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Peptide and protein nanoparticle conjugates:

versatile platforms for biomedical applications
Cite this: Chem. Soc. Rev., 2018,
47, 3574
Christopher D. Spicer, *a Coline Jumeaux, bcd
Bakul Guptabcd and
Molly M. Stevens *abcd

Peptide– and protein–nanoparticle conjugates have emerged as powerful tools for biomedical applications,
enabling the treatment, diagnosis, and prevention of disease. In this review, we focus on the key roles played
by peptides and proteins in improving, controlling, and defining the performance of nanotechnologies.
Within this framework, we provide a comprehensive overview of the key sequences and structures
utilised to provide biological and physical stability to nano-constructs, direct particles to their target and
Received 16th December 2017 influence their cellular and tissue distribution, induce and control biological responses, and form
DOI: 10.1039/c7cs00877e polypeptide self-assembled nanoparticles. In doing so, we highlight the great advances made by the field, as
well as the challenges still faced in achieving the clinical translation of peptide- and protein-functionalised
rsc.li/chem-soc-rev nano-drug delivery vehicles, imaging species, and active therapeutics.

1. Introduction biomedical technologies. Whether as drug-delivery vehicles,

high-contrast imaging agents, or active therapeutics, NPs enable
The use of nanoparticles (NPs) provides new opportunities for the new approaches to be taken for the treatment, diagnosis, or
development of more effective, safe, and commercially-viable monitoring of disease within the human body. Unfunctionalised,
naı̈ve NP constructs are often able to fulfil their desired function
a
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, under controlled in vitro settings. However, in the more complex
Scheeles Väg 2, Stockholm, Sweden. E-mail: christopher.spicer@ki.se environment of the human body many questions still remain –
b
Department of Materials, Imperial College London, Exhibition Road, London, UK. how can the particle be targeted to the site of disease? Can
E-mail: m.stevens@imperial.ac.uk
c
clearance be avoided to ensure a suitable therapeutic lifetime?
Department of Bioengineering, Imperial College London, Exhibition Road,
London, UK
Will the accumulation of a biomolecular corona diminish activity?
d
Institute of Biomedical Engineering, Imperial College London, Exhibition Road, In recent decades, the formation of peptide– or protein–NP
London, UK conjugates has emerged as a vital tool for addressing many of

Chris Spicer is currently a Project Coline Jumeaux studied chemistry

Leader in Prof. Molly Stevens’ at the Ecole Nationale Supérieure
group and based at the Karolinska de Chimie of Montpellier, France,
Institutet in Stockholm. Following where she was awarded a Diplôme
undergraduate studies at the d’Ingénieur Chimiste in 2011. The
University of Cambridge, Chris same year, she graduated with a
was awarded his PhD in the group MSc in biochemistry from the
of Prof. Ben Davis at the University University of Montpellier. In 2012,
of Oxford, studying the site-selective Coline was awarded a Rosetrees
chemical modification of proteins. Trust Scholarship to undertake
He then moved to Imperial College her PhD with Prof. Molly Stevens
London, where he undertook post- at Imperial College London, using
Christopher D. Spicer doctoral research in the group of Coline Jumeaux nanotechnology to develop novel
Prof. Stevens, before moving to biosensing and cancer therapy
Stockholm in January 2017. His interdisciplinary research focuses on platforms. Upon completing her thesis in 2017, she was awarded the
the use of organic chemistry and chemical biology to form functionalised Larry Hench Biomaterials Prize by Imperial College London for best
biomaterials, for biosensing and tissue engineering applications. PhD in a Biomaterials related subject.

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the difficulties that arise as a result of these considerations. drugs often suffer from poor pharmacokinetics, exhibiting rapid
These hybrid materials enable the favourable characteristics clearance and difficulties reaching the desired site-of-action
of nano-sized structures to be combined with the biological in vivo.1 As a result, severe side-effects may accompany any
activity, biocompatibility, and versatility of both naturally derived therapeutic benefit, while it is common for in vitro efficacy to be
and synthetic polypeptides. poorly translated to a clinical setting.7 The ability of NPs
In this review, we provide a comprehensive overview of to solubilise therapeutic molecules, enhance retention and
the development and use of peptide/protein–NP conjugates in circulation, and promote accumulation in the target tissue
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biomedicine. While many reviews have previously been pub- makes them attractive vehicles for overcoming these limita-
lished on methods to create NP conjugates, or their subsequent tions, which hinder the drug discovery process.8 These effects
applications within the body, the specific features imparted can be further enhanced through the incorporation of peptide
by the peptide or protein on the NP conjugate have been or protein coatings, as will be the focus of this review, by further
less widely discussed. We will therefore focus on the distinct improving pharmacokinetics, enabling tissue targeting, and
roles played by the peptide/protein in improving, controlling, or promoting cell and tissue penetration.9 Similarly, NP drug
defining the performance of nano-technologies (Fig. 1). In doing delivery vehicles offer several benefits over the use of protein–
so, we will deliver a detailed reference for both experts and those and antibody–drug conjugates (ADCs), which have emerged over
new to the field of NP technologies alike. Furthermore, we hope the last 10 years as promising clinical tools for the treatment of a
to stimulate discussion and innovation within the field, in order range of diseases.10 In particular, NP platforms offer the possibility
to overcome many of the difficulties that continue to hinder the to incorporate multiple functionalities within a single con-
clinical translation of these potentially powerful tools. struct. As a result, problems such as the poor tumour penetra-
tion often exhibited by ADCs can be overcome, as described in
Section 5.10,11 Furthermore, the encapsulation of the therapeutic
2. The benefits of peptide/protein–NP agent allows the need for a cleavable linkage to be avoided, and
conjugates in biomedicine enables high levels of drug to be delivered for every recognition
event.12
The unique properties and size regime of NPs offer many benefits The benefits of both bare NPs and those decorated by
over small molecules and larger micrometre sized particles. These polypeptides, have also been widely exploited in the field of
have been widely exploited within the biomedical field and in vivo imaging. When compared to small molecule imaging
reviewed extensively elsewhere.1–6 By way of context for this agents, NPs often offer the advantages of greatly improved
review, we will briefly summarise here some of the key factors signal-to-noise ratios, stable signal generation, high spectral
that make NP-based technologies particularly attractive, and resolution for multiplexed detection, and the ability to display
the roles in which they have predominantly been applied. multimodal signal generation.13,14 As a result, NPs have found
One of the most prominent realms in which NP systems have increasing utility for imaging in a range of modalities, including
found utility is as vehicles for drug delivery. Small molecule near-infrared (NIR) fluorescence,15 magnetic resonance imaging

Bakul Gupta is a Postdoctoral Molly Stevens is currently Professor

Research Associate with Prof. Molly of Biomedical Materials and
Stevens at Imperial College London Regenerative Medicine at Imperial
working towards understanding College London and the Research
cell–material interactions. She Director for Biomedical Material
obtained a First Class Honours Sciences in the Institute of Bio-
degree in Nanotechnology and a medical Engineering. She also
Diploma in Innovation Management holds a Professorship at the
in 2009 from UNSW Australia before Karolinska Institutet in Stockholm.
completing her PhD with Professor She is a Fellow of six academies,
Justin Gooding and Dr Peter Reece including the Royal Society of
at the same institution. During her Chemistry and the Royal Academy
Bakul Gupta PhD, she researched the use of sili- Molly M. Stevens of Engineering. She has received
con based nanomaterials for appli- over 20 major awards, such as the
cations in the detection of protease activity in vivo, and was awarded her 2016 Clemson Award for Basic Research from the Society for
doctorate in 2014. Some of her research interests include engineering Biomaterials, the European Life Sciences 2014 Research Group of the
nano-interfaces, drug discovery, targeted drug delivery, and diagnostics. Year Award, in addition to the 2010 Norman Heatley Award for
Interdisciplinary Research and the 2011 CRS Young Investigator
Award. She is currently President-Elect of the RSC’s Division of
Materials Chemistry.

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Fig. 1 Polypeptides can play an important role in determining NP functionality and fate. In this review, we will focus on the features imparted by the
peptide/protein and their influence on NP behaviour.

(MRI),16 and positron emission tomography (PET).17 Indeed, others have also been utilised in more niche settings, such
many NP-based imaging technologies are now routinely used in as peptide-based NPs as discussed in Section 8). Each will be
a clinical setting.18 briefly introduced in order to contextualise the discussion that
Stimuli responsive NPs are also finding increasing utility follows (Table 1):
within the biomedical field, often leading to the localised destruc- (i) Gold nanoparticles (AuNPs). AuNPs are attractive struc-
tion of pathological tissue.19 Whether as a result of magnetic or tures for biomedical applications.22 As one of the most stable
light induced hyperthermia, or NP-mediated photoablation, such and least toxic metal NP formulations, AuNPs offer a safe and
technologies are strongly reliant on adequate accumulation at the effective diagnostic and therapeutic tool.23 The ability of thiols to
site of treatment. Many systems rely on inorganic cores, from form stable linkages with the surface of AuNPs allows versatile
which it is vital to reduce the leaching of metal components due surface chemistry to be achieved.24 Decoration of the particle
to their associated toxicity. As a result, peptide or protein coatings surface with a wide range of biomolecules enables the biodistribu-
that can direct, stabilise, and eventually lead to the clearance tion, physical properties, and intracellular fate to be modulated.
of therapeutic NPs are a key component of these emerging Furthermore, AuNPs exhibit unique optical and electronic
technologies as they approach clinical application.20 properties, with concerted electron oscillation following excitation
leading to strong light emission via localised surface plasmon
resonance (LSPR).25 Their interaction with light is strongly depen-
3. Commonly utilised nanoparticles dent on size, shape, surface chemistry, aggregation and environ-
ment. These properties enable the use of AuNPs in a range of
NPs have found widespread use across the material, physical, imaging modalities, including NIR fluorescence, photoacoustic
engineering, and biological sciences, with the number of new imaging, and X-ray computational tomography, as well as finding
technologies increasing at an exponential rate.21 The particle utility in photoablation and hyperthermic therapies.22,26
structures which enable these applications are equally diverse, (ii) Magnetic nanoparticles (MNPs). MNPs are composed
with precise control over material, architecture, and design predominantly of magnetically-responsive elements, such as iron,
enabling a broad spectrum of tunable properties dependent on nickel, and cobalt in various different forms, and are finding
the end application.4 Despite this large variation, a number of increasing utility in the biomedical field.27,28 Iron oxide particles
NP formats have found particular utility in the biomedical field. have found most widespread use, particularly as high contrast
Much of the research to be discussed within this review has imaging agents for MRI.29 The application of alternating magnetic
focussed on the use of 6 main categories of NPs (though many fields can also be utilised as a minimally invasive stimuli for

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Table 1 Most commonly used NPs in biomedical applications4

Particle type Advantages Disadvantages

Gold NPs  Ease of surface modification  Non-biodegradable
 Active in range of imaging modalities  Potential for low colloidal stability
 Size dependent properties
 Can be utilised for photodynamic therapy
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Magnetic NPs  Active for high-contrast MRI imaging  Cytotoxic and poor biocompatibility
 Can be guided magnetically  Non-biodegradable
 Can be utilised for photodynamic therapy

Semi-conducting  Size tunable, high quantum yield emission  Fluorescence sensitive to surface functionalisation
NPs and QDs  Resistant to photo-bleaching and degradation  Toxicity of metal components
 Broad wavelength excitation  Non-biodegradable
 Narrow emission allows multiplexing

Mesoporous silica NPs  Controllable porosity  Potential toxicity of degradation products

 Biodegradable
 High loading capacity for cargo delivery
 Ease of surface modification

Liposomes  Low immunogenicity and high biocompatibility  Low loading efficiency of valuable cargo
 Ease of functionalization  Poor stability and prone to leakage
 Flexibility of formulation for tuning of structure
 Can encapsulate hydrophilic and hydrophobic cargoes

Polymer NPs  Versatile function and structure  Potential toxicity of both NP and degradation products
 Ease of modification and tunability
 Can be made to be degradable or stimuli responsive

generating hyperthermia as described above.30 Finally, the to their applications in drug delivery, MSNPs have also found
inherent magnetism of MNPs enables their spatial distribution widespread use as imaging agents, as a consequence of their
to be easily manipulated, allowing the guided delivery of drug- ability to encapsulate and concentrate contrast agents for a wide
containing vehicles.31 range of modalities.38
(iii) Semi-conducting NPs and quantum dots (QDs). In order (v) Liposomes. Liposomes are spherical vesicles, most commonly
to undertake fluorescence imaging in vivo it is advantageous composed of at least one bilayer of self-assembled phospholipids.
for emission to be in the NIR to allow tissue penetration.32 The similarity of the liposome bilayer to that of the cell membrane is
Conventional small molecule NIR fluorophores suffer from particularly attractive for drug delivery applications, and indeed
poor photo-stability, high hydrophobicity which hinders dis- liposomes are perhaps the most widely used and investigated NP
tribution, and weak signal-to-noise generation. In contrast, carriers for drug delivery, and were amongst the first NP-based
semi-conducting NPs (and in particular QDs) possess narrow, technologies to enter the clinic.2 Liposomes are able to encapsulate
size-tunable, and high quantum yield emission.33 Furthermore, both hydrophilic cargo, within the aqueous interior, and hydro-
they can be excited with broad wavelength light and display phobics, within the membrane bilayer. Furthermore, liposomes
greatly improved resistance to photobleaching and chemical can be readily functionalised with lipid, or hydrocarbon func-
degradation. They are therefore highly attractive structures for tionalised ligands, making them a stable, cost-effective, and
undertaking biomedical imaging.34 However, the high toxicity attractive tool for biomedical applications.
of cadmium metal, common in many QD formulations, neces- (vi) Polymer nanoparticles. The versatility of synthetic polymers
sitates the use of stable coatings able to prevent leaching into enables a wide range of particle architectures and end-
the biological milieu. applications to be explored. Polymer vesicles (commonly referred
(iv) Mesoporous silica nanoparticles (MSNPs). MSNPs possess to as polymersomes) and micelles, composed of self-assembled
tunable pore sizes, large surface areas, and high pore volume. amphiphilic block copolymers, are particularly attractive due to
They are therefore attractive drug-delivery vehicles, with the their ability to encapsulate cargoes in an analogous fashion to
ability to encapsulate a large payload of cargo ranging from their corresponding lipidic analogues.39,40 The NP size, membrane
small molecules up to large proteins.35,36 The ease of surface thickness, porosity, and many other factors can be tuned by
modification, and cost-effective and scalable production adds to adjusting block length and pendant group functionalisation.
the attractiveness of MSNPs. Furthermore, the classification of Furthermore, the design flexibility enabled by synthetic polymer
silica as a ‘Generally Recognized as Safe’ substance by the United chemistry enables the introduction of stimuli-responsive or
States Food and Drug Administration (FDA) greatly facilitates biologically active functionalities, which are able to modulate
regulatory approval of MSNP technologies, though questions on particle properties in a smart, predictable manner.41 As such,
the long-term effects, biodegradability, and biocompatibility of polymer NPs are finding increasing use across a number of
many silica nanotechnologies remain unanswered.37 In addition biomedical disciplines.

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4. Enhancing stability 4.1 Biological stability

It is striking that the number of NP systems that have found In order to reach the desired site-of-action, NPs must navigate their
successful application in biomedical applications pales in way around an ensemble of biological obstacles. These include
insignificance when compared to the vast body of literature defined biological surfaces, endothelial and cell membrane barriers
on the use of NP constructs under controlled model settings.42 to permeation, and circulating monocytes and macrophages of
This is predominantly due to the significant challenges the body’s immune system.44 To achieve their function, NPs
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associated with translating NP stability under idealised condi- must circulate in the bloodstream for long enough to reach
tions to the complex environments of biologically relevant their target cells, tissues, or organs, whilst avoiding removal by
scenarios.43 Peptide and protein coatings play a major role in active phagocytosis or renal clearance.44,45 The nanoscale
enabling NP based technologies, and their ability to provide dimensions in themselves can lead to greatly altered pharmaco-
both biological and physical stability will be summarised in kinetic properties when compared to small molecules in vivo.
this section (Fig. 2, Tables 2 and 3). Importantly, these two For example, for ‘hard’ inorganic particles, renal clearance is
factors are not necessarily independent or complementary. As greatly reduced above a diameter of B5.5 nm due to the size
will be discussed, a coating which prevents NP aggregation may cut-off of the kidneys for urinary excretion (Fig. 3).46 This ability
also impact on the rate at which the construct is cleared from of NPs to modulate pharmacokinetics has been widely utilised as a
the circulation. It is therefore important to carefully consider means to improve the retention, biodistribution, or in vivo stability
design criteria in order to ensure that the end-application can of an encapsulated small molecule.47 However, these differences
be achieved. do not come without their own challenges – dependent on the

Fig. 2 Methods by which peptide/protein–NP coatings can be designed to influence biological stability and decrease susceptibility to clearance:
(a) recognition and clearance can be limited by providing balanced charge or surface hydrophobicity; (b) ‘self-peptides’ can be recognised by
macrophages, and used to inhibit phagocytosis; and (c) the formation of a protein corona can be modulated by providing a stable peptide/protein
coating, promoting the absorption of dysopsonins, or by tuning NP properties such as size, shape, or charge (i–iii).

Table 2 Peptides mediating biological and physical stability discussed in this review

Common name Sequence (N–C) Mw Charge pI Origin Role

CLPFFD 741 1.1 3.4 b-Amyloid peptide derived Biological stability
CD47 peptide GNYTCEVTELTREGETIIELK 2399 3.1 4.3 CD47 derived Escape phagocytosis
CALNN 534 0.1 5.2 Synthetic Physical stability
CCVVVT 623 0.1 5.1 Synthetic Physical stability
Phytochelatin (gE)C(gE)C(gE)CG 772 3.1 3.4 Natural metal chelator Metal NP stabilisation
GCK15 GCGGCGGKGGCGGCG 1129 +0.7 7.1 Synthetic Metal NP stabilisation
Hexahistidine HHHHHH 841 +0.6 7.8 Synthetic Metal affinity
pI estimated using the online tool at http://isoelectric.ovh.org/.

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Table 3 Proteins mediating stability discussed in this review

Protein name PDB # Mw pI Role

CD47 2JJS 32 000 6.0 Escape phagocytosis
BSA 3VO3 66 500 4.8 Escape phagocytosis
Hydrophobin 2B97 14 500 4.2 Modulate protein corona
Clusterin/ApoJ P10909a 75 000 3.8 Minimise cell uptake
Fibrinogen 3GHG 340 000 5.5 BBB penetration
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a
UniProt; PDB – protein data bank; pI estimated using http://isoelec
tric.ovh.org/.

Fig. 4 Upon entering the body, NPs quickly acquire a protein corona. The
exact composition of the corona is dynamic and highly dependent on the
environment: (a) an initial corona is formed of typically highly abundant
proteins; (b) weakly bound proteins are gradually removed by proteins with
a higher affinity for the NP surface; (c) adsorption of proteins can be
dependent on the already existing coating, with inter-protein as well as
protein–NP interactions determining affinity; and (d) gradually a stable
coating of strongly adsorbed proteins is formed, creating a ‘hard’ protein
corona. Reproduced from Monopoli et al. with permission from Nature
Fig. 3 Retention of different size QDs 4 h after intravenous injection. Publishing Group.49
Particles of o5 nm diameter are rapidly cleared to the kidney, while larger
particles are retained. Adapted from Soo Choi et al. with permission from
Nature Publishing Group.46 lipoproteins (often referred to as dysopsonins) recognition is
blocked and circulation times are increased.53,58,59 Amongst the
other NP properties known to promote clearance, surface charge
size, structure, or functionalisation of a NP, detrimental tissue and hydrophobicity are particularly important. Although conflicting
accumulation can occur, while the uptake of particles by macro- reports exist on the relative clearance rates of positively or
phages can not only lead to rapid removal from the circulation, but negatively charged particles, it has become apparent that a near-
also the induction of an unfavourable inflammatory response.48 neutral charge may in fact be most favourable.60 At the same
As soon as NPs are introduced into a biological environment time, a reduction in hydrophobicity has also shown to be a key
further complications arise, as a mixture of biomacromolecules parameter in reducing the uptake of NPs by macrophages.45,60
interacts with the particle surface, often masking the effect While beneficial from a biological perspective, it is important to
of the particle and strongly influencing the pharmacokinetic note that particles which exhibit neutral or low surface charge
properties.49–51 In protein-rich media, such as blood, this new often exhibit reduced colloidal stability, as discussed in the
coating is referred to as the protein corona. The ‘hard’ corona is subsequent section.61 It is therefore important to consider
made up of tightly-bound proteins, forming thermodynami- the delicate balance that determines NP fate when designing
cally favoured interactions with the NP surface, followed by a peptide/protein coatings.
‘soft’ corona of rapidly exchanging proteins (Fig. 4).52,53 The One widely utilised method to limit opsonisation and NP
nature of this biomolecule coating is highly dependent on the clearance from the circulation is the use of stealth polymers,
environment and thus difficult to predict, often resulting in a such as poly(ethylene glycol) (PEG), which form a protective
dramatic loss of activity or function and strongly influencing layer around the NP, neutralizing surface charge, conferring
the nanomaterial fate in vivo.45,54,55 hydrophilicity, and providing a steric barrier to adsorption.56,62
Peptide and protein coatings can be used to improve upon However, there is a fine line between providing a coating which
the biological stability of delivered NPs and address these improves biodistribution and diminishing interactions with target
difficulties. By altering the interactions with host cells and cells and tissues.61 The coating of NPs with PEG has been associated
circulating biomolecules, peptide/protein coatings can modulate with reduced cellular uptake and thus therapeutic efficacy,63,64
and control tissue accumulation to ensure NP activity (Fig. 2). In the generation of PEG-specific antibodies which accelerate
order to understand how this can be achieved, it is important to clearance from the blood,65,66 and preferential accumulation
first understand the factors which induce clearance. The adsorp- in the liver and spleen.67 The versatility of peptide design thus
tion of opsonins (plasma proteins such as immunoglobulin G, offers an attractive alternative to such coatings (Fig. 2a). The use of
complement factors, and fibrinogen) to the NP surface plays a non-ionic peptides, or those offering zwitterionic balanced charge
prominent role in triggering clearance, by inducing macrophage may be particularly useful for reducing clearance. Following this
recognition and subsequent elimination via phagocytosis.56,57 By strategy, Guerrero et al. conjugated the amphipathic peptide
inhibiting this process, so too can the recognition and removal CLPFFD, previously shown to mediate transport across the
of NPs by the immune system be reduced.56 In contrast, blood–brain barrier, to AuNPs with the aim of increasing particle
when the protein corona is enriched with serum albumins or delivery to the central nervous system.68 By reducing the negative

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charge relative to citrate-capped AuNPs, not only was delivery to

the brain improved but accumulation in the spleen was also
reduced. However, this approach is not generally applicable. In
a similar study, AuNPs were coated with the weakly negatively
charged cell-penetrating peptide VG-21.69 Although enabling
improved cell delivery due to the presence of VG-21, preferential
accumulation was also observed within the spleen.69 Similarly,
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Morais et al. reported that the coating of AuNPs with CALNN led
to increased clearance and liver accumulation compared to
citrate-capped particles.70 These results highlight the difficulty
in predicting pharmacokinetic properties – often apparently
similar peptide sequences can have drastically different effects
on clearance. This conclusion is strongly supported by the
research of Poon et al., who revealed the complexity of designing
peptide–NP conjugates for escaping the body’s reticuloendothelial
system.71 By coating AuNPs with a mixture of PEG and either
the negatively charged therapeutic peptide Myx or the positively
charged cRGD targeting peptide, it was found that the effect
of the PEG/peptide coating could act independently or syner-
gistically, depending on the sequence employed. As such, it
is important to evaluate NP pharmacokinetics on a case-by-
case basis.
An alternative approach to avoiding clearance is to rely on
biological signalling to enhance retention. This has been most
prominently achieved by using the membrane protein CD47. By
interacting with the signal-regulatory protein alpha (SIRPa) and
triggering downstream anti-phagocytic processes, CD47 acts in
effect as a ‘marker of self’, sending out a ‘do not eat me signal’
Fig. 5 (a) Near-infrared imaging of mice injected with control NPs, or
(Fig. 2b).72–75 Peptides derived to mimic the effect of CD47 have thus those coated with human CD47 or a CD47-derived peptide. An increase
been designed and shown to reduce phagocytosis.76 Importantly, in tumour accumulation is observed as a result of reduced clearance;
peptide coated NPs displayed greatly reduced accumulation in (b) quantification of tumour fluorescence intensity. Adapted from Rodriguez
the liver and spleen, and enhanced accumulation in cancerous et al. with permission from The American Association for the Advancement
of Science.78
tissues following intravenous injection (Fig. 5). Similarly,
Qie et al. recently demonstrated that NPs functionalised with
full-length CD47 were also able to modulate clearance time, surfaces and can also alter the composition of the protein
enabling the evasion of different macrophage populations.77 corona when NPs are exposed to bodily fluids.52 The high
However, this work also highlighted the need to better under- affinity of hydrophobin enables the protein to remain strongly
stand the interaction of nanomaterials with macrophages dis- associated with the NP surface, even when in competition with
playing distinct phenotypes, in order to pave for the way for other plasma proteins. Apolipoprotein coatings have also been
truly immunologically inert nanomaterials.77 shown to increase NP circulation time. A study of polystyrene–NP
The adsorption of dysopsonins is known to enhance retention. coatings demonstrated that amino and sulfonate functionalised
A third approach to improve NP pharmacokinetics is therefore to particle coatings led to the accumulation of high levels of
promote the formation of a favourable dysopsonin-protein corona apolipoprotein.59 These results highlight the importance of
(Fig. 2c). This can be achieved either through the pre-coating of surface charge and functional groups on influencing protein
NPs prior to in vivo application,79 or by tuning the NP surface corona formation. Notably, the binding of specific apolipoprotein
properties to promote the enrichment of dysopsonins over subtypes was also shown to strongly influence subsequent cell
opsonins in situ.53,80 Serum albumin, the most abundant uptake in vitro.59 Schöttler et al. demonstrated that the apolipo-
protein in blood, plays a wide range of roles including acting protein clusterin (also known as ApoJ) reduced the non-specific
as a molecular and protein transporter, maintaining oncotic cellular uptake of sterically protected polymer-NPs.80 A recent
pressure, and buffering blood pH. Pre-formation of an albumin study highlighted the effect of initial polymer coverage on the
corona is also able to act to prevent the attachment of alter- downstream effects of clusterin, which was able to shield NPs
native proteins and improve NP stability.53 Peng et al. showed from opsonisation at low PEG densities but did not effect NP
that such a strategy was able to limit phagocytosis and prolong clearance at higher PEG coverages.82 The downstream effects of
circulation time in vivo, through simple NP pre-incubation in a protein corona formation must also therefore be considered.
solution of bovine serum albumin (BSA).58,81 Hydrophobin, an Fibrinogen is a circulating glycoprotein in the bloodstream
exogenous protein expressed by fungi has high affinity for NP and a well known opsonin able to inhibit cell adhesion at

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suitable concentrations. When low fibrinogen concentrations for which the high susceptibility to aggregation must be care-
are adsorbed on NP surfaces, a single highly adhesive monolayer fully mitigated. For example, depending on the capping layer
is formed.83 In contrast, dense coatings at high concentrations utilised, unfunctionalised AuNPs are incredibly sensitive to
reduce cell adhesion under both static and flow conditions, via environmental changes, with small changes in pH, salt concen-
the formation of a nanoscale multilayer matrix.84 However, tration, temperature, or the presence of biomolecules often
translating fibrinogen to designed NP coatings for increased leading to rapid and irreversible aggregation.26,33,99–103 Through
circulation remains challenging, as fibrinogen can also lead to combinatorial design, peptide sequences have been identified
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NP aggregation as a consequence of the formation of inter- that are able to find a careful balance of charge, polarity,
particle bridges.85 functionality, hydrophobicity, and length.104 In 2004, Lévy et al.
identified the pentapeptide CALNN through such libraries, as a
4.2 Physical stability water-soluble peptide able to provide extremely stable AuNPs
The physical stability of a NP construct is an important con- following surface functionalisation.105 The presence of the
sideration when designing a biomedical technology. Not only N-terminal cysteine enables facile functionalisation of the particle
are suitable stabilities required to ensure that the particle is surface, with the rest of the sequence providing a densely packed,
able to fulfil its function, but the leaching of small molecules negatively charged peptide corona which is able to withstand
from the particle structure or the formation and accumulation aggregation.
of larger aggregates must also be controlled to minimise The ability of thiols to bind to metal surfaces has led to
toxicity. In order to demonstrate the ability of NPs to be applied cysteine capped peptides being widely utilised as capping agents
both in vitro and in vivo they must form a dispersed, stable for metal NPs.106–109 Indeed, peptides such as CALNN can be
suspension under physiological conditions. Several reports used as a core peptide to which additional biological function-
have demonstrated that agglomerated NPs have drastically ality can be attached, providing a stable peptide-functionalised
altered properties, in particular toxicity, which can lead to large inner corona bearing pendant peptides which can then direct
discrepancies in experimental output.86–90 Indeed, aggregated and influence NP activity.110–112 However, although cysteine–
particles display a greatly decreased surface area and reduced metal binding is sufficient to provide stabilised NPs in vitro, the
cellular uptake when compared to dispersed particles, often reversibility of thiol–metal bond formation means that ligand
leading to an underestimation of toxicity prior to application exchange can occur.113–115 The concentration of free thiols in the
in vivo. In contrast, sedimentation and decreased diffusion can blood is far lower than that observed intracellularly, but remains
significantly impact the ‘effective dose’ experienced by cells high enough to induce loss of monothiol coatings during
in vitro, leading to large discrepancies in experimental readout circulation.116 This is in part due to the possibility for dissocia-
and significantly decreased efficacy upon in vivo translation.91,92 tive ligand exchange (SN1-like pathway), in which ligands are
NP aggregation can also trigger opsonisation in the bloodstream, rapidly diluted within serum upon dissociation, and replaced by
as discussed above, making particles more visible to the phago- circulating small molecule thiols.117 In addition, the high levels
cytotic system.63,64 Thus, physical stability and biological stability of serum albumin in the blood, containing a single reduced
are intimately linked. cysteine residue, have been shown to mediate ligand exchange in
The flocculation of particles in aqueous solution is governed by biological samples.118,119 This loss of NP coating can lead to two,
the Derjaguin–Landau–Verwey–Overbeek theory (DLVO theory).93,94 often concurrent outcomes in the dilute regime provided by
The stability of the dispersion depends on the balance between in vivo settings: (i) replacement of the initial thiol coating by
attractive and repulsive forces – a particle becomes unstable and thiolated biomolecules, leading to a loss of any function imparted
starts to agglomerate when the repulsive energy is not sufficient to by the capping layer, as discussed throughout this review; and
counteract the van der Waals attractive energy.88 At biological salt (ii) loss of any stabilization effects provided by the capping layer as
concentrations the situation becomes more complicated, however ligands are gradually lost.120 As a result, increasingly stabilised
the basics for stability remain similar.95 Sufficient electrostatic peptide coatings have been explored in recent years. This is often
and steric stabilization is required to provide the repulsive achieved through the use of multi-dentate binding sites – that is,
energy to prevent agglomeration.88 For biomedical applica- to use multiple cysteine residues or unnatural amino acids
tions, coatings must not only provide colloidal stability, but bearing dithiol motifs, to provide multiple anchoring points to
also aqueous solubility, while conserving the NP functionality. the metal surface for each individual peptide (Fig. 6).121–124 In
The formation of a protein corona upon application in vivo may doing so, even with reversible desorption of one thiol group from
either enhance or compromise NP colloidal stability. Most the metal surface, the other is still attached maintaining stability.
commonly, the steric bulk provided by the protein coating Due to the proximity of the unbound thiol, this will then simply
can provide significant stabilisation, limiting inter-particle reattach and reform the stabilised NP construct.
attractive forces.49,96,97 This phenomena is able to offset the low An alternative approach, particularly widely employed for the
contribution of stabilising electrostatic repulsion forces which are functionalisation of QD surfaces, is the utilisation of poly-histidine
largely negated in the presence of the high electrolyte concentra- sequences, able to form multi-dentate NP interactions. The ability of
tions of physiological conditions.97,98 histidine residues to bind a variety of metals is well established
Peptides have been particularly widely used to provide throughout biology, and the hexa-histidine motif in particular
physical stabilisation to metal and semi-conducting particles, has been found to form stable metal coordinates (Fig. 6).126–128

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Fig. 6 Monothiol ligands are prone to ligand exchange on the surface of metal NPs, and coatings can therefore be rapidly lost in biological
environments. In contrast, bidentate dithiol ligands offer increased stability, as two simultaneous exchange events are required in order to disrupt the
coating. Alternatively, hexahistidine ligands offer strong and stable metal binding.121,125

Hexahistidine tagged peptides and proteins have therefore Furthermore, even when NPs are taken up by cells, for example via
been widely used to provide stable biomolecule AuNP and QD endocytosis, the challenges are not over – up to 99% of particles
coatings which are resistant to desorption for a wide variety of may be sequestered from the cytosol or nucleus in endosomes and
applications.125,129–132 organelles, depending on their exact properties, the target cell type,
and the uptake mechanism.135,136 There is therefore a pressing
need to develop NP constructs that can be directed to the desired
5. Promoting cell and tissue site-of-action, in order to maintain functionality.
penetration In recent years the decoration of NP surfaces with peptides able
to promote uptake has come to prominence, with many so-called
For a NP to be useful in biomedical applications, it is vital that ‘cell-penetrating peptides’ (CPPs) and sequences able to trigger
the particle is able to reach the necessary site of action – the receptor-mediated endocytosis being studied in detail.137,138
ability of a NP to fulfil its specified purpose is not sufficient. Furthermore, sequences able to efficiently promote endosomal
This is particularly true for particles that are delivered systemically, escape following internalisation have also been identified and
rather than being applied directly at the desired site of action. Even used to great effect. We will here first briefly discuss the key
once a particle has overcome the significant challenge of avoiding mechanisms of peptide-mediated NP uptake, before highlighting
rapid clearance, the body places formidable barriers in the way the main sequences that have been most widely utilised in
of subsequent distribution and delivery of NPs at both the biomedical applications. As a word of caution, it is important
tissue and cellular level.1 These restrictions are the body’s way to note that the efficacy of both penetration and subsequent
of protecting and maintaining homeostasis, preventing the endosomal escape is highly dependent on the experimental
uncontrolled transport of material into and out of sensitive conditions.139,140 While a particular sequence may successfully
environments. Penetrating these barriers remains one of the mediate the transport of one NP cargo, it may fail to enable
biggest challenges in biomedicine, both for small molecule penetration of another.141 Similarly, while uptake may be effi-
therapeutics and the NPs that are the focus of this review. The cient with a cell line in vitro, such results may not be readily
decoration of particles with peptides or proteins able to mediate replicated in the corresponding tissue upon in vivo translation. It
transport or penetration is at the forefront of efforts to enable NP is therefore paramount to test and characterise a newly designed
technologies to reach the areas they need to perform their NP–CPP construct under biologically relevant conditions, rather
function. In this section, we will discuss each barrier in turn than relying on literature precedence for the transport of related,
and give a critical overview of the key sequences that have been but essentially distinct NP cargoes.
utilised to modulate NP transport across them (Tables 4 and 5). Mechanism of uptake. In general, penetration mechanisms
can be split into two major categories: (i) direct, energy-
5.1 Cell penetration independent pathways; and (ii) those relying on active uptake
The cell membrane is a selectively permeable barrier, which via endocytosis (Fig. 7). Often, multiple uptake pathways will be
efficiently protects the cell interior from its environment. Some exploited in parallel, with the exact environmental conditions
specific small molecules may be efficiently transported across the determining which process dominates.142 As such, it can prove
membrane, however the transfer of NPs into the intracellular space complicated to deduce the exact mechanism of internalisation
is far more challenging.133 Although in some cases the uptake of and conflicting reports are therefore commonplace.143
unfunctionalised particles is possible, cell membrane imperme- Direct uptake mechanisms are typically initiated by the
ability often results in a highly detrimental lack of activity.134 interaction of the peptide–NP conjugate with the cell membrane,

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Table 4 Peptides mediating penetration discussed in this review

Common name Sequence (N–C) Mw Charge pI Origin Role

TAT GRKKRRQRRRPQ 1622 +8 12.5 HIV protein derived CPP
R8 RRRRRRRR 1267 +8 12.7 Synthetic CPP
Penetratin RQIKIWFQNRRMKWKK 2247 +7 12.1 Drosophila Antennapedia CPP
homeodomain
HA2 peptide GDIMGEWGNEIFGAIAGFLG 2054 3 3.2 Influenza virus HA-2 derived Endosomal escape
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GALA WEAALAEALAEALAEHLAEALAEALEALAA 3023 6.9 3.7 Synthetic Endosomal escape

Pas FFLIPKG 821 +1 9.1 Cathepsin B substrate Endosomal escape
THRPPMWSPVWP 1491 +1.1 10.5 Phage display BBB penetration
Angiopep-2 TFFYGGSRGKRNNFKTEEY 2301 +2 9.3 Kunitz domain BBB penetration
Glutathione (gE)CG 307 1.1 3.6 Natural anti-oxidant BBB penetration
CDX FKESWREARGTRIERG 1978 +2 10.3 Snake toxin candoxin derived BBB penetration
Chlorotoxin MCMPCFTTDHQMARKCDDCC 4004 +2.6 7.3 Snake venom peptide BBB penetration
GGKGRGKCYGPQCLCR
MiniAP-4 c(DLATEPAL[Dap]) 911 2 3.3 Bee toxin apamin derived BBB penetration
g7 GFTGFLS(Glucose) 889 0 N/A Simil-opiod glycopeptide BBB penetration
RV29 YTIWMPENPRPGTPCDIFTNSRGKRASNG 3266 +1.9 9.1 Rabies glycoprotein derived BBB penetration
iRGD CRGDKRGPDEC 1235 0.1 5.9 Phage display Tumour penetration
IL-13p TAMRAVDKLLLHLKKLFREGQFN 4334 +4 9.5 IL-13Ra2 binding domain Tumour penetration
RNFESIIICRDRT
CGEMGWVRC 1040 0.1 5.8 Phage display Tumour penetration
Lyp-1 c(CGNKRTRGC) 993 +3 9.1 Phage display Tumour penetration
c – cyclic; Dap – diaminopimelic acid; pI estimated using the online tool at http://isoelectric.ovh.org/.

Table 5 Proteins mediating penetration discussed in this review This is followed by permanent or transient destabilization of the
cell membrane, for example through pore or inverted micelle
Protein name PDB # Mw pI Role
formation.142,145 Although many CPPs were at first thought to be
Transferrin 1D3K 76 000 5.5 BBB penetration uptaken via such means, subsequent reports have demonstrated
OX26 N/A N/A N/A BBB penetration
Lactoferrin 1LFG 77 302 8.7 BBB penetration that experimental artefacts in fact led to initial discrepancies and
it is no longer thought that direct uptake is the major contributing
PDB – protein data bank; pI estimated at http://isoelectric.ovh.org/.
factor in the penetration of CPP-coated NPs.145,146 In contrast,
during endocytotic uptake, interactions with cell membrane
most commonly via electrostatic interactions of positively components lead to the engulfment of the peptide–NP construct,
charged surfaces with negatively charged phospholipids.110,144 which is then transported intracellularly in endosomes.

Fig. 7 Schematic demonstrating the main mechanisms by which NPs are uptaken and subsequently intracellularly processed by cells. The exact
mechanism taken by a particular particle is highly dependent on the precise NP characteristics, polypeptide coating layers, target cell type, and
environment. The situation is further complicated by the ability of particles to exploit multiple different uptake pathways in parallel. Adapted with
permission from Zhu et al.149 Copyright 2013 American Chemical Society.

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Endocytosis occurs predominantly via macropinocytosis, or peptides are able to deliver their cargo – it is difficult to
clathrin- or caveolin-dependent receptor-mediated uptake, improve upon the penetration efficiency, even if other proper-
but in all cases subsequent escape of the cargo from their ties leave a lot to be desired, as described below.
resultant endosomal location is a key step, as described in the The most commonly utilised method to mediate internalisa-
subsequent section. tion is to rely on highly cationic peptide sequences, able to first
The exact mechanism by which a particular NP is internalised is strongly bind negatively charged cell membranes and then
a vast area of research, and one which often remains contentious. induce transport via either a direct or endocytic pathway. This
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Exact details are therefore outside the scope of this review and group includes the now ubiquitous human immunodeficiency
readers are instead directed to a number of excellent reviews on virus (HIV) derived TAT peptide, as well as synthetic polyarginine
the topic.133,139,147,148 It has become increasingly evident that it and penetratin (derived from the Drosophila Antennapedia
is difficult to draw generalisations. Importantly, the precise homeodomain) sequences. Indeed, the CPPs utilised for the
means by which a particular construct is taken up is highly in vivo delivery of NP substrates fall almost exclusively into this
dependent on not only the peptide sequence used to promote category. In 1999 Schwarze et al. demonstrated that the con-
uptake, but also on the target cell type, and perhaps most jugation of TAT to a protein substrate could be used to deliver
strongly the exact nature of the NP cargo, including size, the cargo to all parts of the body.152 Since this initial report, a
structure, and surface properties. number of NP cargoes have been systemically delivered to all cells
Key peptide sequences. By some estimates, in excess of indiscriminately, with preferential localisation determined merely
800 unique peptides have been identified which are able to by the NP pharmacokinetics.153,154 The passive accumulation of
promote cell penetration in in vitro experiments.150 Of these, NPs in tumours has also been exploited to allow the preferential
only a handful of prominent sequences or closely related delivery of TAT-labelled, drug-loaded micelles155 and chitosan
analogues have been translated to biomedical applications. NPs,156 as well as anti-tumour silver NPs157 to tumour cells in
Although there are many possible explanations for the paucity animal in vivo models (Fig. 8). Similarly, arginine-rich peptides158,159
of peptides that have been used in such settings, 2 factors are of and penetratin160,161 have been used to both penetrate tumour
particular importance. Firstly, many sequences induce toxicity, cells, following passive accumulation, and to target the brain as
particularly amphipathic peptides which often induce membrane covered in Section 5.3. Interestingly, the non-natural D-enantiomer
disruption in order to promote uptake.151 Secondly, and perhaps of TAT has also been shown to mediate cell penetration. This
more importantly, is the efficiency with which the best CPP strongly suggests that NP uptake as a result of TAT decoration

Fig. 8 Cellular uptake of: (a) phosphate-buffered saline (PBS); (b) bare silver NPs; and (c) TAT-functionalised silver NPs. TAT-particle accumulation
in tumours enables a reduction in tumour growth when compared to unfunctionalised particles, due to increased intracellular delivery. Adapted from
Liu et al. with permission from Elsevier.157

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is not due to a specific biological interaction – rather it is the environment, the TAT peptide was unmasked enabling cell
density or distribution of positive charges along the peptide uptake. Alternatively, ultra-violet (UV) light-cleavable groups
backbone that are responsible for internalisation.154,162 Impor- can be used to trigger cleavage of appended lipids with spatial
tantly, despite the ability of cationic peptides to increase uptake precision.168 Finally, hydrazone-linked PEGs that are cleaved in
efficiency, cytosolic delivery of the NP cargo often remains the mildly acidic tumour environment have also been reported
low.163 The strong affinity of positively charged peptides towards for the selective unblocking of TAT peptides at the desired site-
negatively charged endosomal membrane components may of-action.169,170 Importantly, hydrazone structure has been
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hinder endosomal escape. As a result, additional factors able shown to play a vital role in the rate of hydrolysis, and thus
to mediate this process may need to be incorporated during NP the ability of the NP construct to undergo acid-mediated
design, as discussed in Section 5.2. cleavage. By suitable choice of coupling partners, hydrazone
Increasing cell and tissue specificity. In all of the cases noted half-life can be tuned from a matter of minutes, all the way up
above, the low specificity of CPPs for a particular cell type or to months at neutral pH.171,172 This therefore represents an
target organ leads to widespread tissue delivery. CPPs cross cell important consideration during construct design. For example,
membranes in a largely indiscriminate manner, leading to the aliphatic hydrazones may display insufficient stability to allow
uptake of NPs by almost all cells that are encountered. As such, application in vivo,170 while diaryl linkages may prove too stable
technologies relying on these sequences are often associated to allow acid responsive behaviour to be displayed.173
with significant side-effects, particularly when they are used to Role of peptide/protein density. The density of CPP surface
deliver a therapeutic payload. In order to address this limitation, coverage is an important determinant of NP behaviour. Sufficient
a number of methods for CPP ‘screening’ have been reported ligands must be presented to enable efficient penetration, while
that enable NP targeting prior to unmasking of the penetrative also avoiding overcrowding which can diminish bioactivity and
sequence. In an important early demonstration of this principle prevent the presentation of dual-functionalities.174 In the early
the Tsien group attached a complementary polyanionic peptide, 2000s, Zhao et al. demonstrated that the efficiency of MNP uptake
able to electrostatically bind and thus block the activity of a was highly dependent on the number of conjugated TAT
polyarginine CPP, via a matrix metalloproteinase (MMP) cleavable peptides.175 Penetration was greatly enhanced when more than
linker region.164 In the presence of the corresponding protease, 10 peptides were displayed on the particle surface, with a non-
typically upregulated within the tumour environment, peptide linear response suggesting multi-valent interactions were at least
cleavage resulted in the unmasking of the cationic CPP and partially responsible for the increase in efficiency. This has been
intracellular payload delivery. Such systems have been subsequently validated by a number of subsequent reports – a lower coverage of
utilised for the intracellular delivery of QDs165 and more recently peptides bearing multiple CPP arms often results in a higher cell
for the selective in vivo delivery of PEG–polycaprolactone (PCL) uptake than that observed for particles coated with a high density
NPs to tumours166 (Fig. 9). of mono-valent peptides, though the effect is not universal.176,177
A related approach is to screen the activity of the CPP using Indeed, in a recent report Breger et al. demonstrated that QDs
steric bulk. Harris et al. reported the coating of TAT-functionalised decorated with a single ligand could be efficiently uptaken if a
iron oxide NPs with a shield of PEG, via a MMP-2 cleavable multivalent dendrimeric CPP was presented.177 A number of
peptide linker.167 Upon action of the protease within the tumour hypotheses have been made to explain the origins of these effects,

Fig. 9 (a) Schematic demonstrating the electrostatic blocking of polyarginine cell-penetrating ability. An R8 peptide is blocked by electrostatic binding
of an octa-glutamic acid peptide, connected via an MMP cleavable linker. Upon protease activity in the tumour environment, cleavage of the linker will
lead to release of the glutamic acid blocking group, and activation of R8-mediated penetration. (b) NIR imaging of mice injected with NPs coated with R8
or E8–R8 peptides, in the presence or absence of an angiopep-2 glioma targeting sequence (see Section 5.4). Targeted R8–E8 functionalised NPs display
the greatest enhancement at the tumour site. Adapted with permission from Gao et al.166 Copyright 2014 American Chemical Society.

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and, as is often the case, the exact answer is probably depen- and the presence of highly active efflux pumps makes even such
dent on the precise cell type, NP cargo, and construct design. transcellular movement difficult.188
While receptor clustering and crosslinking is known to play a Four main strategies have been exploited in order to allow
major role in many cell–surface recognition events and may BBB penetration, each of which can be exploited by peptide or
also be important in mediating cell uptake, an increase in protein decorated NPs: (i) receptor-mediated transport, whereby
membrane curvature and pore formation as a result of greater NP interactions with over-expressed receptors at the BBB trigger
localised electrostatic binding has also been proposed to play a internalisation; (ii) transporter-mediated movement, hijacking
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role.177,178 Intriguingly, the Jana group has recently demon- the natural uptake of nutrients such as glutathione by the brain;
strated that the valency itself is a key determinant in not only (iii) adsorptive transport, as a result of the strong binding of
uptake mechanism, but also subsequent sub-cellular location, positively charged particles to the negatively charged BBB; and
and indeed the rate at which particles are subsequently ejected (iv) the exploitation of CPPs, which have also been demon-
by exocytosis.179 A high valency of TAT peptides on the surface of strated to mediate transport across the BBB (Fig. 10). For an in
QDs was found to lead to an increased rate of initial cell uptake. depth discussion on these processes and the mechanisms by
However, the same particles were then rapidly processed and which NPs can be transported across the BBB the reader is
exocytosed, leading to an overall drop in penetration efficiency.180 referred to excellent recent overviews by the groups of Teixidó,
Gao, and Tosi.187,188,190
5.2 Endosomal escape Importantly, the ‘leakiness’ of the BBB is often increased during
As described above, the endocytosis of NPs (both with and pathogenesis. This is particularly true for tumours originating in
without peptide functionalisation) leads to internalisation within the brain, more commonly referred to as gliomas.191 Gliomas are
endosomes. Unless for tissue imaging purposes, this location is therefore commonly used as a model system on which to
typically not the end target, with delivery to the cytosol or nucleus demonstrate the ability of a particular NP construct to penetrate
usually necessary for activity. As such, the endosomal escape of the BBB. While this is not in itself problematic, it is important to
the NP cargo is an essential yet often overlooked factor in have in mind the possible effects of impaired cellular junctions
enabling function. While a number of small molecule additives on NP transport in such systems.
such as chloroquine may act as transduction enhancers, their use Key peptide sequences. The key CPP sequences utilised for the
is not a plausible solution for cell penetration in vivo. As an in vivo delivery of NPs, as outlined in Section 5.1, have all also been
alternative, a number of peptide sequences have been identified utilised to deliver NP cargoes across the BBB. Their ability to
that are able to promote escape, typically through the formation penetrate the cells of the brain endothelium enables transcellular
of disruptive a-helices, which can be triggered selectively within transport into brain tissue. QDs,192 peptide self-assemblies,193 and
the acidic environment of endosomal compartments.181 The AuNPs186,194 have all been shown to penetrate the brain following
N-terminal domain of influenza virus haemagglutinin HA2 TAT functionalisation. Similarly, the delivery of polyarginine-
was one of the earliest sequences identified, with Plank et al. functionalised liposomes195 and penetratin-labelled PEG–PLA
demonstrating that HA2 functionalisation of DNA-based NPs particles160 has also been reported. However, as described above,
greatly enhanced cell transduction.146,181,182 Other sequences, the promiscuity of CPPs limits any specificity for brain targeting
such as oligomers of the tetrapeptide GALA183 and Penetration and these reports are also associated with systemic delivery
Accelerating Sequence (Pas)184,185 have also been utilised to to other organs and tissues. As a result, strategies improving
promote the endosomal escape of functionalised liposomes and specificity for brain targeting represent a more attractive solution.
QDs respectively. An alternative approach to enhance escape has The most common technique to promote BBB specific trans-
recently been reported by Morshed and co-workers. By attaching port is to target receptors at the blood–brain interface, which are
TAT peptides to AuNPs via an acid labile hydrazone linkage, they able to induce receptor-mediated endocytosis. The transferrin-
were able to promote endosomal escape through cleavage of the receptor (TfR) is overexpressed on brain endothelial cells, playing
CPP. Using a non-cleavable linkage resulted in the strong binding a crucial role in the transport of iron across the BBB by mediating
of the peptide to the negatively charged endosomal membrane the endocytosis and transport of the iron-binding protein trans-
being retained and thus hindered particle escape.186 ferrin (Tf). The attachment of Tf itself to NPs has therefore been
widely used as a means to mediate BBB penetration, transporting
5.3 Crossing the blood–brain barrier a range of structures including drug-loaded serum albumin
The blood–brain barrier (BBB) is a formidable and restrictive NPs,196 liposomes,158,197,198 polymersomes,199 and polymer
obstacle, able to exclude over 98% of small molecule drugs dendrimer particles,200 as well as RNA-based structures.201
and almost all nanoscale objects in order to maintain brain Alternatively, anti-TfR antibodies, such as OX26 can be utilised
homeostasis.187,188 The endothelial cells of the brain capillaries to mediate transport.202–205 However, possible difficulties
form continuous tight junctions that preclude paracellular with the species-specificity of antibodies may partially hinder
transport.188,189 While it is possible to disrupt this barrier such technologies. For example, since OX26 is targeted to the
and allow passage between cells, more commonly strategies rat TfR, NPs coated with this antibody are unable to be readily
which aim to overcome the BBB rely on transport through translated into human systems.206
the cells that make up the barrier (Fig. 10). However, the The use of the TfR as a target for BBB penetration has
downregulation of receptors that mediate vesicular transport, significant limitations in spite of its attractiveness as a target.

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Fig. 10 Schematic demonstrating the key mechanisms by which peptides and proteins can mediate the transport of cargo across the BBB. NPs are most
commonly transported via transcellular mechanisms, rather than passing through the tight endothelial cell junctions. For a detailed overview, readers are
directed to the excellent recent review by Oller-Salvia et al. from which this graphic is reproduced, published by The Royal Society of Chemistry.188

Firstly, the TfR is commonly expressed on the surface of many

tissues, in particular the liver and spleen, lowering organ
specificity.158,207 Secondly, and more significantly, due to the
importance of transporting iron to all areas of the body the
physiological levels of Tf circulating in the blood are high,
leading to virtual saturation of the TfR and preventing the
binding of exogenous protein.208 This greatly limits the ability
of Tf-labelled NPs to cross the BBB.
Receptors for the closely related iron-binding protein lacto-
ferrin (Lf) are also commonly over-expressed at the BBB.187
Since the endogenous circulating level of Lf is far lower than Fig. 11 Fluorescence microscopy images of brains from mice intra-
that of Tf, receptor targeting is greatly facilitated.209 As a result, venously injected with QDs (red channel): (a) unfunctionalised QDs;
(b) QDs functionalised with a retro-enantio TfR binding peptide identified
a wide range of Lf-conjugated NPs have been reported, and
from phage display screening. Scale bar 30 mm. Reproduced from Prades
demonstrated to cross the BBB.208,210–214 An alternative et al. with permission from John Wiley and Sons.217
solution to the high physiological levels of circulating Tf has
been offered by Lee et al.215 Through phage display, they were
able to identify a short peptide sequence capable of binding the The short peptides angiopep-2 and glutathione (GSH) repre-
TfR in a non-competitive manner with Tf. As a result, efficient sent two of the most promising candidates for the clinical
receptor mediated endocytosis can be instigated even at translation of BBB-penetrating NP technologies. Both peptides
saturating levels of Tf.216 Recently Prades et al. demonstrated have the ability to target and be internalized by receptors
that N-methylated, enantio, and retro-enantio derivatives of this overexpressed on brain endothelium, specifically low-density
peptide maintained or even enhanced the ability to transport lipoprotein receptor-related protein (LRP) and the GSH receptor
NPs across the BBB, while decreasing susceptibility to protease respectively. These two peptide sequences have been widely
mediated-degradation and particle clearance (Fig. 11).217 This studied for their ability to mediate transport. Importantly,
work represents an impressive demonstration of the synthetic they both display extremely low associated toxicities and are
versatility of peptides and their ability to greatly improve the therefore key components of a number of technologies
characteristics of NP cargoes. currently in clinical trials.188,218 Angiopep-2 was first identified

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rodents suggested that the endothelial barriers lining the

vasculature displayed increased permeability to large species, while
reduced lymphatic drainage enhanced tumour retention.245,246
These two factors combined form the basis for the enhanced
permeability and retention (EPR) effect.247 However, the
impact of the EPR effect on nanomedicine remains highly
controversial.248–251 While studied extensively in rodents, the
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clinical relevance of the EPR effect in human patients remains

uncertain. Even within a single model species the porosity of
the vasculature can vary dramatically depending on the tumour
type, growth stage, microenvironment, and even individual.252
Furthermore, even with accumulation, the subsequent trans-
port of particles across the blood-tumour barrier and penetra-
tion of the poorly vascularised, hypoxic environment of densely
cellularised solid tumours remains problematic.
A number of peptide sequences have been identified that are
able to first target NPs to the tumour site, and then also lead
to their distribution across the entire mass. Amongst these, the
Fig. 12 Electroencephalographs from epileptic mice, injected with the C-end rule (CendR) class of peptides identified and pioneered
anti-epileptic drug PHT. When supplied in solution, little benefit is by the Ruoslahti group are particularly prominent. CendR
observed. However, delivery within angiopep-2 functionalised hydrogel peptides were first isolated from phage display libraries by
NPs enables penetration of the BBB and a therapeutic output. Adapted
Teesalu et al.253 By screening peptides against cancer cells they
from Ying et al. with permission from John Wiley and Sons.226
were able to identify a consensus (R/K)XX(R/K) sequence which
promoted cell uptake. This sequence shares homology with the
by Demeule et al. from the LRP binding Kunitz domain of a neuropilin-1 binding domain of vascular endothelial growth
number of proteins able to cross the BBB.219 In the subsequent factor (VEGF) (with which CendR peptides are thought to
10 years, angiopep-2 has been utilised to deliver a wide range of compete for uptake).253 Importantly, this motif must be present
NP cargoes across the BBB, including liposomes, polymer-, at the C-terminus of the peptide in order to cause penetration,
upconverting-, and gold–NPs (Fig. 12).166,220–229 Similarly, GSH hence the name CendR. Internal or reversed sequences have no
decoration has been used extensively to deliver NPs, particularly biological effect.
drug-loaded liposomes, across the BBB.230–232 Indeed, technologies Importantly, the requirement for the CendR sequence to
based on these systems for the delivery of doxorubicin to gliomas be placed at the peptide C-terminus enables it to be used as a
have recently completed phase I/IIa clinical trials using the cryptic internal sequence, blocked by a protease-sensitive
tradename G-technologys formulation.188 directing motif for specific delivery to a target tissue. This
In addition to these prominent examples, a number of other was exploited by Sugahara et al. to produce the peptide iRGD,
peptide sequences have been identified which are able to able to both target a variety of NP substrates and promote their
mediate transport across the BBB. Many of these are derived uptake after protease processing.254,255 This is achieved through
from pathogens or toxins, mimicking the ability of the parent a multi-step mechanism, in which binding is first induced by
protein/peptide to elicit damage within the brain by either interaction of a terminal RGD motif with integrins overexpressed
disrupting the endothelial membrane or by receptor-mediated on the surfaces of many tumours (see Section 6.1). This targeting
endocytosis. The snake venom-based peptides CDX233 and moiety is then proteolytically cleaved exposing a CendR peptide,
chlorotoxin,234,235 bee venom-derived MiniAp-4,236 opioid glyco- which induces cell penetration following interaction with
peptide g7,237,238 and rabies virus-derived peptide RV29239,240 nueropilin-1. Combining the RGD targeting and CendR penetra-
have all been shown to enable the delivery of NP cargoes across tion motifs within a single modular peptide construct enabled
the BBB. Finally, the attachment of cationic proteins has also greatly enhanced tumour penetration of lipid micelles, iron oxide
proved to be an effective method for promoting NP transport. In nanoworms, and albumin NPs, when compared to particles
particular, the cationization of serum albumin proteins and bearing either motif in isolation (Fig. 13).254 iRGD NP decoration
subsequent NP decoration has been shown to promote BBB has subsequently been utilised to deliver polyethylenimine (PEI)–
penetration. Initial binding is induced by electrostatic interactions PEG NP gene therapies to glioblastomas,256 iron oxide imaging
with the highly negatively charged brain endothelium, and is agents to detect breast cancer metastasis,257 and doxorubicin
followed by subsequent adsorptive-mediated transcytosis.241–244 coated AuNPs to deep tumour sites.258
As will be discussed in Section 6.1, a number of peptide
5.4 Tumour penetration sequences which target NPs to cancerous regions have been
The structural and functional abnormality of tumour tissues identified and used to home particles to the desired site. Most
offers many opportunities for NP delivery and penetration, but sequences often merely lead to NP distribution in the periphery of
also many challenges which must be overcome. Early studies in the tumour vasculature, rather than actively enabling penetration

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a cryptic CendR motif, greatly increasing cell penetration as

well as distribution throughout the tumour mass, and thus
increasing the efficacy of therapeutic delivery.266,267

6. Targeting particles for therapeutic

and diagnostic purposes
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Directing a NP construct to the organ or tissue in which its

action is required is crucial in ensuring the successful function
of both therapeutic and diagnostic tools. The ability of peptides
and protein coatings to modulate distribution within the body
has been widely exploited. When combined with motifs that
reduce clearance by the reticuloendothelial system and success-
fully deliver particles intra-cellularly, targeted delivery can lead
to highly potent nano-technologies.
Targeting approaches can be broadly classified into two
modes, ‘passive’ and ‘active’, though these names can be
misleading. They imply the rational targeting of particles through
Fig. 13 Mice bearing tumours were injected with iron oxide nanoworms design rather than natural distribution, and active guiding to the
functionalised with iRGD, or the analogue CRGDC which retains targeting target site rather than localisation through chance encounters
ability but with no CendR motif for penetration. (a) MRI imaging after 3 h; respectively.247 Passive targeting exploits the normal NP bio-
(b) MRI at 7 h; (c) fluorescence imaging at 7 h. Penetration away from the distribution, as typified by the observed accumulation of parti-
tumour vasculature into the tissue is only observed for iRGD functionalised
particles. Scale bars 100 mm. Adapted from Sugahara et al. with permission
cles in the vasculature of some tumours via the EPR effect, as
from Elsevier.254 discussed above.268 While this may also enable targeting of the
liver and spleen, in which NPs are naturally deposited following
clearance from the blood, other organs and tissues require more
into the deeper tissue. However, a smaller number of peptide sophisticated ‘active’ means of directing delivery.269 This is most
sequences have been identified that are able to promote both commonly achieved through the introduction of a recognition
processes, first targeting NP delivery to the tumour, then motif around the corona of the NP, that can preferentially bind
penetrating away from the vasculature via receptor mediated cell–surface receptors and other biomolecules exposed within
transcytosis. One such peptide sequence is IL-13p, a truncated the target area. Small molecules and vitamins, carbohydrates,
derivative of the inflammatory cytokine interleukin-13 (IL-13). peptides, proteins, and aptamers can all be utilised to target
IL-13 has a promiscuous receptor activating profile throughout delivery.270 In this section, we will discuss the most common
the body, yet IL-13p has been found to be selective for the polypeptide sequences utilised to direct NP transport, and their
receptor IL-13Ra2, which is strongly overexpressed on the impact on biomedical technologies.
surface of many tumour cells and thus represents an interesting
target for tumour-directed therapies.259 In addition to providing 6.1 Peptide targeting motifs
receptor specificity, IL-13p overcomes the low stability and high Short peptides have emerged as the preferred agent for influencing
immunogenicity of the parent protein. Furthermore, it has been NP distribution due to a number of advantageous properties
found to subsequently promote cell and tumour penetration, (Table 6). Firstly, when compared to full size protein targeting
making it an effective tool for NP delivery.260 In a series of agents peptides are able to form compact NP coatings due to their
papers, Gao and co-workers demonstrated that IL-13p decorated small size, limiting disruption of the NP hydrodynamic diameter.
PEG–PCL NPs could be used to efficiently deliver anti-cancer Furthermore, their surface loading can be controlled, enabling
therapeutics and suppress glioma growth in vivo.259,261,262 Wang the simultaneous presentation of multiple targeting sequences,
et al. have also demonstrated that the targeting of IL-13Ra2 providing high affinity and synergistic binding. Peptides of less than
with a phage-display derived binding peptide enables glioma 30 amino acids can also be accessed in a straightforward manner via
targeting and penetration, allowing the delivery of poly(lactic-co- solid-phase peptide synthesis, enabling the facile introduction of
glycolic acid) (PLGA)–PEG NPs.263 functional chemical handles and non-natural residues. This may
In a similar manner, the cyclic peptide Lyp-1, first identified provide both ease of conjugation, and sequences with increased
from phage-display libraries by Laakkonen et al., is a tumour- binding and thus targeting efficiencies. Finally, peptides offer
homing peptide targeting the over-expressed p32 receptor.264 the possibilities of lowered immunogenicity, increased stability
Karmali et al. demonstrated that delivery of the clinically approved of presentation, and reduced binding to physiological bio-
paclitaxel NP delivery vehicle Abraxanes could be significantly molecules when compared to full length proteins.271
improved through Lyp-1 decoration.265 Subsequent investigations Many of the peptide-targeting ligands utilised in biomedical
have further developed this peptide, by combining Lyp-1 with applications were first identified from phage display panning

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Table 6 Targeting peptides discussed in this review

Name Sequence (N–C) Mw Charge pI Origin Role

GFE CGFECVRQCPERC 1530 0.3 5.8 Phage display Membrane dipeptidase targeting
F3 KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK 3433 +8 9.9 High mobility group Blood vessel and tumour targeting
protein 2 derived
Lyp-1 CGNKRTRGC 993 +2.9 9.1 Phage display Tumour lymphatic vessel targeting
CREKA 605 +0.9 8.1 Phage display Clotted plasma/tumour targeting
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RGD RGD 346 0 6.5 Integrin binding sequence Tumour targeting

c(CNGRC) 550 +1 11.2 Phage display CD13/tumour targeting
Bld-1 CSNRDARRC 1080 +1.9 8.5 Phage display Bladder cancer targeting
AHNP YCDGFYACYMDV 1450 2.1 3.2 Trastuzumab derived Breast cancer targeting
SP204 KQFSALPFNFYT 1462 9.6 9.1 Phage display Prostate cancer targeting
CKGGRAKDC 937 8.2 9.2 Phage display White fat targeting
GGGGYDRVTIHPF 1375 7.4 7.8 Angiotensin II derived Infarcted cardiac tissue targeting
PLGLAGGWGERDGS 1371 4.2 3.9 MMP cleavable peptide Infarcted cardiac tissue targeting
CAQK 448 8.1 9.1 Phage display Extravascular brain tissue targeting
D(KLAKLAK)2 1523 9.8 11.4 Synthetic Mitochondria directing
Eriss MRYMILGLLALAAVCSA 1796 8.3 8.6 ER insertion sequence ER directing
c – cyclic; pI estimated using the online tool at http://isoelectric.ovh.org/.

experiments against a target tissue, receptor, or cell type.272–275 multiplexed imaging of both the blood and lymphatic vessels
First reported in 1985 by Smith, phage display relies on the within the same tumour (Fig. 14). The exquisite sensitivity and
presentation of short amino acid sequences on the protein target specificity offered by these sequences is in part due to
coating of filamentous phage.276 Random peptide libraries of a their multivalent presentation on the QD surface. This highlights
defined length can be expressed, and then screened for binding the power of combining peptide display with nanomaterials for
against the target. Following several rounds of increasingly biomedical imaging. In a similar manner, the tumour-homing
stringent screening, coupled with the amplification of binding pentapeptide CREKA was also identified from phage libraries,
phage, peptides with high target affinity can be identified.277 and found to target the clotted plasma proteins that accumulate
Phage display has been most widely used to screen binding to a within the interstitial tissue and vessel walls in cancerous
target protein or cell–surface receptor. However it is also masses (Fig. 15).285,286 This peptide was able to deliver both
possible to systemically deliver phage libraries in vivo. Using iron oxide NPs and liposomes to the desired site, where they
this technique, phage binding to a target tissue or organ have were observed to induce additional clotting and thus amplified
been isolated and peptide sequences mediating cargo transport accumulation.
to the desired location identified.274,278–280 Poor perfusion of the tumour vasculature has been shown
The Ruoslahti group have utilised in vivo bio-panning to to limit the penetration of NPs into the tumour tissue and
identify a number of sequences able to target NP delivery, presents a major obstacle to effective tumour treatment. In
particularly for cancerous tissue.264,281–283 Åkerman et al. demon- Section 5 we introduced a number of sequences which address
strated that 3 of these sequences, GFE (targeting membrane these difficulties, mediating transcellular transport deep into
dipeptidase on the endothelial cells of lung vasculature),281 the tumour mass. The Lyp-1 peptide described above is one
F3 (binding blood vessels and various tumour cell types),282 such sequence, playing a dual-functional role in mediating
and Lyp-1 (targeting tumour lymphatic vessels, as discussed in both targeting and penetration. As discussed in Section 5.4,
Section 5.4),264 could be used to selectively deliver QDs in vivo.284 Karmali et al. demonstrated that combining the CREKA and
Interestingly, F3 and Lyp-1 labelled particles can be used for the Lyp-1 peptide sequences within a single NP construct enabled

Fig. 14 Fluorescence imaging of breast cancer xenografts in mice, following intravenous injection of peptide labelled QDs. (a) F3 labelled QDs (red)
colocalise with tumour vasculature (green); (b) Lyp-1 (red) labelled particles have a distinct, lymphatic distribution from the vasculature (green); (c) F3 (red)
and Lyp-1 (green) labelled QDs label different portions of the tumour. Original magnifications are 400 (a) and 600 (b and c). Adapted from Åkerman
et al., copyright 2002 National Academy of Sciences.284

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Fig. 15 CREKA coated superparamagnetic iron oxide NPs were intra-

venously injected into cancer xenograft containing mice where they
targeted clotted plasma proteins. (a and b) Fluorescent imaging of
melanoma xenograft in fibrinogen deficient mice, no labeling is observed;
(c) NIR imaging of mice treated with or without heparin. In the presence of
heparin, clotting is inhibited and therefore no tumour labeling is observed.
Original magnifications 200 (a and b). Adapted from Simberg et al.,
copyright 2007 National Academy of Sciences.285
Fig. 16 NIR fluorescent imaging of mice intravenously injected with
fluorescently labelled peptides targeting brown adipose tissue (PEP3), or
both the targeted delivery and tumour penetration of paclitaxel a control untargeted peptide (PEP1). Reproduced from Azhdarinia et al.
containing Abraxanes particles.265 Accumulation and tumour with permission from Nature Publishing Group.301
penetration were greatly enhanced when both peptides were
present, compared to NPs functionalised with a single species,
demonstrating the ability of CREKA and Lyp-1 to act in a number of clinical breast cancer treatments.296–298 Additional
synergistic fashion to minimise tumour growth. The phage-display imaging and therapeutic NP–peptide conjugates have been
derived CendR family of peptides, introduced in Section 5.4 as developed for specifically targeting prostate cancer,299,300 pre-
tumour-penetrating sequences, can also be combined with dominantly using sequences identified through biopanning
targeting motifs to provide dual-functional linear peptides. against cancerous cells in vivo.
Indeed, as discussed previously the internalizing iRGD sequence Although peptides targeting tumours have been most widely
makes use of one of the most widely studied and utilised studied, other sequences specific for healthy tissue have also
targeting groups, RGD, to direct cargo to cells overexpressing av been identified and used to deliver NP cargoes. Adipose tissue
integrin.253–255 iRGD coated NPs have been shown to be efficient has been particularly widely targeted as a consequence of its
vehicles for drug-delivery and imaging applications.256–258 In important role in obesity and related pathologies. Kolonin et al.
contrast, RGD peptides lacking the proteolytic sites required to first identified the white fat directing peptide CKGGRAKDC
expose the CendR sequence, do not enable the penetration of from phage-display libraries and demonstrated its ability to carry
deep tumour tissue in vivo, despite their ability to instigate pro-apoptotic peptides for the reversal of obesity (Fig. 16).301,302
tumour delivery.254,287,288 A number of other peptide sequences Hossen et al. subsequently demonstrated the delivery of drug-
able to target NPs to tumour tissue have been identified, including loaded PEGylated NPs for the control of adipose behaviour and
cyclic- and linear-NGR (binding the tumour vasculature receptor weight gain.303 This peptide has also been reported to mediate
CD-13).289–293 the delivery of pro-angiogenic agents, able to stimulate the
Many peptides have been identified that target a wide range conversion to brown adipose tissue which is more readily
of tumour types. Sequences also exist for directing particles to expended during normal energy usage.304
a specific cancer, through binding to over-expressed tissue- The heart and brain are major organs of interest for the
specific receptors. For example, the bladder cancer binding delivery of NPs for therapeutic and diagnostic purposes. Following
peptide Bld-1 was identified from whole cell phage-panning myocardial infarction, the leakiness of blood vessels in the left
by Lee et al.294 This peptide has subsequently been shown to ventricle promotes passive NP accumulation.268 Particles can
mediate the directed delivery of doxorubicin-loaded MSNPs also be selectively delivered by targeting the angiotensin II type 1
for the treatment of bladder cancer.295 Similarly, peptides able receptor, commonly overexpressed in infarcted tissue. Dvir et al.
to bind human epidermal growth factor receptor-2 (HER2) demonstrated that a truncated 9 amino acid peptide mimicking
have been utilised to selectively deliver NPs to breast cancer the behaviour of angiotensin II was thus able to direct the
tissue, mimicking the activity of anti-HER2 antibodies used in a delivery of therapeutic liposomes.305 Alternatively, Nguyen et al.

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reported that the overexpression of MMPs within the heart As described in Section 5.3, the iron-binding proteins Tf and
following infarction could be used to direct the aggregation of Lf mediate the transport of this key nutrient around the body.
drug-loaded NPs, following proteolytic degradation of hydro- NPs coated with these structures can therefore be targeted to
philic surface peptides and thus accumulation at the infarct sites where their relevant receptors are expressed, as epitomised
site.306 In order to mediate NP delivery to the brain, Mann et al. by their ability to deliver particles to, and indeed induce trans-
recently developed the short tetrapeptide CAQK, which despite port across, the BBB.187 The TfR has a particularly widespread
its relatively short length, is remarkably able to target the distribution throughout the body – almost all cells are thought to
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delivery of both small molecule and NP cargoes.307 Importantly, express cell surface TfR, albeit at significantly varying levels.312
this peptide displayed affinity for the extravascular tissue, However, the over-expression of TfR in certain cancers as a
rather than the blood vessels themselves. As a result, delivery result of rapid cell growth kinetics, and thus a demanding need
was enabled throughout the site of acute brain injury for the for iron, enables the use of Tf as a targeting agent when
distribution of therapeutic payloads. combined with potential particle accumulation within the
In addition to peptides which can direct the delivery of particles tumour vasculature via the EPR effect.313 Interestingly, a number
to certain parts of the body, several sequences able to drive NP of papers have demonstrated that TfR targeting does not occur
accumulation within specific subcellular locations following cell as a result of altered biodistribution, with similar accumulation
uptake have also been identified. Distinct from peptides that kinetics being observed for both functionalised and non-
induce cell penetration or endosomal escape, these sequences functionalised NPs. Instead, it is the ability of the targeting
enable intracellular trafficking to the site at which their action is group to induce penetration and uptake within the target tissue
required.271 Although such sequences have been most commonly that leads to an enhancement of the therapeutic efficiency of NP
utilised for the in vitro study or targeting of intracellular processes, drug-delivery vehicles.314,315
more recent studies have also demonstrated their application in a The degree of Tf modification plays an important role in
biomedical setting.271 Agemy et al. combined the mitochondrial determining the level of NP uptake, whether through multi-
targeting peptide (KLAKLAK)2 with tumour-targeting CGKRK valent interactions or increased chance of recognition.315,316
and penetrating iRGD sequences, to form a tri-functional iron Salvati et al. have also highlighted the importance of carefully
oxide NP coating, able to efficiently deliver particles within designing NP coatings in order to maintain maximal activity
mouse glioblastoma models. In addition to its targeting role, in vivo. Following the incubation of Tf-functionalised silica NPs
(KLAKLAK)2 also promoted apoptosis through disruption of the in serum, they showed that rapid loss of TfR targeting occurred
mitochondrial membrane, increasing the therapeutic effect.308 due to the accumulation of a blocking protein corona.317
In another example, the endoplasmic reticulum (ER) retention Despite this sensitivity, Tf functionalisation has been the most
signal ‘Eriss’ (derived from the adenovirus E13-19K protein) has widely utilised strategy for directing tumour delivery. Such
been used to direct NPs to the ER of lymphocytes, improving the systems have been shown to mediate the transport of a wide
processing and presentation of NP-displayed antigens. By doing range of NP cargoes, as well as being at the core of several
so, the efficiency of synthetic vaccines can be greatly enhanced liposomal based therapeutic technologies currently in clinical
in vivo as discussed in Section 7, leading to an improvement in trials (Fig. 17).313,314,316,318–321
the generation of immunity.309,310 Many other protein ligands have also found widespread use
for the selective targeting of NPs to cell surface receptors within
6.2 Protein targeting motifs a specific tissue or organ. An important consideration is the
The recognition of protein binding partners by cell–surface ability of many such ligands to trigger cellular responses and
receptors is one of the most important interactions in biology. downstream effects. While this may be favourable in certain
The vast number of hormones, growth factors, cytokines, cell applications, in such cases the protein substrate is not merely
adhesion proteins, and cell-signalling structures are involved in acting as a targeting motif but is also controlling or inducing
virtually all intracellular interactions that enable the formation a biological effect.322 For example, growth factors such as
and function of complex tissues.311 It is therefore not surpris- epidermal growth factor (EGF) and VEGF have been used to
ing that protein–NP coatings have become an important means target NPs to their relevant receptors, when overexpressed on
to drive targeted delivery to a certain area of the body where a the surface of tumour cells and tissues.270,322–327 However, the
particular receptor is likely to be overexpressed (Table 7).270,275 potency of growth factors as cell signalling moieties and their

Table 7 Targeting proteins discussed in this review

Protein name PDB # Mw pI Role

Transferrin 1D3K 76 000 5.5 Tumour targeting
EGF 1NQL 134 000 5.3 Tumour targeting
VEGF 2VPF 27 400 8.1 Tumour targeting
LFA-1 N/A N/A N/A Tumour/inflammation targeting
Apolipoprotein AI 3R2P 31 000 5.4 Tumour targeting
WGA 2UVO 21 200 6.4 Alveoli targeting
UEA-1 1JXN 27 000 4.5 Intestinal targeting

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lipids around the body. The interaction of HDLPs with a

number of receptors commonly implicated in pathological
conditions can be exploited to deliver NP based therapeutic
agents and imaging tools.270 The scavenger receptor type B-1
(SR-B1) has been most widely targeted. For example, Yang et al.
demonstrated that AuNP templated HDLP particles could be
used to selectively sequester cholesterol in vivo (Fig. 18). These
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structures could be targeted to B-cell lymphoma cells, due to

their need for cholesterol and thus upregulated expression of
SR-B1.329 Similarly, apolipoprotein A-I, the major component of
HDLPs, has been demonstrated to enable the selective delivery
of fluorescent NPs to cancerous tissue in vivo.330
Although cell surface receptors are the most commonly utilised
target for NP delivery, other cell or tissue specific markers can also
be utilised to mediate NP targeting. Most prominently, lectins,
or carbohydrate-binding proteins, can direct NPs to tissues
displaying particular cell–surface glycans.270 Glycoproteins
and glycolipids play a vital role in cell biology, providing
stability to the cell membrane, facilitating cellular recognition,
and adding an extra level of complexity and functionality to the
proteins that determine cell behaviour. With rapidly improving
tools for studying the role of glycans in many normal and diseased
states, so their biological importance is being increasingly
appreciated. As a result, specific glycoforms have now been
identified to be more prominently displayed during certain
Fig. 17 Fluorescence imaging of mice bearing red fluorescent protein
pathologies, making them attractive targets for NP delivery.
(RFP) expressing tumours. Treatment with free siRNA failed to impact
tumour growth. However, Tf labelling of poly-siRNA (psi) particles enabled
Lectins commonly display exquisite sensitivity for a particular
efficient tumour delivery and growth suppression. Adapted with permis- sugar structure and conformation, often analogous to that
sion from Yhee et al.318 Copyright 2013 American Chemical Society. shown by antibodies. Wheat germ agglutinin (WGA) binds
specifically to N-acetylglucosamine residues commonly found
on the alveolar epithelium, and has thus been utilised by Surti
often promiscuous activation profiles must be carefully con- and Misra to deliver corticosteroid loaded NPs to lung tissue.331
trolled, particularly following systemic delivery. Furthermore, A number of groups have subsequently used WGA to induce the
cell–surface adhesion motifs can also be targeted. Chen et al. intra-nasal transport of functionalised NPs, due to its ability
demonstrated that iron oxide NPs could be functionalised with to both bind and promote transcellular delivery across the
lymphocyte function-associated antigen (LFA)-1 proteins, able epithelial barriers of the nasal passageway.332–334 Ulex europaeus
to target intercellular adhesion molecule-1 (ICAM-1) on the agglutinin I (UEA-1) can also be used to selectively bind
surface of both tumour cells and inflamed tissue in vivo.328 a-fucose residues on the surface of microfold cells (M-cells) within
High density lipoproteins (HDLPs) have also found utility for the small intestine.335 Oral delivery of UEA-1 labelled antigen
selective NP delivery. These lipid–protein hybrids naturally containing NPs enables the initiation of a mucosal immune
form spherical particles in order to mediate the transport of response and vaccination of the recipient.336,337 Mucosal M-cells

Fig. 18 AuNP templated HDLP particles were injected into xenograft tumour bearing mice: (a) particles selectively target and are uptaken by B-cell
lymphoma cells. Disruption of cholesterol flux as a result limits tumour growth; (b) in contrast, particles are not targeted to T-cell lymphoma cells and
have no effect on tumour growth. Reproduced from Yang et al., copyright 2013 National Academy of Sciences.329

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can also be targeted via intranasal delivery, providing a facile

means to undertake needle-free NP immunization.338

6.3 Antibody targeting motifs

The ability of antibodies (Abs) to bind with high specificity and
affinity for their target antigen makes them ideal directing
groups for the delivery of NP cargoes. It is therefore unsurprising
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that Ab–NP conjugates have found widespread utility in the

biomedical field.12,275,339–342 The ability of Abs to neutralise the
effect of their target or to induce a favourable biological response
allows their use as therapeutic agents in their own right. They
have also commonly been exploited to deliver a therapeutic
cargo, in the form of an Ab–drug conjugate (ADC).10,343–345
At the start of 2017, 52 monoclonal Ab technologies were in
clinical trials, with a further 16 awaiting or having been granted
marketing approval.346 The importance of tumour delivery is
highlighted by the fact that 40% of these systems were targeted
towards cancer therapy. The generation of Abs for a target
protein, cell type or tissue of interest is outside the scope of this
review, and the reader is directed instead to a number of
comprehensive reviews for further details.275,347,348
Most Abs are composed of 4 chains (2 light and 2 heavy)
each of which has a constant and a variable region. Specificity
towards the desired target is generated predominantly by the
variable region. Ab fragments may therefore retain binding
affinity of the full length construct, though in some cases bis-
antigen binding is necessary to maintain full affinity. These
smaller targeting groups can be produced via genetic engineering
or careful protease-mediated Ab digestion, generating motifs
which overcome the disruptive size, potential immunogenicity,
and high cost of the parent Ab.342 Both full length and fragment
Abs have been extensively used to target the delivery of NP Fig. 19 Fluorescence imaging of xenograft bearing mice systemically
cargoes, as have naturally occurring single domain camelid injected with Ab labelled iron oxide NPs against HER2. Targeting is enabled
Abs.349 Tumours have been particularly widely targeted due to by the use of full length Ab, or half-chain and single-chain Ab fragments
the frequency with which cell–surface receptors are overexpressed with little difference in efficiency. Adapted with permission from Fiandra
and can thus be used as a site for selective delivery.12 The most et al.357 Copyright 2013 American Chemical Society.

common realisations of Ab–NP strategies deliver a therapeutic

payload via a liposomal drug-delivery vehicle, though other NP is implicated in many cancers, especially those with epithelial
formats for both therapeutic and diagnostic purposes have also origins, and thus serves as a useful target for immunoliposome
been the subject of significant interest.342 targeting.361–363 Indeed, a single antigen-binding fragment of
Overexpression of HER2 is implicated in over 30% of breast the monoclonal Ab cetuximab conjugated to doxorubicin
cancers and thus represents an important target for delivery.350 loaded liposomes has been tested in phase I clinical trials for
The HER2 selective Ab trastuzumab (sold under the tradename the treatment of patients with solid malignancies.362 A number
Herceptin) and other related structures are probably the most of other cancer markers have been widely targeted via Ab–NP
widely studied and utilised targeting groups for NP delivery. In conjugates (both for therapeutic and diagnostic purposes)
a series of early papers, Park and co-workers demonstrated that including the TfR,204–206,364,365 B-lymphocyte associated anti-
the labelling of liposomes with an anti-HER2 Ab resulted in an gen CD19 for the treatment of lymphoma,366,367 the melanoma
efficient delivery vehicle for targeting breast cancer xenografts and carcinoma marker endoglin,368–370 nucleosomes released
with chemotherapeutics or DNA.351–353 HER2 targeted iron oxide by proximal apoptotic tumour cells,364,371 death-receptor 5
NPs have also been widely reported as a means to induce directed using the commercial Ab Conatumumab,372 and the adhesion
hyperthermia and for MRI imaging (Fig. 19).354–360 NDong et al. receptor VCAM-1.373
recently reported that a single antigen-binding fragment of The choice of Ab, the use of full length protein or fragment,
trastuzumab could also be used for targeting in vivo, leading and the NP cargo are all important considerations when
to effective delivery of iron oxide particles to tumour sites.359 designing an Ab-labelled technology. However, the widespread
The epidermal growth factor receptor (EGFR) is another interest in Ab–NP conjugates has led to the identification
popular target for Ab-mediated delivery. EGFR overexpression of a number of other key design criteria. The orientation of

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the Ab on the NP surface is an important consideration – non- NP vaccines can offer the benefits of increasing antigen
specific protein modification techniques can lead to Ab con- stability over soluble delivery. Furthermore, their nanoscale
jugates with many different NP binding points, some of which size may promote scavenging by dendritic cells and therefore
may result in an antigen-binding domain that is positioned in a improve T-cell presentation, minimising the activation of alter-
hindered or blocked orientation. Indeed, a recent study by native immune response pathways.382,385 Reddy et al. demon-
Herda et al. demonstrated that as little as 5% grafted protein strated that when NPs were of a sufficiently small size (o100 nm)
possessed an accessible epitope following non-specific conju- preferential drainage and accumulation in the lymph nodes was
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gation to silica NPs.374 The use of site-specific modification enabled.386 However, other reports have suggested that particles
strategies able to selectively form Ab–NP conjugates away from up to 1 mm in diameter may also preferentially accumulate under
the active recognition site are likely to improve the efficiency of certain delivery conditions, highlighting the need for further
NP targeting in vivo.342,375 To this extent, Greene et al. recently investigation into this phenomena.387 Preferential exploitation
reported a strategy to functionally re-bridge distal disulphide of the lymphatic system brings particles into closer contact with
bonds within a trastuzumab fragment–Ab, enabling orientated the residing dendritic cells and leads to an enhanced activation
Ab display on the surface of PLGA–PEG NPs and a corresponding of the immune system maximising vaccine efficiency. However,
increase in binding efficiency when compared to non-specific Ab the major advantage of NP-based vaccines lies in their ability to
conjugation.376 enable the multivalent display of antigens, promoting the
In a related manner, the Ab loading density on the NP interaction of multiple ligand–receptor pairs. Such processes
surface can play an important role in targeting efficiency. At often play an essential role during T-cell activation, rather than
high levels, hindered antigen-binding may result leading to an relying on individual recognition events.388 As such, the pre-
initially counter-intuitive drop in targeting efficiency. Indeed, sentation of multiple antigens on the NP surface can play a far
Jiang et al. demonstrated that the size of the NP template plays more effective role in modulating the immune response than
an important role in determining hindrance and thus binding presentation of free peptide or protein motifs. Interestingly,
efficiency, with increasing curvature leading to an increase in it has been shown that the antigen patterning plays a crucial
separation and thus decrease in steric inhibition of antigen role during activation. As a result, antigen grafting is a key
recognition.377 Although multi-valent Ab display may be desirable design feature during the production of NP vaccines. The Yu
in some cases in order to promote receptor recognition, recent group demonstrated that enhanced antigen clustering elicits a
papers by the groups of Davis and Prosperi have highlighted stronger immune response than uniformly distributed ligands
that this may not always be the case.378,379 In both cases, a at both the micron and nano scale.388,389 Similarly, particle
singly Ab labelled NP was found to be sufficient, and in the case surfaces that closely mimic the natural context of the antigen,
of Colombo et al. superior to multivalent Ab display for effective such as liposomes able to mimic cell membranes, have been
tumour targeting. found to improve activation efficiency.390,391
Amongst the NPs used for synthetic vaccines, ‘hard’ NPs have
generally been more widely applied for the surface presentation of
7. Mimicking biological species biomimetic peptide/protein antigens. In contrast, ‘soft’ particles
able to encapsulate a payload have commonly been utilised for
Many biological entities fall within the nanometre size regime, the delivery of antigen following particle recognition and sub-
displaying multi-valent peptide or protein motifs on their surface. sequent release.384 AuNPs in particular have found widespread
NP–polypeptide conjugates are able to effectively mimic the use as NP antigen carriers due to their ease of surface function-
behaviour of these structures, stimulating signalling pathways and alisation, high surface-area, biocompatibility, and tunable size.392
eliciting cellular responses. The interactions induced by these parti- Importantly, recent research suggests that AuNPs may also act as
cles are responsible for many of the applications discussed in this size- and shape-dependent adjuvants, stimulating the immune
review. The promotion of receptor clustering is often implicated in system and enhancing antigen recognition.393,394 As a result, the
receptor-mediated endocytosis and cell or tissue penetration,380,381 attachment of peptide and protein ligands to AuNPs has emerged
and NP targeting may be enhanced by multi-valent interactions at as an effective means to stimulate the production of Abs against a
the nanoscale. As these topics have already been covered in detail wide range of pathologies and pathogens, including malaria,395
above, in this section we will focus on the ability of NP–polypeptide foot and mouth disease,396,397 the Yersinia pestis bacteria
constructs to mimic the multi-valent display of antigens present on responsible for plague,398 cancers via the mucin-1 (MUC-1) glyco-
the surfaces of pathogens and tumour cells. By doing so these protein,399,400 influenza A virus,401 Streptococcus pneumoniae,402
conjugates are able to induce a controlled immune response and respiratory syncytial virus,403 encephalitis causing viruses,404
thus generate immunity in the recipient. Technologies which rely on and HIV via gp120 derived peptides.405
the encapsulation, rather than surface presentation of antigens, are Both single- and multi-walled carbon nanotubes (CNTs) have
outside the scope of this review and the reader is instead also been widely explored as antigen carriers due to their largely
directed to a number of comprehensive reviews on this biologically inert nature, facile surface modification, and ability
topic.382–384 Furthermore, the use of self-assembling peptides/ to induce cell penetration. However, recent reports on their
protein NPs as vaccine candidates will be detailed in Section 8 toxicity may limit further development towards applications in
below and so will also not be discussed in detail here. patients.406 Since being first introduced by Pantarotto et al.,407

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peptide- and protein-modified CNTs have been utilised known to be targeted by auto-immune responses in patients
to generate immune responses against a range of antigens suffering from multiple sclerosis, with tolerogenic small mole-
including tumour lysate,408 Plasmodium vivax apical membrane cules that stimulate Treg proliferation on the surface of AuNPs,
antigen-1 derived peptide,409 Mycobacterium tuberculosis protein has subsequently been shown to suppress symptoms within
derivatives,410 Wilm’s tumour antigen,411 and foot and mouth mouse models of the disease.425 More recently, Hess et al.
virus derived antigens.412 ‘Hard’ NPs such as polystyrene nano- utilised QDs as a scaffold for the display of myelin peptides
beads,413,414 polyacrylate dendrimers,415 and calcium phosphate allowing the authors to monitor the distribution and activated
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particles416,417 have all also been shown to enable Ab generation pathways of the NP–peptide complexes in vivo.426
in vivo. The Baneyx group have recently reported that a calcium
phosphate binding peptide epitope can be attached to the
termini of the desired antigen, inducing biomineralization and 8. Playing a structural role
particle assembly at a late stage of the formulation process.416,417
By doing so, the low stability of calcium phosphate particles can In much of this review, we have focussed on systems in which
be mitigated, allowing ‘on-demand’ production and application the peptide or protein defines or modulates NP function or
to take place. performance. In addition to these roles, peptide/proteins have
Although ‘soft’ organic NPs have been more rarely used for emerged as key structural motifs, which are integral to the
the formulation of vaccines, a number of examples exist and in formation as well as eventual end application of the NP. Indeed,
many cases they reveal interesting facets of vaccine design that in many of the scenarios to be discussed in this section the
should be carefully considered, regardless of the particle type. particle is composed entirely of peptide or protein components.
For example, liposome formulations have been commonly Peptide self-assembly into complex nano-architectures can
utilised for the encapsulation of peptide or protein antigens, be instigated by a combination of intra- and inter-molecular
enabling delivery to immune cells typically following targeted non-covalent interactions, including hydrogen bonding, electro-
delivery. Virosomes, liposomes functionalised with virus static or hydrophobic interactions, and p–p stacking.427 Although
components (often influenza virus derived) able to mimic viral individually these interactions may be weak, cumulatively they are
envelopes, have been particularly widely used as safe, self- able to define the secondary and tertiary structures of complex
adjuvanted, and stable antigen delivery vehicles.418,419 However, native protein architectures, and can be exploited to produce a vast
Guan et al. have demonstrated that the surface-presentation of array of self-assembled structures on both the nano- and micro-
ligands within liposome formulations is able to mediate activa- scale. The formation of architectures, ranging from fibres and
tion of alternative branches of the immune system in vivo, when tubes, through to vesicles and micelles, and on to more elaborate
compared to encapsulated antigen.420 Careful NP design is structures such as crystals and donuts can all be instigated.427–431
therefore key in ensuring that the desired response is triggered. While the fundamental driving forces behind the formation of a
Similarly, peptide–lipid amphiphiles have also been studied, particular architecture are becoming increasingly well understood,
as discussed in Section 8, for their ability to self-assemble in many cases the route of assembly remains a dynamic process
into peptide-decorated micelles. The use of peptides as a key which can be affected by seemingly minor modifications of
structural component results in an extremely high density of peptide primary structure or growth conditions.432–436
surface antigen coverage, enhancing recognition and activation The formation of self-assembled peptide architectures has
efficiency.421 Importantly, the crowded environment provided by found widespread application across the biomedical field, and
such a set-up has been found to induce peptides to adopt a further afield in the wider materials research community. Here, we
secondary structure more akin to their natural presentation will focus on the formation of 3D spherical NPs, particularly
within the parent protein.415,422 As a result, the Abs generated peptide/protein micelles and vesicles. The conditions for the
downstream are better able to produce an effective response formation of these structures are often strict, requiring precise
when subsequently challenged. control over composition, assembly conditions, and handling. For
While applications using peptide– and protein–NP conju- an in depth overview of the wider field, and in particular the use of
gates as vaccines to stimulate a protective immune response self-assembling peptides in the formation of 2D nano-fibres,
to previously unencountered antigens have been most widely hydrogels, and nanotubes, the reader is referred to excellent recent
studied, technologies which modulate the immune system, and reviews on the subject.427,429,430,437–440 It should be noted that we
in particular mitigate auto-immunity, have begun to emerge. In will not cover nano-sized aggregates of globular proteins in this
a healthy individual, tolerance of self-antigens is maintained review, such as those formed by serum albumins or gelatin, which
by the activity of regulatory T cells (Tregs). Modulation of have found increasing clinical use in recent years. Such systems do
Treg activity and addressing deficiencies is thus an attractive not rely on the specific self-assembly of polypeptide components,
target for the treatment of autoimmune diseases.423 Tsai et al. and thus fall outside the scope of this review. A number of reviews
demonstrated that the presentation of recombinant major focussed on these topics have recently been published.441–444
histocompatibility complexes bearing type 1-diabetes associated
peptides on the surface of iron oxide NPs resulted in the in vivo 8.1 Dipeptides
expansion of Tregs, and ultimately the restoration of normoglycemia Dipeptides represent the simplest self-assembling peptide
in diabetic mice.424 Furthermore, the co-delivery of myelin peptides, motif. Since the breakthrough discovery by Reches and Gazit

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that di-phenylalanine assembled into nanotubes at high was first reported by Vauthey et al.452 1 or 2 C-terminal aspartic
concentrations,445 a wide-range of nano-architectures have acid residues, bearing 2 or 3 negative charges respectively, were
been reported, making use of both natural and non-natural found to be sufficient to drive the assembly of an N-capped
amino acids.446 Often, the presence of a di-aromatic motif is hydrophobic peptide chain of 6 alanine, valine, or leucine
vital, providing the driving force for assembly via p–p stacking. residues. Dynamic heterogenous mixtures of nano-tubes,
The ease with which dipeptides can be accessed synthetically vesicles, and micelles were observed, depending on the exact
makes them particularly attractive structures for biomedical peptide sequence. Subsequent reports on the formation of
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applications, with researchers from diverse backgrounds able vesicles from glycine-tail anionic peptides453 or cationic peptides
to exploit their use. bearing lysine or histidine termini432,433 validated this approach,
The first dipeptide shown to form spherical particles was the with the hydrophobic ‘tail’ and charged ‘head’ group in effect
unnatural structure di-phenylglycine, in stark contrast to the mimicking the structure of lipid surfactants. Interestingly, it has
nanotubes formed by the closely related di-phenylalanine.447 recently been reported that rather than forming tail-to-tail
The significance of seemingly minor differences and subtle arrangements analogous to those observed in lipid membranes,
changes in structure highlight the surprising versatility of such the hydrophobic regions of amphiphilic peptides form inter-
simple structures. The situation is further complicated by the digitated b-strand-like assemblies, leading to greatly reduced
dynamic nature of dipeptide nanostructures, which have been membrane thickness.454
observed to result in reversible transitions between architec- Despite these important fundamental studies on the ability
tures in response to stimuli or incubation conditions.446,448,449 of short amphiphilic native-peptides to form vesicles, bio-
Despite this apparent plasticity, dipeptide NPs exhibit remark- medical applications have to date been limited by the relative
able stability. Indeed, di-phenylglycine particles were shown to instability of self-assembled constructs. Linear peptides typi-
be stable to both acid and base treatment with no observed cally exhibit high critical aggregation concentrations (CACs) in
change in particle number.447 aqueous solution, below which particle formation does not
Despite the simplicity of dipeptide assemblies, their applica- occur, and a dynamic equilibrium with free peptide therefore
tion in biomedicine has been limited to just a few reports. In an often exists. This situation is further complicated in complex
early example, Alam et al. demonstrated that H2N–methionine– biological fluid.433–435 A number of different approaches have
dehydrophenylalanine–CO2H NPs could be loaded with the anti- been taken to address this issue, yet often CACs or dissociation
cancer drug curcumin and used to induce tumour regression in a constants are not reported, obscuring the analysis of particle
mouse melanoma model.450 Importantly the unnatural amino stability. Gudlar et al. showed that branched peptides derived
acid dehydrophenylalanine not only increased packing efficiency, from natural transmembrane helices can enhance vesicle stability
and thus enhanced physical stability through increased p–p as a result of enhanced hydrophobic interactions.455 These
stacking, but also promoted biological stability by providing branched structures were found to be a key driving force for the
protease resistance. More recently, Fan et al. reported the assembly preferential formation of vesicles over fibres, closely mimicking
of H2N–tryptophan–phenylalanine–CO2H, to produce fluorescent the di-hydrophobic tail of native lipids and enabling the delivery
NPs.451 By combining p–p stacking with peptide–zinc interactions of cargoes to cells in vitro. In order to stabilise vesicles post-
they were able to produce particles with visible light emission, formation, van Hell et al. incorporated cysteine residues into the
mimicking the red-shifted emission exhibited by fluorescent pro- primary structure of the amphiphilic peptide SA2.456,457 SA2 is a
teins upon metal-binding. Subsequent modification of the particle rationally-designed amphiphilic sequence containing sequential
surface with a MUC-1 binding aptamer enabled biocomptability to hydrophobic residues of decreasing bulk, leading to a cone-
be enhanced when compared to other fluorescent NPs such as shaped monomer that promotes the formation of spherical
quantum dots.451 Although this promising system has only so far architectures, with a CAC of 0.5 mM prior to crosslinking.434 The
been demonstrated in vitro, it offers an attractive means through formation of interchain disulphide linkages between adjacent
which to produce simple, photo-stable, biocompatible NPs for cysteines led to the production of stable vesicles, and enabled
in vivo imaging in the future. cellular delivery of encapsulated photosensitizers.457
An alternative approach to stabilise self-assembled peptide
8.2 Peptide amphiphiles architectures is to incorporate non-native functionalities. In its
Amphiphilic peptides, containing both a hydrophobic and simplest form, this can involve the incorporation of unnatural
hydrophilic domain, can be broadly split into three categories: amino acids, as reported by Tanisaka et al., providing increased
(i) those composed only of native amino acids (with or without hydrophobic interactions as well as resistance to proteases
minor modifications at the termini); (ii) those containing in vivo.458 Stable vesicles composed of hydrophilic sarcosine
unnatural amino acids; and (iii) lipid- or polymer-conjugated and hydrophobic methyl glutamate residues were shown to
peptide hybrids. In all three groups, self-assembly into spherical accumulate in cancer tissue as a result of the EPR effect in
particles is typically driven by a mixture of hydrophobic and animal xenografts. Taking this concept further, the formation
electrostatic interactions, with the formation of secondary struc- of lipid- or polymer-hybrid peptides has become an attractive
tures possible in some cases.427,431 means by which to drive peptide self-assembly.459,460 Liu et al.
The formation of nano-vesicles composed entirely of native demonstrated that addition of cholesterol to the end of a hydro-
amino acids, referred to in some instances as ‘peptosomes’, philic hexaarginine-TAT peptide block drove micelle formation

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(CAC = 10.1 mM).193 These structures were able to preferentially

disrupt bacterial cell membranes and thus act as antimicrobial
agents in vivo, though it is important to note that host haemo-
lysis is a likely side-effect at higher concentrations. Similarly,
Lv et al. have shown that triblock PEG–polyphenylalanine–
polyglutamic acid polymer–peptide hybrids are able to form
stable stealth nanoparticles, for the delivery of encapsulated
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doxorubicin to tumours (CAC = 2.6 mM).461 Upon glutamic acid

protonation in the increasingly acidic environment of maturing Fig. 20 (a) Linear peptide, composed of a pentameric coiled-coil repeat
from COMP and a de novo designed trimeric coiled-coil repeat, bearing a
endosomes, disassembly and subsequent cargo release led to
terminal SARS antigen; Peptides can self-assemble into an antigen dis-
tumour apoptosis. playing NP in a 60 (b) or 180 (c) peptide chain icosahedron. Adapted from
Pimentel et al. with permission from John Wiley and Sons.474
8.3 Dendrimers
As an extension to the use of linear peptide amphiphiles,
higher order, repetitively branched peptide dendrimers with designed peptide that formed a self-assembled trimer. This
increased bulk and a unique globular architecture have also peptide was able to undergo self-assembly into regular, poly-
found utility. On their own, dendrimers do not typically interact hedral NPs, and display multiple copies of a bioactive species
sufficiently to form stable self-assembled structures. In a series on the particle surface.473 As described in Section 7, such a
of papers the Gu group have developed an elegant solution to display is capable of effectively mimicking pathogen antigen
this barrier, through the use of a cooperative self-assembly presentation. Coiled-coil NPs have subsequently therefore been
process. Hydrophilic polylysine dendrimers are first electro- demonstrated to act as novel immunogens for the production
statically bound to a linear hydrophobic peptide bearing a of vaccines, for diseases including severe acute respiratory
negatively charged C-terminal glutamate residue. This amphiphilic, syndrome (SARS),474 malaria,475 and HIV476,477 (Fig. 20). Further
supramolecular dendrimer can then further self-assemble into developments which enhance NP stability, including the addition
nano-sized micelles, capable of encapsulating a hydrophobic drug of lipid tails,477,478 and the formation of elastin-like peptide
or DNA cargo.462 Under weakly acidic endosomal conditions hybrids (as discussed below),327 have additionally been reported,
following uptake, glutamate protonation leads to disassembly of bringing CACs into the low nM range. Importantly, the coiled-coil
the nanostructure, and release of the cargo.463 These dendritic core has been shown to only be weakly immunogenic itself. As a
systems have been used to successfully deliver the anti-tumour result, Abs generated against these NPs are predominantly
drug doxorubicin in vivo through passive targeting,464 and more targeted towards the displayed antigen, though low titres against
recently to allow directed delivery by exploiting the modularity of the particle have also reportedly been generated.476,478,479
dendrimer assembly to enable surface functionalisation.465
8.5 Peptide–nucleic acid complexes
8.4 Coiled-coil peptides The non-viral delivery of DNA or RNA to cells is a powerful
Along with b-sheets, a-helices are the most common secondary emerging technique for the treatment of disease. A number
structures adopted by polypeptides. Hydrogen bonding between of treatments based around the delivery of plasmid DNA, or
the backbone amide oxygens and the N–H bond 4 residues away antisense, silencing, or micro RNAs are currently in the clinic,
induces a right-handed helical structure in which the amino acid with an even greater number undergoing advanced clinical
sidechains are directed away from the core. Further stabilisation trials.480–483 Positively-charged peptides, able to self-assemble
can be achieved through the subsequent assembly of multiple into compact NPs with negatively charged nucleic acids, are
peptides to form coiled-coil motifs, whereby multiple helices particularly attractive. By exploiting the research discussed
wrap around each other into a superhelical bundle.466 Assembly throughout this review, peptide vectors have been shown to
is driven first by the exclusion of hydrophobic residues from protect the DNA/RNA cargo from damage, enable targeting of
the aqueous exterior, and then stabilised and enhanced by inter- the desired cell type, and perhaps most importantly facilitate
strand electrostatic interactions between polar and charged intracellular delivery and endosomal escape, as described in
amino acids.467 The resultant assemblies offer a powerful means Section 5.484 Indeed, the same cationic residues that enable
to produce complex bioactive materials that have found use NP formation, such as lysine and arginine, are also those able
across a range of disciplines.466,468–470 to promote cell penetration.
Coiled-coil peptides were first used in the nanotechnology Wu and Wu were amongst the first to report peptide–nucleic
field by Stevens et al. to drive the assembly of AuNPs into higher acid NP mediated gene delivery. Complexes of plasmid DNA
order aggregates.471 The formation and application of NPs and polylysine were shown to efficiently deliver their cargo to
composed entirely of coiled-coil peptide structures was sub- cells in vitro.485,486 Many subsequent reports have focussed on
sequently driven by the group of Burkhard. In 2006 they the ability of peptide–nucleic acid complexes to transfect iso-
reported the rational structure-based design of a linear peptide, lated cells, rather than being applied in vivo. This is in part due
composed of the pentameric repeat forming coiled-coil domain to the trade-off between the toxicity associated with longer
of cartilage oligomeric matrix protein (COMP),472 and a de novo cationic peptides, and the low particle stability when shorter

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leading to a decrease in Tt, and conversely, hydrophilic residues

leading to a corresponding increase.
The aggregation of ELPs can be exploited to form spherical,
nano-sized cargo-delivery vehicles via a number of different
strategies. The simplest realisation of such technologies
relies on the passive accumulation of soluble ELP monomers
within tumours via the EPR effect. Through suitable design
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of the sequence, Tt can be modulated to be slightly higher

than body temperature. Peptide self-assembly can then be
driven at the site of interest through the application of localised
hyperthermia.498–500 During this process, NP assemblies are
first formed which are able to promote cellular uptake, followed
by the subsequent formation of larger aggregates which lead to
retention at the site of heating.500 Importantly, such strategies
require a precise control over the concentration of systemically-
injected ELPs – too high, and off-target aggregation can result,
too low, and no assembly will be observed upon heating.501 In
spite of this sensitivity, the hyperthermia induced assembly of
ELP NPs can still be used enhance the activity of attached drugs
Fig. 21 (a) Self-assembly of tumour-homing arginine-rich peptide such as doxorubicin, promoting both retention and uptake at
hybrids with siRNA creates targeted siRNA encapsulated NPs; (b) particle the site of heat application.502
delivery enables accumulation and siRNA delivery to tumour tissue. The use of amphiphilic ELP structures that can form stable
Adapted from Wang et al. with permission from The Royal Society of
micelles above the Tt has more recently been reported, bringing
Chemistry.495
CACs into the high nM–low mM range. In an early example,
Dreher et al. demonstrated that the attachment of hydrophobic
sequences are used instead.487 Stabilisation through both doxorubicin (through an endosome cleavable hydrazone bond)
dialdehyde crosslinking487 and disulphide formation488,489 has could itself drive micelle formation.503 MacKay et al. subsequently
been reported, however the most common means of enabling showed that by tuning Tt, stable structures with low CACs could
in vivo application is the use of more complex peptide substrates be generated and used to effectively treat tumours in vivo
capable of lowering CACs sufficiently to overcome these problems. (Fig. 22).504,505 As an alternative, the formation of diblock
In an early example, Rittner et al. replaced acidic residues in the ELP polymer can be used to drive assembly, with each block
amphiphilic cell-penetrating peptide JTS1 with cationic lysines possessing a unique Tt. When the temperature is above the Tt
or arginines, producing a sequence able to both condense DNA of the first block but beneath that of the second, micelle
and promote efficient systemic transfection in vivo.490 Other formation will occur, which can be further stabilised by the
peptides, such as MPG,491 RALA (an arginine-rich analogue introduction of disulphide crosslinkers.506
of the more commonly utilised endosomal escape peptide ELP-based NPs have been applied in a number of applications
GALA),492 and amphiphilic arginine-containing triblock peptides493 in recent years. In addition to early reports on drug-delivery,
have all found use for the in vivo delivery of nucleic acid ELPs have subsequently been utilised as NIR fluorescent
cargoes. More recently, the Kaplan494 and Zheng495 groups imaging agents,507 microPET contrast agents,508 and synthetic
have reported the targeted delivery of peptide–nucleic acid vaccines.509 Furthermore, the attachment of multi-valent tumour
NPs, via the introduction of a tumour-specific homing peptide targeting sequences,510 cell-penetrating peptides,511,512 and
(Fig. 21). In both cases, preferential accumulation and sub- pH-responsive ELP cores513 have all been shown to enhance
sequent nucleic acid delivery in the tumour tissue was achieved the applicability of ELP-nanostructures in biomedicine.
following intravenous injection. This was observed to prevent
the potential side-effects that can occur as a result of systemic 8.7 Casein micelles
transfection. Casein is the collective term for a family of phosphorylated
proteins commonly found in milk. These proteins have a well-
8.6 Elastin-like polypeptides defined, hydrophilic N-terminal domain, and a hydrophobic
Elastin-like polypeptides (ELPs) are biosynthesised polymeric- C-terminal domain, creating a structure that can in essence be
peptide repeats, typically of the pentameric sequence (VPGXG)n, viewed as an amphiphilic diblock copolymer.514 Unlike other
where X and n can be varied to ultimately determine the proper- proteins which may form globular aggregates, caseins are able
ties of a particular ELP construct. All ELPs possess an ‘inverse to undergo controlled self-assembly to form stable micelle-like
transition temperature’ (Tt), above which they undergo a sharp structures in aqueous solution with a CAC of 20–80 mM.515 This
phase transition from a highly solvated peptide monomer to a is particularly true when individual casein family members,
desolvated aggregate.496,497 The exact properties are determined most notably b-casein, are used in isolation rather than as the
by the so-called ‘guest-residue’, with hydrophobic residues naturally occurring mixture.515

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Casein micelles have recently been the subject of increasing

interest, as naturally occurring nano-delivery vehicles for a
range of hydrophobic cargoes. The use of casein NPs to improve
the bioavailability of supplemented vitamin D, prior to release
following protease action in the stomach, has been the subject of
human clinical trials.516 Similarly, the delivery of hydrophobic
therapeutics, particularly for the treatment of stomach cancers,
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has been widely studied by the Livney group.515,517–519 Finally,

the tumour accumulation of intravenously injected, cisplatin
containing casein-micelles has been shown to result in improve-
ments in therapeutic outcome in vivo (Fig. 23).520 These promising
early reports, coupled to the ease of use, inexpensive production,
bio-degradable, non-toxic and non-immunogenic nature of casein
micelles makes them particularly appealing as delivery-vehicles. It
is likely that the coming years will see such structures finding
increasing utility in biomedicine.

9. Sensing analytes and biomarkers

NP biosensing complexes offer a powerful means by which to
detect and monitor disease, and to understand pathological
conditions. This is usually achieved using optically active NPs,
though other sensing modalities can also be utilised.33 The
most common sensing platforms rely on a change in optical
properties in the presence of the desired analyte or biomarker, or
Fig. 22 (a) Conjugation of multiple doxorubicin groups at the terminus selective binding of a NP complex within a ‘detection-region’.521,522
of a hydrophilic ELP generates an amphiphilic linear peptide that can Peptides and proteins are vital components of such systems,
self-assemble into stable micelles with a drug rich hydrophobic core; inducing NP binding or mediating a responsive output to the
(b) tumour growth in mice is greatly reduced when doxorubicin (Dox) is
presence of the desired analyte. NP biosensors are most widely
conjugated to ELP in the form of micelles via an acid cleavable hydrazone
bond, when compared to treatment with free doxorubicin. Adapted from utilised for the ex vivo analysis of biofluids, as epitomised by the
Mackay et al. with permission from Nature Publishing Group.504 use of anti-human chorionic gonadotropin (HCG) antibody coated

Fig. 23 NIR fluorescence imaging of tumour xenograft bearing mice, injected with cisplatin loaded casein NPs. Gradual accumulation within the tumour
is enabled by the EPR effect and long circulation time of casein particles. Reproduced from Zhen et al. with permission from Elsevier.520

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AuNPs in commercially available lateral flow pregnancy tests.523 nanoworms.532 Due to their size, these particles were blocked
The development of NP-based biosensors has become a vast from renal clearance and displayed accumulation in the liver.
field of research, and a detailed overview of ex vivo technologies However, following protease activity, the peptide cleavage
is outside the scope of this review. The reader is instead referred products were excreted in the urine, and could subsequently
to a number of excellent comprehensive reviews on the topic for be detected by mass spectrometry, giving a panel-readout of
further details.33,524–527 Instead, we will here focus on the far in vivo protease levels. Subsequent iterations of this technology
smaller body of literature that focusses on the application of have demonstrated the detection of thrombosis and colorectal
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NP-biosensors in vivo, and the challenges that have so far cancer using peptides terminated with recognition elements
limited their widespread implementation. Such systems allow that can subsequently be detected in urine by enzyme-linked
the true complexity of tissues to be captured in a way that immunosorbent assays, or point-of-care lateral flow detection
in vitro testing of biofluids cannot provide.528 systems.533,534 In order to overcome the challenges presented
Sensing complexes provide a means to interrogate biological by non-specific activation by circulating proteases, Dudani et al.
systems and probe differences in activity and temporal distribu- reported the use of photo-protected peptide–NP coatings which
tions of a desired analyte, as opposed to the spatial localisation could be activated towards protease sensitivity with spatial
provided by imaging modalities. Rather than being detected and temporal control, following the application of UV or two-
within a specific area, in vivo sensors offer a responsive platform photon light.535 Furthermore, the sub-cutaneous implantation
to monitor small molecules, biomacromolecules, and diseased of 8 nm peptide-functionalised PEG–NPs, which enable gradual
states in a continuous manner.528,529 Although implanted electro- leaching of a particle reservoir into the blood stream, has
chemical sensors have been widely described, in particular for the recently been shown to allow the continuous monitoring of
monitoring of blood glucose levels, problems with induced protease activity in vivo, although detection was still limited to a
fibrosis and foreign body responses can limit sensitivity and 24 h time period.535
accuracy.528 NP based systems offer a viable alternative, but also
have their own complications – in addition to the challenges of
generating a signal that can be actively transduced for detection, 10. Outlook and conclusions
NPs must overcome background signal generation within the
complex environment of the body, be retained at the desired site The past 20 years have seen a rapid increase in the development
of detection, and ideally provide a reversible and dynamic of nanotechnologies, and the exploitation of polypeptides in
response. The use of peptide/protein–NP conjugates in particular this context has also found increasing favour. The structural
must address the sensitivity of biological components to degra- and functional versatility of peptides and proteins allows
dation, clearance, and unfavourable interactions.529 As a result, them to play an important role in modulating, instigating,
systems in which peptides and proteins play an active sensing and defining the activity of NP constructs. In this review we
role, rather than mediating imaging or targeting, have only have outlined the key roles played by polypeptide coatings and
recently begun to emerge. structural components, and demonstrated how they can be
In one realisation of such technologies, environmentally utilised to improve the efficacy of NP tools in biomedical
sensitive polymer NPs have been exploited to detect differences applications. In many cases, both peptides and proteins can
in analyte concentration as a result of conjugated enzyme activity. fulfil the desired function. Each offers important advantages
More specifically, polymer NPs incorporating platinum based over the other (though exceptions to these generalisations exist)
fluorescent sensors have been shown to display phosphorescence that should be carefully considered during NP-conjugate design
dependent on environmental oxygen concentrations. Cash and (Fig. 24). For example, the ease with which peptides can be
Clark demonstrated that by conjugation of the histamine- produced via solid-phase peptide synthesis (SPPS) is highly
metabolising, oxygen-consuming enzyme diamine oxidase to the beneficial. As well as offering cost-effective, quick, and scalable
NP surface, biologically relevant levels of this key inflammatory- production, the versatility of SPPS enables unnatural chemical
and neuro-modulator could be detected by a change in space to be explored. As such, the facile introduction of reactive
phosphorescence.530 This response was reversible upon diffusion handles and protease resistant residues, or structures able to
of oxygen back into the biological milieu, with only a limited promote biological interactions, is enabled (Fig. 24b and c).
dropoff in polymer dynamic range observed upon repeated Furthermore, the small size of peptides enables the disruption of
applications. Similarly, Sun et al. monitored glucose levels NP hydrodynamic diameter and detrimental effects on activity to
in vivo using a glucose oxidase-functionalised oxygen–polymer be minimised, while creating a dense, accessible, and flexible
NP transducer.531 Subcutaneous implantation enabled reten- coverage of active sequences (Fig. 24a and d). In contrast, the
tion of the particles for up to a month, enabling reproducible increased structural complexity offered by proteins enables
signal generation over this extended period. NP-conjugates to typically attain increased activity and recogni-
As an alternative, the Bhatia group has pioneered the use of tion, albeit potentially at the cost of decreased biological
protease-sensitive NP coatings, which release peptide cleavage stability and increased recognition by the reticuloendothelial
products into the urine upon enzymatic activity in vivo. In their system (Fig. 24e). In addition, small structural modifications
original report, Kwong et al. conjugated peptides sensitive to a to proteins are usually well tolerated, without leading to major
range of common proteases onto the surface of iron oxide disruptions in activity. Corresponding changes in peptide

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Fig. 24 Making the choice between a peptide– or protein–NP conjugate is an important decision during the design of biomedical nanotechnologies.
Both have significant advantages over the other, as well as drawbacks which may limit their applicability in certain scenarios. It is therefore imperative to
consider the interaction of the polypeptide component with the NP surface, core, and cargo, as well as the end application, interplay with natural
systems, and route of administration: (a) the small size of peptides allows a high surface density of coating to be achieved; (b) solid-phase peptide
synthesis enables the straightforward production of large volumes of short peptides, as well as (c) enabling the facile introduction of unnatural amino
acids, backbones, and architectures; (d) multiple peptides can be introduced within a single NP construct, enabling multi-functional coatings to be
accessed; (e) proteins typically display enhanced bioactivity and binding affinity when compared to peptide substrates, though many examples of potent
peptide substrates also exist; (f) the high molecular weight of proteins leads to strong non-covalent interactions which can drive the formation of stable
self-assemblies; (g) single amino acid alterations to protein sequences lead to significantly less structural disruption when compared to the analogous
peptide substrate.

sequence have a stronger influence on conformation and by the nanotechnology field.536 Within the realm of peptide/
activity, potentially leading to a significant drop in the efficacy protein–NP conjugates, we believe that addressing the follow-
of the nanotechnology (Fig. 24g). The situation is further ing issues is vital to enable translation of these naı̈ve systems to
complicated by the complex interplay between peptide/protein a biomedical setting:
components and the particular NP core, cargo, application, and (i) The use of native amino acids brings with it the threat of
delivery route. As such, consideration of the precise polypeptide protease sensitivity, and activity can often be rapidly lost in the
component is a vital step during NP-conjugate design. complex environments faced in vivo. In this review, a number of
Despite the advances that have been made in enabling approaches have been introduced towards addressing these
effective and targeted delivery to the desired site of action, difficulties. Unnatural amino acids are particularly effective,
providing an integral means to maintain activity, and acting to providing peptide bonds which are not recognised and processed
hijack and exploit native biological pathways, there remains by native enzymes. This is most easily achieved with synthetic
significant scope for the improvement of polypeptide–NP tech- peptides, as discussed above – relatively simple modifications
nologies. In a recent editorial, Leroux highlighted that while the such as N-methylation, enantio- or retro-enantio amino acids,
number of papers reporting increasingly creative and complex or extended b- or g-linkages can be introduced.217,537,538 While
NP systems increases exponentially, their therapeutic transla- increased stability is harder to achieve with protein substrates,
tion remains disappointing, highlighting the challenges faced the advent of technologies which allow the introduction of

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non-natural stabilising residues,539,540 the addition of protec- (v) Proper and standardised characterisation of NP–peptide/
tive polymer coatings,541,542 or the formation of additional protein conjugates is essential to not only allow progression in the
cross-links543,544 are now allowing such issues to be addressed. field, but to enable regulatory and clinical standards to be met.172
In order to fully facilitate the translation of NP-based systems, it This challenge must be addressed by both the development of new
is important that the research community begins to actively tools which are able to reflect the true nature of NP complexes, as
embrace such technologies. well as increased uptake by researchers of existing technologies, and
(ii) The small size of peptides allows them to form compact NP a more thorough approach to NP characterisation being taken.552,553
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coatings, which lead to minimal disruption of desired function. At present, many systems reported in the literature are inadequately
They are therefore often the preferred choice when designing a NP– characterised, leading to large discrepancies in results, conflicting
polypeptide conjugate, with the major drawback being reduced reports, and incorrect interpretations that subsequently propagate
bioactivity (Fig. 24a and e). Nature has had millions of years to throughout the field. The inherent complexity of NP-bioconjugates
slowly evolve proteins with complex 3D structures and potent makes them undoubtedly challenging to study – even what appears
activity, and it remains challenging for designed, screened, or a simple measurement such as the concentration of particles in
derived peptides to fully recapitulate this. Nowhere is this more solution is often difficult.172 This becomes even more significant for
strongly felt than in the reduced ability of short synthetic peptides more complex calculations such as the density or orientation of a
(o30 amino acids) to target and bind a particular protein or cell type protein on a NP surface, or the origins of biological effects arising
when compared to Abs or other binding proteins. Although strongly from heterogeneous samples. However, novel tools that can answer
binding long peptides such as affibodies (450 amino acids) can be these questions are beginning to emerge, and although often highly
accessed synthetically, more typically they are produced recombi- specialised, are beginning to be readily accessible to researchers,
nantly and still result in a bulky coating on the NP surface.545 There through national and international facilities for NP research and
is a therefore a pressing need for technologies which can bring short characterisation.553–556
peptide binding affinities down from the mM to the nM range.546 A big leap towards enabling the clinical translation of many
Recent advances in the screening, design, and exploitation of peptide/protein–NP conjugate technologies will be taken as the
bicyclic peptides are enabling such levels to be reached, and will biomedical community addresses these issues. As a result, our
surely be exploited in the near future to enhance the ability of ability to treat, diagnose, and understand disease will be greatly
NP-targeting strategies to achieve their goals.547–549 enhanced, allowing nano-technology to fulfil its undoubted
(iii) The bioactivity of a peptide or protein–NP conjugate is promise in the biomedical sciences.
highly dependent on achieving an appropriate and accessible
orientation of the bioactive domain. Again, this can be easier to
achieve using peptides rather than protein substrates – tethering Conflicts of interest
residues can be easily introduced at the termini during solid There are no conflicts to declare.
phase peptide synthesis. Often protein substrates are attached
either by non-specific conjugation techniques or simple adsorp-
tion, leading to a detrimental loss of structure, heterogenous Acknowledgements
coatings, and steric hindrance of the active site.375 It is essential
that recent developments in the field of site-specific or selective Drs Mattias Björnmalm and Michael Thomas are thanked for
protein modification are exploited to overcome these problems, to critical evaluation of the manuscript. C. D. S. and M. M. S.
enable the controlled orientation of proteins on the NP surface. acknowledge support from the Swedish Research Council (VR
Strategies for genetically or chemically manipulating proteins to 4-478/2016) and the Swedish Foundation for Strategic Research
introduce and exploit uniquely reactive natural and non-natural (SSF 4-3713/2016). C. J. and M. M. S. acknowledge financial
amino acids, or to incorporate hexahistidine and other affinity support from the Rosetrees Trust. B. G. and M. M. S. acknowledge
tags, are becoming increasingly widespread and offer powerful support from GlaxoSmithKline through the Imperial College
tools to the biomedical research community.342,550,551 London Engineered Medicines Laboratory Project. M. M. S.
(iv) Many of the applications for peptide/protein–NP conjugates acknowledges support from the ERC Seventh Framework
discussed in this review rely on processes that are still poorly Programme Consolidator grant ‘‘Naturale CG’’ (616417), the
understood at a fundamental level. Controversy still exists about Engineering and Physical Science Research Council (EPSRC) grant
the precise mechanisms by which peptides mediate transport across ‘‘Bio-functionalised nanomaterials for ultrasensitive biosensing’’
biological barriers,145 coatings and particle components which were (EP/K020641/1), the i-sense EPSRC IRC in Early Warning Sensing
previously thought to be biologically inert are becoming increasingly Systems for Infectious Diseases (EP/K031953/1), and the Well-
found to induce an immunological response,66 and the design come Trust Senior Investigator Award (098411/Z/12/Z).
criteria required to produce stable self-assembled polypeptide NPs
are still being developed.436 In order to provide NP technologies that References
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