temperature-
responsive [171,173] have been prepared via ATRP method. In the second group, temperature has
been recognized as one of the most widely used physical stimuli in environmentally responsive polymer
systems. Not only can the changes of temperature be easily controlled, but it is also readily applicable
both in vivo and in vitro. Poly (N-isopropylacrylamide) ( PNIPAAm) is the most broadly employed
thermo-responsive polymer, which exhibits a lower critical solution temperature(LCST) of about 32 ̊ C
in aqueous media. The state of PNIPAAm can be changed; it undertakes hydrophilic state below the
LCST while a hydrophobic state is happening above the LCST due to rapid, reversible chain dehydration
and aggregation [174,175]. Because of this remarkable property, precise PNIPAAm brushes have been
extensively used in the preparation of stimuli-responsive surfaces in order to control the cell adhesion
[171, 172,173].
In tissue engineering without the participation of biochemical or releasing of chemical reagents, it is
desirable to do cell culture and then detach the cells at an appropriate stage to be harvested.
PNIPAAm-based thermo-responsive polymers have been extensively used as surface mediators for cell
attachment [176,177]. The adhesion and detachment of cultured cells on these surfaces can be
controlled using only temperature variation. On the PNIPAAm surfaces, cells can adhere followed by
growth and proliferation at 37 ̊ C. However, at temperatures below the LCST of PNIPAAm, the cultured
cells can be detached at the same time from the hydrophilic surfaces. For the purpose of controlling
cell response, many PNIPAAm surfaces have been prepared via conventional graft polymerizations,
made by electron beam [177] or plasmas [178]. These methods offered less control over chain density,
length, and flexibility of the grafted PNIPAAm on the substrate surfaces. To get control of the cell
adhesion/detachment, ATRP has been used to prepare well-defined PNIPAAm brushes [176,179,180].
Xu et al. [179] prepared well-defined functional PNIPAAm brushes via surface-initiated ATRP from
silicon surfaces resulting in steady development of chains of PNIPAAm in controlled process. By
adjusting the cell culture temperature, the cell adhesion/detachment on the PNIPAAm-grafted silicon
surfaces can be changed.
Nanomedicine 502
Rapid detachment of cultured cells from substrates plays a vital role in fabrication of functional tissue-
mimicking structures. Hence, the hydration of the underlying PNIPAAm grafted on the surface can
affect the rate-limiting step to cell substrate recovery. It is also reported that the incorporation of
PEGMA units into the NIPAAm chains could enhance the hydration of the cell-cultured surfaces
resulting in quick cell detachment during the temperature transition. However, the introduction of PEG
derivatives has also resulted in a dramatic decrease in growth or adhesion of cells at temperatures
above the LCST [181]. Accordingly, to accelerate cell detachment at lower temperatures, without
affecting cell adhesion and growth at temperatures above the LCST, attempts have been made to
prepare novel thermo-responsive copolymer brushes from silicon surfaces via ATRP[182]. Figure 20.6
illustrates the mechanism of preparation of precise poly (glycidyl methacrylate) (PGMA) brushes on
silicon surfaces via surface-initiated ATRP of glycidylmethacrylate (GMA) from the 4 vinylbenzyl
chloride (VBC)-coupled silicon surface. In fact, the epoxy groups of the PGMA brushes were employed
for the direct coupling of 3-chloropropionic acid (CPA) followed by ATRP of NIPAAm.
The PGMA main chains with hydroxyl groups provided a local hydrophilic microenvironment for
accelerated hydration of PNIPAAm side chains at temperatures below the LCST, while the grafted
PNIPAAm chains acted as the thermo-responsive side chains of the comb copolymer brushes. The comb
copolymer brushes assisted cell recovery below LCST without restraining cell attachments and growth
at 37 ̊ C due to its unique microstructure. The well-defined PNIPAAm brushes were examined to
investigate the complicated relations between the biophysical response of cells and the physiochemical
properties of PNIPAAm brushes [180,176]. The longer polymerization time can cause a higher initial
rate of cell detachment below the LCST [180]. Increasing the density of PNIPAAm brushes during initial
cell recovery caused a reduction in the degree of cell deformation and average adhesion energy. In
addition, to control cell adhesion and separation of biomolecules, PNIPAAm-functionalized surfaces
were also used to get control of drug release [183]. In fact, PNIPAAm brushes were prepared inside the
pores for the control of drug release in response to temperature. It was speculated that the PNIPAAm
brushes inside the pores could form internal holes for loading the drug molecules, while responding to
external temperature stimuli.
FIGURE 20.6
Process of preparing comb-shaped copolymer brushes via successive surface-initiated ATRPs of GMA and NIPAAm
for accelerated cell detachment below the LCST of PNIPAAm [182].
Nanomedicine 503
Hydrogel scaffolds via ATRP

Hydrogels with cross-linked polymer networks are appropriate materials for tissue engineering
scaffolds because of their similar mechanical and mass transfer properties with native tissues. Due to
their cross-linked polymer network, this class of materials allows small molecules such as proteins to
diffuse in and out of the matrix of hydrogel. On the other hand, larger molecules as plasmid DNA are
often entrapped within their network and can only be set free subsequent to the degradation of
hydrogel. Ratner et al. [184] recently reported the synthesis of biodegradable PHEMA hydrogels for
tissue engineering using ATRP technique, a degradable cross-linker and a macro-initiator. Since PCL is a
hydrolytically and enzymatically degradable polymer, they used oligomeric blocks of PCL as a cross-
linking agent and a degradable macro-initiator that also contained oligomeric PCL to initiate the ATRP,
in order to prepare biodegradable scaffold. As a result, they have observed that the degradation rate is
a variable of the cross-linking density, the PCL chain length, and the PHEMA backbone chain length. The
combination of a macroscopic hydrogel and nanogel to form nanostructured hybrid hydrogels could
lead to enormous progress in the field of tissue engineering to synthesize scaffolds that are able to
deliver single growth factor or a combination of different growth factors simultaneously. These
biodegradable nanostructured hydrogels containing growth factors, drug and other useful low
molecular weight biomolecules could be used as an artificial extra cellular matrix (ECM) for tissue
regeneration.
In another study, Matyjaszewski et al. [185] prepared nanostructured hybrid hydrogels by synthesizing
POEO300MA nanogels using activators generated electron transfer atom transfer radical polymerization
(AGET-ATRP) in cyclohexane inverse miniemulsion at ambient temperature and subsequent FRP of
hyaluronic acid-glycidyl methacrylate (HAGM). In the synthesis procedure of POEO300MA nanogels with
ATRP approach, they have used hydroxyl-containing water-soluble ATRP as an initiator (oligo(ethylene
oxide)-functionalized bromoisobutyrate, HO–EO–Br) in order to produce functional nanogels with the
capacity for further chemical modifications. As shown in Figure 20.7, they functionalized hydroxyl-
containing nanogels with methacrylated groups to generate photo reactive nanospheres. They also
introduced disulfide moieties into the polymerizable groups to gain a controllable release of nanogels
from cross-linked HAGM hydrogels under a reducing environment.
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FIGURE 20.7
Synthesis of well-defined fluorescent dye-loaded GRGDS-POEO300MA nanogels by AGET-ATRP in inverse
miniemulsion of water/cyclohexane at ambient temperature. The nanogels were subsequently substituted with
MAH or coupled with DTPA/HEMA to incorporate cleavable photopolymerizable groups. Nanostructured hybrid
hydrogels were prepared by covalent incorporation of methacrylated POEO 300MA nanogels into macroscopic
HAGM or PEODM hydrogels via FRP under ultra-violet (UV) irradiation [185].
Nanomedicine 505
In order to monitor the cytotoxicity and cell adhesion, they covalently incorporated GRGDS (Gly–Arg–
Gly–Asp–Ser) contained MA-nanogelsinto PEODM scaffolds. The results showed that GRGDS in the
nanogel structure promoted the cell–substrate interactions within 4 days of incubation and cells could
recognize the integrin-binding motif whether GRGDS grafted to nanogels or to a macroscopic scaffold.
They concluded that the cells did not show any reaction to the variations of molecular architecture
(Figure 20.8).
FIGURE 20.8
Mouse myoblast cells (C2C12) seeded on a series of 10% (wt/v) hydrogels; PEODM (a), GRGDS-modified PEODM
(b), PEODM-co-GRGDS-MA-nanogels (nanogel content: 10 mg/mL) (c), and PEODM-co-GRGDS-MA-nanogels
(nanogel content: 50 mg/mL) (d). The cells were cultured for 4 days and stained with the live stain on top (green)
and dead cell stain on the bottom (red) [185].
Following the aforementioned study, Matyjaszewski et al. [186] synthesized a nanostructured HA

hydrogel by a combination of ATRP and Michael-type addition reaction. The intent of this was to take
advantage of having a second delivery carrier in addition to the macroscopic scaffold matrix for the
controlled delivery of growth factors, drug and other useful low molecular weight biomolecules. With
this perspective, they have applied AGET-ATRP in cyclohexane inverse mini emulsion in the presence of
a hydrolytically labile cross-linker to create biodegradable POEO300MA-co-PHEMA nanogels with
pendent hydroxy groups. These hydroxy groups were subsequently altered with acrylated segments to
produce reactive nanogels that could make covalent bonds with nucleophilic thiols via a Michael-type
addition reaction. Carbodiimide-mediated coupling reaction of HA with cysteamine hydrochloride
occurred leading to thiol-derivatization of HA (HA-SH) to prepare nanostructured hybrid hydrogel by
mixing HA-SH with acrylated-nanogels under physiological conditions. As shown in Figure 20.9, the HA-
based nanostructured hybrid hydrogel was obtained by the nucleophilic thiolene addition between the
thiol functionalized HA chains and the vinyl moieties contained in grafted acrylic nanogels.
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FIGURE 20.9
Gel formation via Michael-type addition reaction under physiological conditions. Formation of the nanostructured
hybrid hydrogel was visually observed with digital images before and after gelation [186].
In addition, as shown in Figure 20.10, the SEM photomicrographs of the prepared nanostructured HA
hydrogel demonstrated uniform distribution of nanogels on the surface and in the interior structure of
the nanostructured hydrogel and porous three-dimensional structure of scaffolds.
FIGURE 20.10
SEM photomicrographs of nanostructured HA hydrogel. Morphology and structure of SEM images: (a) overall
image, (b) crosssection of interior, and surface hybridized with nanogels (c,d) at different magnifications. Dashed
circles (d) denote nanogels homogeneously dispersed in the macroscopic HA hydrogel. The images are shown at
the following scales: 1 mm (a), 50 μm (b), 1 μm (c), and 500 nm (d) [186].
Nanomedicine 507
Besides using the nanogels to make the properties of scaffolds more and more similar to extracellular
matrix, adding inorganic nanomaterials such as hydroxyapatite to the matrix of scaffolds can improve
applicability of engineered bone tissue scaffolds. However, hydroxyapatite (HAP) is widely used as
inorganic nanomaterial fillers to prepare scaffolds for bone-like tissues because of its great properties
such as high bioactivity, biocompatibility and osteoconductivity; the intrinsic brittleness and low
mechanical strength of pure HAP and the low compatibility of HAP with the surrounding matrix compel
its application without any further surface modification. In order to overcome this issue, Qing Cai et al.
[187] have reported a new strategy to prepare HAP–poly (l-lactide) (PLLA) nano hybrids, where the
oligomers were grafted from the nanoparticle surfaces via SI-ATRP of methylacrylate group terminated
PLLA macro monomers (PLLA-MA).
As shown in Figure 20.11, by increasing the compatibility of the matrix, after the surface modification
of HAP with PLLA segments through SI-ATRP, mineralized depositions occurred much faster in
comparison to the control samples. This is due to better dispersibility of the modified HAP
nanoparticles in the matrix of polymer, which causes more nucleation sites for apatite formation on the
film surface.
FIGURE 20.11
Surface morphologies of different composites cultured in 1.5 SBF at 37 ◦C for 2 days: (a) pure PLLA, (b) PLLA/HAP
(containing 10 wt% of HA), (c) PLLA/HAP–PLLA (containing 10 wt% of HAP), (d) PLLA/HAP (containing 30 wt% of
HAP), and (e) PLLA/HAP–PLLA (containing 30 wt% of HAP). The insets show the morphologies of different
composites before biomineralization [187].
Nanomedicine 508
PMMA is repeatedly used for tissue engineering purposes as bone cement or orthopedic devices.
Incorporation of HAP with PMMA can generate synergistic effects of both materials but, as HAP has low
compatibility and very poor interfacial adhesion with PMMA, the preparation of PMMA composites and
pristine HAP may result in agglomeration of HAP. Due to the agglomeration of HAP inside the matrix,
the mechanical properties of the prepared composites may dramatically reduce (because of stress
concentration). In order to eliminate this phenomenon, it is necessary to modify the surface of HAP
with PMMA chains to prevent HAP nanoparticles from aggregating. For the surface modification of HAP
by grafting PMMA to avoid the agglomeration of HAP particles, Lang et al. [188] reported grafting of
PMMA chains using “grafting from” approach on the HAP surfaces through ATRP method. They first
modified the HAP particles with aminopropyltriethoxysilane and then with 2-bromoisobutyryl bromide
to introduce the appropriate amount of Br groups on the surface of HAP particles. Then, the
polymerization of methyl methacrylate (MMA) on the surface of HAP particles was carried out using SI-
ATRP. Results showed an increase in the compatibility with PMMA and improvement in the
dispersibility and hydrophobicity of HAP particles.
In comparison to normal ATRP, components of the initial system (transition metal complex) such as
Cu(II) of reverse ATRP, are more tolerant of exposure to air. In a recent study, the feasibility of reverse
ATRP on the HAP surface and the control of polymerization has been investigated [189]. Surface-
initiated reverse ATRP has been employed by covalent attachment of peroxide initiator moiety to the
surface of HAP through the surface hydroxyl groups (g-HAP). In the next step, end bromide groups of
grafted PMMA initiated normal ATRP of MMA to form further modification of HAP nanoparticles (b-
HAP). The TEM images of HAP, g-HAP and b-HAP show that pristine HAP tends to be aggregated as a
result of inter-particle van der Waals interactions, although the surface modification of HAP showed a
strong improvement in dispersion of the nanoparticles (Figure 20.12).
FIGURE 20.12
TEM micrographs of (a and d) HAP, (b and e) g-HAP-8h and (c and f) b-HAP-12h dispersed in chloroform [189].
Nanomedicine 509
Injectable thermo-responsive scaffolds via ATRP

Over the last two decades, stimuli-responsive macromolecules (i.e., pH-, thermo-, photo-, chemo-, and
bio-responsive polymers) have attracted the scrutiny and considerable interest of materials science,
nanotechnology and biotechnology scientists. Stimuli-responsive polymers provide a wide range of
design and application of smart scaffolds for tissue engineering and regenerative medicine. They can
adapt to surrounding environments and rapidly change their microstructure, wettability, dimension or
physical properties, etc. Thermo-responsive polymers for biomedical applications, including drug
delivery, tissue engineering and gene delivery, have drawn much attention among all the other smart
polymers. In particular, thermo-responsive polymers that have cloud point or LCST around 32°C in
water, which is close to physiological temperature, are useful for biomedical applications. Thermo-
responsive polymers switch from hydrophilic to hydrophobic states above LCST in response to
temperature changes, which makes them dehydrate and aggregate. Besides the advantages of thermo-
responsive polymers, disadvantages of other stimuli-responsive polymers offer a wealth of reasons to
use thermo-responsive polymers in the tissue engineering field. Whereas light is not a suitable stimulus
for turbid biological fluids like blood, and changes in ionic strength and/or pH require transport of
matter and can provoke sensitive reactions of biological systems, moderate temperature changes can
be applied to most specimens without an adverse effect [190-199].
In recent years, development of in situ curing gels, also called injectable scaffolds, has been introduced,
heralding in a new era. This is because they facilitate the injection of even large implant hydrogels
directly into cavities with irregular shapes and sizes via minimally invasive surgery. Another advantage
of in situ hydrogels is their ability to encapsulate cells, nutrients and other important biomolecules
throughout the scaffolds. In situ cross-linking of stimuli-responsive hydrogels in the body generally
occurs in two steps. First, with response to external stimuli, such as temperature, macromolecules form
hydrogels via physical cross-linking. In this stage the physical interactions help the scaffold hold its
shape, but it would still have weak mechanical properties. In the next step, gelation via chemical cross-
linking, initiated by either photo-cross linking or chemical compounds, should be conducted to exhibit
much better mechanical performance.
Using thermo-responsive copolymers to produce injectable scaffolds, Tai et al. [200] synthesized water-
soluble thermo-responsive copolymers containing multiple methacrylate groups via one-step
deactivation enhanced ATRP (DE-ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA,
Mn=475), poly(propylene glycol) methacrylate (PPGMA, Mn=375), and ethylene glycol dimethacrylate
(EGDMA) to form covalent cross-linked hydrogels by photo-polymerization. The copolymers can also be
cross-linked by photo-polymerization through their multivinyl functional groups. In their study, they
have used photo-polymerization, which is a mild cross-linking method, since chemical cross-linking can
cause a harsh reaction. In fact, by using thermo-responsive polymers besides the photo-polymerization
method advanced injectable biomaterials could be obtained. To prove the biocompatibility of the
synthesized samples, mouse C2C12 myoblast cells were cultured in the presence of the copolymers.
Figure 20.13 indicates that the majority of the cells remained viable, as assessed by Alamar Blue,
lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays [201,186].
Nanomedicine 510
FIGURE 20.13
Light phase control microscope images for the cells cultured (a) in PEGMEMA-PPGMA-EGDMA copolymer (1 in
Table 1) culture media solutions (750 μg/mL); (b) in the culture media without polymers, (c) on the photo-cross-
linked polymer films. (d) Live/Dead viability assay for the cells cultured in the copolymer/culture media solutions
after 5 days. The viable cells fluoresce green, whereas the nonviable cells fluoresce red (pointed by the arrow)
[200].
In another similar work by Tai et al. [202] a thermo-responsive hyper-branched copolymer system of
poly(ethylene glycol) methyl ether methacrylate-co-2-(2-methoxyethoxy) ethylmethacrylate-co-
poly(ethylene glycol) diacrylate (PEGMEMA475–MEO2MA–PEGDA258) has been developed using DE-ATRP
approach. Figure 20.14 demonstrates the procedure of forming this new injectable and in situ cross-
linking hybrid hydrogel from the combination of thiolated hyaluronan and PEG-based thermo-
responsive hyper-branched functional groups. These functional groups can react with HA–SH via
efficient Michael-type thiolene reaction at physiological condition. In order to investigate the biological
response of the hydrogels, 3D cell culture study was done and the results demonstrated good cell
viability after the cells were embedded inside the samples (see Figure 20.15).
Nanomedicine 511
FIGURE 20.14
Synthetic route and chemical structures of PEGMEMA475–MEO2MA–PEGDA258and HA–SH hydrogel. (i) modification
of HA with cysteamine (EDAC–NHS) carbodiimide coupling; (ii) ‘One-pot and One-step’ DE-ATRP in butanone at
50 C
̊ ; (iii) hydrogel from in situ crosslinking of components via Michael-type thiolene addition at pH 7.4 [202].
Vinyl polymers made by ionic or radical polymerization cannot efficiently degrade. In order to
demonstrate the concept of preparing degradable polymers based on a combination of controlled
radical polymerization of vinyl monomers and radical ring-opening polymerization (RROP),
Matyjaszewski et al. [203], reported the development of an injectable thermo-sensitive hydrogel
consisting of polyacrylamides with degradable units as an injectable scaffold to improve fracture repair.
They have synthesized poly (N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane)
(poly(NIPAAm-co-BMDO)) by ATRP and RAFT polymerization.
Nanomedicine 512
FIGURE 20.15
LIVE/DEAD viability assay for 3D cell embedded study. The hydrogels were formed from 5 wt% polymer solution
5 −1
and the HA–SH (2%, w/v) was added with molar ratio of thiol to vinyl group 4:1. 5 10 cells mL of 3T3 fibroblast
cells and rabbit ADSCs were embedded into hydrogels separately. After 7 d, the hydrogels fluorescently labeled
with a dye that fluoresces green upon the presence of intracellular esterase activity in living cells (calcein- AM) and
a dye that fluoresces red when bound to the DNA of dead membrane compromised cells. The samples were
directly visualized on an Olympus IX81 inverted microscope [202].
After conducting the in vitro cell viability assay by culturing C2C12 cells on the top of a surface coated
with the synthesized polymer and by adding the polymer directly to growing cells, they showed that
both methods had no apparent cytotoxicity by live/dead and CyQUANT assays, as is clearly shown in
Figure 20.16 and Figure 20.17.
Nanomedicine 513
FIGURE 20.16
Combined fluorescence microscope images of C2C12 cells at 37 C ̊ . 95% ±1% viability was measured for the control
(no polymer on surface) after 2 days (left image). 94% ± 2% viability was measured for cells cultured on top of
poly(NIPAAm-co-BMDO) (Table I, expt. 3) after 2 days (center image) and 90% ± 2% viability was observed after 5
days (right image) [203].
FIGURE 20.17
Combined live/dead fluorescence microscope images of C2C12 cells after 24 h without added polymer (control),
showing 96% ± 1% cell viability (left), and with added poly(NIPAAm-co-BMDO) (Table 2, expt. 6), showing 94% ± 2%
cell viability (right). [203].
Nanomedicine 514
Furthermore, the synthesized polymer was degraded completely in Dulbecco’s modified Eagle medium
(DMEM) and was added directly to the culturing cells confirming that the degradation products are not
toxic in biological environments. The degradation products in the medium were also nontoxic
indicating the high potential of this material for tissue engineering applications.
Conclusion
Advanced polymer chemistry allows careful tailoring of new biomaterials for different applications.
New approaches in this field have led to the formation of complex architectures of defined molecular
weight and polydispersity. As a new technique, ATRP has recently gained much attention for the design
and synthesis of functional surfaces due to its ability to produce antifouling, antibacterial, or stimuli
responsive surfaces. This technique has been recognized as a robust and versatile tool for the
development of different kinds of functional bioactive scaffolds used in tissue engineered constructs
which we discussed in this article. It is expected that these sophisticated polymeric structures with
specific characteristics for clinical applications will lead to the development of new techniques for
synthesis of tissue engineering scaffolds in the future.
Acknowledgements
This work was partially supported by AFOSR under Grant no. FA9550-10-1-0010, the National Science
Foundation (NSF) under Grant no. 0933763 and Oklahoma Center for Advancement of Science and
Technology under Grant no. AR131-054 8161.
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International Standard Book Number (ISBN): 978-1-910086-01-8(EBook)

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Chapter 2 In Vivo Applications of Quantum Dot Nanoparticles for Optical

Chapter 3 Conjugation of Gold nanoparticles and liposomes for combined

Chapter 4 Multifunctional Core-Shell Nanostructures

Chapter 5 Biomaterial surface modification of titanium and titanium alloys for

Chapter 6 Experimental and Clinical Therapeutic Uses of Low-Molecular-

Chapter 7 Nanophysical approach to diagnosis of epithelial tissues using Opto-

Chapter 8 Solid lipid nanoparticles – a promising drug delivery system

Chapter 9 Quantum Developments in Nanomedicine: Nanocurative Actions by

Chapter 10 Optimization of perfluorocarbon nanoemulsions for molecular

Chapter 11 Designing polymeric nanoparticles for targeted drug delivery system

Chapter 12 Surface modification of metallic implants with anodic oxide

Chapter 13 Unsupervised medical image segmentation using stochastic

Chapter 14 Biological applications of Semiconductor Nanoparticles

Chapter 15 Biosensors and nanobiosensors: design and applications

Chapter 16 Titanate Nanotubes as a Versatile Platform for Nanomedicine

Chapter 17 Bismuth nanoparticles: antimicrobials of broad-spectrum, low cost

Chapter 18 Surface Modification of Porous Anodic Alumina for Medical and

Chapter 19 Tissue visualization mediated by nanoparticles: from tissue staining

Chapter 20 Recent trends in tissue engineering applications of atom transfer

Molecular-nano fabrications, surface modifications, quantum developments, nanoformulations are

Professor Alexander Marcus Seifalian

Alexander M. Seifalian (AMS) who is Professor of

i. commercialisation of bypass grafts for vascular access and haemodialysis;

Achala de Mel – Bio

Achala de Mel is a lecturer in Regenerative Medicine at UCLs Division of

Deepak M. Kalaskar – Bio

Dr Deepak Kalaskar is Team Leader in development of artificial organs at

1. Department of Chemistry, University of Calgary

Blood Type Antigens and Immunology

Variable Compatible Blood Groups Incompatible Blood Groups

tacrolimus, mycophenolate, and steroids for pretransplant immunosuppression, administration of

Nanoparticles for Tolerogenesis

Two-photon excitation (TPE)-FCS is a non-invasive fluorescence technique commonly used to analyze

Material Functionalization Dye Incorporation Dye

Two-Photon Excitation Fluorescence Correlation Spectroscopy

TPE-FCS measures the fluctuations of fluorescence in an optically-defined interrogation volume over

= 1/N * <F> Equation (3)

Nanoparticle Characterization in Solution

Chicken Embryo Chorioallantoic Membrane (CAM) Model for Nanoparticle Characterization

S2.5. Zeta-Potential Measurement

Results and Discussion

Approximate time in solution until aggregation

Nanoparticles in the CAM

Data on a Hemolysis assay?

Approximate time (post (SD, mV)

Quantum Confinement and Size-tunable Properties of QDs

Bulk Semiconductor Physics

Synthesis and Bioconjugation of QDs

Photophysical Properties of QDs and Comparison with Organic Dyes

Property Organic dyes QDs

Absorption spectra Narrow, discrete bands Broad, steadily increasing

IN VIVO STUDIES of QDs

Non-targeted In Vivo Imaging

Sentinel Lymph Node (SLN) Mapping

Passive Tumour Targeting

Targeted In Vivo Imaging