Nano For Biomimetics

Journal of Nanomaterials
Nano for Biomimetics

and Biomaterials
Guest Editors: Il-Kwon Oh, Anchal Srivastava, In-Kyu Park,
and Michael Z. Hu
Nano for Biomimetics and Biomaterials
Guest Editors: Il-Kwon Oh, Anchal Srivastava, In-Kyu Park,

and Michael Z. Hu
Copyright © 2014 Hindawi Publishing Corporation. All rights reserved.
This is a special issue published in “Journal of Nanomaterials.” All articles are open access articles distributed under the Creative Com-
mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Editorial Board
Katerina E. Aifantis, Greece Alan K. T. Lau, Hong Kong Somchai Thongtem, Thailand
Nageh K. Allam, USA Burtrand Lee, USA Alexander Tolmachev, Ukraine
Margarida Amaral, Portugal Benxia Li, China Valeri P. Tolstoy, Russia
Xuedong Bai, China Jun Li, Singapore Tsung-Yen Tsai, Taiwan
Lavinia Balan, France Xing-Jie Liang, China Takuya Tsuzuki, Australia
Enrico Bergamaschi, Italy Shijun Liao, China Raquel Verdejo, Spain
Theodorian Borca-Tasciuc, USA Gong Ru Lin, Taiwan Mat U. Wahit, Malaysia
C. Jeffrey Brinker, USA J. -Y. Liu, USA Shiren Wang, USA
Christian Brosseau, France Tianxi Liu, China Yong Wang, USA
Xuebo Cao, China Songwei Lu, USA Ruibing Wang, Canada
Shafiul Chowdhury, USA Daniel Lu, China Cheng Wang, China
Kwang-Leong Choy, UK Jue Lu, USA Zhenbo Wang, China
Cui ChunXiang, China Ed Ma, USA Jinquan Wei, China
M. A. Correa-Duarte, Spain Gaurav Mago, USA Ching-Ping Wong, Hong Kong
Shadi A. Dayeh, USA Santanu K. Maiti, India Xingcai Wu, China
Claude Estourns, France Sanjay R. Mathur, Germany Guodong Xia, Hong Kong
Alan Fuchs, USA Vikas Mittal, United Arab Emirates Zhi Li Xiao, USA
Lian Gao, China Weihai Ni, Germany Ping Xiao, UK
Russell E. Gorga, USA Sherine Obare, USA Shuangxi Xing, China
Hongchen Chen Gu, China Atsuto Okamoto, Japan Yangchuan Xing, USA
Mustafa O. Guler, Turkey Abdelwahab Omri, Canada N. Xu, China
John Zhanhu Guo, USA Edward Andrew Payzant, USA Doron Yadlovker, Israel
Smrati Gupta, Germany Kui-Qing Peng, China Yingkui Yang, China
Michael Harris, USA Anukorn Phuruangrat, Thailand Khaled Youssef, USA
Zhongkui Hong, China Mahendra Rai, India Kui Yu, Canada
Michael Hu, USA Suprakas Sinha Ray, South Africa William W. Yu, USA
David Hui, USA Ugur Serincan, Turkey Haibo Zeng, China
Y.-K. Jeong, Republic of Korea Huaiyu Shao, Japan Tianyou Zhai, Japan
Sheng-Rui Jian, Taiwan Donglu Shi, USA Renyun Zhang, Sweden
Wanqin Jin, China Vladimir Sivakov, Germany Bin Zhang, China
Rakesh K. Joshi, UK Marinella Striccoli, Italy Yanbao Zhao, China
Zhenhui Kang, China Bohua Sun, South Africa Lianxi Zheng, Singapore
Fathallah Karimzadeh, Iran Saikat Talapatra, USA Chunyi Zhi, Hong Kong
Alireza Khataee, Iran Nairong Tao, China
Do Kyung Kim, Republic of Korea Titipun Thongtem, Thailand
Contents
Nano for Biomimetics and Biomaterials, Il-Kwon Oh, Anchal Srivastava, In-Kyu Park, and Michael Z. Hu
Volume 2014, Article ID 485642, 1 page
A Facile Nanodelivery Platform Based on Functionalized Hyperbranched Poly(ether-ester) for

Individualized Antitumor Drugs: Pingyangmycin as a Model, Xing-ai Jin, Yan-wu Li, Guo-lin Li,
Shao-hua Lv, Ying-qun Liu, and Li-min Mao
Volume 2014, Article ID 601272, 10 pages
Nanostructural Colouration in Malaysian Plants: Lessons for Biomimetics and Biomaterials,

S. Zaleha M. Diah, Salmah B. Karman, and Ille C. Gebeshuber
A Dual-Functional [SBA-15/Fe3 O4 /P(N-iPAAm)] Hybrid System as a Potential Nanoplatform for

Biomedical Application, Andreza de Sousa, Karynne Cristina de Souza, Paula Maria da Silva Leite,
Ricardo Geraldo de Sousa, and Edésia Martins Barros de Sousa
Characterization of the Casein/Keratin Self-Assembly Nanomicelles, Su Xiao-Zhou, Wang Hong-Ru,

and Huang Mian
Nanopigmented Acrylic Resin Cured Indistinctively by Water Bath or Microwave Energy for Dentures,
L. S. Acosta-Torres, M. C. Arenas, R. E. Nuñez-Anita, F. H. Barceló-Santana, C. A. Álvarez-Gayosso,
J. Palacios-Alquisira, J. de la Fuente-Hernández, Marcos Cajero-Juárez, and V. M. Castaño
A New Method for Fabrication of Nanohydroxyapatite and TCP from the Sea Snail Cerithium vulgatum,
O. Gunduz, Y. M. Sahin, S. Agathopoulos, B. Ben-Nissan, and F. N. Oktar
Cationic Gelatin Nanoparticles for Drug Delivery to the Ocular Surface: In Vitro and In Vivo
Evaluation, Ching-Li Tseng, Ko-Hua Chen, Wen-Yu Su, Yen-Hsien Lee, Chi-Chang Wu, and Fen-Huei Lin
Development of Antibiotics Impregnated Nanosized Silver Phosphate-Doped Hydroxyapatite Bone

Graft, Waraporn Suvannapruk, Faungchat Thammarakcharoen, Phetrung Phanpiriya,
and Jintamai Suwanprateeb
Iron Oxide Magnetic Nanoparticles: Characterization and Toxicity Evaluation by In Vitro and In Vivo
Assays, Alina Mihaela Prodan, Simona Liliana Iconaru, Carmen Steluta Ciobanu,
Mariana Carmen Chifiriuc, Mihai Stoicea, and Daniela Predoi
Electrospun Hyaluronan-Gelatin Nanofibrous Matrix for Nerve Tissue Engineering, Hau-Min Liou,
Lih-Rou Rau, Chun-Chiang Huang, Meng-Ru Lu, and Fu-Yin Hsu
Investigation of the In Vitro Degradation of a Novel Polylactide/Nanohydroxyapatite Composite for

Artificial Bone, Jianghong Huang, Jianyi Xiong, Jianquan Liu, Weimin Zhu, and Daping Wang
A Study on the Effect of Plasma Treatment for Waste Wood Biocomposites, MiMi Kim, Heung Soo Kim,
and Joong Yeon Lim
Water Absorption and Diffusion Characteristics of Nanohydroxyapatite (nHA) and

Poly(hydroxybutyrate-co-hydroxyvalerate-) Based Composite Tissue Engineering Scaffolds and
Nonporous Thin Films, Naznin Sultana and Tareef Hayat Khan
Hindawi Publishing Corporation
Volume 2014, Article ID 485642, 1 page
http://dx.doi.org/10.1155/2014/485642
Editorial
Il-Kwon Oh,1 Anchal Srivastava,2 In-Kyu Park,3 and Michael Z. Hu4

1
Graphene Research Center, School of Mechanical, Aerospace and Systems Engineering, Division of Ocean Systems Engineering,
Korea Advanced Institute of Science and Technology (KAIST), 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
2
Department of Physics, Banaras Hindu University, Varanasi 221005, India
3
Department of Biomedical Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu,
Gwang-Ju 500-757, Republic of Korea
4
Oak Ridge National Laboratory, 4500N, A34, MS 6181, Oak Ridge, TN 37831-6181, USA
Correspondence should be addressed to Il-Kwon Oh; ikoh@kaist.ac.kr
Received 3 April 2014; Accepted 3 April 2014; Published 29 June 2014
Copyright © 2014 Il-Kwon Oh et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This special issue has been considered as a necessary Acknowledgment

technology which can overcome the limitations and problems
of other modern technologies and lead to a new industrial The guest editors and other colleagues from various institu-
revolution. The future promise of nanotechnology depends tions offered invaluable support for peer-reviewing and the
on its high multidisciplinary nature and shared knowledge successful completion of this special issue. We sincerely thank
and information from various fields which can be merged all reviewers and authors of this special issue.
and used to evolve as a revolutionary new technology. Con-
Il-Kwon Oh
sidering the potential impact of nanotechnology on future
Anchal Srivastava
industry, many countries are investing huge research funds
In-Kyu Park
and resources in the field of nanotechnology as one of their
Michael Z. Hu
top research priorities.
The aim of the special issue is to share the novel
knowledge covering the state of the art on biomimetics and
nanobiomaterials providing an overview on their potential
applications in the industrial, biomedical, and robotic fields.
The research topics covered in the special issue include
bioinspired materials, devices, structures, and graphene-
based materials. The special issue will present current status
of the fields of biomimetics and biomaterials. This special
issue will be a necessary platform for ongoing studies between
researchers from different areas (chemistry, physics, biology,
medicine, engineering, robotics, etc.) within biomimetic and
biomaterial technologies.
We hope that this special issue reflects the current state
of nanotechnology for biomimetics and biomaterials and will
be a useful reference for researchers working in this research
field.
http://dx.doi.org/10.1155/2014/601272
Research Article
A Facile Nanodelivery Platform Based on Functionalized
Hyperbranched Poly(ether-ester) for Individualized Antitumor
Drugs: Pingyangmycin as a Model
Xing-ai Jin,1 Yan-wu Li,2 Guo-lin Li,3,4 Shao-hua Lv,3,4 Ying-qun Liu,1 and Li-min Mao3
1
Department of Pediatrics, College of Stomatology, Harbin Medical University, 23 Youzheng Street, Harbin 150001, China
2
Department of Periodontology, College of Stomatology, Harbin Medical University, 23 Youzheng Street, Harbin 150001, China
3
Department of Oral and Maxillofacial Surgery, College of Stomatology, Harbin Medical University, 23 Youzheng Street,
Harbin 150001, China
4
State Key Laboratory of Metal Matrix Composites, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,
800 Dongchuan Road, Shanghai 200240, China
Correspondence should be addressed to Li-min Mao; hrbmlm@126.com
Received 14 December 2013; Revised 18 February 2014; Accepted 7 March 2014; Published 26 June 2014
Academic Editor: In-Kyu Park
Copyright © 2014 Xing-ai Jin et al. This is an open access article distributed under the Creative Commons Attribution License,
Nanodelivery of antitumor drugs is a new treatment mode for cancer. The aim of this investigation was to construct and evaluate
a facile nanodelivery platform for individualized antitumor drugs based on functionalized hyperbranched poly(ether-ester)s.
Poly(ether-ester)s, as a kind of hyperbranched polymers, have received extensive attention. Three terminal-functionalized (OH–,
NH2 – and COOH–) hyperbranched poly(ether-ester)s were prepared and characterized by dynamic light scattering and attenuated
total reflectance Fourier transform infrared spectroscopy. The relationship between chemical terminal variation and physical
surface charges was investigated. Biocompatibility of these polymers was confirmed by methyl tetrazolium assays and scanning
electron microscopy. As a model drug, pingyangmycin has antitumor and antiangiogenic effects. In the paper, pingyangmycin
was mixed with carboxyl-modified hyperbranched poly(ether-ester) through ionic binding. Polymer-mixed pingyangmycin
exhibited significant inhibition of HN-6 head and neck cancer human cells in vitro. These studies demonstrate that functionalized
hyperbranched (ether-ester)s can be exploited as a facile nanodelivery platform for antitumor therapy.
1. Introduction methacrylate, polyphenylene, poly(ether ketone), polyester,

and polyurethane [11]. As a novel generation of dendritic
Although nanodelivery of antitumor drugs has numerous polymers, hyperbranched polymers (HBP), which have char-
advantages, such as improved solubility [1], accurate target- acteristic incomplete branching and irregular dimensions,
ing [2–5], increased permeability of tumor vasculature to provide a facile substitute for the preparation and screening
macromolecules, and decreased lymphatic drainage from the of nanocarriers. Distinct from their linear analogues, hyper-
tumor; the complicated techniques involved in nanodelivery branched polymers have structures and topologies similar
development have impeded individualized nanodelivery of to dendrimers and possess some strikingly superior material
numerous antitumor drugs. Even dendritic polymers, which properties [9, 10]. Due to their low dispersity and excel-
have been used for nanodelivery for years, involve compli- lent biocompatibility and biodegradability [1, 12–16] hyper-
cated techniques and high costs associated with the crystal branched polymers have been applied in the field of phar-
architecture [6, 7]. maceutical delivery. In our previous studies, hyperbranched
Over the past two decades hyperbranched polymers have poly(ether-ester) (HPEE) was prepared, characterized [17,
received a great deal of attention [8–10]. Since the first inten- 18], and applied as a nanocarrier of antitumor drug [1, 19]. In
tional preparation of hyperbranched polymers, many types the present study, a series of terminal-functionalized deriva-
have been synthesized, including polyimide, polyether, poly- tives were established based on the HPEE backbone, thus
2 Journal of Nanomaterials
providing a facile platform for optimization of individualized 50 units/mL streptomycin) at 37∘ C in a humidified atmo-
antitumor drug delivery. Pingyangmycin is a water-soluble sphere containing 5% CO2 . After 48 h logarithmic growth the
glycopeptide produced by Streptomyces pingyangensisn. It is attached cells were collected by enzymatic digestion (0.25%
chemically similar to bleomycin with antitumor and antian- pancreatin and 0.02% EDTA) for further assay. HN-6 cancer
giogenic effects. In this paper pingyangmycin is used as an cells (a human head and neck squamous carcinoma cell
example to assess the efficacy of this nanodelivery platform. line) were cultured in PRMI-1640 supplemented with 10%
FBS and antibiotics (200 units/mL penicillin and 50 units/mL
2. Materials and Methods streptomycin) at 37∘ C in a humidified atmosphere containing
5% CO2 . Using enzymatic digestion (0.25% pancreatin and
2.1. Synthesis of End-Functionalized HPEE Derivatives. A 0.02% EDTA), cells were passaged with a 1 : 3 ratio every 2-3
suspension of potassium hydride (KH, Aldrich) in min- days for numerous cell generations.
eral oil (30% in weight) was placed in a dry preweighed
100 mL Schlenk flask under nitrogen. The mineral oil was 2.4. In Vitro MTT Assay for Cytotoxicity Assessment. In vitro
removed by three extractions with tetrahydrofuran (THF), cytotoxicity of a serial dilution of polymer solution against
which was added to the flask by syringe. Completely dried NIH/3T3 cells was measured by the MTT viability assay. Syn-
KH (0.58 g, 14.46 mmol) was added to 40 mL dimethyl thesized HPEE-OH, HPEE-NH2 , and HPEE-COOH were
sulfoxide (DMSO) and tetra (ethylene glycol) (TTEG; 5.62 g, compared with their structural analogues (HPE-OH, HPE-
28.92 mmol) (Aldrich, ShangHai). The solution was stirred NH2 , and HPE-COOH). All solutions were dissolved in PBS
for 30 min to form the alcoholate potassium. Subsequently, with serial dilutions of 0.001 mg/mL, 0.01 mg/mL, 0.1 mg/mL,
glycidyl methacrylate (GMA; 4.12 g, 29.98 mmol) (Sigma, 1 mg/mL, and 10 mg/mL. The same concentration series of
USA) was added and polymerization was conducted for 24 h dextran and PEI were also prepared as negative and positive
at 80∘ C. The resultant mixture was precipitated in 1000 mL controls, respectively. NIH/3T3 cells were seeded into 96-
of acetone/diethyl ether (V/V 1/4) and then redissolved in well plates at a seeding density of 4.0 × 103 cells per well
methanol and neutralized by filtration over cation-exchange in 50 𝜇L. After 24 h of incubation, the culture medium
resin. The polymer was precipitated twice from methanol was removed and replaced with 50 𝜇L polymer solution at
solution into cold diethyl ether and subsequently dried different concentrations. After treatment with polymers for
under vacuum at 50∘ C for 24 h. The purified HPEE-OH 24 h, 48 h, and 96 h, 20 𝜇L of 5 mg/mL MTT stock solution
was obtained as a highly viscous polymer. For HPEE-NH2 in PBS was added to each well. After addition of 200 𝜇L
synthesis, 2 g of HPEE-OH was dissolved into 25 mL of DMF. DMSO to each well and shaking for 5–10 min, the absorbance
Fmoc-glycine (2.97 g, 10 mmol), dicyclohexylcarbodiimide was measured at a wavelength of 490 nm using BioTek
(DCC; 4.13 g, 20 mmol), 4-dimethylaminopyridine (DMAP; SynergyH4. Cytotoxicity was determined by the absorbance
0.61 g, 5 mmol), and hydration p-toluene sulfonic acid (PTSA; relative to the blank control.
0.95 g, 5 mmol) were added to the solution. The mixture The in vitro inhibitory effect of pingyangmycin-mixed
was dissolved in 20% piperidine to remove fmoc-protected polymers against HN-6 cells was also evaluated by MTT
groups. For HPEE-COOH synthesis, 1 g HPEE-OH was dis- assay. After incubation of HN-6 cells (8.0 × 103 cells/well)
solved in 15 mL dichloromethane (CH2 Cl2 ) under moderate for 24 h, the culture medium was removed and replaced
stirring at room temperature. When it was completely dis- with 200 𝜇L of medium containing pingyangmycin-mixed
solved, 1 g of succinic anhydride and 360 𝜇L dried piperidine polymer. Pingyangmycin was tested at serial concentrations
were added to the flask under the same conditions. For com- of 0.01, 0.1, 1.0, 10, and 100 𝜇g/mL.
parison and confirmation purposes, the nonbiodegradable
structural analogue hyper-branched poly(ether) (HPE) and 2.5. Surface Morphological Features of 3T3 Cells. 3T3 cells (2
its functionalized derivatives were concurrently synthesized × 105 /mL) were separately cultured with solutions of three
and analyzed (details are provided in Supplementary Material end-modified HPEE derivatives and three end-modified HPE
available online at http://dx.doi.org/10.1155/2014/601272). The analogues. To strengthen the results, each polymer was tested
same synthesis protocols were used for preparation of HPE and at low concentration (10 𝜇g/mL) and high concentration
its functionalized derivatives. (1 mg/mL) and the corresponding data were compared. Cells
that were simultaneously incubated with HPEE derivatives
2.2. Characterization. Nuclear magnetic resonance (NMR): for 1 h are shown in Figure 4(a) (low concentration sub-
1
H NMR spectra of the polymers were recorded on an group) and Figure 4(b) (high concentration subgroup). At
Advanced III 400 M spectrometer (Bruker, Germany) in D2 O the same time, two control experiments were performed:
as the solvent. Fourier infrared spectra were mefasured on polyethyleneimine (PEI), an accepted cell toxicant, was used
an EQUINOX55 (Bruker, Germany). Respective potentials as a positive control and dextran with polymer structure was
were tested using NaCl titrant (25∘ C, 100 mmol/L, pH = used as a negative control. Normal 3T3 cells incubated with
7) on Malvern Instruments Zesasizer 2000. Dynamic light PBS are also shown as a blank control. Cells of each subgroup
scattering (DLS) was assessed on Zetasizer Nano S (Malvern were collected and fixed with 2.5% glutaraldehyde for 24 h.
Instruments Ltd., Malvern, Worcestershire, UK) at 25∘ C. Morphological features of the cell surface were observed by
scanning electron microscopy (SEM) (FEI Corp. USA).
2.3. Cell Cultures. NIH/3T3 normal cells (a mouse embryonic
fibroblast cell line) were cultured in DMEM supplemented 2.6. Self-Assembly of Pingyangmycin-Mixed Micelle Prepa-
with 10% FBS and antibiotics (50 units/mL penicillin and ration. Based on pingyangmycin’s physical and chemical
Journal of Nanomaterials 3
PEE-COOH PE-COOH
PEE-NH2 PE-NH2
PEE-OH PE-OH
6 5 4 3 2 1 6 5 4 3 2 1
(ppm) (ppm)
Figure 1: Comparison of 1 H NMR spectra between three PEE-derivatives and three PE-analogues.
characteristics, anionic carboxyl-functionalized HPEE was group was grafted onto HPEE. When a glycine residue
selected for preparation of micelles. Two functionalized (–OCOCH2 NH2 –) was grafted onto HPEE, a novel 3.9 ppm
mixtures (HPEE : carboxyl = 1 : 1 or 1 : 1.02) were separately peak that indicated the methylene proton from the amino
added to 10 mL ultrapure water. Pingyangmycin (Bolai Phar- group could be observed. Similar variation of 1 H NMR peak-
macy Company, China) was then added to each flask with to-terminal could be observed when the same terminals
increasing polymer/pingyangmycin ratios of 1 : 1, 1 : 2, 1 : 3, were added to the HPE backbone (Figure 1, Right). The
1 : 4, and 1 : 5. All mixtures were stirred at room temperature degree of (–OCH2 CH2 CH2 CH2 O–) in HPE was found to be
for 24 h to form a transparent aqueous solution. 1.59 ppm. 1 H NMR distributions were 0.70–0.85.17–4.12 ppm
(hydroxyl protons), 0.70–0.85 ppm (methyl protons), and
2.7. Visualization of Self-Assembled Micelles by Transmission 3.15–3.70 ppm (methylene and methane adjacent to ether
Electron Microscopy. Prepared micelle samples that had been oxygen and alcohol oxygen).
dried for 24 h in a vacuum were observed by transmission
electron microscopy (TEM) (JEOL2010) at 200 KV. 3.3. Fourier Transform Infrared (FT-IR) Spectra of the
Poly(ether-ester) Derivatives. FT-IR spectra of PEE-OH,
PEE-NH2 and PEE-COOH are shown in Figure 2 (Left).
3. Results Characteristic ether bond and ester bond absorption bands
3.1. Synthesis of End-Modified Hyper-Branched Poly(ether- were shown at 1100 cm−1 and 1725 cm−1 , respectively. A
ester)/HPEE Derivatives. Characterization of the HPEE bac- band at 1668 cm−1 could be seen after grafting of amidogen,
kbone has been intensively documented in a previous study whereas the 1557 cm−1 band could only be seen after the
[20], therefore terminal-modified hyper-branched poly(eth- carboxyl group was grafted. For comparison purposes, FT-
er-ester)s could be easily synthesized based on the HPEE R spectra of HPE-analogues are also shown in the same
backbone according to Scheme 1. Supplemental data address figure (Figure 2, Right). Similarly, the 1100 cm−1 FTIR band
the scheme for preparing structure-matched HPE derivatives. in the top HPE-OH figure was produced by asymmetric
stretching vibration originating from C–O–C groups. An
3.2. Characterization of Functionalized Hyper-Branched amidogen absorption band at 1660 cm−1 and a carbonyl
absorption band at 1740 cm−1 appeared when –NH2 was
Poly(ether-ester)s/HPEE by 1 H NMR. According to the
polymerized into HPE-OH. For HPE-COOH, the carbonyl
quantitative 1 H NMR spectrum of HPEE (Figure 1, Left),
and carboxyl bands could be observed at 1730 cm−1 and
the shift of methyl protons was found to be 1.05 ppm. The
backbone of HPEE was observed at 1.80–2.10 ppm and 1555 cm−1 , respectively.
revealed a spectrum of –CH2 –CH(CH2 –)–O– or –CH2 –
C(CH3 )– structural subunits. A proton peak of methine 3.4. Correlation between Surface Potentials and Terminal
adjacent to alcohol oxygen and methylene adjacent to Groups. Surface charge varied with chemical terminal func-
ether oxygen was observed at 4.2–3.3 ppm. Novel peaks tionalization, thus reflecting an interrelationship between
could be observed at 2.50 ppm (–OCOCH2 CH2 COOH–) physical and chemical variations. As shown in Table 1, the
and 2.30 ppm (–OCOCH2 CH2 COOH–) after the carboxyl negative potential carried by hydroxyl on the surface of
R1 R O
OO O
OH O
O OH
R1 OO O R
R1 R1 O
HO O O OH
R HO O
O R O
1 O HO
OH O
OH O O
R O R HO
O O O
O HO
HO R R1 O OHR
R O
HO OH + HO O R HO O
O O HO O O OH OH
O R1 O O R O
KH O R1 O O
O
O Cl 2 HO O HO O R 1 OH
H2 OH
e/C
din
R O P yr i R1 = –COCH2 CH2 –
O
OH O
OH
O O R
HO OH O R O
R HO H2 N O
OH OH HO O OH O NH2
O O O
O O O OH
R O O R NH2 O
O O HO (i) H2 N R
OH OH HO DC Fmoc NH2 O R O O
C/D -
HO O R R R O MA glycine HO O O
O OH O OH P/P O NH2
HO O (ii) TSA O O OH O
HO Pip O NH2
O R erid R R O
HO O ine O O O O
HO HO O OH OH OHO
R R O
HO O R
HO O O OH O OH
R = –CH2 OCH2 CH2 CH2 CH2 OCH2 – O O
O R
NH2 HO O O O NH2
NH2
O O
Scheme 1: End-functionalization between HPEE and amino group/carbonyl group.
PEE changed to a positive charge when the hydroxyl was lost their integrity during the whole incubation period. As
replaced by amidogen; conversely, the negative potential of a positive control, severely damaged 3T3 cells cultured in a
PEE-OH decreased to a more negative value when hydroxyl high concentration of PEI totally lost their fibroblastic shape
was replaced by carboxyl. The PE analogues exhibited similar and the surface topography appeared to be very irregular and
variation in charge-terminal relationships. destroyed. Similar SEM observations of HPE-derivatives are
shown in Supplementary Data 2.1.
3.5. In Vitro Cytotoxicity against 3T3 Cells. The results of
MTT assays for 3T3 cells following incubation for 24 h, 48 h, 3.7. Self-Assembly of Pingyangmycin into Functionalized
and 96 h with various concentrations of the HPEE end- HPEE. To verify the nanocarrier platform based on HPEE
functionalized derivatives are shown in Figure 3. In general, functionalized derivants for individualized antitumor drug
HPEE derivatives demonstrated low cytotoxicity against 3T3 delivery, we used pingyangmycin as a model drug. Based on
cells. Even 3T3 cells treated with 10 mg/mL HPEE-NH2 , the physical and chemical characteristics of pingyangmycin,
which was predicted to have the greatest cytotoxicity, for carboxyl-terminated hyperbranched poly(ether-ester) was
a long incubation time of 96 h retained good viability. The selected for bioconjugation as shown in Scheme 2.
experiment was repeated using the HPE analogues at the After screening, the self-assembly protocol was perfor-
same concentrations. Similar MTT results were obtained for med. As shown in Figure 5 and Figure 6, successfully conju-
HPE-derivatives, as shown in Supplementary Data 2.1. gated spherical micelles (Sample 3 and Sample 5) with average
diameters of 156 ± 9.6 nm (Sample 3/S3) and 173 ± 12.4 nm
3.6. Visualization of 3T3 Cell Surface Morphological Changes (Sample 5/S5) were observed by DLS and TEM visualization.
by SEM. Cell surface morphological features were visual-
ized by scanning electron microscopy (SEM). As shown in 3.8. In Vitro Cytotoxicity of Carboxyl-HPEE-Pingyangmycin
Figure 4, we observed an extraordinarily smooth surface in Micelles against HN-6 Cells. To evaluate the potential thera-
normal 3T3 cells, except for a scattering of microvilli and peutic efficiency of the carboxyl-terminal-HPEE-pingyang-
some ruffles at the ends of the pseudopodia. Compared mycin nanocarrier, an in vitro MTT assay was performed
with PBS controls, cells treated with hyperbranched polymers using HN6 human neck and head carcinoma cells. Both
showed notable changes in scattered microvilli and irregular carboxyl-terminated-HPEE-pingyangmycin nanocarriers
ruffle distributions on cell surfaces that reflected mild adap- (S3 and S5) displayed concentration-dependent and time-
tation rather than cell injury. None of the cell membranes dependent cytotoxicity as shown in Figure 7. For 24 h
Table 1: Correlation between surface potential and end-functionalized polymers.
Delivery vehicle (PEE) Potential Comparable analogue (PE) Potential

PEE-OH 2.1 ± 1.2 PE-OH −1.3 ± 1.1
PEE-NH2 5.4 ± 2.5 PE-NH2 4.2 ± 0.8
PEE-COOH −11.8 ± 3.5 PE-COOH −11.7 ± 2.2
PEE-OH PE-OH
PE-NH2
1725
PEE-NH2
1740 1660
1728 1668
PE-COOH
PEE-COOH
1730
1726
1557 1555
2000 1500 1000 2000 1500 1000

Wavenumber (cm−1 ) Wavenumber (cm−1 )
Figure 2: Comparison of FT-IR spectra between three PEE-derivatives and three PE-analogues.
treatment, cell viabilities were decreased by 25% for both confirmation purposes, HPEEs and their HPE structural
pingyangmycin alone and pingyangmycin-nanocarrier analogues were simultaneously observed. The similar results
complexes at concentrations of 0.1 mg/mL to 1 mg/mL. As confirmed a correlation between physical surface charge and
the concentration increased to 100 mg/mL, cell viabilities chemical terminal structure; thus, tunable modification of
significantly reduced. When the time of treatment extended physical charges could be easily achieved by chemical func-
to 48 h, the viability of HN-6 cells was slightly lower than tionalization. Based on this physical-chemical correlation,
that observed for 24 h treatment. a tunable nanodelivery platform based on a functionalized
HPEE backboned was initially constructed for individualized
antitumor drug delivery.
4. Discussion Although good biocompatibility of hyperbranched poly-
mers has been reported [27], the potential cytotoxicity of
Theoretically, a facile platform could be constructed using functionalized HPEE has not been documented. According
functionalized hyperbranched polymer derivatives such that to the MTT assays, all HPEE derivatives demonstrated
a series of nanocarriers could be obtained for optimization excellent biocompatibility even when high concentrations of
of individualized antitumor-drug delivery. Hyperbranched polymers were used. Furthermore, the biocompatibility of
polymers are characterized by three-dimensional cavities end-modified HPEEs was confirmed by SEM visualization.
with abundant surface terminals and therefore represent an Previous studies have found that cationic charge of polymers
ideal candidate backbone [1, 21–23]. As shown in Scheme 1, was one of the risk factors for increased cytotoxicity. Due
ion-transfer polymerization was observed during HPEE ter- to complicated mechanisms including interactions between
minal functionalization. A hydroxyl group initially reacted cell membranes [28], generation-related clearance [29], and
with butanedioic anhydride to form a carboxyl group [24, 25]. inherent toxicity [30], a strong positive charge of primary
The carboxyl end then reacted with the glycine that was amino groups significantly increased the cytotoxicity [28].
protected by the 9-carbonyl methoxycarbonyl group to gen- Overall, the greatest cytotoxicity of the entire platform
erate an amino group by subsequent esterification [26], after was indeed observed for high concentrations of the HPE-
which methoxycarbonyl protection was rapidly removed. NH2 subgroup, consistent with the previous study. Similarly,
Using the facile polymerization procedure presented here, mildly enhanced cytotoxicity was observed when the ionic
a series of terminal-functionalized polymers were simul- HPEE-COOH subgroup was substituted by the cationic
taneously prepared. Subsequent studies revealed physical- HPEE-NH2 subgroup. However, relatively good biocompati-
chemical interrelationships during functionalization. For bility was demonstrated for all HPEE-derivatives in the plat-
180 180
150 150
Cell viability (% control)

120 120
90 90
60 60
30 30
0 0
HPEE-OH
HPEE-COOH
HPEE-NH2
PEI
Dextran
HPEE-OH
HPEE-COOH
HPEE-NH2
PEI
Dextran
3T3 cell viability for 24 h incubation 3T3 cell viability for 48 h incubation
180
150
120
90
60
30
0
HPEE-OH
HPEE-COOH
HPEE-NH2
PEI
Dextran
3T3 cell viability for 96 h incubation
Concentration 0.001 mg/mL Concentration 1 mg/mL

Concentration 0.01 mg/mL Concentration 10 mg/mL
Concentration 0.1 mg/mL
Figure 3: 3T3 cell viability following treatment with various concentrations of HPEE-derivatives.
form, including cationic HPEE derivatives despite previous pingyangmycin has only been used in situ to treat head and
reports of time- and concentration-dependent cytotoxicity of neck cancers [31–34]. The specific benefits of nanocarriers,
cationic polymers [28]. The mechanism of the reduced cyto- such as passive and accurate targeted therapy with decreased
toxicity of cationic HPEE functionalization was attributed to systemic toxicity and long circulation [16], could increase
scattered surface charge density and the unique uncompleted the clinical application of pingyangmycin. As shown in
hyperbranched architecture. Scheme 2, taking into account the amino-bonding surface of
To evaluate potential application of the present nan- pingyangmycin, electrostatic interactions would theoretically
odelivery platform, an anticancer drug, pingyangmycin be formed between the positive-charged protonated amines
(Bleomycin A5), was used as a model agent to assess assembly of pingyangmycin and the surface carboxyl groups on the
capability. Pingyangmycin is an antibiotic that was initially oxidized HPEE [35]; therefore, a self-assembled micelle could
isolated from the culture medium of Streptomyces pingyan- potentially be constructed in water. Although it is well docu-
gensisn spp. in China and has been known for a long time to mented that hyperbranched polymers possess great capability
exhibit significant cytotoxicity to tumor cells [31–34]. Despite for self-assembly in solution, the issue of interfacial self-
its effective antitumor activity, the systemic toxicity and assembly and hybrid self-assembly [18, 36–38] and whether
short half-life time of pingyangmycin have largely prevented and how functionalized HPEE could successfully mix with
its widespread clinical application. Therefore, hydrophilic pingyangmycin was unknown. In addition, some properties
Cell incubated in low-concentration Cell incubated in low-concentration Cell incubated in high-concentration Cell incubated in high-concentration
HPEE-OH for 1 h HPEE-COOH for 1 h HPEE-OH for 1 h HPEE-COOH for 1 h
Cell incubated in low-concentration Cell incubated in PBS (as Cell incubated in high-concentration Cell incubated in PBS (as
HPEE-NH2 for 1 h blank control) for 1 h HPEE-NH2 for 1 h blank control) for 1 h
Cell incubated in low-concentration Cell incubated in low-concentration Cell incubated in high-concentration Cell incubated in high-concentration
PEI (as positive control) for 1 h dextran (as negative control) for 1 h PEI (as positive control) for 1 h dextran (as negative control) for 1 h
(a) (b)
Figure 4: (a) SEM visualization of surface morphological features of 3T3 cells following incubation with low concentration (10 𝜇g/mL) of
functionalized HPEE-derivatives and controls. (b) SEM visualization of surface morphological features of 3T3 cells following incubation with
a high concentration (1 mg/mL) of functionalized HPEE-derivatives and controls.
R1 R
O O O O
OH O OH
O OH NH2 N
R1 O HN
O R1 O R R1 O OH
O NH2 H N O
HO R HO O O OH H
O H2 N N N
R1 O O N O OH
OHHO O
O O H2 N O O O
OH R O O NH OH
O R
O OO HO
O
O OH O HO HO
R1 R N S O HO OH
HO R O H
HO O R N O NH2
O O OH S N N
HO O OH O H
O O O
R1 O R HO
O O HN
HO R1 O O
O HO O R1 OH
OH NH2
HN
R1 = –COCH2 CH2 –
(a) (b)
Scheme 2: Structure of COOH-HPEE (a) and pingyangmycin (b).

Size distribution by intensity Size distribution by intensity

25 30
20
Intensity (%)
Intensity (%)
15 20
10
10
5
0 0
0.1 1 10 100 1000 10000 0.1 1 10 100 1000 10000
Diameter (nm) Diameter (nm)
Record 1: S3 Record 7: S5
(a) (b)
Figure 5: (a) Diameter of Sample 3 analyzed by dynamic light scattering assay. (b) Diameter of Sample 5 analyzed by dynamic light scattering
assay.
(a) (b)
Figure 6: Transmission electron microscopy (TEM) visualization of two micelles of pingyangmycin conjugated into carboxyl-terminated
HPEE that were successfully self-assembled in water ((a), S3; (b), S5).
100 HN6 cell viability for 24 h treatment 80 HN6 cell viability by 48 h treatment
80
60
60
40
40
20
20
0 0
0.1 1 10 100 0.1 1 10 100
Concentrations (𝜇g/mL) Concentrations (𝜇g/mL)
Pingyangmycin Pingyangmycin
S3(HPEE-PYM) S3(HPEE-PYM)
S5(HPEE-PYM) S5(HPEE-PYM)
(a) (b)
Figure 7: (a) HN6 cell viability following treatment with various concentrations of HPEE-pingyangmycin nanocarriers or pingyangmycin
alone for 24 h. (b) HN6 cell viability following treatment with various concentrations of HPEE-pingyangmycin nanocarriers or
pingyangmycin alone for 48 h.
of hyperbranched polymers have been found to be modulated facile platform presented here provides an attractive pathway
by terminal-backbone interactions [39]; therefore, confir- for individualized drug delivery based on functionalized-
mation of successful self-assembly was required. For the HPEEs.
purpose of comparison, in the present study functionalized
HPEE was initially prepared at two different dissolving Abbreviations
ratios to give appropriately modified polymer and excessively
modified polymer. Accordingly, comparison between these HPEE: Hyper-branched poly(ether-ester)
subgroups allowed us to understand the contribution of HPE: Hyper-branched poly(ether)
physical charges. After preparation of functionalized-HPEE PEE: Poly(ether-ester)
with distinct terminal/backbone ratios, pingyangmycin solu- PE: Poly(ether)
tions of various concentrations were added for nanocar- HBP: Hyper-branched polymers
rier copolymerization. Based on the present results, stable PYM: Pingyangmycin.
micelles could only be successfully mixed at a ratio of
carboxyl-HPEE : pingyangmycin of 5 : 1, irrespective of ter- Conflict of Interests
minal/backbone functionalized ratios, suggesting that the
terminal-backbone interaction exerted a mild effect on sur- This work is sponsored by the National Natural Science Foun-
face charge modification. The polymer : drug ratio meant that dation of China (20974062) and China Postdoctoral Scientific
one pingyangmycin molecule was appropriately combined Foundation (20100470692), Shanghai Jiao Tong University
with five HPEE-COOH molecules in the water. Notably, Med-Science Cross Research Foundation (YG2007MS11),
numerous multimolecular compounds sized approximately and China National Funds for Distinguished Young Scientists
10 to 50 nm were also observed in the coconjugated solution. (21025417). No conflict of interests has been claimed.
The size of these smaller particles was approximately that of
HPEE [1], and the particles were considered to be redundant
Authors’ Contribution
carboxyl-terminal HPEE. The final assembled delivery parti-
cles were equivalent to nanoparticles, which can be efficiently Xing-ai Jin and Yan-wu Li are joint first authors.
taken up by tumor cells due to passive targeting, known as the
enhanced permeation and retention (EPR) effect. Therefore,
appropriately sized HPEE-pingyangmycin nanocarriers have References
high potential for targeted antitumor therapy in vivo. A previ- [1] G. Li, J. Liu, Y. Pang et al., “Polymeric micelles with water-inso-
ous study revealed that acute cytotoxicity could be eliminated luble drug as hydrophobic moiety for drug delivery,” Biomacro-
by chemical modification involving replacement of cationic molecules, vol. 12, no. 6, pp. 2016–2026, 2011.
amino-dendrimers on the surface by neutral copolymers [2] D. Colombo, L. Franchini, L. Toma et al., “Anti-tumor-prom-
[40]. The in vitro cytotoxicity of nanocarrier against tumor oting activity of simple models of galactoglycerolipids with bra-
cells was assessed by MTT assays. Slight differences in tumor nched and unsaturated acyl chains,” European Journal of Medic-
cell inhibition were observed between pingyangmycin-HPEE inal Chemistry, vol. 40, no. 1, pp. 69–74, 2005.
conjugation and pingyangmycin alone, indicating that the [3] O. Schiavon, G. Pasut, S. Moro, P. Orsolini, A. Guiotto, and F. M.
HPEE-COOH used in the nanodelivery system based on Veronese, “PEG-Ara-C conjugates for controlled release,” Euro-
electrostatic interaction exerted little pharmacological effect, pean Journal of Medicinal Chemistry, vol. 39, no. 2, pp. 123–133,
at least in an in vitro assay. 2004.
[4] C. Boulanger, C. di Giorgio, and P. Vierling, “Synthesis of acri-
dine-nuclear localization signal (NLS) conjugates and evalua-
5. Conclusions tion of their impact on lipoplex and polyplex-based transfec-
Due to the facile synthesis and inexpensive costs, indi- tion,” European Journal of Medicinal Chemistry, vol. 40, no. 12,
pp. 1295–1306, 2005.
vidualized nanodelivery systems for antitumor drugs could
[5] A. Kim and J. H. Hong, “Synthesis and antiviral activity of C-flu-
be optimized using the present platform of functionalized
oro-branched cyclopropyl nucleosides,” European Journal of
hyperbranched poly(ether-ester)s. Based on the observed
Medicinal Chemistry, vol. 42, no. 4, pp. 487–493, 2007.
relationship between the chemically modified end group and
[6] B. Guo, Z. Shi, Y. Yao, Y. Zhou, and D. Yan, “Facile preparation of
physically variations in surface charge, tunable drug delivery novel peptosomes through complex self-assembly of hyperbra-
seems easily achievable. After the physical-chemical inter- nched polyester and polypeptide,” Langmuir, vol. 25, no. 12, pp.
relationship between surface terminal and charge loading 6622–6626, 2009.
of HPEE was revealed, pingyangmycin, a potent antitumor [7] M. Ji, W. Yang, Q. Ren, and D. Lu, “Facile phase transfer of hyd-
drug, was used as a model drug to assess the HPEE- rophobic nanoparticles with poly(ethylene glycol) grafted hype-
dependent nanocarrier system. Negative-charged carboxyl- rbranched poly(amido amine),” Nanotechnology, vol. 20, no. 7,
functionalized HPEE was chosen because it was expected to Article ID 075101, 2009.
form ionic bonds with the positive-charged pingyangmycin. [8] R. Duncan, “Polymer conjugates as anticancer nanomedicines,”
Mixture between carboxyl-HPEE and pingyangmycin was Nature Reviews Cancer, vol. 6, no. 9, pp. 688–701, 2006.
successfully achieved by self-assembly and confirmed by DLS [9] C. Gao and D. Yan, “Hyperbranched polymers: from synthesis
and TMS. The resultant nanoparticles exhibited promising to applications,” Progress in Polymer Science, vol. 29, no. 3, pp.
antitumor cytotoxicity as assessed by MTT assays. The 183–275, 2004.
[10] B. Voit, “New developments in hyperbranched polymers,” Jour- [26] Y. B. Lim, S. M. Kim, H. Suh, and J. S. Park, “Biodegradable, end-
nal of Polymer Science A: Polymer Chemistry, vol. 38, no. 14, pp. osome disruptive, and cationic network-type polymer as a
2505–2525, 2000. highly efficient and nontoxic gene delivery carrier,” Bioconjugate
[11] S.-J. Park, K. Li, and F. L. Jin, “Synthesis and characterization of Chemistry, vol. 13, no. 5, pp. 952–957, 2002.
hyper-branched polyimides from 2,4,6-triaminopyrimidine [27] R. S. Kumar, S. Arunachalam, V. S. Periasamy, C. P. Preethy, A.
and dianhydrides system,” Materials Chemistry and Physics, vol. Riyasdeen, and M. A. Akbarsha, “DNA binding and biological
108, no. 2-3, pp. 214–219, 2008. studies of some novel water-soluble polymer-copper(II)-phena-
[12] C. Kontoyianni, Z. Sideratou, T. Theodossiou, L. A. Tziveleka, D. nthroline complexes,” European Journal of Medicinal Chemistry,
Tsiourvas, and C. M. Paleos, “A novel micellar PEGylated hype- vol. 43, no. 10, pp. 2082–2091, 2008.
rbranched polyester as a prospective drug delivery system for [28] D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, and T. Kissel, “In
Paclitaxel,” Macromolecular Bioscience, vol. 8, no. 9, pp. 871–881, vitro cytotoxicity testing of polycations: influence of polymer
2008. structure on cell viability and hemolysis,” Biomaterials, vol. 24,
[13] J. Zou, W. Shi, J. Wang, and J. Bo, “Encapsulation and controlled no. 7, pp. 1121–1131, 2003.
release of a hydrophobic drug using a novel nanoparticle- [29] J. C. Roberts, M. K. Bhalgat, and R. T. Zera, “Preliminary
forming hyperbranched polyester,” Macromolecular Bioscience, biological evaluation of polyamidoamine (PAMAM) Starburst
vol. 5, no. 7, pp. 662–668, 2005. dendrimers,” Journal of Biomedical Materials Research, vol. 30,
[14] S. Chen, X. Z. Zhang, S. X. Cheng, R. X. Zhuo, and Z. W. Gu, no. 1, pp. 53–65, 1996.
“Functionalized amphiphilic hyperbranched polymers for tar- [30] N. Malik, R. Wiwattanapatapee, R. Klopsch et al., “Dendrimers:
geted drug delivery,” Biomacromolecules, vol. 9, no. 10, pp. 2578– relationship between structure and biocompatibility in vitro,
2585, 2008. and preliminary studies on the biodistribution of 125I-labelled
[15] W. Daniel, S. E. Stiriba, and F. Holger, “Hyperbranched poly- polyamidoamine dendrimers in vivo,” Journal of Controlled
glycerols: from the controlled synthesis of biocompatible poly- Release, vol. 65, no. 1-2, pp. 133–148, 2000.
ether polyols to multipurpose applications,” Accounts of Chem- [31] X. Meisheng, “Histopathologic study of esophageal squamous
ical Research, vol. 43, no. 1, pp. 129–141, 2010. cell carcinoma treated preoperatively with pingyangmycin,”
[16] Y. Wang, M. Gou, C. Gong et al., “Pharmacokinetics and dis- Chinese Medical Journal, vol. 92, no. 5, pp. 343–348, 1979.
position of nanomedicine using biodegradable PEG/PCL poly- [32] K. W. Tai, Y. C. Chang, L. S. S. Chou, and M. Y. Chou, “Cytotoxic
mers as drug carriers,” Current Drug Metabolism, vol. 13, no. 4, effect of pingyangmycin on cultured KB cells,” Oral Oncology,
pp. 338–353, 2012. vol. 34, no. 3, pp. 219–223, 1998.
[17] Z. Jia, G. Li, Q. Zhu et al., “Hybrid polymerization of vinyl and [33] K. Adriane, J. Huang, G. Ding, J. Chen, and Y. Liu, “Self assem-
hetero-ring groups of glycidyl methacrylate resulting in ther- bled magnetic PVP/PVA hydrogel microspheres; magnetic drug
moresponsive hyperbranched polymers displaying a wide range targeting of VX2 auricular tumours using pingyangmycin,”
of lower critical solution temperatures,” Chemistry, vol. 15, no. Journal of Drug Targeting, vol. 14, no. 4, pp. 243–253, 2006.
31, pp. 7593–7600, 2009.
[34] P. Chen, B. Liu, and M. Hu, “The effect of hydroxycamptothecin
[18] Y. Zhou, W. Huang, J. Liu, X. Zhu, and D. Yan, “Self-assembly of
and pingyangmycin on human squamous cell carcinoma of the
hyperbranched polymers and its biomedical applications,”
tongue,” Oncology Letters, vol. 5, no. 3, pp. 947–952, 2013.
Advanced Materials, vol. 22, no. 41, pp. 4567–4590, 2010.
[35] M. L. Chen, M. L. Chen, X. W. Chen, and J. H. Wang, “Func-
[19] P. Li, G. Zhou, X. Zhu et al., “Photodynamic therapy with hype-
tionalization of MWNTS with hyperbranched PEI for highly
rbranched poly(ether-ester) chlorin(e6) nanoparticles on
selective isolation of BSA,” Macromolecular Bioscience, vol. 10,
human tongue carcinoma CAL-27 cells,” Photodiagnosis and
no. 8, pp. 906–915, 2010.
Photodynamic Therapy, vol. 9, no. 1, pp. 76–82, 2012.
[36] Y. Shi, C. Tu, R. Wang, J. Wu, X. Zhu, and D. Yan, “Preparation of
[20] Z. Jia, H. Chen, X. Zhu, and D. Yan, “Backbone-thermo-
CdS nanocrystals within supramolecular self-assembled nano-
responsive hyperbranched polyethers,” Journal of the American
reactors and their phase transfer behavior,” Langmuir, vol. 24,
Chemical Society, vol. 128, no. 25, pp. 8144–8145, 2006.
no. 20, pp. 11955–11958, 2008.
[21] J. Liu, W. Huang, Y. Pang, X. Zhu, Y. Zhou, and D. Yan, “Hype-
rbranched polyphosphates for drug delivery application: des- [37] Y. Zhou and D. Yan, “Supramolecular self-assembly of amp-
ign, synthesis, and in vitro evaluation,” Biomacromolecules, vol. hiphilic hyperbranched polymers at all scales and dimensions:
11, no. 6, pp. 1564–1570, 2010. progress, characteristics and perspectives,” Chemical Communi-
cations, no. 10, pp. 1172–1188, 2009.
[22] H. Zhang, C. Zhao, H. Cao et al., “Hyperbranched poly(amine-
ester) based hydrogels for controlled multi-drug release in [38] H. Jin, W. Huang, X. Zhu, Y. Zhou, and D. Yan, “Biocompatible
combination chemotherapy,” Biomaterials, vol. 31, no. 20, pp. or biodegradable hyperbranched polymers: from self-assembly
5445–5454, 2010. to cytomimetic applications,” Chemical Society Reviews, vol. 41,
no. 18, pp. 5986–5997, 2012.
[23] Y. Xia, Y. Wang, Y. Wang et al., “A tumor pH-responsive com-
plex: carboxyl-modified hyperbranched polyether and cis-dic- [39] L. Song, C. Tu, Y. Shi et al., “Controlling the optical properties of
hlorodiammineplatinum(II),” Colloids and Surfaces B: Biointer- hyperbranched conjugated polyazomethines through terminal-
faces, vol. 88, no. 2, pp. 674–681, 2011. backbone interactions,” Macromolecular Rapid Communica-
tions, vol. 31, no. 5, pp. 443–448, 2010.
[24] I. J. Majoros, A. Myc, T. Thomas, C. B. Mehta, and J. R. Baker Jr.,
“PAMAM dendrimer-based multifunctional conjugate for can- [40] N. A. Stasko, C. B. Johnson, M. H. Schoenfisch, T. A. Johnson,
cer therapy: synthesis, characterization, and functionality,” Bio- and E. L. Holmuhamedov, “Cytotoxicity of polypropylenimine
macromolecules, vol. 7, no. 2, pp. 572–579, 2006. dendrimer conjugates on cultured endothelial cells,” Biomacro-
[25] Y. Luo and G. D. Prestwich, “Synthesis and selective cytotoxicity molecules, vol. 8, no. 12, pp. 3853–3859, 2007.
of a hyaluronic acid-antitumor bioconjugate,” Bioconjugate
Chemistry, vol. 10, no. 5, pp. 755–763, 1999.
Review Article
Nanostructural Colouration in Malaysian Plants:
Lessons for Biomimetics and Biomaterials
S. Zaleha M. Diah,1 Salmah B. Karman,1,2 and Ille C. Gebeshuber1,3

1
Institute of Microengineering and Nanoelectronics, Universiti Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor, Malaysia
2
Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia
3
Institute of Applied Physics, Vienna University of Technology, Wiedner Hauptstra𝛽e 8-10/134, 1040 Vienna, Austria
Correspondence should be addressed to Ille C. Gebeshuber; gebeshuber@iap.tuwien.ac.at
Received 3 September 2013; Revised 10 February 2014; Accepted 10 February 2014; Published 14 April 2014
Academic Editor: Il-Kwon Oh
Copyright © 2014 S. Zaleha M. Diah et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Plant tissues include leaves, flower petals, and fruits. These can provide us with variety of design inspirations. Biomimetics allows
us to learn from nature and transfer the knowledge we gain from studying sophisticated and amazing biological structures,
materials and processes to engineering and the arts. The microstructures of morphology and anatomy of plant tissue have potential
applications in technology through bioinspired design, which can mimic the properties found in nature or use them as inspiration
for alternative applications. Many applications have been developed as a result of studying physical properties of plant tissues.
Structural colours, for example, have been applied in the design of thin films both with regard to single or multilayer thin film
interference, scattering, and diffraction gratings. Iridescent, metallic, or greyish colouration found naturally in plants is the result
of physical structures or physical effects and not pigmentation. Phenotypical appearance of plants with structural colouration in
tropical Malaysia is correlated with environmental parameters such as location (shady understory rainforest, sunny conditions)
and altitude (highlands, lowlands). Various examples of bioinspired technical innovations with structural colours highlight the
importance of inspiration by structural colours in living nature.
1. Introduction deter herbivores, and help in light management (e.g., UV

protection or focusing of light on the chloroplasts). Acquiring
Ornamental plants are commercially popular. They are attrac- a detailed understanding of these structural colours can help
tive and help to relieve stress. Beautiful plants and/or flowers scientists and engineers to develop new materials that offer
make houses, restaurants, and offices more attractive. In similar tailored properties while remaining benign and sus-
the process of photosynthesis, plants produce the oxygen tainable. A number of engineering devices and applications
we need to breathe and absorb the carbon dioxide that have been developed based on the mechanisms of structural
we exhale, using it as their own source of food. Plant colour production that are observed in nature. Examples of
diversity is broad and a variety of patterns, colours, and these include thin-film multilayers and photonic crystals,
types can be found in nature. Furthermore, plants can which are both formed in nanoscale structures.
also represent a source of inspiration for nanoscience and Understanding nanostructures for colouration in plants
nanotechnology. and their correlation with the respective function can yield
Some plants produce nanostructures that yield coloura- increased appreciation of the structure-function relationship
tion, either in the visible range or in the UV range that is in such functional nanostructures and could potentially
only visible to certain species of insects. The nanostructures inspire nanoscientists and nanotechnologists to develop
employed to achieve this colouration serve various functions more integrated, multifunctional applications that are both
in plants; for example, they make them even more attractive, biodegradable and benign.
Colour is a property of both the colour of the object the actual absorption of electromagnetic radiation by a leaf
(body colour) and the perception of the observer. Structural is significantly different from the absorbance spectrum of
colouration is caused by the interaction of light with minus- chlorophyll in solution. The packaging of pigments into
cule structures with spatial dimensions of some hundreds organelles (as with chlorophylls and carotenoids) leads to
of nanometers (i.e., the wavelength of visible light) or less, sieving effects that increase light capture in the regions
down to some tens of nanometers. Iridescent colour is colour of the greatest absorbance by these pigments. The path-
that changes according to the angle from which it is viewed lengthening effect of air spaces in tissues promotes much
and everyday examples of this can be seen in the appearance greater absorption of electromagnetic radiation in normally
of DVDs, CDs, and soap bubbles. Iridescent colouration is weakly absorbed wavelengths. Plant cells have dimensions of
not caused by pigmentation but is an optical effect. Studies ∼50 𝜇m, and the shapes and distributions of cells profoundly
on structural colours date back to the seventeenth century, influence the optical properties of the leaves and other plant
when the earliest scientific description of structural colours organs.
appeared in “Micrographia,” written by Robert Hooke in 1665. Optical properties in plant tissues were first studied in
As early as 1704 in his book “Opticks,” Sir Isaac Newton had 1917 by Richard Willstätter and Arthur Stoll [3] (Nobel
already related iridescence to optical interference: “The finely Prize to Willstätter in 1915). They published a model of the
colour’d feathers of some birds, and particularly those of the optics of a leaf. Their model considered complex patterns of
peacocks’ tail, do in the very same part of the feather appear of internal reflectance within the leaf and disclosed how this was
several colours in several positions of the eye, after the very same impacted by odd cell angles and accompanying air spaces.
manner that thin plates were found to do.” Following his work, As a result of the presence of chlorophyll all plant leaves
a plethora of articles and books that examined structural are basically green; however, some of them appear in other
colours in organisms were published (see Kinoshita 2008 and colours. Furthermore, leaves have variegation patterns that
the references therein) [1]. In Malaysia (formerly known as are highly influenced by cell division patterns at early growth
Malaya) pioneering research on structural colours in tropical [4]. Plant cells have a specialized anatomy and morphology
understory plants was conducted by Lee and Lowry [2]. that make them function properly and for survival. In this
Professor David W. Lee was, at that time, a lecturer at the respect, it is worth briefly examining the components of plant
Faculty of Science, University of Malaya. We will discuss this cells. Cellulose is secreted by the cytoplasm and forms the cell
in more detail in Section 2. wall. Cellulose has interesting optical properties: its refractive
index is higher than that of water and depends on the angle
at which light passes through the layer and the degree of
2. Plants with Nanostructural absorption of water. If cellulose is fully moist, it may have
Colouration in Malaysia a reflective index as great as 1.40. The cell wall becomes
rigid and protects the plant from the loss of water due to
2.1. Some Basic Information on Plants drought. Inside the cytoplasm is the nucleus, where genetic
information is stored, transcribed, and then translated into
2.1.1. The Anatomy and Morphology of Plants. Plants, just products that run the cell and the whole organism. Besides
like animals, have parts that provide specific structures and that, cells also have other organelles, such as chloroplasts,
functions; however, their way of life is much different to the sites of photosynthesis [5, 6]. Mitochondria are found in
that of animals. Animals run, walk, hop, and move to both animal and plant cells. Some plants have characteris-
get their food and protect themselves, their offspring, and tic iridescent colouration, especially in tropical understory
mates. Plants produce their own food through the process forests. The parts that are of interest exhibit iridescent
of photosynthesis, and they use colourful flowers, exhibit a colouration [2, 5, 7–9] and/or metallic lustre [10] in leaves,
variety of leaf patterns and colours that attract pollinators, fruits [11, 12], and flowers [13]. Examples of iridescent leaves
and produce a variety of fruits. Some plant cells have lens include Begonia rex, B. pavonina, Selaginella willdenowii, and
structures that interact with light in a manner that influences Danaea nodosa. The occurrence of iridescence (change of the
the reflection and absorption properties of the cell surface colouration according to the angle from which it is viewed)
[3]. Plant tissues, including leaves, flower petals, and fruits, is always an indication of nanostructures responsible for the
have their own distinct functions. The leaf functions as an colour rather than the pigmentation itself [1]. Early studies
optical organ in plants and uses a complex tissue organization on silvery plants with metallic lustre were performed on
that facilitates the distribution of light to tissues according to peas [14], tomatoes [15], and marrow [16, 17]. The physical
their differing physiological requirements for light in pho- basis and detailed phenomena will be discussed later in
tosynthesis, while at the same time facilitating appropriate Section 2.2.
levels of gas exchange and water and nutrient delivery to
those tissues. Figure 1 presents the anatomy of a leaf in
terms of the structures through which the plant interacts 2.1.2. Iridescence in Plants in Malaysia. Plants with iri-
with light (Figure 1). Pigmentation, both by chlorophylls and descent properties are generally found in shaded forests
accessory pigments, makes an important contribution to and tropical latitudes and are associated with lowland and
these optical properties, as also does the distribution of air vegetated environments. In Malaysia, these plants adapt to
spaces, which cause optical scattering and path-lengthening the shady, humid conditions of the forest understory. Blue-
effects. A consequence of these physical properties is that green iridescent plants are widely distributed across tropical
Cuticle
Upper epidermis
Palisade
parenchyma
Spongy Vascular bundle

parenchyma
Bundle sheath
Lower epidermis
Figure 1: Typical anatomy of a leaf structure. A leaf contains a waxy cuticle, an epidermal cell as a cover for the upper and lower surface.
Function of each tissue: the cuticle repels water, prevents rapid desiccation, acts as a selective filter in reflecting harmful ultraviolet light,
and allows the absorption of visible light for photosynthesis. Epidermis cells are specially adapted for light absorption. In some plants the
pigmentation is located in the mesophyll cell, immediately beneath the palisade layer. Figure modified from [6].
Southeast Asia and Africa and some iridescent plants belong Iridescent or structural colour phenomena are not lim-
to the pteridophytes genus Selaginella (Selaginellaceae). ited to leaves but can also be found in fruits and flower
Among the common species and predominant blue iridescent petals. However, at present, plants found in Malaysia only
plants is Selaginella willdenowii (peacock fern, Malay name exhibit an iridescent effect on their leaves. Several struc-
“pakis merak”). Besides, S. willdenowii blue iridescent plants tural fruit colourations with brilliant blue iridescence have
include Begonia pavonina (Begoniaceae), Diplazium tomento- been reported for Elaeocarpus angustifolius (Elaeocarpaceae)
sum (Athyriaceae), Danaea nodosa (Marattiaceae), Lindsaea [21, 22], Delarbrea michieana (Araliaceae) [12], and Pollia
lucida (Lindsaeaceae), and Phyllagathis rotundifolia (Melas- condensata (Commelinaceae) [11]. D. michieana is a small
tomataceae). 52 native species of Begonia are known in Penin- understory tree that is endemic of the tropical region of
sular Malaysia and over 1500 species are recorded in wild Queensland, Australia. Vukusic and Stavenga [20] investi-
tropical and subtropical Asia, Africa, and America. The native gated the structural colouration of this species.
species of Begonia, which is characterised by leaves that some-
times have a bluish tinge, include B. alpina, B. carnosula, B.
integrifolia, and B. thaipingensis [18]. A species with green iri- 2.1.3. Phylogenetics of Plants: Genotype and Phenotype. The
descence is the cave moss Schistostega pen- nata (Schistoste- physical appearance of an organism is phenotypic, while its
gaceae), which can be found in shady and dark places near the internal coding is genotypic. A single genotype can produce
cave entrance at the Batu Caves near Kuala Lumpur [7]. The different phenotypes in different environments; that is, seeds
colour of many understory tropical rainforest plants can be from the same plant might very well yield completely different
described as iridescent because of the intensity and metallic looking offspring plants, with the nanostructures responsible
quality [7] of their colouring and the fact that they appear to for colouration varying according to environmental param-
change colour when viewed from different angles. Iridescent eters such as altitude and light intensity. The patterns in
leaves generally distribute the reflected granules on epidermal plants, for example, those found in a leaf, are under genetic
cells, for example, in the case of S. willdenowii (Figure 2). control and may involve different mechanisms for control-
Green iridescence occurs via the refraction of diffused light ling pigments and structure. This fundamental property of
onto specially oriented chloroplasts by lens-shaped cells, organisms is known as phenotypic plasticity. Recent intensive
while blue occurs when, for example, a thin film acts as an study has shown that plants are plastic for a remarkable array
interference filter in or on the epidermis [7]. Iridescence can of ecologically important traits, ranging from diverse aspects
span two or more different colours and can appear in regions of morphology and physiology to anatomy, developmental
of the spectrum that are visible to a variety of animals includ- and reproductive timing, breeding system, and offspring
ing ultraviolet (UV) [19] or the range visible for humans [20]. developmental patterns. Comparative, quantitative genetics
There is a distinct need for researchers to complete further and molecular approaches are leading to new insights into
studies of the phenomenon of iridescence in plant aspects the adaptive nature of plasticity, its underlying mechanisms,
such as structures, functions, distribution, and behaviours. and its role in the ecological distribution and evolutionary
(a) (b)
Figure 2: Iridescent plants. Selaginella willdenowii leaf (a), Figure © one of authors (SZMD). Surface details of S. willdenowii leaf (b), showing
the sites of its unusual colour production. Figure from [23] Permission pending.
diversification of plants [24]. We suggest performing stud- physiological constraints that either induce or block the
ies on phenotypical variations of nanostructures yielding production of pigments [26]. Existing research on phyloge-
colouration. Such studies shall give important insights into netics and physiology of coloration in plants is much less
the function of the respective nanostructures and facilitate common than that on animals. Lee et al. [12] examined the
understanding of the structure-function relationship and phylogenetics of two different taxa of the family Araliaceae,
related biomimetics. namely, Delarbrea and Mackinlaya. Genetic diversity and
Inheritance is the acquisition of traits that are genet- variation in Begonia species was studied by Matolweni et al.
ically transmitted from parents to offspring. In individual [27].
cells, information that controls the division of cells and the
formation of tissues, organs, and the complete organism
is maintained in the nuclei. Nucleic acids are very large 2.1.4. Distribution and Environmental Factors (Biotic and
structures that include DNA (deoxyribose nucleic acid) Abiotic): Understory and Nonunderstory Forest. Individual
and RNA (ribose nucleic acid) and are composed of four organisms live in different habitats and localities. They can
nucleotides (adenine, thymidine, cytosine, and guanine, but be modified or altered by development, physiology, and
in RNA guanine is replaced with uracil). The nucleotides life history according to environmental conditions. Phys-
follow a specific sequence in this basic information and have a ical environmental conditions, such as water availability,
double helix structure. Each sequence of nucleotides contains humidity, temperature, soil chemistry, light exposure, and
information for the production of a unit of heritance or a locality, affect the distribution, pattern, and abundance of
gene. Nucleic acids are the main constituents of the genes plant species.
that contain hereditary information within long polymers at
specific locations on chromosomes. However, it is possible Light Condition. Plant growth in the understory rainforest is
that the alteration of genes may occur at an individual level much different to that in the open air or with direct exposure
and will be expressed in the form of different characteristics in to sunlight. In shaded areas, the light intensity is less and can
a trait such as flower colour and leaf shape. These are known be only 3–5% of the fully sun-exposed value [5].
as alleles. Gregor Mendel established the laws of expression of
genes and alleles in the nineteenth century through Mendel’s Elevation. The majority of plants that are found at high
Principles of Heredity or Mendelian Inheritance and he found elevations are covered with hairs or wax that protect them
that some alleles are dominant in their expression while against the increased light intensity. Furthermore, some of the
others are recessive. Afterwards, Mendel found many excep- plants found in these conditions are capable of converting the
tions to the rules governing the inheritance for combining sun’s light rays into heat (with the help of red pigments) [5].
genes and alleles [5]. For example, it would be interesting Tropical rainforests with their high humidity and low
to compare the nanostructures of the peacock begonia when light intensity provide a rather specific environment for the
grown in different conditions and start to correlate the herbaceous ground flora. Here, many iridescent plants can be
structure and function from such investigations. In such a found. In most cases, the iridescence of the leaves and fruits
way, templates for the transfer of such colours to engineering is in the blue and blue-green range of the light spectrum. The
could be generated [25]. Despite recent developments in first study on the physical basis and ecological significance of
molecular genetics in terms of understanding gene action, iridescence in plants, on Selaginella willdenowii (changing the
still little is known about the physiology of colouration. colouration of the top of its leaves from green to blue and back
Greater knowledge is needed about the developmental and to green when viewed at different angles), was conducted by
Lee and Lowry [2] and revisited in Thomas et al. [28]. Accord-
ing to these authors, iridescent leaves are mainly found in
shaded tropical rain forest: when growing in sunlight, their
iridescence was lost [2, 28]. Recent research on the structural
colours found in plants and other nonanimals (summarized
by Gebeshuber and Lee, 2012) [29] indicates that the most
common mechanisms in plants that cause structural coloura-
tion are multilayer interference and diffraction gratings [29].
Multilayer interference is found predominantly in shade-
plant leaves, suggesting a role either in photoprotection or in
optimizing the capture of photosynthetically active light (Lee
2007 and references therein) [5]. Diffraction gratings may be
a common feature of petals, such as those found on tulips or Figure 3: Iridescent plant. A tropical Asian understory herb,
Mapania caudata. The colour comes from nanoparticles of biogenic
hibiscus.
silica. Image from http://bioserv.fiu.edu/∼leed/research.html. Fig-
Structural colours may be surprisingly frequent in the ure reproduced with permission.
plant kingdom, and still much remains to be discovered
about their distribution, development, and function [19]. The
scattering of light yields, for example, the blue appearance of
the needles of the blue spruce, and photonic crystals yield Begonia species have a variety of natural foliar variegated
interesting and beautiful effects in high altitude plants such patterns that include leaf structure and pigment-related var-
as Edelweiss or in viruses and diatoms. Cholesteric liquid iegation [31]. The silvery spots that are sometimes present in
crystals might be the reason for the structural colouration in Begonia are not caused by pigments but by increased air-filled
some Malaysian iridescent understory tropical fern species cells. These spots on the leaves mimic insect’s eggs, preventing
such as Danaea nodosa, the necklace fern Lindsaea lucida, butterflies from laying eggs on these leaves because “it is
and Diplazium tomentosum. Gebeshuber and Lee [29] also already taken by other insects.” B. pavonina is found in
described a multitude of plants and other nonanimals where altitudes above 1,000 meters in hill forests such as those
the nanostructural origin of the colouration effects has not yet found in the Cameron Highlands, Malaysia. Zhang et al. [10]
been described and/or identified. There might be very inter- studied the metallic appearance of B. rex. The leaves of this
esting nanoscience waiting in some of these organisms and species have two regions that reflect light: spotted patterns
some surprises too! In animals, the interaction of light with and polygonal patterns. They showed that the polygonal
the nanostructures of biological tissue produces iridescence patterns influence the metallic colour. Interior air spaces are
either via thin-film interference, multilayer interference, the most important factors in the formation of the polygonal
scattering, diffraction, or photonic crystals [1]. pattern [10].
Some benthic marine algae produce blue to violet iri-
descence. The moss Schistostega shows iridescence in the
golden-green part of the spectrum [31]. Surprisingly, for
2.2. Colouration in Plants
colouration in certain plants, the presence of nanoparticles of
2.2.1. Principles of Colouration. Colour is a property of both biogenic silica provides the basis for the colour; an example
the coloured object and the perception of the observing for this is the tropical Asian understory herb, Mapania
animals or people [19]. According to Mott (1893), the colours caudata (Figure 3), and two Malaysian tropical rainforest
of animals and plants have three causes: (1) physical causes herbs, Diplazium crenatoserratum and Phyllagathis rotundi-
such as diffraction and interference from striated surfaces, folia (Figure 4). In 1975, Lee and Lowry published an article
as in some iridescent feathers and shells; (2) pigments whose in the journal Nature on their research regarding the brilliant
function seems to be especially to give colour; (3) the molec- iridescent blue in Selaginella, which is caused by structure,
ular structure of the tissues themselves [30]. Colouration not pigments. They found that the leaves lose their colour
in organisms is normally caused by pigments (chemical) or when immersed in water but that the blue colour reappears
optical effects (structural) or a combination of both. In plants, when the leaves are dried [2]. Prior to their research little was
the majority of colouration is produced by a variety of pig- understood about the iridescent phenomena found in plants.
ments such as anthocyanin, flavonoids, and carotenoids. In
chemical colours, light is selectively reflected, absorbed, and 2.2.2. Pigmentation (Chemical Colours). Pigmentation phe-
transmitted. Pigments reflect the wavelengths of light that nomena are associated with the most fundamental elements
produce a certain colour and absorb the other wavelengths. of organic life. The colour of tissue or pigments depends on
While in structural colours, the incident light is reflected, the portion of white light that is reflected from them. Sunlight
scattered, and deflected on structures with negligible energy is white light, and after it hits objects it is partly reflected and
exchange between material and the light, resulting in strong partly absorbed [30]. The majority of colouration observed
and shining colouration [1]. Plants that have waxy coverings in plants stems from pigmentation. In chemical colours,
are whitish blue [5], for example, the Schima wallichii conifer light is selectively reflected, absorbed, and transmitted. The
tree. Shaded lowland tropical rainforests are home to various production of colours occurs when pigments reflect certain
species with leaves that have blue-green iridescence. wavelengths of light and absorb other wavelengths [29]. Some
2.2.4. Functions and Potential Applications. Colours serve a

multitude of functions in nature. Animals use them mainly
Diplazium for signalling, mimicry, mating choice, and protection. Struc-
crenatoserratum tural colour in plants attracts pollinators and helps to protect
and these colour functions are primarily found in flowers and
Phyllagathis leaves. Fruits may attract animals like birds and mammals,
rotundifolia
which then disperse the seeds. Structural colour may be
related to significant functional biology and the physiology
of plants. Structural colouration is relatively permanent and
Figure 4: Iridescent plants. Two Malaysian tropical rainforest generally does not bleach in the same way that pigment
herbs, Diplazium crenatoserratum (on top) and Phyllagathis ro- colours do. A piece of the fruit from Pollia condensata
tundifolia (underneath). Image from http://bioserv.fiu.edu/∼leed/ that was collected in Ghana in 1974 and kept dried as a
research.html. Figure reproduced with permission. herbarium specimen in the herbarium of the Royal Botanic
Gardens, Kew, United Kingdom, ever since still retains its
brilliant blue colour [11]. In Selaginella willdenowii, lamellae
are observed, which might serve as functional antireflective
coating, reflecting light in short wavelengths and enhanc-
sepals (basic parts of a flower, below the petals) change their
ing the absorption of light at longer wavelengths through
colours under different cultivation conditions, such as in
destructive interference [2]. Iridescent blue leaves might also
Hydrangea macrophylla. The colour changes in this species
function to protect leaves via photoinhibition when exposed
from colourless in the early stages of development to blue
to high light levels [37].
and then green and finally red during senescence. This is due
Plants that are coloured by pigmentation are regularly
to a change in anthocyanin biosynthesis [13]. Pigmentation
used for body decoration purposes; for example, henna
colours occur when light is absorbed in materials. Illumi-
(Lawsonia inermis) is used to colour hands [5] and hair.
nating light in materials such as pigments, dyes, and metals
Plants are also utilized for many other vital purposes, such
interacts with electrons within the materials and excites them
as the provision of food, medical treatments, and dyeing of
to a higher state, by virtue of the energy consumption of light
cloth. Further details about the application and biomimetics
[1]. The blue colour in fruits and flowers is mainly caused by
of structural colour will be discussed in Section 4.
modified anthocyanins.
2.2.3. Structural Colours. Studies on structural colour can be 3. Relationship between Nanotechnology and
traced back to the development of electromagnetic theory Optical Properties
by Maxwell 1873 [32], followed by electromagnetic waves by
Hertz 1884 [33] and the upgrading of the electromagnetic 3.1. Light, Vision, and Colour
theory by Lord Rayleigh 1917 [34]. This was, in turn, aug-
mented by the research on surface colours that was completed 3.1.1. Light Perception and Optical Properties. Colours
by Walter 1895 [35] and Michelson 1911 [36], finally resulting undergo changes throughout the day, and these changes are
in the affirmation of the principle of the concept. Structural a function of the amount of light that is available. Daytime is
colours are normally observed in nature in various ani- dominated by bright and clear colours; however, this ceases
mals, plants, and microorganisms. Famous examples of this to be the case when the weather is overcast or cloudy. During
phenomenon include wings of butterflies (such as Morpho nights, the surroundings are colourless and dark and the
sp., Parides sp.), peacocks feathers, and beetle carapaces. light source comes from the moon or artificial lighting.
In plants with structural colouration, chloroplast extraction Humans and animals are dependent upon light as a vital
shows only green chlorophyll and a few carotenoid pigments. sensory signal, while plants are dependent upon light for
The anthocyanins that form the blue colour are not present their growth and physiological responses. Plants capture light
[5, 37]. Various physical mechanisms are responsible for the as a source of energy and depend on it for survival. Colour is
production of structural colour in plant leaves, fruits, and regarded as a visual perception property that corresponds to
others (for overview, see Table 1). humans. Human beings are unable to perceive iridescence in
Many iridescent colours in plants are produced by mul- flowers; however, honey bees are attracted to flowers for that
tilayer thin-film interference, diffraction gratings, and scat- very reason and this culminates in them collecting nectar.
tering (Tyndall scattering) [8] (see Section 3.1). These mech- The understanding of the interaction between plants and
anisms are associated with distinct colouration appearance light and how animals perceive this interaction as form and
properties such as tuneable colours that change according colour is called light perception phenomena [5].
to the viewing angle [38] and retroreflection properties Light can be defined as an electromagnetic wave that
[39]. Structural colours are involved in functions such as contains particles of energy [40]. Light perception is always
display and defence, photoprotection, and photoreception. related to light propagation, incident light, light reflection,
Detailed characteristics, mechanism, and functions of struc- refraction, transmission, and absorption. Light propagating
tural colour phenomena for some species of plants were from one medium to another can be reflected or refracted
reviewed by Glover and Whitney [19]. by a surface or interface. The characteristics of the reflected
Table 1: Noncomprehensive list of physical mechanisms that yield iridescence in plants, with examples and functions [29].
Physical
Visual appearance Examples Function
mechanism
Thin film Iridescent blue. Interference within the peridium,
Slime mold Diachea leucopodia Photoprotection
interference a 200 nm transparent layer in fungi.
Peacock fern Selaginella willdenowii,
S. uncinata, Diplazium
Iridescent blue leaves.
crenatoserratum (Figure 4),
Various layers on the surface, each layer less than Photoprotection
D. tomentosum, Lindsaea lucida,
100 nm thick.
Multilayer Danaea nodosa,and Trichomanes
interference elegans
Iridescent blue leaves. Modified chloroplast Peacock begonia Begonia pavonina,
structures (iridoplasts) with many layers and each Phyllagathis rotundifolia (Figure 4), Photoprotection
layer less than 100 nm thick. and P. griffithii
Iridescent red. Multilayer system with 17 electron
Byproduct of wear-protection
opaque and translucent layers between ten and a Algae Iridaea
mechanism
few hundred nanometers thick.
Blue, green, and yellow
structural colouration in
hibiscus: structural
Diffraction Petals iridescence. Diffractive optics. Surface Hibiscus trionum, Tulipa
colouration in tulips in the
gratings striated one micrometer apart. kolpakowskiana
UV part of the spectrum (not
visible to people, visible for
bees).
Microscopic air spaces in surface hairs Preferential scattering of short
Scattering (trichomes) that reflect the light. Epicuticular wax Blue spruce Picea pungens wavelengths and enhanced
structures. reflectance of UV.
Iridescence in fruits. Iridosomes (secreted by
epidermis cells of fruits, partly cellulosic situated Blue quandong Elaeocarpus
inside cell wall), multilayer system arranged in 3D angustifolius syn. E. grandis Absorption of UV light,
Photonic structure. photoprotection, display and
crystals Internal structure of hollow hairs acts as a 2D Edelweiss Leontopodium nivale subsp. defence.
photonic crystal (optical fiber with photonic alpinum
crystal cladding).
Iridescent blue fruit. Multilayers in cell walls of Curved micro-Bragg reflector,
Pollia condensata
epicarp [11]. used for display and defence.
Cholesteric
liquid Modified chloroplasts with helicoidal structures. Fern Danaea nodosa
crystals
or refracted light are dependent on the optical properties of properties, such as surface condition, multilayer thickness,
the respective surfaces or interfaces. The optical properties of number of multilayers, and refractive index, are the main fac-
surfaces are determined by their structure and morphology tors that contribute to the quality of the reflected or refracted
[41]. Note that, in the terminology of solid-state physics, colours. A smooth surface can produce specular reflections,
“structure” refers to the geometrical arrangement of atoms while rough surfaces will produce diffuse reflections [40].
in a crystal lattice, while “morphology” refers to the macro-
scopic shape of a surface. This usage of the term structure 3.1.2. Structural Colour Mechanisms. The physical phenom-
is different to the usage of structure in “structural colours,” ena involved in producing structural colour in nature and
where it relates to micro- and nanometer-sized features that engineering are identified as follows: thin-film interference,
yield colouration or metallic effects [42]. multilayer thin film, diffraction gratings, and scattering.
Snell’s law [41] can be used to understand the reflection
and refraction of light (Figure 5). These phenomena occur Thin-Film Interference. Interference is described by super-
on every surface and interface in this world. The structural position of two or more waves [40]. When two or more
colours of many animals and plants are generated by the waves overlap, the displacements of waves at any point
reflection and refraction of incident light on nanostructured and any instant in time are combined. For the overlapping
surfaces of periodic biological multilayers [43]. Structural condition where the two waves are coherent, the sum of
Eye
𝜃i Reﬂected light
Incident light
𝜃t
Retina
Medium 1; n1 point
Medium 2; n2
𝜃r
Refracted
light Air
Film n
Figure 5: The principle of Snell’s law.
Figure 6: Thin-film interference (modified from [33]).
the two waves will produce constructive interference. In

contrast, destructive interference is described by the situation
where cancellation or partial cancellation of individual wave
amplitudes occurs. The structural colours of many biological number of slits, and distance between the centres of the
tissues, especially in plants, are produced by interference adjacent slits. The colouration resulting from light interacting
in multilayer thin films [44]. Thin-film interference occurs with diffraction gratings depends on the width and separation
when the incident light is reflected from its upper and of the slits/obstacles [40]. A diffraction grating produces
lower surfaces [40]. The incident light that reaches the upper rainbow colours that are strongly dependent on incident
surface is partly reflected and partly transmitted into the thin angle (e.g., DVDs, CDs). Some diffraction patterns can be
film. The transmitted light that reaches the lower surface observed through a microscope but not with the naked
of the thin film is partly reflected. The constructively or eye [44]. Diffraction gratings in plants were discovered
destructively interfering reflected light beams will reach the pretty late. Whitney and coworkers (2009) published a paper
retina of the eye (Figure 6). on colour-generating diffraction grating-like structures on
Colours produced from thin-film interference are not the surface of petals of Hibiscus trionum in Science [45].
very bright in most cases and examples include soap bubble Nowadays, we know that diffraction gratings occur regularly
colours and colours from oil films on water or gas sheets [43]. in flowering plants.
Increasing the number of layers results in a multilayer that can
produce much more intense, brilliant colours. Scattering. The scattering phenomena that yield structural
colouration in plants can either be coherent or incoherent
Multilayer Interference. The colour produced by this phe- [29]. Due to the constructive relationship between the two
nomenon is caused by sharp periodic boundaries in the scattered waves, coherent scattering produces strong colours.
refractive index [11]. The multilayer could be from liquid, The destructive relationship between the scattered waves
solid, or gaseous material [43]. The sparkling metallic lustre produces weak colours in incoherent scattering phenomena.
in the leaves of Begonia rex has been mimicked to improve Two types of scattering phenomena occur in plants: Mie and
colouration technology. The metallic effect in these leaves is Tyndall scattering. Mie scattering is a scattering phenomenon
formed by light reflection off the surface and the interior of that is caused by particles that are larger than the wavelength,
the leaves [10]. The formation of the light reflection from the while Tyndall scattering is caused by particles that are smaller
interior is associated with the interior structure of the leaf than the wavelength. Whereas the blue colour of the sky is
and is impacted by cell density and the arrangement of air produced by Rayleigh scattering, the Mie scattering produces
spaces. The anatomical structure of the leaf shows stacked the white colour of the clouds [40]. The high concentration
layers of cells, where each layer is one cell thick, in the interior of water particles in the clouds allows the scattering of all
of the leaf. A different type of cell with different cell density wavelengths of light, which results in the white colour of
forms every second layer. Through this structure the plant clouds. The fat globules in fine suspension of milk scatter the
establishes a multilayer system. light in all ranges of wavelengths, resulting in the white colour
of the milk. A milk solution that has low concentrations of fat
Diffraction Grating. The diffraction effect is defined by the globules will be perceived as blue, due to the fact that blue is
interference of many light waves around an obstacle [33]. It scattered more than red. Tyndall scattering phenomena occur
can also be caused by single or multiple slits, which represent in blue spruce and are also producing a blue hue in banana
obstacles. The diffraction pattern depends on the width, leaves [29].
Table 2: Engineering applications that use structural colour based optical devices.
Physical mechanism Device structure Engineering application References

Multilayer interference Multilayer thin film Coating [46]
Multilayer interference Self-assembled nanocrystalline cellulose film Humidity sensor [47]
Multilayer interference Multilayer flakes Coating/painting [48]
Multilayer interference Multilayer thin film Solar collector [49]
Multilayer interference Multilayer structure Solar collector [50]
Multilayer interference Multilayer thin film structure Antireflection coatings for solar cells [51]
Multilayer interference Polypeptide-based LB film Display system [52]
Multilayer interference Elastic optical multilayer fibre Colouration [53]
Multilayer interference Multilayer structure Interference filters [54]
Multilayer interference Multilayer structure Interference filter [55]
Photonic structure Photonic crystals Temperature tuneable photonic crystals [56]
Photonic structure Photonic heterostructures Colour filter [57]
Photonic structure Photonic crystals Calorimetric sensor [58]
Photonic structure Photonic crystals Antibody detection [59]
Diffraction grating Metal-dielectric multilayer Colour shift [60]
Photonic structure Photonic crystals Blast exposure sensor [61]
4. Production of Nanostructures properties as systems known from solid state physics, with
for Colouration photonic bands.
A discussion on the multilayer and 3D photonic crystal
4.1. Existing Products or Devices. As summarized in Table 1, structures used in tuneable colour devices for application
the mechanisms involved in the generation of struc- in coatings, for decoration, sensors, and display systems is
tural colouration in the plant kingdom consist of thin- provided in Sections 4.1.1–4.1.4. Although the mimicking of
film/multilayer interference, scattering, diffraction gratings, plant structures in developing such devices is not mentioned
and photonic structures. In engineering applications, mul- in most of the reviewed papers, due to the applied mecha-
tilayer interference is associated with thin-film technology, nisms, some of them might very well be inspired by structural
while photonic crystals are generally used to produce pho- colouration in plants. The majority of engineering devices are
tonic band gap (PBG) devices [58]. designed in simpler ways than the related plant structures
Besides being used for colouration purposes, devices that [53].
incorporate structural colour are used for applications such
as coatings, sensors, fuel cells, and certain types of display
systems (Table 2). 4.1.1. Multilayer Based Colouration
Man-made structural colour is obtained via the manip-
ulation of the optical properties of thin-film multilayers Multilayer Based Colouration for Coatings. Hirayama et al.
and photonic crystals, such as layer thickness and refractive [46] proposed multilayer film based coatings for illumina-
indices. Multilayer thin films consist of several layers alter- tion models for objects. The film’s primary reflection and
nately stacked one above the other. The improvement of layer- refraction multilayer structure was used to coat smooth and
by-layer (LBL) deposition and fabrication techniques allows rough surfaces by using multilayer film ray tracers (MFRT).
the development of multilayer thin films that offer various The illumination models used in this study were made of
kinds of optical properties [62]. silicon, coated with three layers: a dielectric-silver-dielectric
Photonic crystals (PCs) are materials that have a peri- multilayer or a dielectric-gold-dielectric multilayer. A better
odic modulation in their refractive index. This can be rendering illumination model was obtained in this study,
used to produce intense visible colours through coherent where the rough surface model showed clearer iridescent
Bragg diffraction [63, 64]. Photonic crystals are divided colour than the smooth surface.
into three categories: one-dimensional, two-dimensional, Yasuda et al. [48] developed novel TiO2 /SiO2 multi-
and three-dimensional structures [64]. These three structures layered flakes without cores for application as interference
differ in terms of periodic modulation of the permittivity. flakes that exhibit structural colours. These multilayered
These periodic modulations occur in one-dimensional, two- flakes were proposed to improve effect pigment coating in
dimensional, and three-dimensional structures at one, two, terms of coating thickness reduction (note that the tech-
and three directions of the medium, respectively. Due to this nical term “effect pigments” refers to colouration without
modulation, photonic crystal structures show similar optical actual pigments, but with structures alone. However, this
term is established and generally used, so we also use it, index of the layer could be controlled to obtain the desired
although we state that it is structures that are responsible colour.
for the colouration, not pigments). In this study, seven layers
of TiO2 /SiO2 sol-gel route multilayers were developed for Multilayer Based Colouration for Sensing. Zhang et al. [47]
high/low refractive index layers for interference effects on developed a self-assembled nanocrystalline cellulose-based
flakes for decorative paint. This multilayer reflected cyan and chiral nematic multilayer film that changed colour with
orange and transmitted red and light blue colour. humidity. The film had a helical twist axis of periodic layer
Future tuneable structurally coloured fabrics could be structures that acted as multidomain Bragg reflectors. A
realized through the bioinspired design of multilayer based multilayer that was constructed using a nematic-type phase
soft photonic fibres, as proposed by Kolle et al. [53]. Inspired structure that possessed self-aligned rod-shape molecules
by the structure of the seed coating found in Margaritaria was used to obtain the long-range directional order with
nobilis fruits, band-gap tuneable elastic multilayer fibres were regard to parallel long axes [67]. Through the sorption and
developed by forming a bilayer of two elastomeric dielectrics, desorption of water by the film due to reaction to the
polydimethylsiloxane (PDMS) and polystyrene-polyisoprene humidity, the layer thickness changed and thereby also the
triblock copolymer (PSPI) on a silicon substrate. This bilayer colour. The iridescent colour produced was blue to green in
was then rolled up onto the glass fibre to form a multilayer the dry state and red to orange in the wet state.
cladding with a diameter of ∼15 𝜇m. Removing the glass fibre
from the rolled-up multilayer allows mechanical deforma- Multilayer Based Colouration for Display. Structural coloura-
tion, resulting in tuning of the band gap and spectral blue- tion has also been used in the development of chameleon-
shift, with brilliant colours. like display systems, as proposed by Kinoshita et al. [52].
The display system was formed by polypeptide multilayers
based on Langmuir-Blodgett (LB) films [62]. Kinoshita et al.
Multilayer Based Colouration for Solar Collectors. Schüler et
developed the LB film by transferring the poly (𝛾-hexyl-l-
al. simulated the development of a multilayer based coloured
glutamate) (PHeLG) multilayer onto the silicon substrate in a
solar thermal collector [49, 65]. The issue of colour of
number of layers up to 160 with a thickness of 1.6 nm per layer.
solar thermal collectors for buildings is important due to
Stacks with different thickness of such PHeLG multilayers
architectural limitation of black solar thermal collectors. A
produced different colours: 40–50 layers produced brown,
colour reflecting cover glass was proposed in this study as a 60–70 layers produced dark blue, 80–100 layers produced
means of achieving better appearance without interrupting light blue, yellow was obtained when the number of layers was
the energy absorption of the solar thermal collector sys- 120, and red-purple was obtained at 160 layers. The reflective
tem. The study was conducted through simulation of an VIS spectra of multilayers produce different interference
International Commission on Illumination (CIE) (CIE is a colors depending on the number of layers.
short form of International Commission on Illumination in
French: Commission Internationale de l’Eclairage [66]) based Multilayer Based Interference Filters. Multilayer polymeric
colour coordinated approach on at least two designs, two interference reflectors were reviewed by Nevitt and Weber
layered systems and three layered systems designs. From this [54]. They found that the desired polymeric interference
study, the two-layered design was proposed as a means of reflector with the desired optical properties could be obtained
creating a colourful reflection in the visible spectral region by controlling the thickness and structural uniformity of
and a region of antireflection. The three-layered design was the polymeric multilayer stacks. One popular product that
proposed as a means of creating a strong enhancement of the possesses this kind of structure is the narrow-band visible
reflectance peak. comb filter that is used on a 3D display system. Asghar
Wu et al. [50] fabricated titanium-aluminium nitride et al. [55] modelled multilayer thin-film interference-based
(Ti-AIN) multilayer based solar thermal collectors. Five broad-band-pass filters. The structural modelling was per-
colours, namely, black, purple, yellowish green, red, and formed by layer-matching the quarter-wave-thick layers in
yellowish orange, were obtained by variation in layer number low, medium, and high refractive indices over the visible
and thickness of the Ti-AIN multilayer that was fabricated spectrum. The proposed multilayer based broad-band-pass
through magnetron sputtering. filter was utilized to transmit visible spectra in a smooth man-
Selj et al. [51] proposed a clear coloured, highly efficient ner, while suppressing the unwanted peak of the spectrum.
solar cell with multilayer antireflection coatings. The use of
a multilayer antireflection coating in this study had benefits, 4.1.2. Photonic Structure-Based Colouration. Wang and
not only as an antireflection coating but also as a coloured Zhang [58] reviewed the tuneable structural colour of
coating. The multilayer antireflection layer was made of calorimetric sensors using photonic crystals (PCs). One-
SiNx and silicon oxide via plasma-enhanced chemical vapour dimensional (1D) and three-dimensional (3D) photonic
deposition (PECVD) and nanoporous silicon methods. The crystals are mostly found in plants and always used for
colours resulting from the multilayer antireflection layers such artificial sensing systems. 1D photonics crystals are
prepared using the PECVD method produced a red, green, more popular than 3D crystals as a result of the fact that
and blue. Using nanoporous silicon methods, the colours they incorporate an inherently simple photonic structure.
obtained were green, red, purple, and orange. Through The 1D and 3D photonic crystal-based artificial sensing
nanoporous silicon methods, the thickness and refractive systems reviewed by Wang and Zhang were vapour and
solvent sensors, temperature sensors, ion and pH sensors, markets. Effect pigments are structured platelets. There are
and pressure sensors. three types: (a) substrate-free effect pigments structure, (b)
Liu et al. [56] developed the sol-gel inverse opal structure- effect pigments with layer and substrate structure, and (c)
based temperature tuneable photonic band gap crystals for multilayer effect pigments without substrate structure (note
a temperature sensor. A change in temperature changes the that what is denoted as pigment here actually refers to
liquid-vapour phase that fills the cavities of the inverse opal structures, but, as described above, because the name “effect
film and this precipitates the change of the refractive index. pigment” is well introduced and widely accepted, we use it
They found that changing the refractive index of the inverse here too).
opal film shifted the photonic band gap, which consequently
resulted in a colour shift.
The inverse opal structure film-based labelling of free
specific detection of immunoglobulin G antibody (IgG) by 4.2. Development, Modelling, and Fabrication. As described
using nanoporous hydrogel photonic crystals was proposed in Section 4.1, the fabrication process represents an important
by Choi et al. [59]. Using the proposed sensor, the IgG step towards obtaining a multilayer thin film, a photonic
concentration level could be determined via the naked eye crystal, or a diffraction grating with the desired optical
by looking for changes in colour. In this study, 10 mg/mL IgG properties. The development of optical structures that are
concentration was indicated by a colour change from green used in various applications within this review consisted of
to dark orange. The proposed sensor made a simple and cost- many approaches, such as mathematical modelling, simu-
effective process fabrication possible. lations, and fabrication. Potential fabrication methods for
The heterostructures of 1D photonic crystal-based three- biomimetic animal-inspired optical materials were reviewed
color filters were developed by Li et al. [57]. This photonic by Yu et al. [69]. The methods proposed could be divided
crystal incorporates defect layers that contain Si/MgF2 mul- into two approaches: a biotemplate-based approach and
tilayer films. The thickness of the defect layer was altered to a nonbiotemplate approach. The first approach could be
restrict certain wavelengths of light from entering the pho- used to produce a structure or an inverse template, while
tonic crystal band gap. The restricted light in that wavelength the artificial analogue of the structure could be obtained
was reflected and correspondingly appeared in colour. Li et al. via the second approach. The fabrication techniques that
[57] successfully obtained a blue-green-red colour filter, with are involved are atomic layer deposition, nanoimprinting,
high transmission rates. and the electron beam lithography process, all of which
A structural colour-based display system could also be are well-established techniques that are regularly used to
developed via photonic ink (P-Ink) technology, which is fabricate animal-inspired biomimetic optical materials. The
comprised of photonic crystals. This system was developed fabrication of some photonic crystal structures based on
by Wang et al. [63], who utilized the changes of applied calorimetric sensors was systematically reviewed by Wang
current and voltage in order to reflect a certain band of and Zhang [58].
colour. They found that every single P-Ink material was Through mathematical modelling, the structural colour
capable of reflecting all of the spectral colours in the visible of a plant bioinspired multilayer thin-film model is always
range. The colour switching that occurred in the system was determined using the Fresnel formula, along with reflection
caused by the expansion and contraction of the cross-linked and transmittance phenomena theory [48]. The fabrication
electroactive polymer network. techniques used in the development of the plant bioinspired
multilayer thin film are sol-gel coating [48, 49, 56], spin
coating [53], plasma enhanced chemical vapor deposition
4.1.3. Diffraction Grating-Based Colouration. A colour filter (PECVD), and electrochemical etching, Selj et al. [51].
based on the reflection resonance of metal-dielectric-metal
As stipulated in this section, many applications are made
trilayered structures was proposed by Chen and Liu [60].
viable through the use of engineering-based multilayer thin
The design of the filter mainly focused upon adjusting the
film and photonic structural products. Engineering-based
thickness and refractive indices of the middle layer. Red,
application products always apply the reflected or refracted
green, and blue colours were expected to emanate from this
colours, which are the result of the interference phenomena.
filter.
Due to this requirement, the reflection or refraction features
are regarded as crucial in this context. On the other hand,
4.1.4. Effect Pigment-Based Colouration. The iridescent effect different applications require different optical structures. The
in flower petals is generated by a diffraction grating mech- desired structures, with certain optical properties, could be
anism in combination with pigments. This phenomenon obtained via the manipulation of the stacked layer’s thickness,
entails that the diffraction grating might enhance the the refractive index of each layer, structural uniformity, and
pigment-based colouration in the flower petals. In coloura- the surface condition of the outermost layer of the multilayer.
tion technology, the effect that the pigment technology has is The performance of such optical structure’s designs is very
similar to the iridescent effect found in flower petals. much influenced by fabrication techniques. Due to the high
The progress of effect pigment technology was reviewed cost of the fabrication process, computer simulation and
in detail by Maile et al. [68]. The effect pigments associated mathematical modelling are popular approaches for initial
with special effect colours like angle-dependent ones is in investigations and designs for complex optical structures [46,
high demand in today’s industries and consumer product 48, 55].
Structure
characterization of Optical
optical structures in Fabrication methods
characterization of for optical
biology. Array colour optical structures in biomimetic materials
effect either structural
or pigmentation biology
colour
Figure 7: Three steps in multidisciplinary concept from nature (organism) to optical biomimetic materials (structural colour) [57].
5. Conclusion and Outlook One always needs to bear in mind the fact that one basic
property of the materials and structures of living entities is
5.1. General Ideas in This Paper. The process of studying their multifunctionality, so if it is the intention just to transfer
this subject was intense and the experience resulted in valu- the deep principles of the colouration, then not all structural
able knowledge. Carrying out research involving multidisci- aspects need to be incorporated in the biomimetic system.
plinary fields that includes biology, science, and engineering
is difficult, and understanding the key concepts can be
challenging; however, its impact is rather significant. The
language used in research papers can be difficult to grasp 5.2.2. Concept of Biomimetics. Throughout history, nature
when the work is multidisciplinary in nature. In this context, has inspired various human achievements and has led to
effective communication plays an important role, so that cost, the development of effective materials, structures, tools,
time, and energy can be optimized. Biology is the study of mechanisms, processes, algorithms, and functions that are
living organisms and their interactions with the environ- advantageous to humankind. The use of nature’s designs
ment and other organisms; however, current engineering to solve engineering problems is known as biomimetics.
that learns from living nature just mimics, imitates, or is Biomimetics is an interdisciplinary knowledge field that com-
bioinspired by a part of that study, mainly mechanisms and bines biology, technology, and the arts [71] and it has proven
materials. promising in the development of emerging MEMS (micro-
electromechanical systems). Biomimetics aims to identify the
deep underlying principles of materials, structures (including
5.2. Biomimetics: Transfer from Nature to nanostructures), and processes found in living nature and to
Technological Applications subsequently transfer knowledge about these phenomena to
engineering and the arts. It seeks to apply certain principles
5.2.1. Biology as a Model. Biology represents a large and from biological systems to technological strategies in order
valuable basis model for technologies and applications. The to develop innovative applications. The range of potential
structural properties of the mechanisms found in leaves, uses for biomimetics is enormous and in today’s society these
petals, and some fruits prove to be productive in research include architecture and design and surface and materials
and biomimicry or bioinspired design. In structural colour, technologies as well as sensors, medical engineering, and
the presence of the thickness of filters/layers and refractive management [72–74].
indices is important. The attractive and sometimes really
Structural colour has inspired researchers to study and
striking structural colours found in animals, plants, and
understand the step from organisms to biomimetics in
microorganisms have attracted interdisciplinary interest and
related fields such as optical materials. In this proportion, Yu
many scientists and researchers have strived to understand
and coworkers summarize biomimetic optical materials by
their biological basics and to transfer these into the fields
providing a step in optical biomimetic materials (Figure 7)
of engineering [70]. Key parameters of structural colours
[69]. Nature provided diverse microstructures based on thin-
in biological systems that need to be investigated are angle
film interference, multilayer interference, diffraction grating,
dependence, wavelength dependence, polarization depen-
photonic crystals, and light scattering. Optical biomimetics
dence, and system reflectance properties [53].
as a multidisciplinary field requires collaboration among
Natural structural colour phenomena have inspired tech-
biologists, physicists, chemists, and materials scientists.
nologically exploitable features for light and colour manip-
ulation. The nanoscale photonic architecture that is found in
iridescent plants formed a key element that is used to produce
iridescent colouration in the engineering world. Sometimes Glossary
the structures used in bioinspired artificial systems neglect
many aspects of the complexity found in the related structures LBL: Layer-by-layer
of the biological material. For example, the tuneable elastic IgG: Immunoglobulin G
optical multilayer fibres that were inspired by the Margar- Iridescence: The property of certain surfaces that
itaria fruits have much simpler structures than the related appear to change colour as the angle of
structure in nature [53], which, for example, comprises view or the angle of illumination
elliptical structures in the periodic layers of the fruit cells. changes
Iridosome: Secreted by epidermis cell of fruit, partly [10] Y. Zhang, T. Hayashi, M. Hosokawa, S. Yazawa, and Y. Li,
cellulosic caused situated inside cell wall “Metallic lustre and the optical mechanism generated from the
Iridoplast: Modified colourful chloroplast struc- leaf surface of Begonia rex Putz,” Scientia Horticulturae, vol. 121,
tures of leaf no. 2, pp. 213–217, 2009.
LB: Langmuir-Blodgett [11] S. Vignolini, P. J. Rudall, A. V. Rowland et al., “Pointillist
MFRT: Multilayer film raytracers structural color in Pollia fruit,” Proceedings of the National
PBG: Photonic band gap Academy Sciences, vol. 109, no. 39, pp. 15712–15715, 2012.
PCs: Photonic crystals [12] D. W. Lee, G. T. Taylor, and A. K. Irvine, “Structural fruit
PDMS: Polydimethylsiloxane coloration in Delarbrea michieana (Araliaceae),” International
PECVD: Plasma enhanced chemical vapour Journal of Plant Sciences, vol. 161, no. 2, pp. 297–300, 2000.
deposition [13] K. Yoshida, D. Ito, Y. Shinkai, and T. Kondo, “Change of color
Petal: Part of a flower and components in sepals of chameleon hydrangea during
maturation and senescence,” Phytochemistry, vol. 69, no. 18, pp.
PHeLG: Poly (𝛾-hexyl-l-glutamate)
3159–3165, 2008.
P-Ink: Photonic ink
[14] G. A. Marx, “Argenteum (Arg) mutant of Pisum: genetic control
lp160ptPSPI: Polystyrene-polyisoprena triblock poly-
and breeding behavior,” Journal of Heredity, vol. 73, no. 6, pp.
mer 413–420, 1982.
Sepal: The outmost part in flowering plant
[15] P. E. Grimbly and B. J. Thomas, “Silvering, a disorder of the
Ti-AIN: Titanium-aluminium nitride tomato,” Journal of Horticultural Science, vol. 52, pp. 49–57, 1977.
Understory forest: Plant life growing beneath the forest
[16] Y. Burger, H. S. Paris, H. Nerson, Z. Karchi, and M. Edelstein,
canopy without penetrating it to any “Overcoming the silvering disorder of Cucurbita,” in Cucurbit
extent. Genetics Cooperative Report, 1983.
[17] H. S. Paris, H. Nerson, and Y. Burger, “Leaf silvering of
Conflict of Interests Cucurbita,” Canadian Journal of Plant Science, vol. 67, pp. 593–
598, 1987.
The authors declare that there is no conflict of interests [18] R. Kiew, Begonias of Peninsular Malaysia, Natural History
regarding the publication of this paper. Publications (Borneo) Sdn. Bhd, Sabah, Malaysia, 2005.
[19] B. J. Glover and H. M. Whitney, “Structural colour and irides-
cence in plants: the poorly studied relations of pigment colour,”
Acknowledgment Annals of botany, vol. 105, no. 4, pp. 505–511, 2010.
This work was supported by the Ministry of Higher Edu- [20] P. Vukusic and D. G. Stavenga, “Physical methods for investigat-
cation, Government of Malaysia, Project no. FRGS/1/2013/ ing structural colours in biological systems,” Journal of the Royal
Society Interface, vol. 6, no. 2, pp. S133–S148, 2009.
TK02/UKM/01/1.
[21] E. J. H. Corner, Wayside Trees of Malaya, Malayan Nature
Society, Kuala Lumpur, Malaysia, 3rd edition, 1988.
References [22] D. W. Lee, “Ultrastructural basis and function of iridescent blue
colour of fruits in Elaeocarpus,” Nature, vol. 349, no. 6306, pp.
[1] S. Kinoshita, Structural Colors in the Realm of Nature, World 260–262, 1991.
Scientific Publishing, River Edge, NJ, USA, 2008.
[23] D. W. Lee, “Iridescent blue plants,” The American Scientist, vol.
[2] D. W. Lee and J. B. Lowry, “Physical basis and ecological 85, no. 1, pp. 56–63, 1997.
significance of iridescence in blue plants,” Nature, vol. 254, no. [24] S. E. Sultan, “Phenotypic plasticity for plant development,
5495, pp. 50–51, 1975. function and life history,” Trends in Plant Science, vol. 5, no. 12,
[3] R. Willstätter and A. Stoll, Untersuchungen über die Assimilation pp. 537–542, 2000.
der Kohlensäure, Springer, Berlin, Germany, 1918. [25] S. Zobl, T. R. Martin, B. Y. Majlis, T. Schwerte, M. Schreiner, and
[4] J. A. Tanno and T. R. Webster, “Variegation in Selaginella I. C. Gebeshuber, “Structural colours in the focus of nanoengi-
martensii f. albovariegata. I. Expression and inheritance,” Cana- neering and the arts: survey on state-of-the art developments,”
dian Journal of Botany, vol. 60, pp. 2375–2383, 1982. in Proceedings of the 3rd European Conference on Tribology,
[5] D. W. Lee, Nature’s Palette: The Science of Plant Color, The Vienna, Austria, 2011.
University of Chicago Press, Chicago, Ill, USA, 2007. [26] S. Lev-Yadun, M. Inbar, I. Izhaki, G. Ne’eman, and A. Dafni,
“Colour patterns in vegetative parts of plants deserve more
[6] J. D. Mauseth, Botany: An Introduction to Plant Biology, Jones
research attention,” Trends in Plant Science, vol. 7, no. 2, pp. 59–
and Bartlett, 4th edition, 2009.
60, 2002.
[7] D. W. Lee, “On iridescent plants,” Garden’s Bulletin, vol. 30, pp. [27] L. O. Matolweni, K. Balkwill, and T. McLellan, “Genetic
21–29, 1977. diversity and gene flow in the morphologically variable, rare
[8] R. M. Graham, D. W. Lee, and K. Norstog, “Physical and endemics Begonia dregei and Begonia homonyma (Begoni-
ultrastructural basis of blue leaf iridescence in two neotropical aceae),” The American Journal of Botany, vol. 87, no. 3, pp. 431–
ferns,” The American Journal of Botany, vol. 80, no. 2, pp. 198– 439, 2000.
203, 1993. [28] K. R. Thomas, M. Kolle, H. M. Whitney, B. J. Glover, and U.
[9] K. S. Gould and D. W. Lee, “Physical and ultrastructural basis of Steiner, “Function of blue iridescence in tropical understorey
blue leaf iridescence in four Malaysian understory plants,” The plants,” Journal of the Royal Society Interface, vol. 7, no. 53, pp.
American Journal of Botany, vol. 83, no. 1, pp. 45–50, 1996. 1699–1707, 2010.
[29] I. C. Gebeshuber and D. W. Lee, “Nanostructures for coloration [49] A. Schüler, J. Boudaden, P. Oelhafen, E. de Chambrier, C.
(organisms other than animals),” in Springer Encyclopedia of Roecker, and J.-L. Scartezzini, “Thin film multilayer design
Nanotechnology, B. Bhushan and M. Nosonovsky, Eds., pp. types for colored glazed thermal solar collectors,” Solar Energy
1790–1803, Springer, 2012. Materials & Solar Cells, vol. 89, no. 2-3, pp. 219–231, 2005.
[30] F. T. Mott, “Organic color,” Science, vol. 21, no. 541, pp. 323–325, [50] Y. W. Wu, W. Zheng, L. Lin, Y. Qu, and F. Lai, “Colored solar
1893. selective absorbing coatings with metal Ti and dielectric AlN
[31] C.-R. Sheue, S.-H. Pao, L.-F. Chien, P. Chesson, and C.-I. Peng, multilayer structure,” Solar Energy Materials & Solar Cells, vol.
“Natural foliar variegation without costs? The case of Begonia,” 115, pp. 145–150, 2013.
Annals of Botany, vol. 109, no. 6, pp. 1065–1074, 2012. [51] J. H. Selj, T. T. Mongstad, R. Søndenå, and E. S. Marstein,
[32] J. C. Maxwell, A Treatise on Electricity and Magnetism, vol. 2, “Reduction of optical losses in colored solar cells with mul-
Clarendon Press, Oxford, UK, 1873. tilayer antireflection coatings,” Solar Energy Materials & Solar
Cells, vol. 95, no. 9, pp. 2576–2582, 2011.
[33] H. Hertz, “On the relations between Maxwell’s fundamen-
tal equations of the opposing electromagnetics,” Wiedemann’s [52] T. Kinoshita, S. Hayashi, and Y. Yokogawa, “Preparation of a
Annalen, vol. 23, pp. 84–103, 1884. structural color forming system by polypeptide-based LB films,”
Journal of Photochemistry and Photobiology A: Chemistry, vol.
[34] L. Rayleigh, “On the reflection of light from regularly stratified
145, no. 1-2, pp. 101–106, 2001.
medium,” Proceedings of Royal Society London A, vol. 93, no. 655,
pp. 565–577, 1917. [53] M. Kolle, A. Lethbridge, M. Kreysing, J. J. Baumberg, J. Aizen-
berg, and P. Vukusic, “Bio-inspired band-gap tunable elastic
[35] B. Walter, Die Oberflächen- oder Schillerfarben, Braunschweig,
optical multilayer fibers,” Advanced Materials, vol. 25, pp. 2239–
1895.
2245, 2013.
[36] A. A. Michelson, “On metallic colouring in birds and insects,” [54] T. J. Nevitt and M. F. Weber, “Recent advances in multilayer
Philosophical Magazine Series 6, vol. 21, no. 124, pp. 554–567, polymeric interference reflector products,” Thin Solid Films, vol.
1911. 532, pp. 106–112, 2013.
[37] D. W. Lee, “Plant tissue optics: micro- and nanostructures,” in [55] M. H. Asghar, M. B. Khan, and S. Naseem, “Modeling thin
Biomimetics and Bioinspiration, vol. 7401 of Proceedings of SPIE, film multilayer broad-band-pass filters in visible spectrum,”
August 2009. Czechoslovak Journal of Physics, vol. 53, no. 12, pp. 1209–1217,
[38] J. Xu and Z. Guo, “Biomimetic photonic materials with tunable 2003.
structural colors,” Journal of Colloid and Interface Science, vol. [56] Z. Liu, J. Gao, B. Li, and J. Zhou, “Temperature tunable
406, pp. 1–17, 2013. photonic band gap crystals based on liquid-infiltrated inverse
[39] I. Woo, S. Yu, J. S. Lee et al., “Plasmonic structural-color thin opal structure,” Optical Materials, vol. 35, pp. 1134–1137, 2013.
film with a wide reception angle and strong retro-reflectivity,” [57] H. Li, H. Guan, P. Han, Y. Li, and C. Zhang, “Design for a broad
IEEE Photonics Journal, vol. 4, no. 6, pp. 2182–2188, 2012. non-transmission band gap of three-color filters using photonic
[40] H. D. Young and R. A. Freedman, Sears and Zemansky’s Univer- heterostructures,” Optics Communications, vol. 287, pp. 162–166,
sity Physics: With Modern Physics, Pearson Addison Wesley, San 2013.
Fancisco, Calif, USA, 11th edition, 2004. [58] H. Wang and K. Q. Zhang, “Photonic crystal structures with
[41] V. G. Bordo and H. G. Rubahn, Optics and Spectroscopy at tunable structure color as colorimetric sensors,” Sensors, vol. 13,
Surfaces and Interfaces, Wiley-VCH Verlag GmbH & Co.KGaA, no. 4, pp. 4192–4213, 2013.
Weinheim, Germany, 2005. [59] E. Choi, Y. Choi, Y. H. P. Nejad, K. Shin, and J. Park, “Label-
[42] S. Y. Lee and L. Dal Negro, “Angularly independent structural free specific detection of immunoglobulin G antibody using
color of nanostructured metal surfaces,” in Proceedings of the nanoporous hydrogel photonic crystals,” Sensors and Actuators
16th International Conference on Optical MEMS and Nanopho- B: Chemical, vol. 180, pp. 107–113, 2013.
tonics (OMN ’11), pp. 25–26, August 2011. [60] Y. Chen and W. Liu, “Reflection and color characteristics of
[43] S. Berthier, Iridescences : The Physical Colors of Insects, Springer, tri-layer metal-dielectric structures for generation of distinctive
New York, NY, USA, 2007. color shifts,” Optik, vol. 24, pp. 13–16, 2013.
[44] J. H. McClendon, “The micro-optics of leaves. I. Patterns of [61] D. K. Cullen, Y. Xu, D. V. Reneer et al., “Color changing photonic
reflection from the epidermis,” The American Journal of Botany, crystals detect blast exposure,” NeuroImage, vol. 54, no. 1, pp.
vol. 71, no. 10, pp. 1391–1397, 1984. S37–S44, 2011.
[45] H. M. Whitney, M. Kolle, P. Andrew, L. Chittka, U. Steiner, and [62] G. Decher and J. B. Schlenoff, Multilayer Thin Films: Sequen-
B. J. Glover, “Floral iridescence, produced by diffractive optics, tial Assembly of Nanocomposite Materials, Wiley-VCH Verlag
acts as a cue for animal pollinators,” Science, vol. 323, no. 5910, GmbH & Co. KGaA, Weinheim, Germany, 2003.
pp. 130–133, 2009. [63] H. Wang, F. Kerins, U. Kamp, L. Bonifacio, A. C. Arsenault, and
[46] H. Hirayama, K. Kaneda, H. Yamashita, and Y. Monden, “An G. A. Ozin, “Photonic-crystal display materials,” Information
accurate illumination model for objects coated with multilayer Display, vol. 27, no. 7-8, pp. 26–29, 2011.
films,” Computers and Graphics, vol. 25, no. 3, pp. 391–400, 2001. [64] I. A. Sukhoivanov and I. V. Guryev, “Photonic crystal optical
[47] Y. P. Zhang, V. P. Chodavarapu, A. G. Kirk, and M. P. Andrews, fibers,” Photonic Crystals, vol. 152, pp. 127–161, 2010.
“Structured color humidity indicator from reversible pitch tun- [65] A. Schüler, D. Dutta, E. de Chambrier et al., “Sol-gel deposition
ing in self-assembled nanocrystalline cellulose films,” Sensors and optical characterization of multilayered SiO2 /Ti1−𝑥 Si𝑥 O2
and Actuators B, vol. 176, pp. 692–697, 2013. coatings on solar collector glasses,” Solar Energy Materials &
[48] T. Yasuda, K. Nishikawa, and S. Furukawa, “Structural colors Solar Cells, vol. 90, no. 17, pp. 2894–2907, 2006.
from TiO2 /SiO2 multilayer flakes prepared by sol-gel process,” [66] CIE, “C.B. CIE—International Commission on Illumination
Dyes and Pigments, vol. 92, no. 3, pp. 1122–1125, 2012. CIE Central Bureau,” 2014, http://www.cie.co.at/.
[67] J. A. Rego, J. A. A. Harvey, A. L. MacKinnon, and E. Gatdula,

“Asymmetric synthesis of a highly soluble “trimeric” analogue
of the chiral nematic liquid crystal twist agent Merck S1011,”
Liquid Crystals, vol. 37, no. 1, pp. 37–43, 2010.
[68] F. J. Maile, G. Pfaff, and P. Reynders, “Effect pigments—past,
present and future,” Progress in Organic Coatings, vol. 54, no.
3, pp. 150–163, 2005.
[69] K. Yu, T. Fan, S. Lou, and D. Zhang, “Biomimetic optical
materials: integration of nature’s design for manipulation of
light,” Progress in Materials Science, vol. 58, pp. 825–873, 2013.
[70] P. Vukusic, “Structural colour: elusive iridescence strategies
brought to light,” Current Biology, vol. 21, no. 5, pp. R187–R189,
2011.
[71] Y. Bar-Cohen, Biomimetics: Biologically Inspired Technologies,
CRC Press, Boca Raton, Fla, USA, 2005.
[72] I. C. Gebeshuber, P. Gruber, and M. Drack, “A gaze into the
crystal ball: biomimetics in the year 2059,” Proceedings of the
Institution of Mechanical Engineers C: Journal of Mechanical
Engineering Science, vol. 223, no. 12, pp. 2899–2918, 2009.
[73] I. C. Gebeshuber, Biomimetics and Nanotechnology, UKM Press,
Penerbit UKM Bangi, Bangi, Malaysia, 2011.
[74] I. C. Gebeshuber and M. O. Macqueen, “What is a physicist
doing in the jungle? Biomimetics of the rainforest,” in Advances
in Bionic Engineering, vol. 461 of Applied Mechanics and Mate-
rials, pp. 152–162, 2014.
http://dx.doi.org/10.1155/2014/293624
Research Article
A Dual-Functional [SBA-15/Fe3O4/P(N-iPAAm)] Hybrid System
as a Potential Nanoplatform for Biomedical Application
Andreza de Sousa,1,2 Karynne Cristina de Souza,1 Paula Maria da Silva Leite,1,2

Ricardo Geraldo de Sousa,2 and Edésia Martins Barros de Sousa1
1
Serviço de Nanotecnologia, Centro de Desenvolvimento da Tecnologia Nuclear CDTN/CNEN, Avenida Presidente Antônio Carlos,
6.627, Campus da UFMG, Pampulha, 31270-90 Belo Horizonte, MG, Brazil
2
Departamento de Engenharia Quı́mica, E.E.UFMG, Avenida Presidente Antônio Carlos, 6.627, Campus da UFMG, Pampulha,
31270-90 Belo Horizonte, MG, Brazil
Correspondence should be addressed to Edésia Martins Barros de Sousa; sousaem@cdtn.br
Received 15 November 2013; Revised 7 February 2014; Accepted 23 February 2014; Published 3 April 2014
Academic Editor: Anchal Srivastava
Copyright © 2014 Andreza de Sousa et al. This is an open access article distributed under the Creative Commons Attribution
cited.
The synthesis strategy of a multifunctional system of [SBA-15/Fe3 O4 /P(N-iPAAm)] hybrids of interest for bioapplications was
explored. Magnetite nanoparticles coated by mesoporous silica were prepared by an alternative chemical route using neutral
surfactant and without the application of any functionalization method. Monomer adsorption followed by in situ polymerization
initiated by a radical was the adopted procedure to incorporate the hydrogel into the pore channels of silica nanocomposite.
Characterization of the materials was carried out by using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy
(FTIR), N2 adsorption, transmission electron microscopy (TEM), and Temperature programmed reduction studies (TPR). Their
application as drug delivery system using atenolol as a model drug to assess the influence of the application of low frequency
alternating magnetic fields on drug release was evaluated. The structural characteristics of the magnetic hybrid nanocomposite,
including the effect of the swelling behavior on heating by the application of an alternating magnetic field, are presented and
discussed.
1. Introduction The delivery of these molecules was once considered

impossible, because of the difficulty associated with the
Ordered mesoporous materials have a number of promis- diffusion of large molecules through the materials of con-
ing applications in many fields of technology as advanced ventional drug delivery systems. These organic substances
electronics, catalysis, and nanostructured materials, among are normally very large in size and mesoporous silica is
others [1–3]. The intrinsic uniform porous structure of this a potential candidate to encapsulate such molecules by
class of compounds with their large specific surface area utilizing ordered mesopores [5, 6]. Nevertheless, there is
and pore volume, associated with surface silanol groups, one major problem for the mesoporous systems as far as
give these materials a significant potential for applications drug release is concerned, that is, the limited control of
as matrices of many chemical species, such as organic the drug release whose major mechanism is diffusion [7].
molecules, metals, and polymeric materials. The performance The need to create new materials with optimized, prede-
of these materials in many fields of applications depends termined characteristics has spurred an increasing interest
directly on the silica network porosity. Because of their large in hybrid materials, especially in organic and inorganic
pores, high hydrothermal stability, and easy preparation, nanocomposites.
SBA-15 materials have been considered very promising for For a better release control, some systems can be exter-
hosting and further delivery under appropriate conditions of nally activated to release more drugs when necessary using
a variety of molecules of pharmaceutical interest [4]. external forces such as temperature or magnetism [8].
In the case of temperature aging as an external force In view of the aforementioned, the objective of this study
for diffusion, temperature-responsive hydrogels are a well- was to investigate the synthesis strategy of a dual-functional
studied class of drug delivery systems, as they can respond system of [SBA-15/Fe3 O4 /P(N-iPAAm)] hybrids of interest
pronouncedly to temperature changes. Among these stimuli- for bioapplications. Magnetite nanoparticles coated by meso-
responsive polymers, temperature-responsive hydrogels porous silica were prepared by an alternative chemical route
such as poly(N-isopropylacrylamide) [P(N-iPAAm)] [9, 10] using neutral surfactant and without the application of any
exhibits, in water, a phase transition at a lower critical functionalization method. Monomer adsorption followed by
solution temperature (LCST) of approximately 33∘ C [11]. in situ polymerization initiated by a radical was the adopted
Below the LCST, the hydrogel incorporates water and swells, procedure to incorporate the hydrogel into the pore channels
whereas the release of water in response to an increase of silica nanocomposite. Subsequently, its physicochemical
in temperature causes shrinkage. Thus, the development characteristics were investigated by using different techniques
of hybrid functional nanosystems based on silica-[P(N- and the drug release profile of the system was studied in the
iPAAm)] has drawn much attention to the control of presence of magnetic field-induced heating. In addition, the
molecular transport, including drug release, because effects of the gel swelling behavior on heating by the appli-
self-regulated delivery allows for drug release when it is cation of an alternating magnetic field were also presented
needed [5]. Such a thermosensitive polymer with reversible and discussed in terms of drug release and heat generation
phase transition characteristics is attractive as a polymeric capacity.
material for the temperature responsive drug release systems
[12].
In the case of magnetism, the magnetite nanoparticles 2. Experimental
can be used to target localized heating in vivo when an
alternating current (AC) magnetic field is applied, like in 2.1. Material Synthesis. SBA-15 was synthesized in accor-
the hyperthermia treatment for anticancer therapy due their dance with the published procedure [17] using Pluronic
unique magnetic properties [13]. This treatment consists P123-PEO20 PPO70 PEO20 (poly(ethylene glycol)-block-poly
in dispersing the magnetic nanoparticles in the diseased (propylene glycol)-blockpoly (ethylene glycol), 𝑀av = 5800,
tissue and applying an alternating magnetic field to cause Sigma-Aldrich) as a template in acidic conditions. In a typical
local heating. Temperature around 43–45∘ C lysis the tumor experiment, 4 g of triblock copolymer was dissolved in water
cells with no damage to normal cells. However, nonsurface- and HCl (37 wt. % solution) under stirring at 40∘ C. After 1 h,
modified magnetic nanoparticles with a large surface-area- 8.2 g of tetraethyl orthosilicate (TEOS, Sigma-Aldrich) was
to-volume ratio tend to agglomerate and form large clus- added to the solution. After aging under continuous stirring
ters, with the consequent loss of interesting characteristics. at 100∘ C, the solids were collected by filtration and dried in
Therefore, a suitable coating is essential to prevent such air at 40∘ C. The surfactant was removed by calcination, which
limitations, what can be obtained by using mesoporous was carried out by increasing the temperature to 550∘ C under
silica like SBA-15. For SBA-15/Fe3 O4 , it is possible to obtain nitrogen flow for 2 h and then in O2 atmosphere for another
magnetite nanoparticles embedded into mesoporous silica, 1 h. SBA-15/Fe3 O4 was prepared by adding 4 g of iron oxide
preventing the agglomeration [14]; these coatings provide precursor (Fe2 (SO4 )3 ⋅6H2 O) before the addition of TEOS in
not only the stability to the nanoparticles in solution but the synthesis of silica SBA-15. The step of surfactant removing
also helps in binding the various biological ligands at the was carried out by heating the material in a nitrogen atmo-
nanoparticle surface for various biomedical applications sphere at a rate of 5∘ C min−1 to 550∘ C. At this temperature
[15]. the material was kept under nitrogen flow for 8 hours. The
Considering a multifunctional system composed by sil- nitrogen flow remained constant throughout the heating
ica/magnetite/gel, a magnetic field with an alternating cur- process and followed up to complete cooling of the sample.
rent (AC) can be used to target localized heating in vivo The hybrid was prepared using the following procedure:
through the magnetohyperthermia treatment for anticancer 0.5 g of SBA-15/Fe3 O4 nanocomposite was added to 3.5 mL
therapy, which in turn causes a phase change in the host solution of 0.245 g of monomer (N-isopropylacrylamide—
polymer to allow diffusion and release of drugs. In this N-iPAAm) and 0.005 g of crosslinking agent (N,N,N󸀠 ,N󸀠 —
case, the thermosensitive grafts collapse, opening pathways methylene-bisacrylamide—MBA). The mixture was trans-
for an imbedded drug into the system to escape [16]. Thus, ferred to an INNOVA 4200 (150 rpm) shaker and the mixture
an important improvement in cancer therapy would allow was continuously purged with nitrogen. The solution was
two modes of treatment with synergistic effects of magnetic then allowed to polymerize for 24 h in a water bath at 9∘ C.
hyperthermia and drug release using a single hybrid system The obtained multifunctional hybrid material was dried at
consisting of silica/magnetite/poly(N-isopropylacrylamide). 60∘ C for 24 h and subsequently washed to remove the excess
Even though there have been significant advances, studies monomers, crosslinking agent, initiator, and accelerator. In
involving the potential use of responsive hybrids in the area the washing stage, the material was disaggregated, suspended
of controlled release of drugs in synergism of magnetohyper- in water, and continuously stirred. The hybrid was then
thermia are still incipient, many properties of these materials collected by centrifugation at 3600 rpm for 3 min and dried
are in the process of analysis, and synthesis procedures are at 60∘ C for 24 h. It was designated [SBA-15/Fe3 O4 /P(N-
being modified in order to gain greater control over these iPAAm)] and the gel composition studied was 5 × 1 [7]. The
morphological and structural materials. monomer: SBA-15 mass ratio used was 1 : 2 (wt/wt).
2.2. Characterization. The samples were characterized by soaking nanocomposites [SBA-15/Fe3 O4 ] and hybrid [SBA-
X-ray diffraction (XRD), Fourier-transform infrared spec- 15/Fe3 O4 /P(N-iPAAm)] samples in 30 mL of simulated body
troscopy (FTIR), N2 adsorption, transmission electron fluid (SBF) [22]. The temperature was maintained con-
microscopy (TEM), and Temperature programmed reduc- stant (37∘ C) and the solutions were continually stirred. UV
tion studies (TPR). Low-angle XRD measurements were spectrometry (UV-Vis Shimadzu, model 2401) was used to
obtained using synchrotron radiation with 𝜆 = 1.488 nm. monitor the amount of drug delivered as a function of time.
Synchrotron radiation measurements were carried out at the The concentration of atenolol in SBF was found from the
D11A-SAXS beamline of the LNLS (Campinas, Brazil) using a intensity of the absorption band at 274 nm.
Huber-423 3-circle diffractometer. The high-angle XRD pat-
terns were obtained using a Rigaku Geigerflex-3034 diffrac-
tometer with a Cu-K𝛼 tube. Specific surface area and pore 2.5. Hyperthermia. The capacity of heat generation of the
size distribution were determined by N2 adsorption using obtained nanocomposites and hybrid system was mea-
the BET and BJH methods, respectively [18], in Autosorb- sured in a custom-designed magnetic-induction hyperther-
Quantachrome Nova 1200. The samples were outgassed for mia chamber. Heating was measured of [SBA-15/Fe3 O4 ]
2 h at 120∘ C before analysis. FTIR measurements were con- and hybrid [SBA-15/Fe3 O4 /P(N-iPAAm)] samples dispersed
ducted in a Perkin-Elmer 1760-X spectrophotometer in the in water with sonication. The sample concentration was
range 4000–400 cm−1 at room temperature using KBr pellets. 20 mg⋅mL−1 and the solution was sonicated for 30 min before
TEM characterization was performed through a Tecnai- measurement. A three-loop coil with resonant frequency of
G2-20-FEI 2006 electron microscope with an acceleration 222 kHz was used in the experiments. In order to study the
potential of 200 kV, of the Microscopy Center of UFMG, correlation between the applied magnetic field and the AC
Belo Horizonte, Brazil. TPR experiments were performed in a magnetically induced heating temperature, the heat produced
CHEMBET 3000 equipment with 20 mg sample under 25 mL was measured at a fixed frequency in magnetic 126 Oe until
min−1 H2 (8%)/N2 with heating rate of 5∘ C min−1 . the temperature was nearly steady. A digital thermometer was
placed above the sample inside the coil and the temperature
measurements were taken at the center of the sample. The
2.3. Calculations. BET specific surface area, SBET , was calcu- results were the average of triplicate measurements. Tests
lated from adsorption data in the relative pressure interval have also been performed in pure deionized water without
p/p0 = 0.045–0.25. A cross-sectional area of 0.162 nm2 was any magnetic material and the temperature increase from this
used for the nitrogen molecule in the BET calculations. The blank sample has been subtracted of the results obtained for
total pore volume, 𝑉𝑝 , was calculated from the amount of the solution.
N2 adsorbed at the highest p/p0 (p/p0 = 0.99) [19]. The
micropore volume, 𝑉𝜇 , of SBA-15 silica was estimated from
nitrogen adsorption data using the 𝛼s plot method [20] in
the standard reduced adsorption, 𝛼s , interval from 0.75 to 2.6. Statistical Analysis. The results were calculated and
1 (relative pressure range from 0.15 to 0.40). The external presented as the mean for each group ± the standard error of
surface area, 𝑆ext , was evaluated using an 𝛼s interval from 1.6 the mean (mean ± SD). Statistical evaluation of the data was
to 3.0. The primary mesopore volume, 𝑉meso , was estimated performed using analysis of variance (ANOVA), followed by
as the difference between the total pore volume and the Bonferroni’s test (Post hoc), where 𝑃 ≤ 0.05 was considered
micropore volume. The 𝛼s -plot was analysed by standard to be statistically significant.
reduced adsorption for nonporous hydroxylated silica [21].
The mesopore size distributions were calculated from the
adsorption branches of the nitrogen isotherms employing the 3. Results and Discussion
BJH algorithm.
3.1. Material Characterization. Figure 1 shows the X-ray
diffractograms of the pure magnetite prepared via oxidation-
2.4. Model Drug Adsorption and Delivery Assays. All samples precipitation route, and SBA-15/Fe3 O4 sample treated at
were loaded with atenolol as a model drug as follows: 0.5 g 550∘ C. The broad diffraction peak at high angles (Figure 1(a))
of the powder sample was added to 150 mL of an atenolol at 2𝜃 between 20∘ and 30∘ is attributed to the peak of the
solution (10 mg⋅mL−1 ) and shaken for 48 h at 25∘ C (200 rpm). siliceous material. In addition, several XRD peaks indicate
A 3 : 1 weight ratio of atenolol/solid sample was used. Powder the formation of well-crystallized Fe3 O4 . The sharp main
atenolol loaded samples were recovered by filtration, washed diffraction peak and other weak diffraction peaks at 2𝜃 =
with distilled water, and left to dry for 24 h at 60∘ C. The 30.0∘ , 35.4∘ , 56.9∘ , and 62.4∘ , respectively, can be assigned to
same procedure was used to load the hybrid with atenolol. (220), (311), (511), and (440) reflections, which can be indexed
Small atenolol loaded sample disks with 7 mm diameter and to the spinel structure of pure stoichiometric magnetite
2 mm thick were obtained under uniaxial pressure. TGA was (Fe3 O4 ) (JCPDS file 19-0629).
performed to evaluate the percentage of atenolol adsorbed by SBA-15/Fe3 O4 sample has been analyzed by temperature
each sample. programmed reduction employing H2 as reducing gas and
The in vitro study of release of atenolol from the materials the results are presented in Figure 2. TPR profile shows
was performed as follows. The release profile was obtained by mainly three sets of reduction process. The consumption
1200 than 700∘ C that are characteristic of phase transformation of

1000 (311) ferrisilicate.
Intensity (a.u.)
800
Figures 3(a) and 3(b) show the profile at low angle
600 (440)
(511) for SBA-15/Fe3 O4 and the [SBA-15/Fe3 O4 /P(N-IPAAM)],
400 (220) (400)
(422)
respectively. The three diffraction peaks of the former, at 2𝜃 =
200 (111) (222)
0
0.85∘ , 1.51∘ , and 1.75∘ , can be indexed as (100), (110), and (200)
reflections, respectively, which are typical of hexagonally
10 20 30 40 50 60 70 structured SBA-15 silica with highly ordered mesoporous
2𝜃 (deg) channels, as reported by Zhao et al. [24]. However, a slight
Fe3 O4 shift of these diffraction peaks to higher 2𝜃 values is identi-
fied for [SBA-15/Fe3 O4 /P(N-IPAAm)], Figure 3(b), possibly
(a)
due to the contraction of the support framework with the
4200 (311) formation of the polymer phase in the silica channels. The
Intensity (a.u.)
4000 (220) XRD peaks of SBA-15/Fe3 O4 can be indexed to a hexagonal

3800 (440) lattice structure with d(100) spacing of 9.5 nm and unit cell
3600 (400) (511) parameter (a = 2d/√3) of 10.94 nm, as reported by Souza et al.
(422)
3400 [25]. On the other hand, the d spacing of [SBA-15/Fe3 O4 /P(N-
3200 IPAAM)] shifted slightly to 9.2 nm, corresponding to a unit
10 20 30 40 50 60 70
cell parameter of 10.62 nm. Despite these small differences
2𝜃 (deg)
in the reflections of the XRD peaks, it is clear that the
mesostructure was still ordered after the polymer phase was
SBA-15/Fe3 O4 loaded.
(b) Figure 4 shows the TEM images of the [SBA-15/Fe3 O4 ]
and [SBA-15/Fe3 O4 /P(N-iPAAm)] hybrid samples. In accor-
Figure 1: X-ray diffraction patterns of the pure magnetite and silica-
dance with the low angle XRD results, the mesoscopic order
magnetite nanocomposite.
of the host can be clearly identified in the TEM image of both
samples, which shows a well-defined hexagonal arrangement
Fe3 O4 FeO of uniform pores when the incident electron beam was
SBA-15/Fe3 O4
parallel to the main axis of the mesopores (Figure 4(a)),
Fe2 O3 Fe3 O4
and unidirectional channels, when the electron beam was
H2 consumption (a.u.)
perpendicular to the channel axis (Figure 4(b)). The Fe3 O4

nanoparticles appear as dark dot-like objects between the
channel walls (Figure 4(c)). These nanoparticles are evenly
distributed in the channels with a uniform size, which is close
to the pore diameter of SBA-15. Thus, the TEM investiga-
tion gives consistent evidence that the ordered structure is
preserved in the approach proposed in this work to obtain
a nanocomposite and hybrid systems.
Figure 5 shows the FTIR spectra of the SBA-15/Fe3 O4 and
the hybrid [SBA-15/Fe3 O4 /P(N-IPAAm)] samples before and
0 200 400 600 800 1000 after washing procedure to remove residual monomers. For
Temperature (∘ C) SBA-15/Fe3 O4 sample, the infrared spectrum shows absorp-
tion bands concerning to fundamental vibrations network of
Figure 2: TPR profiles of the SBA-15/Fe3 O4 sample. silica. The amount of water in the samples can be monitored
by observing the adsorption lines at 3500 and 1640 cm−1 ,
whereas SiOH can be seen as a shoulder at 960 cm−1 for
profile of H2 for this sample shows characteristic peaks of all spectra. The band at about 810 cm−1 is related to the
reduction transformation of the following phases [23]: symmetric stretching of the Si–O–Si and the band about
460 cm−1 is related to the vibration mode deformation Si–
Fe2 O3 󳨀→ Fe3 O4 (maghemite 󳨀→ magnetite) O–Si. A broad and very intense band in the range 1000–
(1)
Fe3 O4 󳨀→ FeO (magnetite 󳨀→ wustite) 1200 cm−1 corresponding to ]Si–O–Si modes of the siliceous
matrix of SBA-15 is also present. The spectrum of SBA-
However, the largest nitrogen consumption was observed 15/Fe3 O4 is dominated by ]O–H modes, presenting a broad
in the temperature range of 485–650∘ C, regarding the con- and intense band at 3440 cm−1 assigned to hydroxyl groups.
version of magnetite to wustita, confirming the hypothesis It can be seen in the spectrum of hybrid [SBA-
that the phase present in higher concentration is magnetite. 15/Fe3 O4 /P(N-IPAAM)] systems before the washing pro-
It is worth mentioning that this sample does not contain cedure, bands corresponding to the ]C–H modes of P(N-
ferrisilicate since it has no peaks at temperatures higher iPAAm) at 2972–2875 cm−1 , the bands attributed to isopropyl
2500 2500
300 2000 300
2000 250 250
Intensidade
Intensidade
Intensity
200 (110) 200 (110)
Intensity
1500 (100) 150 (200) 1500 (100) 150 (200)

100 100
1000 50 1000 50
0 0
1.4 1.6 1.8 2.0 1.4 1.6 1.8 2.0
500 2𝜃 (graus) 500 2𝜃 (graus)
0 0
0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0
2𝜃 (deg) 2𝜃 (deg)
SBA-15/Fe3 O4 SBA-15/Fe3 O4 /P(N-iPAAm)
(a) (b)
Figure 3: X-ray diffraction patterns of small-angle region of (a) SBA-15/Fe3 O4 and (b) [SBA-15/Fe3 O4 /P(N-iPAAm)].
(a) (b) (c)
Figure 4: Transmission electron micrographs of [SBA-15/Fe3 O4 /P(N-iPAAm)]: (a) viewed along the pore axis, (b) viewed perpendicular
to the pore axis, and (c) high resolution images showing the magnetite nanoparticles (dark region) covered by the mesoporous silica of
SBA-15/Fe3 O4 .
Absorbance (a.u.)
Absorbance (a.u.)
𝛿CH3 (isopropil)
SBA-15/Fe3 O4 /P(N-iPAAm) before washing process SBA-15/Fe3 O4 /P(N-iPAAm) before washing process
0.3 0.3
C=O
C=C
0.2 0.2
0.1 0.1
4000 3500 3000 2500 2000 1500 1000 500 1800 1600 1400
𝛿C–H (CH2 , CH3 )
Absorbance (a.u.)
Absorbance (a.u.)
1.2 1.2 SBA-15/Fe3 O4 /P(N-iPAAm) after washing process

SBA-15/Fe3 O4 /P(N-iPAAm) after washing process
𝛿N–H
0.8 0.8
0.4 0.4
4000 3500 3000 2500 2000 1500 1000 500 1800 1600 1400
Absorbance (a.u.)
Absorbance (a.u.)
3.0 3.0
2.5 SBA-15/Fe3 O4 2.5 SBA-15/Fe3 O4
2.0 2.0
1.5 1.5
1.0 1.0
0.5 0.5
4000 3500 3000 2500 2000 1500 1000 500 1800 1600 1400
(a) (b)
Figure 5: (a) FTIR spectra of SBA-15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-iPAAm)] before and after the washing procedure to remove the residual
monomers. (b) FTIR spectra of SBA-15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-iPAAm)] before and after the washing procedure to remove the
residual monomers in the expanded scale of 2000–1400 cm−1 .
Adsorbed volume (cc/g)
0.16
dV(r) (cm3·nm−1·g−1 )
500 3.8 SBA-15/Fe3 O4
400 6000 0.12
300 0.08
4000
200 6.6
100 2000 0.04
0 0 0.00
0.2 0.4 0.6 0.8 2 4 6 8 10 12 14
P/P0 Pore diameter (nm)
SBA-15/Fe3 O4
dV(r) (cm3·nm−1·g−1 )
Adsorbed volume (cc/g)
0.06 SBA-15/Fe3 O4 /P(N-iPAAm)

3.8
200 2500
0.04
150 2000
1500 0.02 5.6
100
1000
50 500 0.00
0 0
2 4 6 8 10 12 14
0.2 0.4 0.6 0.8 Pore diameter (nm)
P/P0
SBA-15/Fe3 O4 /P(N-iPAAm) Figure 7: Pore size distribution of SBA-15/Fe3 O4 and [SBA-
15/Fe3 O4 /P(N-iPAAm)] hybrid system.
Figure 6: N2 adsorption-desorption isotherms and respective
derived curves of SBA-15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-iPAAm)]
hybrid system.
Antochshuk and coauthors [26] attributed this stepwise

−1 desorption phenomenon due to the presence of constrictions
group are located at 1386 and 1368 cm . The band cor-
in the porous structures. According to the authors, in the
responding to the bending vibration of CH3 is located at
case of a pore connected to neighboring pores or its sur-
1456 cm−1 , while the bands arising from C=O stretching and roundings through entrances (constrictions) with diameter
N–H bending vibrations are observed at 1645 and 1550 cm−1 , smaller than the pore diameter, capillary evaporation from
respectively. the pore interior is delayed until the capillary evaporation
The monomer characteristic bands (]C=C 1620 cm−1 , from the constrictions takes place. This phenomenon can be
𝛿CH2 = 1409 cm−1 , 𝛿H2 C=C– 1305, and 1325 cm−1 , 𝛿vinyl more easily seen in the curves derived from the adsorption
group at 990 and 916 cm−1 ) are not present in the hybrid sam- and desorption branches (Figure 6). After polymerization,
ple spectra, as can be seen in the scale-expanded FTIR spec- the shape of the hysteresis loop in the N2 -sorption isotherm
trum in the 1800–900 cm−1 region (Figure 5(b)). These results displays a broadening suggestive of a reduction in pore size
prove the presence of P(N-iPAAm) in SBA-15/Fe3 O4 pores uniformity.
with no other significant synthesis components (monomer, All samples present a bimodal pore size distribution due
initiator, or accelerator) and demonstrate the successful to the presence of mesopores of different diameters, for
conversion of the monomers into polymer and the removal example, primary mesopores, secondary, or pores with con-
of the residual monomers. In addition, the stretching bands strictions. In Figure 7, it is possible to observe two maximum
of carbonyl groups and N–H bending vibrations from sec- points for SBA-15/Fe3 O4 : one about 6.6 nm, corresponding to
ondary amide around 1648 and 1550 cm−1 , respectively, in the the primary mesoporosity, and the second corresponding to a
spectrum of the sample after washing, are broader compared diameter of 3.8 nm equivalent to the secondary mesoporosity.
to the spectrum before washing, likely due the intramolecular In the hybrid multifunctional, the first maximum point is
interactions such as hydrogen bond (C=O ⋅ ⋅ ⋅ HN) which can shifted to lower values of diameter: 5.6 nm and no change
occur between the polymer chains after the polymerization were observed in the secondary mesopores, which kept on
reaction. 3.8 nm.
The nitrogen adsorption isotherms for SBA-15/Fe3 O4 It can be seen that the pore size distribution is affected
and the [SBA-15/Fe3 O4 /P(N-iPAAm)] hybrid are shown in by the incorporation of polymeric gel in the SBA-15/Fe3 O4
Figure 6. In both cases, the isotherms were type IV, accord- sample. After incorporation of the gel, it fills the pores with
ing to the IUPAC classification, which is associated with higher diameters (peaks at 6.6 nm), reducing its statistical
the presence of mesopores [19]. These H1 type hysteresis contribution and causing a shift of the maximum point for
isotherms are related to materials with pores of uniform cross lower values of pore diameter. Notably, the incorporation of
section (e.g., cylindrical or hexagonal). However, these mate- the gel does not cause complete filling of the pores of the
rials exhibit the phenomenon of stepwise desorption, often final material. Even after incorporation of the gel into SBA-
referred to as blocking phenomenon of pores and is typically 15/Fe3 O4 samples, significant values of surface areas can be
associated with the bottle-shaped pores (ink bottle pores) observed, which may be convenient to use this material as
(Figure 6). drug release devices (Table 1).
42 75
40 70
38 65
36 60
34
Cummulative (%)
55
Cummulative (%)
32
50
30
45
28
26 40
24 35
22 30
20 25
18 20
16 15
14 10
0 20 40 60 80 100 120 5
Time (min) 0 20 40 60 80 100
SBA-15/Fe3 O4 Time (min)
SBA-15/Fe3 O4 /P(N-iPAAm) SBA-15/Fe3 O4
SBA-15/Fe3 O4 /P(N-iPAAm)
Figure 8: Release kinetic profiles of atenolol from SBA-15/Fe3 O4 and
[SBA-15/Fe3 O4 /P(N-iPAAm)] samples. Figure 9: Release kinetic profiles of atenolol from SBA-15/Fe3 O4 and
[SBA-15/Fe3 O4 /P(N-iPAAm)] systems in an alternating field.
Table 1: N2 adsorption results.
𝑆BET 𝑉𝑝 𝐷𝑝 (BJH)
Sample to mention here that the drug release rate can be enhanced
(m2 ⋅g−1 ) (cm3 /g) (nm)
using magnetic field, which would subsequently reduce the
SBA-15/Fe3 O4 413 0.76 3.8/6.5
period for the drug release. Considering this fact, and in
[(SBA-15/Fe3 O4 /P(𝑁-iPAAm)] 171 0.35 3.8/5.6 order to evaluate the multifunctionality of the SBA-15/Fe3 O4
and [SBA-15/Fe3 O4 /P(N-iPAAm)] systems as release device
via magnetic hyperthermia treatment, a preliminary test was
Table 1 presents the results of N2 adsorption, which show carried out where the samples were subjected to an external
the differences in the specific surface area (𝑆BET ), pore volume magnetic field over the time. The study was based on the
(V p ), and pore diameter (Dp ) of the samples. A significant model proposed by Dash and Cudworth [27], in which the
difference was observed for the 𝑆BET , V p , and Dp for SBA- application of an alternating magnetic field provokes the
15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-IPAAM)] samples, which vibration of the magnetic particles and the quick release
may indicate the presence of the polymer in the pore struc- of large quantities of drugs. The measures presented here
ture. Regarding the nitrogen adsorbed volume, it was found are designed to assess the influence of the application of
that the formation of P(N-IPAAM) into the mesoporous low frequency alternating magnetic fields (168 Oe) on drug
support provoked on a slight decrease in the pore volume and release [28].
in the pore diameter. Figure 9 shows the result of this experiment for SBA-
15/Fe3 O4 . The presence of magnetic field provokes a heat-
3.2. Atenolol Incorporation and Release Profile Study. In ing of the nanocomposite material due to the presence of
vitro atenolol release properties from mesoporous SBA- magnetic particles; thus, the diffusion of atenolol molecules
15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-iPAAm)] were investi- was enhanced by increasing the vibration of the nanoparticles
gated as a function of time and are shown in Figure 8. and, consequently, increasing the amount of drug released.
The resulting drug loading into samples was approximately The same behavior can be observed for hybrids systems.
the same, 41 wt% for SBA-15/Fe3 O4 and 44 wt% for [SBA- Similar to the experiment of release without the influence of
15/Fe3 O4 /P(N-iPAAm)], as no statistically significant differ- the magnetic field, the presence of the gel into the nanocom-
ences could be observed (𝑃 > 0.05) in the loading percentage. posite structure increased the amount of drug released, and
The release profiles of both samples exhibited no initial burst these results can be explained by the possible interaction
release effect during the first minutes. However, as can be of the drugs with the magnetite nanoparticles, maybe due
observed from Figure 8, SBA-15/Fe3 O4 sample did in fact to the incorporation of atenolol into the mesopores, and its
release a smaller percentage of atenolol than did the hybrid interaction with the surface of the magnetic nanoparticles.
sample. The maximum release level achieved in 2 hours for Moreover, the application of an alternating magnetic field in
the SBA-15/Fe3 O4 sample was 32% and for hybrid system was the hybrid system containing magnetic nanoparticle leads to
43%. heat generation, which could drive the swelling transition of
As a result, the presence of the gel increased the amount the polymer.
of drug released, which shows the potential application of Comparing the results obtained during about 100 minutes
this material as a controlled release of drugs. It is relevant of both assays (with and without the influence of an external
42 field for 30 min, the temperature ranged from 24 to 39∘ C and

40 from 24 to 40∘ C for SBA-15/Fe3 O4 and [SBA-15/Fe3 O4 /P(N-
iPAAm)], respectively, showing no statistically significant
38 differences in the temperature variation.
36 The measured temperatures of the hybrid system sus-
Temperature (∘ C)
pension after sonication and under 126 Oe AC magnetic

34
fields after 30 min of assay, presented a Δ𝑇max of 16∘ C. For
32 all evaluated samples, (𝑇 + Δ𝑇max ) was minor than the
30 recommended hyperthermia treatment temperature, which
is reported between 40 and 45∘ C [30]. At these temperatures,
28 the growth of cancerous cells can be halted and any damage to
26 healthy cells can be avoided by using magnetic nanoparticles
with controlled temperatures. It is available treatment when
24
tumors have not metastasized and their locations are known.
22 However, based on such results, it does not mean that the
0 300 600 900 1200 1500 1800 obtained material cannot be used for such application, but
Time (s) the experimental conditions employed to evaluate its heating
SBA-15/Fe3 O4 might be improved.
SBA-15/Fe3 O4 /P(N-iPAAm) Thus, a temperature variation of Δ𝑇max = 5–8∘ C would be
sufficient, based on a body temperature of 37∘ C. Significant
Figure 10: Heating induced by magnetic field of SBA-15/Fe3 O4 and results were obtained in our previous work [25]. Hyper-
[SBA-15/Fe3 O4 /P(N-iPAAm)] systems.
thermia tests have demonstrated a good heating capacity
of the powders prepared from SiO2 -Fe3 O4 nanocomposite,
which increased linearly with milling time. In the selected
magnetic field), we can observe that the presence of magnetic experimental conditions, the measured temperatures of the
field affects the release profile of both samples. Even though nanoparticle suspension after sonication and under 168 Oe
there is an increase in the drug release rate for the nanocom- AC magnetic field increased to 47.5∘ C after 30 min of assay,
posites in the presence of the magnetic field ranging from presenting a Δ𝑇max of 24.5∘ C. The effect of frequency for
32 to 40%, this increase is significantly higher for samples CoFe2 O4 nanoparticles dispersed in water on magnetic
containing the polymer. The analysis of the performance of heating was investigated using a magnetic field of 385 Oe at
the hybrid system as a drug delivery device in the presence of frequencies of 195, 231, and 266 kHz. The results show that
alternating magnetic field shows that the release profiles vary the heating rates diminished with time and reached steady-
from approximately 40 to 70%, indicating that the polymer state temperatures around 30–35∘ C and Δ𝑇 increased linearly
expanded and consequently presented a lesser barrier to the with frequency. As a result, heat generation can be controlled
diffusion of atenolol. in selected magnetic particles by adjusting the magnetic field
These observations lead us to suggest that the drug and frequency [31, 32].
release response of hybrid systems depends on temperature The above results show that the sample prepared in this
and the polymer phase behavior. In this case, a synergistic work is important to be used in a medical application, as
effect of hyperthermia and controlled drug delivery for a drug delivery and magnetic hyperthermia. A critical step in
hybrid system composed by the combination of SBA-15, the combined therapy is controlled drug release. Although
Fe3 O4 nanoparticles, and P(N-iPAAm) has occurred. To important, these conclusions need a deeper study of heat
accomplish this assumption, heating generation experiments generation by adjusting the magnetic field, the frequency, and
were performed and discussed below. choosing an appropriated medium with different amounts
of materials in order to increase Δ𝑇max and it is planned
for future work. Due to their mesoporous properties we also
3.3. Magnetic Hyperthermia. The saturation magnetization of envisioned that such system might be further investigated as a
SBA-15/Fe3 O4 is 2 emu/g which is much smaller than that of theranostic device. The in vivo experiments will be conducted
pure bulk magnetite (94 emu/g) [29] because the nanoscale and reported in the due course.
particles form a single magnetic domain that do not com-
municate/interact due to coating of silica. The magnetic
properties obtained by 57 Fe Mössbauer spectroscopy (not 4. Conclusion
shown) showed that the nanocrystals present superparamag-
netic characteristics. The AC magnetic field-induced heating The possibility to prepare hybrid system to be used as
characteristics of the nanocomposite and hybrid system in nanoplatform for drug delivery and heating agent in hyper-
20 mg⋅mL−1 solution were measured to assess its possible thermia was presented and discussed. We developed an
application as a hyperthermia agent in an in vivo-like envi- easy and direct synthesis route to obtain hybrid functional
ronment. Figure 10 shows the time-dependent temperature nanosystems based on responsive polymer synthesized inside
curves of the pure and hybrid systems for 222 kHz and 126 Oe the iron nanoparticle/mesoporous silica nanocomposites.
AC. When the samples were exposed to an AC magnetic The above results show that the composition and morphology
of carrier materials and the external agents are important ery,” Journal of Materials Science, vol. 45, no. 6, pp. 1478–1486,
factors in influencing the drug delivery performance. Drug 2010.
release was more effective and faster in alternating magnetic [8] G. Vilara, J. Tulla-Puchea, and F. Albericioa, “Polymers and
field for both systems. However, a direct comparison of these drug delivery systems,” Current Drug Delivery, vol. 9, pp. 1–28,
results indicates that the performance of the hybrid system is 2012.
more affected by the presence of the magnetic field, resulting [9] L. M. Geever, M. J. D. Nugent, and C. L. Higginbotham, “The
in a greater release of the drug, as the polymer expanded effect of salts and pH buffered solutions on the phase transition
and consequently presented a lesser barrier to the diffusion temperature and swelling of thermoresponsive,” Journal of
of atenolol. Hyperthermia tests have demonstrated that the Materials Science, vol. 42, no. 23, pp. 9845–9854, 2007.
temperature variation achieved under selected conditions [10] W.-F. Lee and Y.-H. Lin, “Swelling behavior and drug release
increased linearly with milling time. The maximum temper- of NIPAAm/PEGMEA copolymeric hydrogels with different
ature obtained is around 40∘ C inferior to that recommended crosslinkers,” Journal of Materials Science, vol. 41, no. 22, pp.
for hyperthermia treatment. Therefore, the magnetic field 7333–7340, 2006.
amplitude, the frequency, or both of them might be reduced [11] R. F. S. Freitas and E. L. Cussler, “Temperature sensitive gels as
in order to optimize the use of the obtained hybrid system. extraction solvents,” Chemical Engineering Science, vol. 42, no.
In spite of this, we have shown that mesoporous silica-coated 1, pp. 97–103, 1987.
magnetite nanoparticles containing stimuli-responsive poly- [12] C. S. Biswas, V. K. Patel, N. K. Vishwakarma et al., “Synthe-
mers with especial structural and magnetic characteristics sis, characterization, and drug release properties of poly(N-
and unobstructed pores seem to be promising material for isopropylacrylamide) gels prepared in methanol-water conon-
use in biomedical application presenting synergistic effects of solvent medium,” Journal of Applied Polymer Science, vol. 125,
no. 3, pp. 2000–2009, 2012.
hyperthermia and controlled drug delivery.
[13] S. Kralj, M. Drofenik, and D. Makovec, “Controlled surface
functionalization of silica-coated magnetic nanoparticles with
Conflict of Interests terminal amino and carboxyl groups,” Journal of Nanoparticle
Research, vol. 13, no. 7, pp. 2829–2841, 2011.
The authors declare that there is no conflict of interests
[14] L. A. Harris, J. D. Goff, A. Y. Carmichael et al., “Magnetite
regarding the publication of this paper.
nanoparticle dispersions stabilized with triblock copolymers,”
Chemistry of Materials, vol. 15, no. 6, pp. 1367–1377, 2003.
Acknowledgments [15] A. K. Gupta and M. Gupta, “Synthesis and surface engineering
of iron oxide nanoparticles for biomedical applications,” Bioma-
The authors wish to thank FAPEMIG (Fundação de Amparo terials, vol. 26, no. 18, pp. 3995–4021, 2005.
a Pesquisa do Estado de Minas Gerais), CNPQ (Conselho [16] G. Bao, S. Mitragotri, and S. Tong, “Multifunctional nanoparti-
Nacional de Desenvolvimento Cientı́fico e Tecnológico), and cles for drug delivery and molecular imaging,” Annual Review
CAPES (Comissão Aperfeiçoamento de Pessoal de Nı́vel of Biomedical Engineering, vol. 15, p. 253, 2013.
Superior) for their financial support. [17] K. C. Souza, G. Salazar-Alvarez, J. D. Ardisson, W. A. A.
Macedo, and E. M. B. Sousa, “Mesoporous silica-magnetite
References nanocomposite synthesized by using a neutral surfactant,”
Nanotechnology, vol. 19, no. 18, Article ID 185603, 2008.
[1] I. W. Hamley, “Nanostructure fabrication using block copoly- [18] M. Choi, F. Kleitz, D. Liu, Y. L. Hee, W.-S. Ahn, and R. Ryoo,
mers,” Nanotechnology, vol. 14, no. 10, pp. R39–R54, 2003. “Controlled polymerization in mesoporous silica toward the
[2] G. J. D. A. A. Soler-Illia, C. Sanchez, B. Lebeau, and J. Patarin, design of organic-inorganic composite nanoporous materials,”
“Chemical strategies to design textured materials: from microp- Journal of the American Chemical Society, vol. 127, no. 6, pp.
orous and mesoporous oxides to nanonetworks and hierarchical 1924–1932, 2005.
structures,” Chemical Reviews, vol. 102, no. 11, pp. 4093–4138,
[19] K. S. W. Sing, “Reporting physisorption data for gas/solid
2002.
systems with special reference to the determination of surface
[3] W. Paul and C. P. Sharma, “Nanoceramic matrices: biomedical area and porosity,” Pure and Applied Chemistry, vol. 57, no. 4,
applications,” American Journal of Biochemistry and Biotechnol- pp. 603–619, 1985.
ogy, vol. 2, p. 41, 2006.
[20] A. Sayari, P. Liu, M. Kruk, and M. Jaroniec, “Characterization of
[4] A. L. Doadrio, E. M. B. Sousa, J. C. Doadrio, J. Pérez Pariente,
large-pore MCM-41 molecular sieves obtained via hydrother-
I. Izquierdo-Barba, and M. Vallet-Regı́, “Mesoporous SBA-
mal restructuring,” Chemistry of Materials, vol. 9, no. 11, pp.
15 HPLC evaluation for controlled gentamicin drug delivery,”
2499–2506, 1997.
Journal of Controlled Release, vol. 97, no. 1, pp. 125–132, 2004.
[5] A. Sousa, K. C. Souza, S. C. Reis et al., “Positron annihilation [21] S. J. Gregg and K. S. W. Sing, Standard Data for Alpha-s Method
study of pore size in ordered SBA-15,” Journal of Non-Crystalline Comment, 2nd edition, 1982.
Solids, vol. 354, no. 42-44, pp. 4800–4805, 2008. [22] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, and T. Yamamuro,
[6] M. Vallet-Regı́, “Mesoporous silica nanoparticles: their projec- “Solutions able to reproduce in vivo surface-structure changes
tion in nanomedicine,” ISRN Materials Science, vol. 2012, Article in bioactive glass-ceramic A-W3,” Journal of Biomedical Materi-
ID 608548, 20 pages, 2012. als Research, vol. 24, no. 6, pp. 721–734, 1990.
[7] A. De Sousa, D. A. Maria, R. G. De Sousa, and E. M. B. De Sousa, [23] A. Pineau, N. Kanari, and I. Gaballah, “Kinetics of reduction
“Synthesis and characterization of mesoporous silica/poly(N- of iron oxides by H2. Part I: low temperature reduction of
isopropylacrylamide) functional hybrid useful for drug deliv- hematite,” Thermochimica Acta, vol. 447, no. 1, pp. 89–100, 2006.
[24] D. Zhao, Q. Huo, J. Feng, B. F. Chmelka, and G. D. Stucky,

“Nonionic triblock and star diblock copolymer and oligomeric
sufactant syntheses of highly ordered, hydrothermally stable,
mesoporous silica structures,” Journal of the American Chemical
Society, vol. 120, no. 24, pp. 6024–6036, 1998.
[25] K. C. Souza, N. D. S. Mohallem, and E. M. B. Sousa, “Meso-
porous silica-magnetite nanocomposite: facile synthesis route
for application in hyperthermia,” Journal of Sol-Gel Science and
Technology, vol. 53, no. 2, pp. 418–427, 2010.
[26] V. Antochshuk, M. Kruk, and M. Jaroniec, “Surface modi-
fications of cage-like and channel-like mesopores and their
implications for evaluation of sizes of entrances to cage-like
mesopores,” Journal of Physical Chemistry B, vol. 107, no. 43, pp.
11900–11906, 2003.
[27] A. K. Dash and G. C. Cudworth II, “Therapeutic applications of
implantable drug delivery systems,” Journal of Pharmacological
and Toxicological Methods, vol. 40, no. 1, pp. 1–12, 1998.
[28] K. C. Souza, J. D. Ardisson, and E. M. B. Sousa, “Study
of mesoporous silica/magnetite systems in drug controlled
release,” Journal of Materials Science: Materials in Medicine, vol.
20, no. 2, pp. 507–512, 2009.
[29] B. D. Cullity and C. D. Graham, Introdution to Magnetic
Materials, IEEE Press Editorial Board, 2nd edition, 2009.
[30] M. Liangruksa, R. Ganguly, and I. K. Puri, “Parametric investi-
gation of heating due to magnetic fluid hyperthermia in a tumor
with blood perfusion,” Journal of Magnetism and Magnetic
Materials, vol. 323, no. 6, pp. 708–716, 2011.
[31] G. Baldi, G. Lorenzi, and C. Ravagli, “Hyperthermic effect of
magnetic nanoparticles under electromagnetic field,” Processing
and Application of Ceramics, vol. 3, p. 103, 2009.
[32] E. A. Périgo, S. C. Silva, E. M. B. De Sousa et al., “Properties
of nanoparticles prepared from NdFeB-based compound for
magnetic hyperthermia application,” Nanotechnology, vol. 23,
no. 17, Article ID 175704, 2012.
http://dx.doi.org/10.1155/2014/183815
Research Article
Characterization of the Casein/Keratin
Self-Assembly Nanomicelles
Su Xiao-Zhou, Wang Hong-Ru, and Huang Mian

College of Resources and Environment, Shaanxi University of Science and Technology, Xi’an 710021, China
Correspondence should be addressed to Wang Hong-Ru; wanghr@sust.edu.cn
Received 12 September 2013; Revised 16 January 2014; Accepted 19 January 2014; Published 25 February 2014
Copyright © 2014 Su Xiao-Zhou et al. This is an open access article distributed under the Creative Commons Attribution License,
Complex nanomicelles were made from casein and keratin through electrostatic self-assembly and transglutaminase fixation that
was proved to be harmless and green. The complex nanomicelles were characterized by dynamic light scattering, scanning electron
microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and steady-state
florescence. The results show that the complex nanomcelles acquired at the neutral pH in the mass ratio of casein to keratin 4 : 1
exhibit an anomalous sphere shape with uniform size which the diameter is about 40–70 nm. The complex nanomicelles in solution
possess excellent dilution and storage stability due to the fixation and their high 𝜁-potential (22.8 mV). The complex nanomicelles
are relatively hydrophilic and have a good potential for industrial application.
1. Introduction Based on these viewpoints, our studies intended for structural

stability and nanoparticles have been conducted. Biomate-
Molecular self-assembly is the spontaneous association of rials used for drug delivery, ecological coating, or cosmetic
molecules under equilibrium conditions into stable, struc- ingredients with desired properties will be developed based
turally well-defined aggregates joined by noncovalent bonds on the complex casein/keratin nanomicelles without toxicity.
[1]. Self-assembly of biomacromolecules is emerging as a
new route to produce novel materials, which can find many
applications in biomedical technology and biomaterials tech- 2. Materials and Methods
nology.
Both casein and keratin have a strong tendency to self- 2.1. Materials. Casein (technical grade) was purchased from
assemble spontaneously into micelles in solution and their Sigma Company Ltd. High glycine-tyrosine keratin powder
spontaneous self-assembly has been studied extensively in was prepared from wool hydrolysate by stepwise precipitation
recent years [2–7]. Casein has an isoelectric point of 4.6, with acid. All the other reagents used were of analytical grade.
and it is relatively hydrophobic, making it poorly soluble
in water. High glycine-tyrosine keratin from wool has an 2.2. Preparation of the Complex Nanomicelles. Casein was
isoelectric point around 6.5, and it is relatively hydrophilic dispersed in ultrapure water by magnetic stirring at 50∘ C and
in water [8, 9]. When these different types of proteins were then stored at 4∘ C at least for 10 days. The casein solution
mixed together, they did not result in precipitation and (5 mg/mL) was prepared and adjusted to pH 7.0 with 1 M
phase separation. Therefore, the hydrophobic effect and the NaOH and 1 M HCl solutions. High glycine-tyrosine keratin
electrostatic interaction can be employed to reassemble and powder was put into ultrapure water and solid NaOH was
fabricate the casein/keratin nanomicelles. added until the keratin completely dissolved by magnetic
The polymer micelles were characterized by their good stirring. The casein and keratin solutions were then filtrated
drug loading capacity, high structural stability, excellent by millipore filters with 2.0 𝜇m pore size to get rid of
water solubility, and tiny particle size (100 nm or smaller). the impurities and microbes. Then the casein and keratin
Table 1: Influence of the mass ratio of casein to keratin on the 𝐷ℎ , used to observe the micelles at 1 kV. The micelles were fixed by
PDI, and scattering light intensity of the casein/keratin complex 2% glutaraldehyde on the silicon surface for at least 30 min in
nanomicelles. order to keep the micelles primitive form, then washed with
C/K 𝐷ℎ (nm) PDI Intensity (counts) deionized water, and dried at the room temperature. Before
1:0 70.85 ± 1.6 0.591 ± 0.04 82.47 ± 12 observation, the specimens were coated with gold.
8:1 65.22 ± 1.4 0.522 ± 0.03 72.26 ± 9
4:1 60.24 ± 1.1 0.425 ± 0.01 96.53 ± 18 2.7. Atomic Force Microscopy (AFM) Measurements. AFM
2:1 64.55 ± 1.8 0.613 ± 0.06 92.31 ± 11 samples were prepared by dropping the solution on freshly
1:1 50.48 ± 1.5 0.625 ± 0.09 66.89 ± 7 cleaved mica surface, and then the micelles were fixed by 2%
glutaraldehyde for at least 30 min. The samples were washed
1:2 46.57 ± 0.8 0.611 ± 0.08 75.25 ± 14
with deionized water and dried at room temperature. Skiko
1:4 42.15 ± 0.6 0.654 ± 0.07 89.95 ± 12
Atomic force Microscopy SPA400-SPI3800N was used to
1:8 48.58 ± 0.8 0.687 ± 0.09 63.25 ± 12 analyze the micelles in tapping mode.
0:1 120.23 ± 2.8 0.712 ± 0.08 60.54 ± 10
Note: C/K represents the casein/keratin nanomicelles. 𝐷ℎ is the hydrody-
namic diameters; PDI is the particle dispersion index. 2.8. Fourier Transform Infrared Spectroscopy Measurements.
FT-IR spectra of the complex nanomicelles and the complex
nanomicelles fixed by transglutaminase were measured with
solutions were dispersed with sodium citrate in the mass ratio an Equinox 55 Spectrometer (Bruker). The analysis was
of protein to sodium citrate 5 : 1. Casein and keratin solutions performed in triplicate.
were then mixed gently under different conditions and kept
at 4∘ C for at least 24 hours to allow the casein and keratin 2.9. Thermal Analysis. Differential scanning calorimetry
to self-assemble completely. The complex micelles were then (DSC) measurements were performed with an instrument
fixed by transglutaminase for 1 hour at 50∘ C and the enzyme (netzsch STA 409) from room temperature to 400∘ C at a
was inactivated at 80∘ C for 10 min. heating rate of 10∘ C/min. The complex nanomicelles and the
complex nanomicelles fixed by transglutaminase were kept at
2.3. Turbidity Measurements. A Shimadzu UV 1705 spec- 105∘ C for 30 min to get rid of the moisture.
trometer was used for turbidity measurements. Solution
was injected into the Quartz cuvettes and the turbidity was
2.10. Steady-State Fluorescence Measurements. The steady-
detected at 600 nm wavelength.
state fluorescence tests were carried out on a fluorescence
spectrophotometer Hitachi F-7000. Recrystallized pyrene
2.4. Dynamic Light Scattering (DLS) Measurements. Hydro- was dissolved in acetone to prepare a concentration of
dynamic diameter of the micelles was measured by a Zeta 2 × 10−5 g/mL stock solution and its final concentration
Sizer Nano ZS90 (Malvern Instrument, Worcs, UK) equipped in micelles solution was 2 × 10−7 g/mL for test. Before
with 4 mW He-Ne Laser. The measurements were performed measurements, the micelles solution was stored at 4∘ C for 24
at a scattering angle of 90∘ at 25.0 ± 0.1∘ C. The apparent 𝑧- hours after the pyrene was added. The excitation and emission
average diameter (𝐷ℎ ), polydispersity index (PDI), and the wavelength were recorded at the 338 nm, 381 nm, and 373 nm
intensity for 𝑧-average were calculated by the Dispersion wavelengths.
Technology Software provided by Malvern. The concentra-
tion of the micelles for DLS measurements was 5 mg/mL in
each sample. 3. Results and Discussion
3.1. Diameter of the Complex Nanomicelles in Solution. The
2.5. 𝜁-Potential Measurements. The 𝜁-potential was measured influence of mass ratio of casein to keratin on complex
on a Zeta Sizer Nano ZS90 (Malvern Instrument, Worcs, nanomicelles diameter was investigated at neutral pH. The
UK) by using laser doppler microelectrophoresis. In this DLS measurements revealed that the two proteins interacted
technique, an electric field was applied to the micelles, which with each other and formed complex micelles with diameter
then moved with a velocity related to their 𝜁-potential. This about 40–65 nm at pH 7.0 (Table 1). It was also found that
velocity was measured using a patented laser interferometric any of the diameters of the complex nanomicelles in different
technique called M3-PALS (phase analysis light scattering). mass ratio of casein and keratin were smaller than that of
This enabled the calculation of electrophoretic mobility. The either casein micelles or keratin particles. This proved that
test was carried out at 25.0 ± 0.1∘ C. The electrophoresis the two protein particles had a strong hydrophobicity in the
mobility (𝑈𝐸 ) was used to calculate the potential (𝜁) by the reassembly process. The casein micelles and keratin particles
Henry equation 𝑈𝐸 = 2𝜀𝜁𝑓(ka)/3𝜂, and 𝜀, 𝜂, and 𝑓(ka) dissociated partly when these two proteins mixed together.
represented, respectively, the dielectric constant, the viscosity The keratin peptides competed with casein peptides and
of the medium, and Henry’s function. bound to the interior sites of the casein micelles and led to the
dissociation of the colloidal calcium phosphate (CCP). Table 1
2.6. Scanning Electron Microscopy (SEM) Measurements. Hit- also showed that the diameters of the complex nanomicelles
achi S-4800 field emission scanning electron microscope was decreased when the keratin content increased, which implied
60
450 −10
50 400
350 −15
40 300
100 − T (%)
𝜁-potential (mV)
−20
250
Dh
30
200
−25
150
20
100 −30
10 50
−35
0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH 8 4 2 1 0.5 0.25 0.125
Mass ratio (casein/keratin)
Figure 1: Turbidity and diameters of the casein/keratin complex
nanomicelles (5 mg/mL) as a result of pH value in the mass ratio Figure 3: 𝜁-potential of the casein/keratin complex nanomicelles
of casein to keratin 4 : 1. prepared at pH 7.0 in different mass ratios.
−5
stabilized when the solution pH value increased from 5.0 to
−10 9.0.
Figure 3 showed the 𝜁-potential of the complex micelles in
−15 different mass ratios of casein and keratin at neutral pH. The
𝜁-potential absolute value of the complex micelles increased
𝜁-potential (mV)
−20 with the increase of the keratin content. It can be inferred that
the introduction of the keratin increased the stability of the
−25
complex nanomicelles.
−30
3.3. Morphology of the Complex Nanomicelles. The morphol-
−35 ogy of the casein/keratin complex nanomicelles was observed
by SEM. The samples in Figures 4(a) and 4(b) were from the
−40 100-fold diluted self-assembly solution, and the samples in
Figures 4(c) and 4(d) were from the 200-fold diluted self-
5 6 7 8 9
assembly solution.
pH
Figures 4(a) and 4(c) represented the nanomicelles’
Figure 2: 𝜁-potential of the casein/keratin complex nanomicelles. image, and Figures 4(b) and 4(d) represented the nano-
The micelles were prepared in the mass ratio of casein to keratin 4 : 1 micelles’ image fixed by transglutaminase. The complex
and at pH 5.0–9.0. nanomicelles made from casein and keratin via self-assembly
had a diameter of about 20–40 nm (Figures 4(a) and 4(c)).
The fixed complex nanomicelles had a diameter of 40–70 nm
(Figures 4(b) and 4(d)). The fixation with transglutaminase
that the keratin particles played a major role in nanomicelle led to the enhancement of the micelles’ diameter. In addition,
dispersion process. two different fold-diluted samples showed almost the same
The data in Table 1 also showed that the nanomicelles pre- diameter and morphology of the micelles (Figures 4(b) and
pared at neutral pH in mass ratio of casein to keratin 4 : 1 had 4(d)), which indicated that the dilution did not result in the
the highest intensity and relatively narrow PDI. Therefore, dissociation of the complex nanomicelles, and the complex
the smallest and stable nanomicelles can be obtained at the nanomicelles were stable on dilution. These implied that
neutral pH in the mass ratio of casein to keratin 4 : 1, of which the crosslinking was formed either in the nanomicelles or
the average diameter is 60 nm (Figure 1). between the nanomicelles during the fixation with transglu-
taminase.
3.2. 𝜁-Potential of the Complex Nanomicelles. 𝜁-potential Figure 5 showed the topography image of the fixed
relates to the net charges on the surface of the particles in complex nanomicelles by the atomic force microscope. The
solution. Figure 2 showed that the absolute value of the 𝜁- morphology of the nanomicelles was of nearly spherical
potential of the complex nanomicelles increased with the shape and had a diameter of 40–70 nm. This result was in
enhancement of pH value from 5.0 to 9.0 in the mass ratio accordance with the result measured by SEM and DLS. The
of 4: 1. This illustrated that the complex nanomicelles became diameter of the nanomicelles measured in solid state (SEM,
(a) (b)
(c) (d)
Figure 4: SEM images of the casein/keratin complex nanomicelles. (a) and (b) were diluted by 100-fold. (c) and (d) were diluted by 200-
fold. (a) and (c) were casein/keratin complex nanomicelles; (b) and (d) were complex nanomicelles treated with transglutaminase (100 k
magnification).
CK+
CK−
0
60
(nm)
200 1025
0 1113
400
)
200
m
600
(n
400
(nm 600 800
) 800
4000 3500 3000 2500 2000 1500 1000 500
Figure 5: AFM images of the fixed casein/keratin complex micelles. Wavenumber (cm−1 )
Figure 6: FT-IR spectra of the casein/keratin nanomicelles mixtures

(CK−) and mixture treated with transglutaminase (CK+).
AFM) and in solution (DLS) had no great difference. This is
attributed to the unique structure of the nanomicelles and the
fixation method.
The FTIR spectrum of the complex nanomicelles showed three absorption bands, respectively, at 1150 cm−1 , 1107 cm−1 ,
an absorption band at 1113 cm−1 (Figure 6, CK−), but the and 1081 cm−1 in the corresponding area (Figure 6, CK+).
complex nanomicelles fixed with transglutaminase showed The absorption in this area is related to the C–N stretching
1.0
Exothermal ↑ TG CK+ 100
0.5
0.0 90
−0.5 DSC
−1.0 80
−1.5
70
−2.0
P8P9
−2.5 P7 60
DSC (mW/mg)
−3.0
P6 50
TG (%)
−3.5
−4.0 P5 40
1.0 TG CK− 100
0.5
90
0.0
DSC 80
−0.5
−1.0 P4 70
−1.5
60
−2 P2 P3
−2.5 50
D1
P1 40
−3
0 50 100 150 200 250 300 350 400 450
T (∘ C)
Figure 7: DSC thermograms of the casein/keratin nanomicelles mixture (CK−) and mixture treated by transglutaminase (CK+).
vibration of amido bonds. This indicated that some different keratin peptide bonds from different components caused by
new amido bonds were formed during the fixation with the fixation with transglutaminase.
transglutaminase. Furthermore, the complex nanomicelles
fixed with transglutaminase exhibited an additional band at
3.5. Hydrophobicity/Hydrophilicity of the Complex Nanomi-
1025 cm−1 (Figure 6, CK+). On the basis of the transglutami- celles. Recrystallized pyrene was used as a probe to detect the
nase spectrum (spectrum not shown) it was determined that hydrophobicity and hydrophilicity of casein/keratin complex
this band came from the saccharide structure of malt dextrin nanomicelles. The intensity ratio of the first to the third
incorporated into preparation of commercial transglutami- peak (𝐼1 /𝐼3 ) in the fluorescence spectrum can reflect the
nase. microenvironmental polarity where the probe exists [12]. The
greater the value of 𝐼1 /𝐼3 was, the weaker the hydrophobic
microenvironment for pyrene was.
3.4. Thermal Performance of the Complex Nanomicelles. The
Figure 8 showed that the value of 𝐼1 /𝐼3 for the complex
TG curve and the DSC curve of the complex nanomicelles
nanomicelles was pH sensitive at pH 5.0–9.0. The value of
were shown in Figure 7 (CK−). The wide heat absorption
𝐼1 /𝐼3 of the complex nanomicelles increased with the increase
band D1 (120–180∘ C) was related to the phase transition of the
of pH value from 5.0 to 9.0. The complex nanomicelle was
hydrated casein/keratin crystal. Peak 1 (238∘ C) corresponded
more hydrophobic near the isoelectric point of the casein
to the cleavage of casein/keratin crystal. Peak 2 (275∘ C)
and keratin, which led to the relative lower value of 𝐼1 /𝐼3 .
corresponded to the breakdown of crosslinks (–s–s– bonds, H
When the pH was increased from 5.0 to 9.0, the micelles’
bonds, and salt links) in keratin. Peak 3 (290∘ C) corresponded
structure became loose because of the increase in electrostatic
to the rupture of peptide bonds of casein. Peak 4 (330∘ C)
repulsion and the more hydrophilic group stretched out from
corresponded to the liquefaction and rupture of peptide bond
the micelles, which led to the relative higher value of 𝐼1 /𝐼3 .
of keratin [10, 11].
Figure 9 showed that the value of 𝐼1 /𝐼3 for the complex
The TG curve and the DSC curve of the complex nanomi-
nanomicelles varied as a function of mass ratio. The value of
celles fixed with transglutaminase were shown in Figure 7
𝐼1 /𝐼3 of the micelles increased when the value of mass ratio
(CK+). In Figure 7 (CK+), the wide heat absorption band
of casein/keratin decreased. When the keratin was added
(120–180∘ C) related to the phase transition of the hydrated
gradually, the micelles disassociated partly and the structure
casein/keratin crystal disappeared, which indicated that the
became loose, and the micelles got more hydrophilic rela-
hydration ability of casein/keratin micelles was reduced
tively.
by the fixation with transglutaminase. The heat absorption
related to the breakdown of crosslinks in keratin and peptide
bonds in casein appeared at 283∘ C (peak 6) as a single 3.6. Storage Stability of the Complex Nanomicelles. Casein
band, which implied that either the crosslinks or the peptide nanomicelles (5 mg/mL) and casein/keratin complex
bonds were altered by the fixation with transglutaminase. nanomicelles (5 mg/mL, mass ratio 4 : 1) at neutral pH were
The cleavage of casein/keratin crystal was scarcely influenced prepared separately and their stability was compared during
by the fixation (peak 1, peak 5). Peak 7 (310∘ C), peak 8 storage at room temperature. The changes in diameters and
(320∘ C), and peak 9 (333∘ C) corresponded to the rupture of PDI were showed in Figures 10 and 11. Figure 10 showed
1.34 350
300
1.32
250
Dh (nm)
I1 /I3
1.30 200
150
1.28
100
1.26
50
5 6 7 8 9 0 10 20 30 40
pH Storage time
ck
Figure 8: 𝐼1 /𝐼3 ratio of pyrene fluorescence for casein/keratin
c
complex nanomicelles solution as functions of pH in the range of
5.0–9.0. The micelles solutions were prepared at pH 7.0 with a protein Figure 10: Plots of 𝐷ℎ variations for the casein micelles and the
concentration of 5 mg/mL. casein/keratin complex nanomicelles in the 40-day preservation
time. The micelles solutions were prepared at pH 7.0 with a protein
concentration of 5 mg/mL.
1.38 0.90
1.36
0.85
1.34
0.80
1.32
0.75
1.30
PDI
I1 /I3
1.28 0.70
1.26 0.65
1.24 0.6
1.22 0.55
1.20
0 10 20 30 40
8 4 2 1 0.5 0.25 0.125
Storage time
Mass ratio (casein/keratin)
c
Figure 9: 𝐼1 /𝐼3 ratio of pyrene fluorescence for casein/keratin com- ck
plex nanomicelles solution as functions of mass ratio. The micelles
solutions were prepared at pH 7.0 with a protein concentration of Figure 11: Plots of PDI variations for the casein micelles and the
5 mg/mL. casein/keratin complex nanomicelles in the 40-day preservation
time. The micelles solutions were prepared at pH 7.0 with a protein
concentration of 5 mg/mL.
that the value of the diameter for the complex nanomicelles 4. Conclusions
almost was constant during the forty days of storage. On
the contrary, casein micelles’ diameter increased greatly in Casein and keratin were used to fabricate complex nanomi-
this process. The PDI value also exhibited similar result in celles through electrostatic self-assembly. In this process, no
Figure 11. These indicated that the nanomicelles’ stability harmful reagents were used. Polydisperse complex nanomi-
was improved dramatically when keratin was added. This celles can be formed in pH range of 5.0–9.0. Stable complex
is because the keratin employed possesses high stability nanomicelles can be acquired at the neutral pH in the mass
in structure [13] and lower molecular weight with intact ratio of casein to keratin 4: 1, and the absolute value of
secondary structure. 𝜁-potential was 22.8 mV. The stable complex nanomicelles
exhibited anomalous sphere shape with uniform size of [12] C. Keyes-Baig, J. Duhamel, S. Fung, and J. Bezaire, “Self-
which the average diameter was about 40–70 nm. The fixation assembling peptide as a potential carrier of hydrophobic com-
with transglutaminase could enhance the dilution stability pounds,” Journal of the American Chemical Society, vol. 126, no.
and reduce the hydration of the complex nanomicelles to 24, pp. 7522–7532, 2004.
some extent by forming crosslinking in the nanomicelles or [13] J. Li, Y. Li, L. Li, A. F. T. Mak, F. Ko, and L. Qin, “Preparation and
between the nanomicelles. The complex nanomicelles were biodegradation of electrospun PLLA/keratin nonwoven fibrous
relatively hydrophilic and had good storage stability. membrane,” Polymer Degradation and Stability, vol. 94, no. 10,
pp. 1800–1807, 2009.
Conflict of Interests
The authors declare that there is no conflict of interests
Acknowledgments
This work was financially supported by the Hongliang
Research Fund (413118) and Doctor Research Fund of Shaanxi
University of Science and Technology (BJ08-15). The material
in this work was supported by Jiangsu Yiming Bio-Products
Co.
References
[1] G. M. Whitesides, J. P. Mathias, and C. T. Seto, “Molecular
self-assembly and nanochemistry: a chemical strategy for the
synthesis of nanostructures,” Science, vol. 254, no. 5036, pp.
1312–1319, 1991.
[2] C. G. De Kruif, “Casein micelle interactions,” International
Dairy Journal, vol. 9, no. 3–6, pp. 183–188, 1999.
[3] C. F. Narambuena, F. S. Ausar, I. D. Bianco, D. M. Beltramo, and
E. P. M. Leiva, “Aggregation of casein micelles by interactions
with chitosans: a study by Monte Carlo simulations,” Journal
of Agricultural and Food Chemistry, vol. 53, no. 2, pp. 459–463,
2005.
[4] C. Garnier, C. Michon, S. Durand, G. Cuvelier, J.-L. Doublier,
and B. Launay, “Iota-carrageenan/casein micelles interactions:
evidence at different scales,” Colloids and Surfaces B, vol. 31, no.
1–4, pp. 177–184, 2003.
[5] M. van de Locht, “Reconstitution of microfibrils from wool and
filaments from epidermis proteins,” Melliand Textilberichte, vol.
10, pp. 780–786, 1987.
[6] P. M. Steinert and M. I. Gullino, “Bovine epidermal keratin fil-
ament assembly in vitro,” Biochemical and Biophysical Research
Communications, vol. 70, no. 1, pp. 221–227, 1976.
[7] H. Thomas, A. Conrads, P. H. Phan, M. van de Locht, and H.
Zahn, “In vitor reconstitution of wool intermediate filaments,”
International Journal of Biological Macromolecules, vol. 8, pp.
258–264, 1986.
[8] J. E. Plowman, “The proteomics of keratin proteins,” Journal of
Chromatography B, vol. 849, no. 1-2, pp. 181–189, 2007.
[9] L. M. Flanagan, J. E. Plowman, and W. G. Bryson, “The high
sulphur proteins of wool: towards an understanding of sheep
breed diversity,” Proteomics, vol. 2, no. 9, pp. 1240–1246, 2002.
[10] W. Xu, W. Guo, and W. Li, “Thermal analysis of ultrafine wool
powder,” Journal of Applied Polymer Science, vol. 87, no. 14, pp.
2372–2376, 2003.
[11] B. Purevsuren and Y. Davaajav, “Thermal analysis of casein,”
Journal of Thermal Analysis and Calorimetry, vol. 65, no. 1, pp.
147–152, 2001.
http://dx.doi.org/10.1155/2014/198572
Research Article
Nanopigmented Acrylic Resin Cured Indistinctively by
Water Bath or Microwave Energy for Dentures
L. S. Acosta-Torres,1 M. C. Arenas,1 R. E. Nuñez-Anita,2 F. H. Barceló-Santana,3

C. A. Álvarez-Gayosso,3 J. Palacios-Alquisira,4 J. de la Fuente-Hernández,1
Marcos Cajero-Juárez,2 and V. M. Castaño5,6
1
Escuela Nacional de Estudios Superiores, Unidad León, Licenciatura en Odontologı́a,
Universidad Nacional Autónoma de México, Boulevard UNAM No. 2011 Predio el Saucillo y el Potrero, 36969 León, GTO, Mexico
2
Facultad de Medicina Veterinaria y Zootecnia, UMSNH, Km. 9.5 Carretera Morelia-Zinapécuaro, Col. La Palma,
58893 Tarı́mbaro, MICH, Mexico
3
Laboratorio de Materiales Dentales, División de Estudios de Posgrado e Investigación, Facultad de Odontologı́a,
Universidad Nacional Autónoma de México, Avenida Universidad No. 3000, Colonia Copilco, 04510 México, DF, Mexico
4
Posgrado de la Facultad de Quı́mica, Universidad Nacional Autónoma de México, Avenida Universidad No. 3000,
Colonia Copilco, 04510 México, DF, Mexico
5
Departamento de Ingenierı́a Molecular de Materiales, Centro de Fı́sica Aplicada y Tecnologı́a Avanzada,
Universidad Nacional Autónoma de México, Campus Juriquilla, Boulevard Juriquilla No. 3001, 76230 Juriquilla, QRO, Mexico
6
Centro de Tecnologı́a Avanzada, (CIATEQ), av. El Retablo 150, 76150 Querétaro, Qro, Mexico
Correspondence should be addressed to M. C. Arenas; carenas@enes.unam.mx
Received 9 July 2013; Revised 25 December 2013; Accepted 27 December 2013; Published 20 February 2014
Copyright © 2014 L. S. Acosta-Torres et al. This is an open access article distributed under the Creative Commons Attribution
cited.
The highlight of this study was the synthesis of nanopigmented poly(methyl methacrylate) nanoparticles that were further processed
using a water bath and/or microwave energy for dentures. The experimental acrylic resins were physicochemically characterized,
and the adherence of Candida albicans and biocompatibility were assessed. A nanopigmented acrylic resin cured by a water
bath or by microwave energy was obtained. The acrylic specimens possess similar properties to commercial acrylic resins, but
the transverse strength and porosity were slightly improved. The acrylic resins cured with microwave energy exhibited reduced
C. albicans adherence. These results demonstrate an improved noncytotoxic material for the manufacturing of denture bases in
dentistry.
1. Introduction the plastic phase and less porosity and excellent adaptation of
a prosthetic material in contrast to conventional heat-water
Poly(methyl methacrylate) (PMMA) is the main commercial polymerization. Despite these advantages, this method has
acrylic resin used in denture fabrication [1]. Advances in limited use in the dentistry field [4].
polymer science for denture bases have developed differ- A few studies regarding the experimental acrylic resin
ent molding and activation techniques [2]. The heat- and have been reported. In our previous works, spherical particles
microwave-generated commercial acrylic resins have similar of an experimental acrylic resin were synthesized by the
chemical formulations [3], but there are specific components polymerization suspension technique using sodium alginate
to the curing of resins for each technique. The microwave or gelatin as suspension agents. A clear PMMA was obtained
method for PMMA denture base polymerization has the fol- and the morphology, particle size, thermal behavior, and
lowing advantages: shorter times for curing and for attaining flexural properties were fully characterized. The result was a
material comparable with the commercial acrylic resins for 4. Characterization of Nanopigmented
dentures when the material was processed by a water bath or PMMA Particles
a microwave technique [5]. Metallic oxide nanoparticles were
included in the synthesis as pigments to obtain a pink PMMA The nanopigmented PMMA, Lucitone 199, and Acron MC
that was similar to the gums in color. These nanopigmented powders were characterized. Fourier transform infrared (FT-
PMMA particles were thermopolymerized with the water IR) spectroscopy was conducted in a Bruker Vector 33
bath technique, and they presented lower porosity and Instrument using the transmittance technique. The samples
solubility compared with the clear PMMA [6]. Different types were prepared in KBr translucent disks and analyzed with 17
of fibers [7] or silver nanoparticles [8] were added in the scans in the wavelength region between 400 and 4,000 cm−1 .
nanopigmented PMMA formulation, but the fibers did not For scanning electron microscopy (SEM) analysis, the
change the flexural strength, and the nanoparticles decreased polymer particles were coated with gold by vacuum evapo-
this value, despite an improvement of the antifungal effect ration, and the observations were carried out with a JSM-
against Candida albicans. 6060LV scanning microscope (JEOL, Peabody, MA). The par-
The nanopigmented PMMA needs to be assessed in all the ticle size distribution and standard deviation were obtained
physical, antimicrobial, and cytocompatible properties when for each acrylic resin.
it is processed indistinctively by a water bath and microwave
thermopolymerization techniques. These methods do not 5. Water Bath and Microwave Polymerization
sacrifice the resin’s physicochemical properties and might
generate a cheap and nontoxic material. The material needs
for Specimen Preparation
to be compared with the commercial acrylic resins available To obtain the PMMA specimens, the nanopigmented PMMA
for each specific technique. The material was compared with powder was separated in two parts to form two experimental
the Lucitone 199 and Acron MC acrylic resins for denture groups. Mixtures were prepared with the powders of each
bases that are commercially available for specific water bath group and were collocated in three molds with the following
and microwave polymerization techniques, respectively. dimensions: 65 × 10 × 2.5 mm, 50 × 0.5 mm, and 10 × 2 mm.
The first group, designated PMMA-wb, was obtained by
mixing the PMMA with a MMA monomer (3 : 1) and benzoyl
2. Materials and Methods peroxide (1%), packing the mixture into metallic molds, and
processing in a water bath for 90 min at 70∘ C and then for
Methyl methacrylate (MMA) monomer and benzoyl perox- 30 min at 90∘ C. The second group, designated PMMA-mw,
ide (both from Sigma-Aldrich, St. Louis, MO, USA) were was obtained by mixing the PMMA particles with a MMA
used as received. Sodium alginate (Manufacturera-Dental- monomer (3 : 1) and benzoyl peroxide (1%) and packing the
Continental, Mexico) was used as a suspension agent. Iron mixture into polyester molds, which was followed by curing
oxide and titanium oxide nanoparticles (Fe2 O3 [R-4511] with microwave energy at 500 W for 3 min. After the curing
and TiO2 [RF-9400] (González-Cano y Compañı́a, Mex- process, the molds were cooled at room temperature for
ico) were used as pigments. The commercial heat-cured 30 min and placed into cold water at 4∘ C for 30 min before
acrylic resins Lucitone 199 (water bath thermopolymerized; opening the molds.
Dentsply/Trubyte, York, PA) and Acron MC (GC Lab Tech- The commercial acrylic resins Lucitone 199 and Acron
nologies, Alsip, IL) were selected for the comparisons. MC were cured following the manufacturers’ instructions.
The specimens obtained were plates of 65 × 10 × 2.5 mm
for the flexural strength and flexural modulus calculations
3. Synthesis of Nanopigmented (𝑛 = 10), discs of 50 × 0.5 mm for the water sorption and
PMMA Particles solubility tests (𝑛 = 10), and discs of 10 × 2 mm for the C.
albicans adherence and cytotoxicity assays (𝑛 = 9). All the
Nanopigmented PMMA, a pink substance similar to the
specimens were trimmed with wet abrasive paper of grit 100
gums, was synthesized by the suspension polymerization
and 300 (Fandeli, Mexico) prior to use.
technique described in previous work [5]. The brief method
Table 1 summarizes the curing technique, batch number,
was as follows. In a five-neck flask, 200 mL of deionized water,
and viscosity molecular weight of each evaluated acrylic
1.5 g of sodium alginate, 200 g of MMA monomer, and 0.2 g
resin. The mentioned molecular weight belongs to the exper-
of initiator were mixed with reflux; nitrogen gas was added;
imental PMMA without the nanopigment particles.
the mixture was stirred (1,200 rpm) and heated at 70∘ C for 2 h.
The TiO2 and Fe2 O3 nanopigments were dissolved in 30 mL
of deionized water and added to the reactor 30 min before 6. Characterization of the Cured
the initiator incorporation. Constant stirring throughout the Nanopigmented PMMA Specimens
reaction was used to ensure equal distribution of pigments.
When the reaction was finished and the PMMA particles had The processed PMMA-wb, PMMA-mw, Lucitone 199, and
sedimented, they were separated by decantation. The PMMA Acron MC samples were tested as follows.
was washed with deionized water four times until the water Thermogravimetrical analysis (TGA) was carried out on a
was clear to eliminate the nonreactant products. The polymer fragment of each group of the nanopigmented PMMA cured
particles were dried at room temperature. resins using a thermogravimetric analyzer (TA Instrument
Table 1: Summary of processing methods for curing of commercial acrylic resin and experimental PMMA.
Acrylic resin Processing method Manufacturer (location) Viscosity mol wt (g/mol)a

Water bath cured at Dentsply/Trubyte (York,
Lucitone 199 19 × 10−5
70∘ C for 90 min and 90∘ C for 30 min PA)
Microwave cured at
Acron MC GC (Alsip, IL) 14 × 10−5
500 W for 3 min
Water bath cured
Experimental
at 70∘ C for 90 min and 90∘ C for 30 min; — 36 × 10−5
PMMA
microwave cured at 500 W for 3 min
a
The viscosity molecular weight was reported previously [5].
Q500 V6.3) at a heating rate of 10∘ C/min up to 900∘ C in an under stirring conditions. The samples were placed in new
N2 atmosphere. 24-well plates, and 100 𝜇L of benzalkonium chloride was
For the characterization of the flexural behavior, the added to each sample to extract the adhered C. albicans
samples were placed in a transverse deflection machine cells. The plate contents were stirred for 15 min, and the
(Mecmesin, Sterling, VA) at 5 N/min until they fractured. samples were removed. A microbial cell viability assay based
The flexural modulus and transverse strength values were on luminescent ATP measurements (BacTiter Glo; Promega,
obtained using the equations reported previously [5, 6]. Fitchburg, WI) was performed to determine the number
For the water sorption and solubility test, 10 discs (𝑛 = of viable cells that had adhered to the composite resins.
10) were weighed (reported in mg), placed in a silica gel Briefly, extract aliquots (20 𝜇L each) were mixed with 30 𝜇L
desiccator, and weighed every 24 h until a constant mass (𝑚1 ) of BacTiter Glo reagent in 1.5-mL clear Eppendorf tubes,
was obtained. The discs were placed in distilled water for 7 and the luminescence was recorded after 5 min using a lumi-
days at 37 ± 1∘ C. The discs were then dried and weighed nometer (Turner Biosystems, Sunnyvale, CA) at an emission
(𝑚2 ). The discs were placed in the desiccator again and wavelength of 590 nm. The relative luminescence intensity, in
weighed every 24 h until a constant mass (𝑚3 ) was reached. 10 sec integration periods, was measured in triplicate.
The area (𝐴) of each sample was calculated (reported in cm2 ). A MTT assay was performed using NIH 3T3 mouse
The water sorption (Ws) and solubility (Sl) were calculated fibroblast-like cells (ATCC No. CRL-1658). The nanopig-
according to ADA 12, 1990 [9], as follows: Ws = (𝑚2 − 𝑚1 )/A; mented and commercial PMMA samples were sterilized
Sl = (𝑚1 − 𝑚3 )/A. by exposing both faces to ultraviolet irradiation for 5 min.
For the porosity test, the fractured plates were adjusted The cells were exposed to acrylic resins specimens, and
to obtain plates of 30 × 10 × 2.5 mm. They were weighed proliferation was assessed by measuring the reductase
to obtain their mass, and the volume of each sample was enzymatic activity based on the transformation of 3-
calculated (𝑉sp ). The samples were weighed every 24 h and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
placed in a silica gel desiccator until a constant mass was (MTT) into the colored, reduced form of MTT [12]. After 24
obtained. The volume of each sample was determined, and and 48 h incubation times, the resins were removed, the MTT
the internal porosity (𝑉ip ) was calculated as follows: assay was performed following the manufacturer’s instruc-
tions (Sigma-Aldrich), and the absorbance was measured in
𝑊𝑎 a microplate reader (Bio-Rad 680) at a wavelength of 655 nm.
𝑉ip = 𝑉sp − . (1) The viability percentage was calculated as follows: [(opti-
(𝑑𝑟 − 𝑑𝑎 )
cal density of the samples)/(optical density of the control
𝑊𝑎 is the weight of the sample (in g), 𝑑𝑟 is the acrylic resin group)] × 100. The control group was cultured without acrylic
density (1.198 g/cm3 ), 𝑑𝑎 (0.00123 g/cm3 ) is the Mexico City resins. The samples were analyzed in triplicate, and three
air density (at 𝑇 = 294 K, 78 kPa), and 𝑉sp is the volume of independent experiments were performed.
the sample (in cm3 ). For the statistical analysis, One-Way Analysis of Variance
One of the broken parts from each group after the flexural (𝑃 < 0.05) and Tukey tests were applied for the water
tests was used to observe the fracture zone by SEM. sorption, solubility, flexural modulus, transverse strength,
The contact angles of the PMMA polymer films have been porosity, cytotoxicity, and C. albicans adherence values.
measured by the spheroidal segment method using a contact
angle measurement system [10]. 7. Results and Discussion
For the biological testing (C. albicans adherence and
cytotoxicity assays), C. albicans strain 90026 (American 7.1. FTIR and Size Distribution of Nanopigmented PMMA.
Type Culture Collection, Manassas, VA) was cultured in Figure 1 shows the infrared spectrum of the nanopigmented
24-well plates at a density of 1 × 105 cells/mL. The PMMA PMMA compared with those of the commercial PMMA
samples were sterilized by exposing both faces to ultraviolet resins (Lucitone 199 and Acron MC) in the wavelength
irradiation for 5 min [8, 11]. Each acrylic resin sample was range of 4,000 cm−1 to 650 cm−1 . All the spectra present
placed in contact with the microorganisms for 24 h, and similar responses, in which the characteristic peaks of the
the samples were removed and washed with distilled water PMMA were observed [9, 10, 13]. The peaks at 2,950 cm−1 and
1.0 acrylic resins, such as plasticizers, comonomers, or cross-

Nanopigmented PMMA linking agents with different thermal behaviors. The manu-
facturer specifications of Lucitone 199 mention the presence
0.9 Lucitone 199
of ethylene dimethacrylate in its liquid composition, and
Transmittance (a.u)
Acron MC presents a copolymer of poly (methyl methacry-

late/ethylacrylate) in the powder formulation. It is clear
Acron MC that microwave energy influences in thermal stability of the
0.8
nanopigmented PMMA specimens.
-CH3 In the flexural modulus, there were no statistically signif-
0.7 icant differences between any of the tested groups (𝑃 > 0.05).
PMMA-wb, Lucitone 199 and Acron MC showed slightly
O higher transverse strength values than the nanopigmented
0.6
C O PMMA processed by microwaves (PMMA-mw) (Table 2),
3500 3000 2500 2000 1500 1000
and this value fulfilled the minimum allowed value according
to ISO 1567 (65 MPa) [14]. These values are still better
Wavenumber (cm-1)
compared with those of other commercial acrylic resins [15].
Figure 1: Infrared spectra of polymer particles of nanopigmented One important property of acrylates is water sorption and
PMMA, Lucitone 199, and Acron MC showing the main stretching release, which allows for dimensional instability, subjecting
bands of the PMMA molecule. the material to internal stresses that might result in crack
formation and eventually might fracture the denture. The
water molecules spread between the macromolecules of the
material, forcing them apart and affecting the dimensional
1,720 cm−1 correspond to the C–H and C=O (ester carbonyl) behavior and denture stability. Water sorption and solubility
stretching vibrations, and the band at 1,434 cm−1 is due to of these materials should be as low as possible [16]. In
the C–H bending vibrations. Other peaks at low frequencies, the water sorption test, there was a marked difference. The
1,139 cm−1 , 840 cm−1 , and 752 cm−1 , are related to the H–C– PMMA-wb had a lower value (0.27 ± 0.02 mg/cm2 ), and
H stretching vibration, to the O–C–O deformation vibration, Lucitone 199 had a higher value (0.37 ± 0.04 mg/cm2 ). The
and to the puckering vibration of PMMA, respectively. The materials processed by microwave energy had similar results.
absence of peaks in the range of 1,680 cm−1 to 1,640 cm−1 All the groups were tested for water sorption and solubility
indicates that the MMA monomer was fully polymerized [12]. according to the ADA number 12-required values [17], which
Despite the similarities in the infrared results for the were lower than 0.8 and 0.04 mg/cm2 , respectively.
PMMA samples, the particle sizes were very different accord- The mechanical properties of denture base materials
ing to the SEM results (not shown here). The nanopigmented decrease as the solubility increases. One study showed that
PMMA particle sizes were between 4.5 and 10 𝜇m, and the water bath polymerization results in enhanced mechanical
sizes for Lucitone 199 and Acron MC were in the range of 30 properties. It has been established that the water sorption
to 60 𝜇m and 60 to 120 𝜇m, respectively. Figure 2 shows the and solubility of polymers depend on the homogeneity of the
distribution of the particles sizes, where the averages for the material; less water absorbed and less solubility are presented
samples were 16.51 𝜇m ± 6.5 𝜇m (nanopigmented PMMA), in a homogeneous material [16]. PMMA-wb presented lower
32.23 𝜇m ± 10.8 𝜇m (Lucitone 199), and 69.69 𝜇m ± 23.88 𝜇m porosity than Lucitone 199, but there were no significant
(Acron MC). differences between the PMMA-mw and Acron MC. The low
porosity of PMMA-wb was in accordance with the expected
7.2. Evaluation of Nanopigmented PMMA Cured Using a Water values, and it is important to emphasize that the porosities
Bath and Microwave Thermopolymerization. The nanopig- of all the nanopigmented PMMA specimens were lower than
mented PMMA particles were cured by a water bath other acrylic resins [18].
and by microwave methods. The thermal stability of the After the flexural behavior evaluations, the topographies
PMMA specimens (PMMA-wb and PMMA-mw, Lucitone of the fractured zone of each specimen were examined
and Acron MC) was determined from the thermogram pro- in transverse sections by SEM (Figure 4). The micrographs
files (Figure 3). As shown in Figure 3(a), abrupt reductions showed irregular surfaces in the four acrylic resins, and
in weight at approximately 300∘ C to 400∘ C (80 to 98% the morphology changed according to the curing method.
weight loss) were observed for all specimens, and these were A slightly nonhomogeneous surface for PMMA-mw was
attributed to the complete degradation of the polymer chain. observed, which was in concordance with the low transverse
As shown in the inset of Figure 3(a), the profile of PMMA-wb strength value.
is slightly later than Lucitone 199, indicating a better stability. The mechanical properties and wear resistance of denture
PMMA-mw is slightly less thermally stable than Acron MC. materials have improved substantially, but their antibacterial
Based on the derived weight profiles (Figure 3(b)), the two properties are still of great interest [19]. In the present study,
main peaks between 276∘ C and 285∘ C (12 to 20% weight a C. albicans adherence assay was performed. Figure 5(a)
loss) and between 364∘ C and 379∘ C (80 to 98% weight loss) shows the results of the adherence assay, in which PMMA-wb
could be attributed to other components in the commercial and Lucitone 199 resulted in slightly higher values than the
100 100 PMMA-wb
80
PMMA-mw
80 12 Lucitone 199
Weight (%)
60 10
Frequency (a.u)
8
40 120 160 200 240 280 320 360
6
4
20
2
0 0
0 200 400 600 800 20 40 60 80 100 120 140
Temperature (∘ C) Diameter size (𝜇m)
PMMA-wb Acron MC
PMMA-mw Lucitone 199
(a) (b)
12 Acron MC
10
Frequency (a.u)
8
6
4
2
0
20 40 60 80 100 120 140
Diameter size (𝜇m)
(c)
Figure 2: Statistical particle size distribution of (a) nanopigmented PMMA compared to the commercial acrylic resins: (b) Lucitone 199 and
(c) Acron MC.
20
100 100 PMMA-wb 364
364 379
80 15
Derive weight (%/min)
PMMA-mw 372
80
Weight (%)
60
10
40 120 160 200 240 280 320 360
5
20 276
0 0
0 200 400 600 800 150 200 250 300 350 400 450
Temperature (∘ C) Temperature (∘ C)
PMMA-wb Lucitone 199 PMMA-wb Lucitone 199
PMMA-mw Acron MC PMMA-mw Acron MC
(a) (b)
Figure 3: (a) TGA profiles and (b) derived weight profiles of PMMA-wb and PMMA-mw compared with the commercial acrylic resins.
Table 2: Physical properties of PMMA polymerized by water bath and with microwave energy.
Candida albicans
Flexural Transverse Water sorption Solubility Porosity Contact angle
Acrylic resin adherence
modulus (GPa) strength (MPa) (mg/cm2 ) (mg/cm2 ) (%) (∘ )
(×105 LRUs)
PMMA-WB 2.5 ± 0.14∗ 77.6 ± 5.1∗ 0.27 ± 0.02∗ 0.03 ± 0.004∗ 4.6 ± 0.4∗ 60.29 ± 1.8 5.8 ± 1.3
PMMA-MW 2.5 ± 0.30 68.1 ± 2.8 0.31 ± 0.06 0.04 ± 0.005 5.5 ± 0.5 32.66 ± 7.4 2.6 ± 0.5
Lucitone 199 2.5 ± 0.20+ 78.2 ± 0.2+ 0.37 ± 0.04 0.02 ± 0.010 6.8 ± 1.0+ 36.02 ± 4.4 6.3 ± 2.2
Acron MC 2.5 ± 0.17 75.8 ± 5.1 0.30 ± 0.10 0.04 ± 0.008 5.4 ± 0.4 53.02 ± 3.1 1.6 ± 0.4
∗
[6]; + [7].
(a) (b)
(c) (d)
Figure 4: SEM images of the fracture zone of (a) PMMA-wb and (b) PMMA-mw compared with the commercial resins (c) Lucitone 199 and
(d) Acron MC.
acrylic resins processed by microwave energy (PMMA-mw, described, including surface roughness, salivary pellicle for-
Acron MC). Acron MC showed the lowest value, followed mation, hydrophobic property, and electrostatic interactions
by nanopigmented PMMA-mw, which was indicative of a [22].
lower C. albicans adherence compared with PMMA-wb and The water contact angle was measured to estimate the
Lucitone 199. C. albicans possesses various virulence factors, hydrophobicity of the nanopigmented PMMA and commer-
including the capacity to form biofilms, which render anti- cial acrylic resins (Table 2). The nanopigmented PMMA-
fungal drugs less efficient. C. albicans has the ability to form wb specimens presented a high water contact angle and
hyphae, which facilitates soft tissue invasion, allowing the a large amount of C. albicans. This is contrary to the
microorganisms to hide from the host defense system [20]. It references, where the adherence was linearly related to high
is difficult to avoid the adhesion of pathogenic microorgan- hydrophobicity [22]. These specimens are composed of small
isms to the surface of dental materials, though some efforts particles and have low porosity, which could influence the C.
toward this have been made [21]. The exact mechanisms by albicans adherence. The Acron MC specimens formed with
which C. albicans adheres to acrylic surfaces are unknown, large particles present low amounts of C. albicans and high
but many factors that affect C. albicans adherence have been hydrophobicity. The PMMA-mw and Acron MC specimens
120
100
80
Cell viability (%)

90
Acrylic resin Candida albicans adherence (×105 LRUs) 60

PMMA-wb 5.8 ± 1.3
40
PMMA-mw 2.6 ± 0.5
Lucitone 199 6.3 ± 2.2 20
Acron MC 1.6 ± 0.4
0
Control PMMA-wb PMMA-mw Lucitone 199 Acron MC
(a) (b)
120
100
80
Cell viability (%)
90
60
40
20
0
Control PMMA-wb PMMA-mw Lucitone 199 Acron MC
(c)
Figure 5: The antifungal effects and biocompatibility of PMMA-wb and PMMA-mw. (a) The adherence of C. albicans as measured in a
luminescent microbial cell viability assay. The luminescence was determined based on light relative units (LRUs). (b) and (c) The viability of
NIH 3T3 cells after exposure to materials for 24 and 48 h, respectively.
cured with microwave energy presented similar porosities leachable substances from the restoration. Another group
and a low amount of C. albicans, and there was a great of side effects are related to cell proliferation. One study
difference in the particle sizes. showed that a PMMA-based denture base polymer triggered
Cytotoxicity tests were designed to determine how the death signals in cell culture [24]. Another study showed
sample material affected a particular cell type. Figures 5(b) that treatment in a water bath postpolymerization reduced
and 5(c) show the results of the cytotoxicity assays for all the the cytotoxicity of Lucitone 550 [25]. In this work, Lucitone
groups when they were in contact with mouse fibroblast-like 199 and Acron MC were used as controls, representing
NIH 3T3 cell cultures for 24 and 48 h. The MTT test was noncytotoxic resins. The results showed that no significant
used because it is based on an evaluation of mitochondrial differences were observed regarding fibroblast cell viability,
function after exposure to potential toxic substances [23]. The therefore PMMA-wb and PMMA-mw can be considered
results were not statistically significantly different (𝑃 > 0.05), biocompatible materials.
indicating that the synthesized PMMA and the commercial
acrylic resins are nontoxic materials.
Consideration must be given to the relative biocompati- 8. Conclusions
bility of all denture base materials. Considerations of the inci-
dence and severity of side effects of denture bases have been Nanopigmented PMMA particles were successfully synthe-
included as parts of some general studies on dental materials. sized and cured by a water bath or by microwaves for denture
Local reactions that have been reported are not severe, and bases. According to results, the particle sizes and the curing
the most common are lichenoid reactions in the oral mucosa process influence the physical properties of the PMMA. The
and skin reactions such as rashes, dermatitis, and eczematous PMMA specimens exhibited good physical and mechanical
lesions. These reactions depend on the chemical composi- properties and were noncytotoxic, similar to commercial
tion of the materials used and their degradation products, acrylic resins. These nanopigmented particles will be applied
absorption, accumulation, and other factors associated with in vivo in the denture field in further work.
Conflict of Interests [11] V. Di Noto, K. Vezzù, G. A. Giffin, F. Conti, and A. Bertucco,

“Effect of high pressure CO2 on the structure of PMMA: a FT-
The authors confirm that they have no conflict of interests IR study,” Journal of Physical Chemistry B, vol. 115, no. 46, pp.
regarding the present paper. 13519–13525, 2011.
[12] T. L. Tsai, C. C. Lin, G. L. Guo, and T. C. Chu, “Effects of
microwave-assisted digestion on decomposition behavior of
Acknowledgments polymethyl methacrylate (PMMA),” Materials Chemistry and
Physics, vol. 108, no. 2-3, pp. 382–390, 2008.
The authors wish to thank the following individuals for their
[13] D. Padalia, U. C. Johri, and M. G. H. Zaidi, “Study of cerium
excellent technical support: Dra. Genoveva Hernández, Dra.
doped magnetite (Fe3 O4 :Ce)/PMMA nanocomposites,” Physica
Marina Vega, Mtra. Ma. Lourdes Palma Tirado, Q. Carmen B, vol. 407, no. 5, pp. 838–843, 2012.
Vázquez, Dr. Antonio Gómez Cortés, Miguel A. Arellano
[14] “Dentistry-Denture Base Polymers,” ISO 1567, International
Rodrı́guez, C. D. Rodrigo Hernández-Medina, Daniel Mon- Organization for Standardization, Geneva, Switzerland, 1999.
dragón, Antonio Prado, and L. E. I. Daniel González Espejel.
[15] A. Sodagar, M. Z. Kassaee, A. Akhavan, N. Javadi, S. Arab, and
M. J. Kharazifard, “Effect of silver nano particles on flexural
References strength of acrylic resins,” Journal of Prosthodontic Research, vol.
56, pp. 120–124, 2012.
[1] M. G. Tu, W.-M. Liang, T.-C. Wu, and S.-Y. Chen, “Improving [16] P. Pfeiffer and E.-U. Rosenbauer, “Residual methyl methacrylate
the mechanical properties of fiber-reinforced acrylic denture- monomer, water sorption, and water solubility of hypoaller-
base resin,” Materials and Design, vol. 30, no. 7, pp. 2468–2472, genic denture base materials,” Journal of Prosthetic Dentistry,
2009. vol. 92, no. 1, pp. 72–78, 2004.
[2] C. Machado, E. Sanchez, S. S. Azer, and J. M. Uribe, “Com- [17] ADA-12, “Revised American Dental Association Specification
parative study of the transverse strength of three denture base No. 12 for denture base polymers,” Reports of Councils and
materials,” Journal of Dentistry, vol. 35, no. 12, pp. 930–933, 2007. Bureaus/ JADA, 1990.
[18] C. P. Lai, M.-H. Tsai, M. Chen, H.-S. Chang, and H.-H. Tay,
[3] J. S. Moura, W. J. da Silva, T. Pereira, A. A. del Bel Cury, and R. C.
“Morphology and properties of denture acrylic resins cured
M. Rodrigues Garcia, “Influence of acrylic resin polymerization
by microwave energy and conventional water bath,” Dental
methods and saliva on the adherence of four Candida species,”
Materials, vol. 20, no. 2, pp. 133–141, 2004.
Journal of Prosthetic Dentistry, vol. 96, no. 3, pp. 205–211, 2006.
[19] M. Z. Kassaee, A. Akhavan, N. Sheikh, and A. Sodagar,
[4] M. J. Azzarri, M. S. Cortizo, and J. L. Alessandrini, “Effect of the “Antibacterial effects of a new dental acrylic resin containing
curing conditions on the properties of an acrylic denture base silver nanoparticles,” Journal of Applied Polymer Science, vol. 110,
resin microwave-polymerised,” Journal of Dentistry, vol. 31, no. no. 3, pp. 1699–1703, 2008.
7, pp. 463–468, 2003. [20] C. Messier, F. Epifano, S. Genovese, and D. Grenier, “Inhibition
[5] L. S. Acosta-Torres, F. H. Barceló-Santana, C. A. Álvarez- of Candida albicans biofilm formation and yeast-hyphal tran-
Gayosso, and J. Reyes-Gasga, “Synthesis and characterization of sition by 4-hydroxycordoin,” Phytomedicine, vol. 18, no. 5, pp.
poly(methyl methacrylate) polymerized by microwave energy 380–383, 2011.
or conventional water bath,” Journal of Applied Polymer Science, [21] L. Zhou, Z. Tong, G. Wu et al., “Parylene coating hinders
vol. 109, no. 6, pp. 3953–3960, 2008. Candida albicans adhesion to silicone elastomers and denture
[6] L. S. Acosta-Torres, L. M. López Marı́n, R. E. Nuñez-Anita, bases resin,” Archives of Oral Biology, vol. 55, no. 6, pp. 401–409,
G. Hernández-Padrón, and V. M. Castaño, “Biocompatible 2010.
metal-oxide nanoparticles: nanotechnology improvement of [22] C. A. Zamperini, A. L. Machado, C. E. Vergani, A. C. Pavarina,
conventional prosthetic acrylic resin,” Journal of Nanomaterials, E. T. Giampaolo, and N. C. Da Cruz, “Adherence in vitro of
vol. 2011, Article ID 941561, 8 pages, 2011. Candida albicans to plasma treated acrylic resin: effect of plasma
parameters, surface roughness and salivary pellicle,” Archives of
[7] V. Moreno-Maldonado, L. S. Acosta-Torres, F. H. Barceló- Oral Biology, vol. 55, no. 10, pp. 763–770, 2010.
Santana, R. D. Vanegas-Lancón, M. E. Plata-Rodrı́guez, and
V. M. Castaño, “Fiber-reinforced nanopigmented poly(methyl [23] G. Meriç, J. E. Dahl, and I. E. Ruyter, “Cytotoxicity of silica-glass
methacrylate) as improved denture base,” Journal of Applied fiber reinforced composites,” Dental Materials, vol. 24, no. 9, pp.
Polymer Science, vol. 126, pp. 289–295, 2012. 1201–1206, 2008.
[24] M. R. Cimpan, R. Matre, L. I. Cressey et al., “The effect of heat-
[8] L. S. Acosta-Torres, I. Mendieta, R. E. Nuñez-Anita, M. Cajero- and auto-polymerized denture base polymers on clonogenicity,
Juárez, and V. M. Castaño-Meneses, “Cytocompatible antifun- apoptosis, and necrosis in fibroblasts: denture base polymers
gal acrylic resin containing silver nanoparticles for dentures,” induce apoptosis and necrosis,” Acta Odontologica Scandinav-
International Journal of Nanomedicine, vol. 7, pp. 4777–4786, ica, vol. 58, no. 5, pp. 217–228, 2000.
2012.
[25] J. H. Jorge, E. T. Giampaolo, C. E. Vergani, A. L. Machado,
[9] R. W. Walker, L. M. Markillie, A. H. Colotelo et al., “Ultra- A. C. Pavarina, and I. Z. Carlos, “Biocompatibility of denture
violet radiation as disinfection for fish surgical tools,” Animal base acrylic resins evaluated in culture of L929 cells: effect
Biotelemetry, vol. 1, article 4, pp. 1–11, 2013. of polymerisation cycle and post-polymerisation treatments,”
[10] S. Wei, J. Sampathi, Z. Guo et al., “Nanoporous poly(methyl Gerodontology, vol. 24, no. 1, pp. 52–57, 2007.
methacrylate)-quantum dots nanocomposite fibers toward
biomedical applications,” Polymer, vol. 52, no. 25, pp. 5817–5829,
2011.
http://dx.doi.org/10.1155/2014/382861
Research Article
A New Method for Fabrication of Nanohydroxyapatite and TCP
from the Sea Snail Cerithium vulgatum
O. Gunduz,1,2 Y. M. Sahin,3 S. Agathopoulos,4 B. Ben-Nissan,5 and F. N. Oktar2,6,7

1
Department of Metal Education, Faculty of Technical Education, Marmara University, Kadıköy, 34722 Istanbul, Turkey
2
Center for Nanotechnology and Biomaterials Applied & Research, Marmara University, Kadıköy, 34722 Istanbul, Turkey
3
Department of Biomedical Engineering, Faculty of Engineering and Architecture, Istanbul Arel University, Buyukcekmece,
34537 Istanbul, Turkey
4
Department of Materials Science and Engineering, University of Ioannina, P.O. Box 1186, 45110 Ioannina, Greece
5
Department of Chemistry and Forensic Science, University of Technology Sydney, P.O. Box 123, Broadway, Ultimo, Sydney,
NSW 2007, Australia
6
Department of Bioengineering, Faculty of Engineering, Marmara University, Kadıköy, 34722 Istanbul, Turkey
7
Department of Medical Imaging Techniques, School of Health Related Professions, Marmara University, Tıbbiye Street No. 49,
Üsküdar, 34688 Istanbul, Turkey
Correspondence should be addressed to O. Gunduz; oguzhan@marmara.edu.tr and F. N. Oktar; foktar@marmara.edu.tr
Received 26 July 2013; Accepted 27 November 2013; Published 2 January 2014
Copyright © 2014 O. Gunduz et al. This is an open access article distributed under the Creative Commons Attribution License,
Biphasic bioceramic nanopowders of hydroxyapatite (HA) and 𝛽-tricalcium phosphate (TCP) were prepared from shells of the
sea snail Cerithium vulgatum (Bruguière, 1792) using a novel chemical method. Calcination of the powders produced was carried
out at varying temperatures, specifically at 400∘ C and 800∘ C, in air for 4 hours. When compared to the conventional hydrothermal
transformation method, this chemical method is very simple, economic, due to the fact that it needs inexpensive and safe equipment,
because the transformation of the aragonite and calcite of the shells into the calcium phosphate phases takes place at 80∘ C under
the atmospheric pressure. The powders produced were determined using infrared spectroscopy (FT-IR), X-ray diffraction, and
scanning electron microscopy (SEM). The features of the powders produced along with the fact of their biological origin qualify
these powders for further consideration and experimentation to fabricate nanoceramic biomaterials.
1. Introduction especially in orthopedic bone surgery [2] and other hard

tissue implantations [3], such as in dental and aesthetic
To date, biomaterials is a rapidly developing interdisciplinary surgery.
field at the interface of engineering, science, and healthcare Powders of HA can be produced with very various
industry; its effect on human health related issues is also chemical techniques, such as precipitation, hydrothermal
obvious and recognized all over the world. The global techniques, hydrolysis of other calcium phosphates, and sol-
biomaterials device market was estimated as $115.4 billion in gel [4] from very pure chemicals or from natural materials.
2008 and is expected to increase to $252.7 billion in 2014. The Calcination is another method to fabricate HA from different
largest market share among all biomaterial products belongs natural sources, like bone (i.e., human [5], bovine [6], sheep
to orthopedic biomaterials [1], like hydroxyapatite (HA) [7], turkey, and chicken) or tooth dentine [8] and enamel [9–
materials. With a chemical formula of Ca10 (PO4 )6 (OH)2 , 11]. In previous work, there are also papers reporting some
HA is the main inorganic component of bone [2] and very interesting sources for HA production, such as crocodile
tooth [3]. Thus, HA is very popular for implant materials bone [12], dear antler [13], and fish wastes.
belong to the species Nassarius hinnia reticulatus (this species

is overnumbered in these beaches). However, the shells of
Cerithium vulgatum are easily recognized and separated from
the shells of Nassarius hinnia reticulatus because the latter
ones are much smaller in length and diameter than the former
ones and have a brownish color.
Empty shells of a local sea snail (Cerithium vulgatum
Bruguière, 1792) were taken from a local beach in Princes
Figure 1: Typical photos of shells of Cerithium vulgatum Bruguière, Islands, Heybeli Island (local beach name; German Beach) in
1792, Mediterranean [30]. Istanbul, Turkey. No living creatures were used in this study
at all. The empty shells were cleaned thoroughly from sand
particles and other foreign materials. Then, the shells were
+ dried and crushed into small particles and finally planetary-
− +
milled in a porcelain jar. The milled powder was sieved using
a 100 𝜇m sieve (i.e., the particle size was <100 𝜇m).
A small sample of the fine powder was analyzed using
differential thermal and gravimetric analysis (DTA/TGA) to
determine the exact CaCO3 content. Batches of 2 g of powder
were suspended in an aqueous solution of distilled water
in a conical flask. Then, according to a previous study [33],
solution of H3 PO4 was added in such an amount as to satisfy
Figure 2: Habitation areas for Cerithium vulgatum [31]. the stoichiometric molar ratio of Ca/P equal to 1.667 (that
corresponds to HA; this sample is hereafter designated as
A) or 1.5 (that corresponds to TCP; this sample is hereafter
Hydrothermal methods are very popular to transform designated as B). Hot-plate stirrer equipment was used with
various sources with a sea origin, such as cuttlefish bone a conical flask in this work. The temperature of the solution
[14], some oysters [15], and corals [16]. In our more recent was set at 80∘ C and the reaction took place for 8 h under
studies, we have presented some very simple mechano- continuous stirring. After that, the powders were removed
chemical methods, which can be conducted with a simple from the liquid by filtration and dried at 100∘ C overnight in an
hot-plate stirrer and with ultrasonic equipment [17, 18]. incubator. The dried powders were calcined using an electric
Various aragonitic structures, such as cuttlefish bone [19], furnace (Nabertherm HT 16/17, Lilienthal, Germany) for 4 h
sea [20] and land snail shells [21], sea urchin shells [17, 18, in air. The powders of the sample A (i.e., Ca/P = 1.667) and
20], various mussel shells [19, 22–24], pearl powder [25], the powders of the sample B (i.e., Ca/P = 1.5) were calcined at
corals [16], and calcite from egg shells [26], were successfully 800∘ C and 400∘ C, respectively.
transformed into various Ca-phosphate bioceramic powders To characterize the materials, in either the raw form
using these novel mechanochemical methods. or the final powders, the following equipment was used.
In this work, a novel and simple chemical method was The thermal analysis was determined using DSC-DTA-TG
utilised to fabricate nanobiphasic powders of HA and TCP equipment (TA SDT Q600 Protherm). The observation of the
from the shells of a local sea snail, Cerithium vulgatum microstructure of the samples was observed in a scanning
Bruguière, 1792 [27]. The Cerithium vulgatum is a species electron microscope (SEM, JEOL JSM 7000F Field Emission
of sea snail, which is a marine gastropod mollusk and Scanning Electron Microscope, equipped with a Hitachi 1000
also belongs to the family Cerithiidae [28]. Generally the Tabletop microscope). The crystalline phases developed in
Cerithium vulgatum shells are occupied by hermit crabs [29]. the calcined powders were used by X-ray diffraction analysis
The typical shape of these shells is revealed in Figure 1 [30]. (Bruker D8 Advance X-ray diffractometer). The Fourier
transform infrared (FT-IR) spectra of the produced powders
were analyzed in a Bruker ALPHA FT-IR spectrometer.
2. Materials and Experimental Procedure
Generally, the habitation areas for Cerithium vulgatum are 3. Results and Discussion
all coastal areas of the United Kingdom, Spain, Portugal,
Greece, and West of Turkey (Figure 2, [31]). These species The typical microstructure at a fracture surface of the shells
can be obtained from the Black Sea in Turkey [32]. Thus, is revealed in the low-magnification SEM image of Figure 3.
it is not surprising to come across with the empty shells of A plate-like structure can be attributed largely to aragonite
the sea snail Cerithium vulgatum Brugui‘ere by the Marmara crystals. The direction of the plates is perpendicular to the
Sea, in Turkey, and specifically by the beaches of Princes outer (upper part in the image) and the inner surfaces of the
Islands. However, the collection of the Cerithium vulgatum shell. The outer surface apparently has a less dense structure.
shells is generally difficult by the beaches of Istanbul since Calcite is expected to be concentrated in the outer layer
their number are quite small and are negligible in comparison of the shell. The inner layer (lower part in the image) has
to those of other collected shells. The other shells mainly apparently a denser structure. Usually, the inner part of the
100 −1
90 −2
Heat flow (W/g)

Weight (%)
80 −3
43.67%
70 −4
60 −5
50 −6
0 200 400 600 800 1000
Figure 3: Microstructure at fracture surface of Cerithium vulgatum Exo up Temperature (∘ C) Universal V4.5A TA instruments
shell.
Figure 5: Differential and gravimetric thermal analysis (DTA/TGA)
of the raw powders (after planetary milling and sieving).
Intensity (a.u.)
(a) HA calcinated at 800∘ C
Figure 4: SEM image of raw powder after planetary milling and

sieving with a sieve of 100 𝜇m.
T calcinated at 400∘ C
(b) TCP
10 20 30 40 50 60 70 80
shells is largely made of aragonite. Therefore, the raw powder 2𝜃 (deg)
that was subjected afterwards to transformation into calcium
phosphates should involve both the phases of aragonite Hydroxylapatite
+ Calcium phosphate
and calcite, which is observed in all regular shells. But the Whitlockite
influence of aragonite-calcite sea conditions on the evolution
of biocalcification remained up to now poorly understood Figure 6: X-ray diffractograms of the produced powders after
[34]. calcination for 4 h in air: (a) powder B (Ca/P = 1.5, 400∘ C); (b)
The nanopowders were spontaneously fabricated after powder A (Ca/P = 1.667, 800∘ C).
crushing, milling, and sieving, as shown in the SEM image of
Figure 4. The powder mainly consisted of prismatic particles
with a semirounded shape and nanosize dimensions of about
200 nm. Some elongated rod-like prismatic particles with a these diffractograms it is concluded that the transformation
length of ca 1–1.5 𝜇m and a width of ca 200 nm are also of CaCO3 , in the form of either aragonite or calcite, was
observed. completely indicated. In both diffractograms, the phase of
The results of the differential and gravimetric thermal HA was clearly observed (JCPDS card 00-009-0432 in sample
analysis (DTA/TGA) of the raw powders after milling and B and JCPDS card 01-089-4405 in sample A; the differences
sieving are plotted in the diagrams of Figure 5. The decompo- between the two cards are negligible). The second major
sition of CaCO3 to CaO was clearly obtained in both curves. phase recorded was TCP (3CaO⋅P2 O5 ) in particular 𝛽-TCP
These curves confirm that the shell was exclusively consisted in the sample B (JCPDS card 00-009-0346) and whitlockite
of CaCO3 . Thus, the calculation of the amount of H3 PO3 (JCPDS card 00-009-0169) in the sample A. Whitlockite is
solution required to satisfy the demanded Ca/P ratios was also known as 𝛽-tricalcium phosphate (𝛽-TCP) [35], which
possible. is used in treatment of defects of cortical and cancellous bone
The X-ray analysis of the powders produced after cal- due to its osteoconductivity and bioresorbability [36].
cination for 4 h in air is shown in the diffractograms of The findings of the X-ray analysis indicate that the pow-
Figure 6(a), for the powder B (Ca/P = 1.5, 400∘ C), and ders produced are biphasic materials, which comprise HA
Figure 6(b), for the powder A (Ca/P = 1.667, 800∘ C). From and TCP. It is well know that the best bioceramic materials
1024.98
560.40
Absorbance units
0.5
0.4
0.3
0.2
0.1
0.0
3500 3000 2500 2000 1500 1000 500
×3.0 k 30 𝜇m
Wavenumber (cm−1 )
(a)
(a)
1025.45
541.78
602.51
0.5
Absorbance units
0.4
0.3
0.2
0.1
0.0
×3.0 k 30 𝜇m 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm−1 )
(b)
(b)
Figure 7: Microstructure of the produced powders after calcination
for 4 h in air: (a) powder B (Ca/P = 1.5, 400∘ C); (b) powder A (Ca/P Figure 8: FT-IR spectra of the produced powders after calcination
= 1.667, 800∘ C). for 4 h in air: (a) powder B (Ca/P = 1.5, 400∘ C); (b) powder A (Ca/P
= 1.667, 800∘ C).
should ideally consist of biphasic materials of HA and 𝛽- FT-IR spectra of the HA powders in the range 4000–
TCP. In such biphasic biomaterials, 𝛽-TCP is the resorbable
400 cm−1 are revealed in Figure 8 for powder B (Ca/P =
and osteoconductive [37] component. Usually, resorbable
1.5, 400∘ C, Figure 8(a)) and powder A (Ca/P = 1.667, 800∘ C,
bioceramics are considered as very active and thus they
Figure 8(b)), after calcination for 4 h in air. They revealed
stimulate a faster formation of the newly formed bone. On the
strong vibrations modes at the following wave numbers: 541,
other hand, HA presents an excellent biocompatibility and
bioactivity. 560, 602, 1024, and 1025 cm−1 . Absorption bands characteris-
All the results indicate that the produced powders are tic of O–P–O bending vibrations can be clearly seen at 541 and
very promising materials. However, these good prospects, 602 cm−1 . The powders appear to lack the O–H vibrational
due to the biphasic crystalline regime of the produced pow- bands indicated by the weak peak at 630 cm−1 (Figure 8(a))
ders, are further enforced because the SEM analysis showed [38]. The sharp bands at 1024-1025 cm−1 correspond to ]3
that this production process resulted in the production of asymmetric stretching modes of (PO4 )3− groups. Moreover,
nanopowders as well. The characteristic microstructure of the the increase of the calcination temperature to 800∘ C caused
powders produced after calcination for 4 h in air is shown the appearance of the peaks at 1500 and 3700–3500 cm−1 .
in the SEM images of Figure 7(a), for the powder B (Ca/P = The addition, IR bands in the range of 3700–3500 cm−1
1.5, 400∘ C), and Figure 7(b), for the powder A (Ca/P = 1.667, (Figure 8(b)) were also observed by Duta et al. [39], which
800∘ C). The powder B comprises prismatic nanoparticles was assigned them to the O–H stretching vibrations of surface
as well as needle-like nanorods with length of 1.5–3 𝜇m P–OH groups. These spectra are in agreement with the XRD
and diameter of 200 nm. The SEM image of Figure 7(b) results.
for the sample A indicates that this powder is apparently
finer than the powder B because there are less prismatic 4. Conclusions
particles; the rod-like (needle-shaped) particles are thinner
(with a diameter of ca 150 nm), and formation of apparently Using a simple mechanochemical method, biphasic bioce-
loosened agglomerations of nanoparticles was also observed, ramic nanopowders of hydroxyapatite (HA) and tricalcium
as revealed in the middle of Figure 7(b). phosphate (TCP) were fabricated. It has been indicated that
these nanopowders can be used as bioceramic for graft [10] N. Akyurt, U. Karacayli, M. Yetmez, S. S. Pazarlioglu, and F. N.
material. The shell of a regular sea snail from the species Oktar, “Microstructure and mechanical properties of sintered
Cerithium vulgatum was successfully transformed into the sheep enamel-derived hydroxyapatite,” International Journal of
target compounds. This new method is very simple, eco- Artificial Organs, vol. 34, no. 8, p. 700, 2011.
nomic, and safe. [11] N. Demirkol, M. Yetmez, U. Karacayli et al., “Mechanical prop-
erties of hydroxyapatite-tantalum composites,” International
Journal of Artificial Organs, vol. 33, p. 468, 2010, (XXXVII
Conflict of Interests Annual ESAO Congress) Skopje, R. Macedonia from 8th to 11th
September 2010.
The authors declare that there is no conflict of interests [12] K. Lewis, U. Boonyang, L. Evans, S. Siripaisarnpipat, and B. Ben-
regarding the publication of this article. Nissan, “A comparative study of Thai and Australian crocodile
bone for use as a potential biomaterial,” Key Engineering
Acknowledgments Materials, vol. 309–311, pp. 15–18, 2006.
[13] M. Bǎciuţ, G. Bǎciuţ, V. Simon et al., “Investigation of deer
This study was carried out partly with the equipment fur- antler as a potential bone regenerating biomaterial,” Journal of
nished (between 2007 and 2008) to Marmara University with Optoelectronics and Advanced Materials, vol. 9, no. 8, pp. 2547–
the support of the Turkish Republic Government Planning 2550, 2007.
Organization in the framework of the Project 2003K120810 [14] J. H. G. Rocha, A. F. Lemos, S. Agathopoulos et al., “Scaffolds for
“Manufacturing and Characterization of Electro-Conductive bone restoration from cuttlefish,” Bone, vol. 37, no. 6, pp. 850–
Bioceramics.” The authors also acknowledge the very valu- 857, 2005.
able support of the Metallurgical and Materials Engineering [15] A. F. Lemos, J. H. G. Rocha, S. S. F. Quaresma et al., “Hydroxya-
Department, Istanbul Technical University, Turkey. patite nano-powders produced hydrothermally from nacreous
material,” Journal of the European Ceramic Society, vol. 26, no.
16, pp. 3639–3646, 2006.
References [16] B. B. Nissan, A. S. Milev, D. D. Green et al., “Processes for treat-
ing coral and coating an object,” US patent no. 2004/0091547
[1] D. Lahiri, S. Ghosh, and A. Agarwal, “Carbon nanotube rein- A1, 2004.
forced hydroxyapatite composite for orthopedic application: a
review,” Materials Science and Engineering C, vol. 32, no. 7, pp. [17] D. Agaogulları, D. Kel, H. Gokce et al., “Bioceramic production
1727–1758, 2012. from sea urchins,” Acta Physica Polonica A, vol. 121, no. 1, pp.
23–26, 2012.
[2] M. R. Foroughi, S. Karbasi, and R. Ebrahimi-Kahrizsangi,
[18] R. Samur, L. S. Ozyegin, and D. Agaogullari, “Calcium phos-
“Physical and mechanical properties of a poly-3-hydroxybuty-
phate formation from dea urchin-(brissus Latecarinatus) via
rate-coated nanocrystalline hydroxyapatite scaffold for bone
modified mechano-chemical (ultrasonic) conversion method,”
tissue engineering,” Journal of Porous Materials, vol. 19, no. 5,
Metalurgija, vol. 52, pp. 375–378, 2013.
pp. 667–675, 2012.
[19] A. U. Tuyel, E. T. Oner, S. Ozyegin, and F. N. Oktar, “Production
[3] F. N. Oktar, M. R. Demirer, O. Gunduz et al., “Sintering effect on
and characterization of bioceramic nanopowders of natural-
mechanical properties of composites of bovine hydroxyapatite
biological origin,” Journal of Biotechnology, vol. 131S, p. S-65,
(BHA) and Li2 O,” Key Engineering Materials, vol. 309–311, pp.
2007.
49–52, 2006.
[20] M. L. Tamasan, L. S. Ozyegin, F. N. Oktar, and V. Simon,
[4] S. J. Roll, Processing of hydroxyapatite by biomimetic process, “Characterization of calcium phosphate powders originating
a thesis submitted in partial fulfillment of the requirement for from Phyllacanthus imperialis and Trochidae Infundibulum
the degree of bachelor of technology [M.S. thesis], Department concavus marine shells,” Materials Science and Engineering C,
of Ceramic Engineering, National Institute of Technology vol. 33, no. 5, pp. 2569–2577, 2013.
Rourkela, Odisha, India, 2006–2010.
[21] D. Kel, H. Gökçe, D. Bilgiç et al., “Production of natural
[5] G. Goller, F. N. Oktar, L. S. Ozyegin, E. S. Kayali, and E. bioceramic from land snails,” Key Engineering Materials, vol.
Demirkesen, “Plasma-sprayed human bone-derived hydroxya- 493-494, pp. 287–292, 2012.
patite coatings: effective and reliable,” Materials Letters, vol. 58,
[22] I. J. Macha, L. S. Ozyegin, J. Chou, R. Samur, F. N. Oktar, and
no. 21, pp. 2599–2604, 2004.
B. Ben-Nissan, “An alternative synthesis method for di calcium
[6] L. S. Ozyegin, F. N. Oktar, G. Goller, E. S. Kayali, and T. Yazici, phosphate (Monetite) powders from mediterranean mussel
“Plasma-sprayed bovine hydroxyapatite coatings,” Materials (Mytilus galloprovincialis) shells,” Journal of the Australian
Letters, vol. 58, no. 21, pp. 2605–2609, 2004. Ceramic Society, vol. 49, pp. 122–128, 2013.
[7] N. Demirkol, F. N. Oktar, and E. S. Kayali, “Mechanical and [23] S. Agathopoulos, L. S. Ozyegin, Z. Ahmad et al., “Nano-
microstructural properties of sheep hydroxyapatite (SHA)- bioceramics production from razor shell,” Key Engineering
niobium oxide composites,” Acta Physica Polonica A, vol. 121, Materials, vol. 493-494, pp. 775–780, 2012.
no. 1, pp. 274–276, 2012. [24] F. N. Oktar, U. Tuyel, N. Demirkol et al., “A new safe method
[8] G. Goller and F. N. Oktar, “Sintering effects on mechanical prop- to produce bioceramic nano-powders from nacre venus ver-
erties of biologically derived dentine hydroxyapatite,” Materials rucosa,” International Journal of Artificial Organs, vol. 33, pp.
Letters, vol. 56, no. 3, pp. 142–147, 2002. 467–468, 2010, (XXXVII Annual ESAO Congress) Skopje, R.
[9] F. N. Oktar, “Microstructure and mechanical properties of Macedonia from 8th to 11th September 2010.
sintered enamel hydroxyapatite,” Ceramics International, vol. 33, [25] A. U. Tuyel, Production and characterization of bioceramic
no. 7, pp. 1309–1314, 2007. nanopowders of natural-biological origin [M.S. thesis], Institute
for Graduate Studies in Pure and Applied Sciences, T.C. Mar-

mara University, 2008.
[26] D. Kel, U. Karacayli, M. Yetmez, L. S. Ozyegin, E. S. Kayalı, and F.
N. Oktar, “Hydroxyapatite production with various techniques
from sea urchin,” International Journal of Artificial Organs, vol.
34, p. 700, 2011.
[27] April 2013, http://www.marinespecies.org/aphia.php?p=taxde-
tails&id=139066.
[28] April 2012, http://en.wikipedia.org/wiki/Cerithium vulgatum.
[29] A. S. Ates, T. Katagan, and A. Kocataş, “Gastropod shell species
occupied by hermit crabs (Anomura: Decapoda) along the
Turkish coast of the Aegean Sea,” Journal of Zoology, vol. 31, pp.
13–18, 2007.
[30] April 2013, http://www.idscaro.net/sci/01 coll/plates/gastro/
pl cerithiidae 1.htm.
[31] April 2012, http://www.eu-nomen.eu/portal/taxon.php?GUID=
urn:lsid:marinespecies.org:taxname:139066.
[32] V. I. Zdun and S. M. Ignat’ev, “Black Sea mollusc, Cerithium
vulgatum (Gastropoda, Cerithiidae), a new intermediate trema-
tode host,” Parazitologiia, vol. 14, no. 4, pp. 345–348, 1980.
[33] L. S. Ozyegin, F. Sima, C. Ristoscu et al., “Sea snail: an alterna-
tive source for nano-bioceramic production,” Key Engineering
Materials, vol. 493-494, pp. 781–786, 2012.
[34] U. Balthasar and M. Cusack, “Aragonite-Calcite seas and evolu-
tion of biocalcification,” in Proceedings of the 22nd V.M. Gold-
schmidt Conference, Earth in Evaluation, Montreal, Canada,
2012.
[35] J. J. Song, An in vitro investigation of the spatial control involved
in collagen mineralization [M.S. thesis], University of Toronto,
2010.
[36] S. Sai and K. Fujii, “ß-tricalcium phosphate as a bone graft
substitut,” Jikeikai Medical Journal, vol. 52, pp. 47–54, 2005.
[37] P. Hernigou, X. Roussignol, C. H. Flouzat-Lachaniette, P.
Filippini, I. Guissou, and A. Poignard, “Opening wedge tib-
ial osteotomy for large varus deformity with Ceraver TM
resorbable beta tricalcium phosphate wedges,” International
Orthopaedics, vol. 34, no. 2, pp. 191–199, 2010.
[38] A. R. Boyd, B. J. Meenan, and N. S. Leyland, “Surface characteri-
sation of the evolving nature of radio frequency (RF) magnetron
sputter deposited calcium phosphate thin films after exposure
to physiological solution,” Surface and Coatings Technology, vol.
200, no. 20-21, pp. 6002–6013, 2006.
[39] L. Duta, F. N. Oktar, G. E. Stan et al., “Novel doped hydroxya-
patite thin films obtained by pulsed laser deposition,” Applied
Surface Science, vol. 265, pp. 41–49, 2013.
http://dx.doi.org/10.1155/2013/238351
Research Article
Cationic Gelatin Nanoparticles for Drug Delivery to
the Ocular Surface: In Vitro and In Vivo Evaluation
Ching-Li Tseng,1 Ko-Hua Chen,2,3,4 Wen-Yu Su,2,5,6 Yen-Hsien Lee,2,7

Chi-Chang Wu,1 and Fen-Huei Lin2,5
1
Graduate Institute of Biomedical Materials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, No. 250,
Wu-Hsing Street, Taipei City 110, Taiwan
2
Division of Medical Engineering Research, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town,
Miaoli County 350, Taiwan
3
Department of Ophthalmology, Taipei Veterans General Hospital, No. 201, Section 2, Shipai Road, Beitou District,
Taipei City 112, Taiwan
4
National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei City 112, Taiwan
5
Institute of Biomedical Engineering, National Taiwan University, No. 1, Section 1, Ren-ai Road, Taipei City 100, Taiwan
6
Institute of Biomedical Engineering and Material Science, Central Taiwan University of Science and Technology, No. 666, Buzih Road,
Taichung City 406, Taiwan
7
Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, No. 250, Wu-Hsing Street,
Taipei City 110, Taiwan
Correspondence should be addressed to Fen-Huei Lin; double@ntu.edu.tw
Received 2 August 2013; Revised 12 November 2013; Accepted 25 November 2013
Copyright © 2013 Ching-Li Tseng et al. This is an open access article distributed under the Creative Commons Attribution License,
To develop an effective ocular drug delivery carrier, we prepared two different charged gelatin nanoparticles (GPs) and evaluated
particle size, surface charge, and morphology. The in vitro biocompatibility of GPs was assessed using human corneal epithelium
(HCE) cells and in vivo safety by administering them as eye drops to New Zealand rabbits. The GPs prepared using type A gelatin
were positively charged (GP(+), +33 mV; size, 180.6 ± 45.7 nm). Water-soluble tetrazolium salt (WST)-1 assay showed that both
GPs were nontoxic to HCE cells. The fluorescence intensity of HCE cells cultured with cationic GPs conjugated with a fluorescent
dye was higher than that of the anionic GP-treated HCE cells. In vivo examination showed no serious irritation to the rabbit eyes.
Furthermore, corneal thickness and ocular pressure in the eyes of the treated rabbits were similar to those in the eyes of normal
rabbits. Microscopic examination of corneal cryosections showed widely distributed fluorescent nanocarriers, from the anterior to
the posterior part of the cornea of the GP(+) group, and higher fluorescence intensity in the GP(+) group was also observed. In
conclusion, GPs as cationic colloidal carriers were efficiently adsorbed on the negatively charged cornea without irritating the eyes
of the rabbits and can be retained in the cornea for a longer time. Thus, GPs(+) have a great potential as vehicles for ocular drug
delivery.
1. Introduction the eye that limit drug retention, (ii) pulse-drug entry with
high variation in dose, (iii) nasolacrimal duct drainage,
The eye poses unique challenges for drug delivery. The main which causes systemic exposure, and (iv) poor entrance to
objective of ocular therapeutics is to provide and maintain the posterior segments of the eye because of the lens-iris
adequate concentration of the drug at the target site. Most diaphragm [1, 2]. The above disadvantages result in clearance
ocular diseases are treated with topical application of solu- of 90% of the eye drops within 2 min, and only 5% of the
tions administered as eye drops. The major disadvantages of administered dose permeates to the eye [3].
this dosage form include (i) poor ocular drug bioavailability Most efforts in ocular delivery have been focused on
because of the anatomical and physiological constraints of increasing the corneal retention of drugs with the final goal
of improving the efficacy of treatments for different ocular Proliferation Assay Kit II was got from BioVision (CA, USA).
diseases. These attempts include the use of colloidal drug A Live/Dead Kit was purchased from Molecular Probes (OR,
delivery systems such as liposomes [4], nanoparticles [5– USA). Single-well cell inserts (PET) were obtained from
8], and nanospheres [9]. The results of different studies Millipore (MO, USA). All other chemicals were of reagent
showed the potential of nanoparticles for either gene or drug grade and obtained from Sigma-Aldrich.
delivery for ophthalmic application. Nanoparticles are able to
encapsulate and protect the gene/drug against degradation, 2.2. Preparation of GPs. The GPs were prepared by a two-
improve tolerance, and increase corneal uptake and intraoc- step desolvation method as described previously with some
ular half-lives [10]. Gelatin nanoparticles (GPs) were selected modifications [20, 21]. Type A and type B gelatin solution
for topical delivery because of their unique properties such (5 wt%) initially underwent desolvation by addition of excess
as biocompatibility and biodegradability [11]. Moreover, the quantity of acetone. Then, the gelatin deposited was redis-
source of gelatin, collagen, which is the major constituent of solved in water. The pH of the type A gelatin solution was
the corneal stroma, has been used for ophthalmic applica- adjusted to 2.5 and that of type B was adjusted to 11. Acetone
tions [12]. was added in a dropwise manner to form nanoparticles.
Although several studies have examined the use of GPs At the end of the process, 250 𝜇L of 8% GA solution was
for gene/drug delivery [13–16], few studies have examined used as a crosslinking agent for preparing nanoparticles, and
the use of GPs for ocular delivery. Vandervoort examined the solution was stirred for 12 h at 1000 rpm. The remaining
GPs encapsulated pilocarpine or hydrocortisone for topical organic solvent was evaporated using a rotary evaporator
ophthalmic delivery [17]. Vandervoot characterized the dif- (EYELA, Tokyo, Japan), and the resultant nanoparticles were
ferent forms of GPs and reported the rates of drug release stored at 4∘ C for further examination.
from these GPs, but they did not perform in vitro or in
vivo tests. In vivo administration of GPs loaded with plasmid
2.3. Characterization and Measurement of Different Parame-
DNA showed significantly higher expression of MUC5AC
ters of the GPs. The size and zeta potential of the GPs were
in the conjunctiva than that in untreated controls, and
analyzed using photon correlation spectroscopy (PCS) and
naked plasmid DNA encapsulated in GPs was beneficial for
laser Doppler anemometry, respectively, using a Zetasizer,
ophthalmic gene delivery [18]. These results show that GPs
3000 HS (Malvern Instruments, UK). Each batch was ana-
may be effectively used as vehicles for topical administration
lyzed in triplicate. The morphology of the nanoparticles was
to the eyes.
obtained by scanning the dried particles deposited on a flat
The cornea and conjunctiva possess negative surface
surface with a cantilever probe model AC240 (Olympus,
charges, and it is expected that cationic colloidal nanoparti-
USA) using tapping mode in an atomic force microscope
cles may penetrate through the negatively charged ocular tis-
(AFM; Asylum Research, MFP-3DTM, USA).
sues more efficiently than anionic carriers [19]. To determine
the importance of these characteristics in the interaction
of nanoparticles with the cornea, we prepared GPs with a 2.4. Human Corneal Epithelial Cells Culture. The SV40-
positive and negative charge for ocular delivery. The GPs with immortalized human corneal epithelial (HCE) cell line was
different charge were selected for ocular drug delivery. We kindly gifted by Dr. Ko-Hua Chen (Taipei Veterans Gen-
examined the particle size, polydispersity index (PDI), shape, eral Hospital, Taiwan). The HCE cells were cultured in
and surface charge and cytotoxicity of the GPs. Fluorescently DMEM/F-12 supplemented with 5% FBS, 100 U/mL peni-
labeled GPs were used in in vitro and in vivo experiments to cillin, 0.1 mg/mL streptomycin, 10 ng/mL EGF, 0.5% DMSO,
observe the distribution of the particles in the eyes of rabbits. and 5 𝜇g/mL insulin. The cells were cultured at 37∘ C in a 5%
In addition, the central corneal thicknesses and intraocular CO2 -95% air atmosphere. Media were changed every other
pressure (IOP) of rabbits were also examined to confirm the day, and the cells were observed daily under a phase contrast
influence of nanoparticles in rabbit eyes. microscope.
2.4.1. Evaluation of Cytotoxicity of GPs. The cytotoxicity of

2. Materials and Methods the GPs was examined in the HCE cells using the Quick Cell
Proliferation Assay Kit II (BioVision). The cells were seeded
2.1. Reagent and Chemicals. Gelatin type A (derived from onto 96-well plates (5 × 103 cells/well) about 16 h before the
porcine skin, bloom 175), gelatin type B (derived from bovine experiment. The HCE cells were incubated with different
skin, bloom 225), 25% glutaraldehyde (GA) solution, and concentrations of the GPs (500 to 0.1 𝜇g/mL) for 2 h. Then,
acetone were purchased from Sigma-Aldrich (MO, USA). the culture medium was discarded, and 0.2 mL water-soluble
Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1 : 1), tetrazolium-8 (WST-8) working solution was added to each
fetal bovine serum (FBS), insulin, trypsin-EDTA, peni- well. WST-8 is reduced by dehydrogenases in the living
cillin/streptomycin, and phosphate-buffered saline (PBS) cells to produce a yellow colored product (formazan). After
were obtained from Gibco/BRL (MD, USA); epithelial growth incubation for 4 h, 100 𝜇L of the working solution was quanti-
factor (EGF) was acquired from Pepro Tech (Rocky Hill, NJ, tatively assessed using a SpectraMAXM5 spectrophotometer
USA). Tetramethyl rhodamine succinyl (TAMRA-NHS) ester (Molecular Devices, CA, USA) at a wavelength of 450 nm.
and rabbit anti-zona occludens (ZO-1) polyclonal antibody The reference wavelength was set at 650 nm. The cells were
were obtained from Invitrogen (CA, USA). The Quick Cell stained with a live/dead stain (Molecular Probe) to observe
cell viability. The live cells emit green fluorescence, and the The cell suspension (100 𝜇L) was added to a 96-well plate
dead cells emit red fluorescence. Images were acquired using and the OD was measured at an excitation wavelength of
an inverted fluorescence microscope (Nikon, TiS, Japan) and 546 nm and an emission wavelength of 576 nm by the micro-
were analyzed using Nikon NIS Element software. plate spectrophotometer (SpectraMAXM5) under fluores-
cence mode.
2.4.2. Evaluation of Transepithelial Electrical Resistance.
About 3 × 105 HCE cells/cm2 were seeded on PET inserts 2.5. Preliminary Animal Study. Male New Zealand rabbits
with a 0.4-𝜇m pore size (Millipore, MA, USA), and the weighing 2.5–3.5 kg and with no signs of ocular inflammatory
medium was replenished every other day. Resistance across or gross abnormalities were used. The in vivo experimental
the insert membrane was measured using the STX2 electrode protocol was approved by the Institutional Animal Care and
set (World Precision Instruments [WPI], Florida, USA). The Use Committee of the Taipei Medical University (IACUC
transepithelial electrical resistance (TEER) of cells grown Approval No. LAC-100-0165). The animals were housed in
on filters was measured with an epithelial voltohmmeter standard cages in a light-controlled room and were given food
(EVOM, WPI). Cells were used only if their TEER was more and water ad libitum. We used 9 rabbits for measurement at
than 100 V/cm2 . The suspension of GPs (100 𝜇g/mL) was each time point, and during the experiments, the rabbits were
added into the media of the insert well. The electrode set allowed to move their heads freely, and their eye movements
was inserted in both the chambers for the indicated times. were not restricted.
The TEER was calculated from the measured resistance and
normalized using the area of the monolayer (ohms per square 2.5.1. In Vivo Tolerance. Positively charged GPs conjugated
centimeter). The background TEER of blank insert filters with the fluorescent dye (GP [+] TAMRA) were used in this
was subtracted from the TEER of the cell monolayers. Chi- study. We administered 50 𝜇L of sterilized GP(+) TAMRA in
tosan nanoparticles (CNP) were used as the positive control the lower conjunctival sac of the right eye of rabbits. The rab-
because of their capacity to disrupt the tight intercellular bits simultaneously received 50 𝜇L of TAMRA in PBS in their
junctions [22]. The size of the CNP was about 180 nm and left eye. The same volume of PBS was administered to another
their zeta potential showed a positive charge (20 mV). group of rabbits as control. This irritation test was performed
using a clinical evaluation scale of 0 (absence) to 3 (highest)
of discomfort, discharge, cornea/conjunctival chemosis, or
2.4.3. Western Blotting. The HCE cells were lysed to extract redness as described in Table 2 using a modification of
the cellular protein, and their absorbance was measured the scoring system established in the 2002 Organization
at OD 260/280 nm before use. Equal amounts of protein for Economic Cooperation and Development guidelines for
(approximately 10 𝜇g) were separated using 6% sodium ocular irritation testing [4, 23]. The test was performed on 5
dodecyl sulfate polyacrylamide gel electrophoresis. Then, eyes of each group; the test was performed in 3 eyes in the
the proteins were transferred onto nitrocellulose membrane, PBS-treated (control) group. Each animal was observed and
and the membranes were blocked in 5% nonfat powdered tested at 0.5, 2, 4, and 16 h after instillation.
milk in Tris-buffered saline (TBS) and 0.05% Tween. The
membranes were incubated with the primary antibody (ZO-1 2.5.2. Clinical Observations. At each study point, we mea-
at 1 : 3000 overnight at 4∘ C) followed by incubation with the sured the intraocular pressure (IOP) using a Schiotz tonome-
appropriate secondary antibodies (horseradish peroxidase ter (AMANN Ophthalmic Instruments, Liptingen, Germany)
[HRP]-conjugated anti-rabbit antibody at 1 : 10,000 for 1 h calibrated according to the manufacturer’s instructions. For
at room temperature). 𝛼-Tubulin was used as the internal determination of IOP, 5 readings were taken on each eye
control. Bands were visualized using an enhanced chemi- alternating between the left and right eyes, and the mean
luminescence reagent and exposed to a Fujifilm LAS1000 was calculated [24]. Central corneal thickness (CCT) was
Intelligent Dark Box and captured digitally. determined using an ultrasonic pachymeter (DGH Technol-
ogy, Exton, PA, USA) with a hand-held solid probe [25].
2.4.4. Cellular Uptake Study. GPs with positive or negative During the measurements, the probe tip of the pachymeter
charge were labeled with red fluorescence via being conju- was held perpendicular to the central cornea. Averages of
gated with TAMRA-NHS ester (Invitrogen) according to the 10 readings were recorded. An ophthalmic table slit lamp
method described by the manufacturer. The concentration of (Topcon Medical Systems Inc., NJ, USA) was used to observe
the fluorescent dye (TAMRA) in the GPs (100 𝜇g/mL) was and record the anterior segment. The rabbits were killed
0.35 𝜇g/mL. In addition, we examined the culture medium 16 h after administration of the eye drops. The eyeballs were
with the dye concentration equal to that in the aqueous harvested and fixed in 3.7% formaldehyde.
formulation. The fluorescent GPs (100 𝜇g/mL) were cul-
tured with HCE cells for 2 h; subsequently, the medium 2.5.3. Fluorescence Quantification. The rabbits were killed at
was removed, and the cells were washed twice using PBS. 0.5, 2, 4, and 16 h after the last instillation. Eyeballs were
Subsequently, 0.2 mL of cell lysis solution (50 mM Tris-HCl, harvested and cleaned using PBS. Fluorescent GPs in the
pH 7.6, 300 mM NaCl, and 0.5% Triton X-100) was added to eyes were quantified using an in vivo imaging system (IVIS
the cell pellets, and they were maintained for 2.5 h on ice with Imaging System 200 Series; Xenogen, USA). The relative
frequent vortexing. Then, the cells lysate was collected into intensity of fluorescence in the eyes was equivalent to the
Eppendorf tubes and centrifuged at 12,500 rpm for 20 min. concentration of fluorescent nanoparticles. The fluorescence
210.8 nm
72.9
105.4
5.0 0
5.0 0
4.0 4.0
4.0 4.0
(𝜇m)
(𝜇m
(𝜇m)
(𝜇m)
3.0 3.0
3.0 3.0
2.0 2.0 2.0 2.0
1.0 1.0 1.0 1.0
0.0 0.0 0.0 0.0

0.0 1.0 2.0 3.0 4.0 5.0 0.0 1.0 2.0 3.0 4.0 5.0
(𝜇m) (𝜇m)
(a) (b)
Figure 1: Morphology and size of charged GPs with (a) positive or (b) negative charge. Image acquired by atomic force microscopy.
Table 1: Size and zeta potential of gelatin nanoparticles (𝑛 = 3).
Size (nm) Zeta (mV)

GPs(+) 180.6 ± 45.7 33.4 ± 10.9
GPs(−) 230.7 ± 84.6 −44.2 ± 7.2
intensity of the PBS-treated group was used as the back- A gelatin was approximately 180.6 ± 45.7 nm. The size of
ground value. The quantified area was restricted to the the negatively charged GPs (prepared using type B gelatin)
cornea, and the fluorescein signal was calculated (𝑛 = 5). was 230.7 ± 84.6 nm; these nanoparticles were larger and
more widely distributed than GPs prepared using type A
2.5.4. Distribution of GPs in the Cornea Observed Using gelatin. The zeta potential of GPs prepared using type A
Confocal Laser Scanning Microscopy. After the fluorescence and type B gelatin was 33.4 ± 10.9 mV and −44.2 ± 7.2 mV
intensity was quantified, the cornea was excised from the (Table 1). The nanoparticles prepared using type A gelatin
eyeball and separated into 2 sections. One section was had a positive surface charge and were abbreviated as GP(+),
directly mounted on a glass slide and examined under a and those prepared using type B gelatin were negatively
microscope without additional processing of the tissue. A 5- charged and were abbreviated as GP(−). The nanoparticles
𝜇m cryosection was prepared using the other section from of both types observed under the AFM showed a smooth
the apical to the lateral end of the cornea. All cornea samples and ball like structure (Figure 1). The particle size was about
were analyzed with a confocal microscope (Nikon, A1, Japan). 200 nm, which was consistent with the findings of photon
correlation spectroscopy (PCS). Type A and type B gelatin
were prepared using by different processes by extracting
2.6. Statistical Analysis. Experiments were performed at least
gelatins from collagen [11]. The amount of free carboxyl or
in triplicate, and the results were reported as mean ± standard
amino groups was different in both types of gelatin. At pH
deviation (SD). All data were analyzed with the Student’s
6∼7, however, type A gelatin has a positive net charge, while
t-test or one-way analysis of variance (ANOVA). Statistical
type B gelatin is negatively charged [17, 27]; thus, the zeta
significance was considered at a level of 𝑃 < 0.05.
potential of these gelatins may also be different. The positively
charged GPs (GP+) may have electrostatic attraction with
3. Results and Discussion the negatively charged corneal epithelial cells, which is more
preferred in ocular drug delivery.
In this study, we prepared charged GPs and performed in
vitro and in vivo studies. The rabbit cornea model was used 3.2. Cytotoxicity of GPs. An important aspect of the devel-
to determine the retention of the cationic GPs because of opment of new carrier for drug/gene delivery is its safety of
the similarity of this model with that of the human cornea interaction with the target cells. The biocompatibility of the
[10, 26]. newly developed materials should be examined to determine
their potential for ophthalmic use. In this study, we evaluated
3.1. Characterization of GPs. GPs can be prepared using type the cytotoxicity of GPs in the HCE cell line by measuring
A or type B gelatin to obtain positively or negatively charged their metabolic activity. The percentage of viable cells in the
nanocarriers (Table 1). The size of GPs prepared using type treated group versus nontreated group (culture medium) is
Table 2: Grading system of the macroscopic signs in the in vivo tolerance study for the colloidal system tested [23].
Grade Discomfort Cornea Conjunctiva Discharge Lids

0 No reaction No alterations No alterations No discharge No swelling
Mild hyperemia Mild discharge without
1 Blinking Mild opacity Mild swelling
Mild edema moistened hair
Enhanced blinking Intense hyperemia
Intense discharge with
2 Intense tearing Intense opacity Intense edema Obvious swelling
moistened hair
Vocalizations Hemorrhage
140.0
120.0
100.0
Cell viability (%)
80.0
60.0
40.0
20.0
0.0
500.0 250.0 100.0 10.0 1.0 0.1
Nanoparticles concentration (𝜇g/mL)
GP(+)
GP(−)
Figure 2: Results of WST-1 assays of human corneal epithelium (HCE) cells after incubation with 2 kinds of gelatin nanoparticles for 2 h.
(GP+, GPs with positive surface charge; GP−, GPs with negative charge. Data were analyzed using the Student’s t-test and are presented as
mean ± standard deviation (SD); 𝑛 = 6, ∗ 𝑃 < 0.05).
shown in Figure 2. No significant difference was observed (GP+/GP−) slightly increased to 110% ± 12.7% (% against
in cell viability even after treatment with 500 𝜇g/mL of GPs initial) and then returned to normal (Figure 4(a)). No signifi-
for 2 h. The images of HCE cells labeled using the live/dead cant difference was observed in the TEER of HCE cells treated
stain are shown in Figure 3; the live cells emit green fluoresce with GPs(+) or GP(−) after 96 h. However, cells treated
and the dead cells emit red fluoresce. Large percentage of with CNP showed a marked decrease in the TEER (70%
live cells was observed in the control group (Figure 3(a)), decrease), which showed that the barrier integrity of the HCE
and nearly all the HCE cells were viable after coculturing monolayer was changed by CNP treatment. The CNP-treated
with GP(+) or GP(−) for 2 h (Figures 3(b) and 3(c)). HCEs cells showed loss of ZO-1, but no variation in the 2 GPs groups
were viable and only a few dead cells were observed after (Figure 4(b)). Chitosan has been widely used for ocular drug
culturing with GPs, which indicated that GPs had adequate delivery [5, 28–30] because of its mucoadhesive property.
safety for application to the ocular surface. de la Fuente et al. Chitosan disrupts the tight intercellular junctions and results
cultured HCE cells with hyaluronic acid-CNP for 1 h and in loss of membrane-associated ZO-1, thus increasing the
showed that this treatment had no effect on cell viability permeability of the epithelium [22]. The anterior part of the
[7]. The viability of HCE cells treated with cationized gelatin eye is constantly exposed to the external environment and
nanovector hybrid with chondroitin sulfate or dextran sulfate thus is vulnerable to a wide range of microorganisms; further,
for 72 h was not significantly different from that nontreated its moist mucosal surface makes the cornea particularly
HCE cells [18]. These results indicate that GPs are not toxic to susceptible to attack. The barrier to avoid microorganism
HCE cells after short-term or long-term exposure. invasion depends on the integrity of the tight junctions in
the cornea. The tight junctions between the neighboring
3.3. Alteration in the TEER across the Tight Junction in HCE epithelial cells prevent the free diffusion of hydrophilic
Cells by GPs Treatment. The presence of tight junctions molecules across the epithelium by the paracellular route
between epithelial cells prevents the flow of the fluids and [31]. However, CNP increases the drug concentration in the
the movement of molecules and ions between cells [22]. cornea via intracellular (uptake by the cells) and intercellular
The epithelial membrane provides a significant barrier to the (opening the tight intercellular junction) routes [31]. Previous
free diffusion of substances from the cornea to the anterior study showed that the tight junction reclosed may be impeded
chamber. The barrier integrity of these monolayers can be by the unremoved chitosan residue on the surface of the
measured directly by measuring the TEER of HCE cells. Caco-2 cells [22]. Therefore, a similar phenomenon may be
The TEER of HCE monolayers cultured with charged GPs observed in the corneal epithelial cells causing continuously
(a)
(b)
(c)
Figure 3: Fluorescent photomicrographs of human corneal epithelial (HCE) cells cultured with (a) culture medium, (b) GP(+), and (c) GP(−)
at a concentration of 100 𝜇g/mL for 2 h. The polyanionic dye calcein-AM is well retained within the live cells, which produced intense uniform
green fluorescence in the live cells. (Magnification: 40x); GP: gelatin nanoparticles.
open of the tight junction. Therefore, a risk for using CNP that cationic nanoparticles could increase the stability of the
for ocular drug delivery is increasing the microorganism nanoparticle system and improve the interaction between
invasion to the cornea via disruption of cornea tight junction. the particles and the eye surface and thus increase the
But, there is no risk for tight junction disruption by GPs. transfection efficiency [18, 32].
3.4. Intracellular Content. We examined the intracellular acc- 3.5. Tolerance and Clinical Evaluation. Gelatin is commonly
umulation of the charged GPs in the HCE cells. We examined used in the preparation of capsules, and GPs are widely
internalization of fluorescence-labeled GPs by measuring the investigated for drug/gene delivery. However, few studies
fluorescence in the cell lysates. Cationic or ionic GPs con- have examined the safety and tolerance of GPs for ocular
jugated with TAMRA were added into the culture medium. drug delivery. We performed an irritation test on rabbits
The intracellular fluorescence of the cell lysates in the GP(+) after single instillation of 50 𝜇L of GPs formulation. The eye
group at 10, 30, and 60 min was higher than that of the GP(−) treated using PBS was used as a control. Each animal was
(Figure 5). After 60 min, the OD value of the GP(+) group observed at 0.5, 2, 4, and 16 h after instillation. An index
was much higher than that of the GP(−) group (𝑃 < 0.05). of overall irritation (Table 2) was calculated by summing up
This finding is consistent with previous study, which showed the total clinical evaluation scores over the observation time
130.00
120.00
110.00
Initial TEER (%)
100.00
Control GP(+) GP(−) CNP
90.00
∗ ZO-1
80.00
70.00 ∗ 𝛼-Tubulin
60.00
0 20 40 60 80 100
Time (min)
GP(+)
GP(−)
CNP
(a) (b)
Figure 4: The transepithelial electrical resistance (TEER) assay showed recovery of human corneal epithelial (HCE) cell layer barrier after
coculturing with gelatin nanoparticles (GPs), but not in chitosan nanoparticles (CNP). 𝑛 = 5 standard error of mean (SEM), ∗ 𝑃 < 0.05. (b)
Western blot analysis of zonula occluden-1 (ZO-1) expression in HCE cells after treatment with different nanoparticles.
350.0
300.0 ∗
OD value (Ex546/Em576 nm)
250.0
200.0
150.0
100.0
50.0
0.0
10 30 60
Culture period (min)
GP(+)
GP(−)
Figure 5: Nanoparticles uptaken by the human corneal epithelial (HCE) cells were evaluated by measuring the fluorescence intensity of the
cell lysate. 𝑛 = 6 standard error of mean (SEM), ∗ 𝑃 < 0.05.
Table 3: Grading system of macroscopic signs in the in vivo tolerance study of the gelatin nanoparticles.
Control∗ Free TAMRA GP(+) TAMRA

Time 0.5 h 4.0 h 0.5 h 2.0 h 4.0 h 16.0 h 0.5 h 2.0 h 4.0 h 16.0 h
Grade
Discomfort 0 0 0 0 0 0 0 0 0 0
Cornea 0 0 0 0 0 0 0 0 0 0
Conjunctive 0 0 0 0 1 0 0 0 1 0
Discharge 0 0 0 0 1 0 1 0 0 0
∗
𝑛 = 3.
GP: gelatin nanoparticles; TAMRA: tetramethyl rhodamine.
(a) (c)
(b) (d)
Figure 6: The appearance of rabbit eyes topically treated with 100 𝜇L of free tetramethyl rhodamine succinyl (TAMRA) solution: (a) 0.5 h
and (b) 16 h after treatment; treated with GP(+)TAMRA solution (100 𝜇L): (c) 0.5 h and (d) 16 h after the application.
800 25.0
Central cornea thickness (𝜇m)
700
Intraocular pressure (IOP)
20.0
600
500
15.0
(mm/Hg)
400
300 10.0
200
5.0
100
0 0.0
−2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 −2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
After treatment (h) After treatment (h)
PBS PBS
Free TAMRA Free TAMRA
GP(+) TAMRA GP(+) TAMRA
(a) (b)
Figure 7: (a) Measurements of central corneal thickness (CCT) and (b) intraocular pressure (IOP) after treatment with eye drops containing
TAMRA solution or GP(+)TAMRA. An asterisk indicates statistically significant differences (∗ 𝑃 < 0.05; 𝑛 = 5) compared to control (PBS-
treated rabbits, 𝑛 = 3).
8.0E + 09
Total radiant efficiency (p/s)/(𝜇w/cm2 )

7.0E + 09
6.0E + 09
∗
5.0E + 09
4.0E + 09
3.0E + 09
2.0E + 09
1.0E + 09
0.0E + 00
0.5 2 4 16
After treatment (h)
Free TAMRA
GP(+) TAMRA
Figure 8: Ex vivo fluorescence imaging of the eyes of rabbits treated with fluorescent dye for different time periods: (a) TAMRA solution and
(b) GP(+)TAMRA. 𝑛 = 5 standard error of mean (SEM), ∗ 𝑃 < 0.05.
EP EP EP EP
ST ST
ST ST
EN EN
EN EN
(a) (b)
Figure 9: ((a), (b)) are images from cryosections of the cornea treated with the free dye and GP(+) fluorescence dye. Free dye: TAMRA/PBS
solution; GP(+) dye: GP(+) with TAMRA conjugation (red). Scale bar: 50 mm. EP: corneal epithelium; ST: corneal stroma; EN: corneal
endothelium. After treatment for 0.5 h.
points; the results are shown in Table 3. Very slight redness groups treated with free TAMRA and GP(+) TAMRA eye
of the conjunctiva was observed in the eyes treated with free drops showed a decrease in the corneal thickness after 0.5 h
TAMRA solution and in GP(+) TAMRA-treated eyes at 4 h, (297 ± 4 𝜇m; 292 ± 6 𝜇m) and 2 h (304 ± 5 𝜇m; 290 ± 5 𝜇m)
but no chemosis was observed after treatment in other groups (Figure 7(a)). After 4 h of treatment, the corneal thickness
and at other time points. No differences were observed in the in the treated group was almost the same as that in the
ocular tissue of rabbits treated with free and GP(+) TAMRA control group. The mean baseline of IOP values ranged from
after 0.5 and 16 h (Figure 6). Treatment with GP(+) was safe 16 to 20 mmHg (Figure 7(b)). The IOP decreased immediately
and caused no irritation to the eyes of the rabbits. Previous after treatment with PBS. The IOP returned to the baseline
studies have shown that the rabbit eye is more sensitive than level within 2 h. In addition, the IOP decreased at 2 h even
the human eye and has a longer time for epithelial repair after treatment with free TAMRA and GP(+) TAMRA. How-
[10, 33]. Therefore, it is reasonable to expect that charged GPs ever, the IOP in these 2 groups returned to the normal range
are well tolerated by the human eye. after 4 h (Figure 7(b)). The corneal thickness and IOP did
One of the risk factors involved in eye disease is increased not change significantly after treatment with GP(+) TAMRA
IOP, which leads to apoptosis and loss of retinal ganglion suspension.
cells [34]. Therefore, we examined the changes in the IOP
and corneal thickness after eye drop treatment to confirm 3.6. Fluorescence Examination to Determine the Distribution
the safety of GPs for ocular drug delivery. The effects of of GPs in the Eyes. The amount of fluorescent nanoparticles
instillation of free TAMRA and GP(+) TAMRA eye drops in the cornea at different time points acquired using the
on corneal thickness and IOP in rabbits are shown in IVIS spectrum imaging system is shown in Figure 8. The
Figure 7. Compared to the control groups (328 ± 21 𝜇m), the number of fluorescent spots obtained after treatment with
GP(+) TAMRA was greater than that obtained treatment with References
free TAMRA/PBS at 0.5, 2, 4, and 16 h. The accumulation
of the fluorescent dye differed significantly between the free [1] V. P. Torchilin and V. S. Trubetskoy, “Which polymers can make
nanoparticulate drug carriers long-circulating?” Advanced
TAMRA and GP(+) TAMRA treated group at 16 h after treat-
Drug Delivery Reviews, vol. 16, no. 2-3, pp. 141–155, 1995.
ment. After IVIS examination, the cornea was removed and
[2] E. E. Binstock and A. J. Domb, “Nanoparticles in locular drug
a cryosection was prepared for examination under a confocal
delivery,” in Nanoparticles for Pharmaceutical Applications, A. J.
microscope. The distribution profile of the fluorescent dye in Domb, Y. Tabata, M. N. V. R. Kumar, and S. Farber, Eds., pp.
the cornea of rabbits after administration of the eye drops 367–376, American Scientific Publishers, Valencia, Calif, USA,
is shown in Figure 9. The cross-section of the cornea from 2007.
the epithelium, stroma to endothelium layer, was observed [3] N. M. Davies, “Biopharmaceutical considerations in topical oc-
under the same magnification. The cornea treated using ular drug delivery,” Clinical and Experimental Pharmacology
the free TAMRA solution showed a weak fluorescent signal and Physiology, vol. 27, no. 7, pp. 558–562, 2000.
located in the posterior region (Figure 9(a)). The cornea [4] Y. Diebold, M. Jarrı́n, V. Sáez et al., “Ocular drug delivery by
treated with GP(+) TAMRA showed a strong fluorescent liposome-chitosan nanoparticle complexes (LCS-NP),” Bioma-
signal in the entire cornea and (Figure 9(b)). Moreover, the terials, vol. 28, no. 8, pp. 1553–1564, 2007.
fluorescence quantification of the cornea treated with GP(+) [5] A. M. de Campos, A. Sánchez, and M. J. Alonso, “Chitosan nan-
TAMRA increased by 4-fold compared to that treated with oparticles: a new vehicle for the improvement of the delivery
TAMRA/PBS solution, which indicated that the dye encapsu- of drugs to the ocular surface. Application to cyclosporin A,”
lated in the GPs could be retained in the cornea for a longer International Journal of Pharmaceutics, vol. 224, no. 1-2, pp. 159–
time and was distributed uniformly across the entire cornea. 168, 2001.
Solid lipid nanoparticles (SLNs) and CNP are retained for [6] J.-L. Bourges, S. E. Gautier, F. Delie et al., “Ocular drug deli-
a longer time on the corneal surface probably because of very targeting the retina and retinal pigment epithelium using
their small size, and further characterization of nanoparticles polylactide nanoparticles,” Investigative Ophthalmology & Vis-
would help in determining the transcorneal absorption [5, ual Science, vol. 44, no. 8, pp. 3562–3569, 2003.
10]. Hyaluronic acid coated poly-3-caprolactone nanospheres [7] M. de la Fuente, B. Seijo, and M. J. Alonso, “Novel hyaluronic ac-
achieved high levels of cyclosporine A (CyA) in the cornea, id-chitosan nanoparticles for ocular gene therapy,” Investigative
Ophthalmology & Visual Science, vol. 49, no. 5, pp. 2016–2024,
which was 10-fold higher than that was achieved with CyA
2008.
solution in castor oil [35]. In our study, the levels of GPs in
[8] E. Başaran, M. Demirel, B. Sirmagül, and Y. Yazan, “Cyclosp-
the corneas treated with GP(+) TAMRA were higher than
orine-A incorporated cationic solid lipid nanoparticles for oc-
those in the cornea treated with free TAMRA at each time ular delivery,” Journal of Microencapsulation, vol. 27, no. 1, pp.
point, which indicated a longer retention time (16 h) of GP(+) 37–47, 2010.
TAMRA compared to that of free TAMRA. Our in vivo results [9] Y. Kawashima, H. Yamamoto, H. Takeuchi, and Y. Kuno, “Mu-
might be explained on the basis of the prolonged retention coadhesive DL-lactide/glycolide copolymer nanospheres
in the precorneal area and cornea because of the small size coated with chitosan to improve oral delivery of elcatonin,”
of GP(+) (180 nm) and also in the uptake/internalization of Pharmaceutical Development and Technology, vol. 5, no. 1, pp.
GP(+) into the corneal epithelium. 77–85, 2000.
[10] E. H. Gökçe, G. Sandri, S. Eǧrilmez, M. C. Bonferoni, T. Güneri,
and C. Caramella, “Cyclosporine A-loaded solid lipid nanopar-
4. Conclusion ticles: ocular tolerance and in vivo drug release in rabbit eyes,”
Current Eye Research, vol. 34, no. 11, pp. 996–1003, 2009.
The aim of this study was to confirm whether cationic GPs [11] K. B. Djagny, Z. Wang, and S. Xu, “Gelatin: a valuable protein for
could be used for topical application, and this was examined food and pharmaceutical industries: review,” Critical Reviews in
in rabbit eyes. Positively charged GPs were prepared with a Food Science and Nutrition, vol. 41, no. 6, pp. 481–492, 2001.
size of about 180 nm. GPs are nontoxic to HCE cells and had [12] M. B. Sintzel, S. F. Bernatchez, C. Tabatabay, and R. Gurny, “Bio-
no influence on the tight intercellular junctions. The corneal materials in ophthalmic drug delivery,” European Journal of
thickness slightly decreased 0.5 h after treatment and then Pharmaceutics and Biopharmaceutics, vol. 42, no. 6, pp. 358–374,
returned to normal. The IOP showed variation in the normal 1996.
range after treatment with GPs. GPs showed retention of [13] J. O.-H. Sham, Y. Zhang, W. H. Finlay, W. H. Roa, and R. Löb-
the fluorescent dye in the cornea for the prolonged period, enberg, “Formulation and characterization of spray-dried pow-
which is beneficial to maintain the dose in the therapeutic ders containing nanoparticles for aerosol delivery to the lung,”
range. Therefore, dye/drug/gene encapsulated in cationic GPs International Journal of Pharmaceutics, vol. 269, no. 2, pp. 457–
nanoparticles is promising new medicines for ocular disease. 467, 2004.
[14] G. Kaul and M. Amiji, “Tumor-targeted gene delivery using
poly(ethylene glycol)-modified gelatin nanoparticles: in vitro
Acknowledgments and in vivo studies,” Pharmaceutical Research, vol. 22, no. 6, pp.
951–961, 2005.
This work was supported in part by a grant from the [15] A. K. Gupta, M. Gupta, S. J. Yarwood, and A. S. G. Curtis, “Effect
Taipei Medical University Startup Grant (TMU100-AE1-B01) of cellular uptake of gelatin nanoparticles on adhesion, mor-
and National Science Council, Taiwan (NSC 101-2221-E-038- phology and cytoskeleton organisation of human fibroblasts,”
002). Journal of Controlled Release, vol. 95, no. 2, pp. 197–207, 2004.
[16] C.-L. Tseng, W.-Y. Su, K.-C. Yen, K.-C. Yang, and F.-H. Lin, “The [32] L. Rabinovich-Guilatt, P. Couvreur, G. Lambert, and C. Duber-
use of biotinylated-EGF-modified gelatin nanoparticle carrier net, “Cationic vectors in ocular drug delivery,” Journal of Drug
to enhance cisplatin accumulation in cancerous lungs via inh- Targeting, vol. 12, no. 9-10, pp. 623–633, 2004.
alation,” Biomaterials, vol. 30, no. 20, pp. 3476–3485, 2009. [33] C. M. Hutak and R. B. Jacaruso, “Evaluation of primary ocular
[17] J. Vandervoort and A. Ludwig, “Preparation and evaluation of irritation: alternatives to the Draize test,” in Ocular Therapeutics
drug-loaded gelatin nanoparticles for topical ophthalmic use,” and Drug Delivery, R. Ik, Ed., Technomic Publishing, Lancaster,
European Journal of Pharmaceutics and Biopharmaceutics, vol. Pa, USA, 1996.
57, no. 2, pp. 251–261, 2004. [34] L. Guo, S. E. Moss, R. A. Alexander, R. R. Ali, F. W. Fitzke, and
[18] G. K. Zorzi, L. Contreras-Ruiz, J. E. Párraga et al., “Expression of M. F. Cordeiro, “Retinal ganglion cell apoptosis in glaucoma is
MUC5AC in ocular surface epithelial cells using cationized related to intraocular pressure and IOP-induced effects on
gelatin nanoparticles,” Molecular Pharmaceutics, vol. 8, no. 5, extracellular matrix,” Investigative Ophthalmology & Visual
pp. 1783–1788, 2011. Science, vol. 46, no. 1, pp. 175–182, 2005.
[19] R. C. Nagarwal, S. Kant, P. N. Singh, P. Maiti, and J. K. Pandit, [35] I. Yenice, M. C. Mocan, E. Palaska et al., “Hyaluronic acid coated
“Polymeric nanoparticulate system: a potential approach for poly-𝜀-caprolactone nanospheres deliver high concentrations of
ocular drug delivery,” Journal of Controlled Release, vol. 136, no. cyclosporine A into the cornea,” Experimental Eye Research, vol.
1, pp. 2–13, 2009. 87, no. 3, pp. 162–167, 2008.
[20] C. J. Coester, K. Langer, H. Von Briesen, and J. Kreuter, “Gela-
tin nanoparticles by two step desolvation–a new preparation
method, surface modifications and cell uptake,” Journal of
Microencapsulation, vol. 17, no. 2, pp. 187–193, 2000.
[21] C.-L. Tseng and F.-H. Lin, “Preparation of gelatin nanoparticles
with EGFR selection ability via biotinylated-EGF conjugation
for lung cancer targeting,” Biomedical Engineering: Applications,
Basis and Communications, vol. 20, no. 3, pp. 161–169, 2008.
[22] J. Smith, E. Wood, and M. Dornish, “Effect of chitosan on epi-
thelial cell tight junctions,” Pharmaceutical Research, vol. 21, no.
1, pp. 43–49, 2004.
[23] Test Guideline 405: Acute Eye Irritation/Corrosion, Organization
for Economic Co-Operation and Development, 2002.
[24] J.-Y. Lai, “Biocompatibility of chemically cross-linked gelatin
hydrogels for ophthalmic use,” Journal of Materials Science:
Materials in Medicine, vol. 21, no. 6, pp. 1899–1911, 2010.
[25] W. M. Hsu, K. H. Chen, J. Y. Lai, and G. Hsiue, “Transplantation
of human corneal endothelial cells using functional bioma-
terials: poly(N-isopropylacrylamide) and gelatin,” Journal of
Experimental & Clinical Medicine, vol. 5, no. 2, pp. 56–64, 2013.
[26] P. van der Bijl, A. H. Engelbrecht, A. D. van Eyk, and D. Meyer,
“Comparative permeability of human and rabbit corneas to
cyclosporin and tritiated water,” Journal of Ocular Pharmacology
and Therapeutics, vol. 18, no. 5, pp. 419–427, 2002.
[27] S. Young, M. Wong, Y. Tabata, and A. G. Mikos, “Gelatin as a
delivery vehicle for the controlled release of bioactive mol-
ecules,” Journal of Controlled Release, vol. 109, no. 1–3, pp. 256–
274, 2005.
[28] A. M. de Campos, Y. Diebold, E. L. S. Carvalho, A. Sánchez, and
M. J. Alonso, “Chitosan nanoparticles as new ocular drug deliv-
ery systems: in vitro stability, in vivo fate, and cellular toxicity,”
Pharmaceutical Research, vol. 21, no. 5, pp. 803–810, 2004.
[29] A. E. de Salamanca, Y. Diebold, M. Calonge et al., “Chitosan
nanoparticles as a potential drug delivery system for the ocular
surface: toxicity, uptake mechanism and in vivo tolerance,”
Investigative Ophthalmology & Visual Science, vol. 47, no. 4, pp.
1416–1425, 2006.
[30] A. M. de Campos, A. Sánchez, R. Gref, P. Calvo, and M. J. Alo-
nso, “The effect of a PEG versus a chitosan coating on the intera-
ction of drug colloidal carriers with the ocular mucosa,” Euro-
pean Journal of Pharmaceutical Sciences, vol. 20, no. 1, pp. 73–81,
2003.
[31] E. Mannermaa, K.-S. Vellonen, and A. Urtti, “Drug transport in
corneal epithelium and blood-retina barrier: emerging role
of transporters in ocular pharmacokinetics,” Advanced Drug
Delivery Reviews, vol. 58, no. 11, pp. 1136–1163, 2006.
http://dx.doi.org/10.1155/2013/542584
Research Article
Development of Antibiotics Impregnated Nanosized Silver
Phosphate-Doped Hydroxyapatite Bone Graft
Waraporn Suvannapruk, Faungchat Thammarakcharoen,

Phetrung Phanpiriya, and Jintamai Suwanprateeb
National Metal and Materials Technology Center (MTEC), 114 Paholyothin Road, Klong 1, Klongluang, Pathumthani 12120, Thailand
Correspondence should be addressed to Jintamai Suwanprateeb; jintamai@mtec.or.th
Received 2 August 2013; Revised 2 October 2013; Accepted 2 October 2013
Copyright © 2013 Waraporn Suvannapruk et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nanosized Ag3 PO4 loaded hydroxyapatite which was prepared by a novel low temperature phosphorization of 3D printed
calcium sulfate dihydrate at the nominal silver concentration of 0.001 M and 0.005 M was impregnated by two antibiotics
including gentamicin and vancomycin. Phase composition, microstructure, antibiotics loading, silver content, antimicrobial
performance, and cytotoxic potential of the prepared samples were characterized. It was found that the fabricated sample
consisted of hydroxyapatite as a main phase and spherical-shaped silver phosphate nanoparticles distributing within the cluster
of hydroxyapatite crystals. Antibacterial activity of the samples against two bacterial strains (gram negative P. aeruginosa and gram
positive S. aureus) was carried out. It was found that the combination of antibiotics and nanosized Ag3 PO4 in hydroxyapatite could
enhance the antibacterial performance of the samples by increasing the duration in which the materials exhibited antibacterial
property and the size of the inhibition zone depending on the type of antibiotics and bacterial strains compared to those contained
antibiotics or nanosilver phosphate alone. Cytotoxic potential against osteoblasts of antibiotics impregnated nanosilver phosphate
hydroxyapatite was found to depend on the combination of antibiotics content, type of antibiotics, and nanosilver phosphate
content.
1. Introduction Apart from antibiotics, silver ions were known to have

inhibitory and antibacterial properties against a broad spec-
Hydroxyapatite is one of calcium phosphate family which is trum of bacterial strains while being relatively low toxic to
widely used for bone repairs and reconstruction due to its human cells. Recently, silver nanoparticles have increasingly
osteoconductive property. Recently, antibiotics impregnated been used for infection treatment due to its unique properties
hydroxyapatite which was prepared by a novel low tempera- resulting from the nanoscale features and the ability to rapidly
ture phosphorization route was developed for bone infection release of several silver species which were seen to improve
treatment by providing local, sustained, and high concentra- the treatment efficiency [7–9]. Silver phosphate (Ag3 PO4 ) is
tion of antimicrobial agents while also function as a restorable one form of silver compounds that could be used as a silver
bone graft for new bone formation in the injured area [1, source for antibacterial applications due to its advantages
2]. The advantages of this approach were that it minimized compared to other silver compounds including its low sol-
systemic complications which would expose patients to ubility in aqueous solutions, its high antibacterial efficiency,
antibiotic levels that often would result in numerous toxic side and its strong photocatalytic activity under visible light
effects, improved clinical efficacy, and eliminated the need [10, 11]. Recently, we successfully applied a low temperature
for additional bone grafting. Furthermore, hydroxyapatite phosphorization process to in situ load nanosilver phosphate
prepared by this low temperature process could resorb faster into hydroxyapatite structure in a single step which enhances
than typically used high temperature sintered hydroxyapatite its antibacterial property [12]. It was shown previously that
[3–6] due to its low crystalline nanostructure which is close the combination of nanosilver and antibiotics could enhance
to that of bone. the antibacterial performance depending on the bacterial
strains, type of antibiotics, concentration of antibiotics, and and a scanning electron microscope (JSM-5410, JEOL, Japan).
nanosilver concentration [13–17]. However, no study on the In the case of TEM analysis, all samples were grinded into
effect of antibiotics and nanosilver that are incorporated powders and then dispersed onto continuous carbon film
into hydroxyapatite matrix on antibacterial activity has been grids prior to observation. Total silver content in each sample
studied. It is thought that the impregnation of antibiotics was determined by acid digestion technique using an atomic
in nanosilver phosphate-doped hydroxyapatite could also absorption spectrometry (AAS) (Analyst 200, Perkin Elmer).
provide the improvement in antibacterial performance of
hydroxyapatite, but the mechanism and the effect might be 2.3.2. Total Antibiotic Loading. The total antibiotics concen-
different. tration in the sample beads were determined by dissolving
In this study, nanosilver phosphate-doped hydroxyapatite them in 2.4 M hydrochloric acid and analyzed by using
which was prepared by low temperature coconversion pro- UV-VIS spectrophotometer (Jasco V-530) in relation to the
cess was, thus, impregnated with two types of antibiotics previously constructed calibration curve.
including gentamicin and vancomycin. Materials properties
and antibacterial performance including phase composition, 2.3.3. Minimum Inhibitory Concentration. Silver nitrate solu-
microstructure, total drug loading, antibacterial activity, tion was employed to represent the silver ions that were
and cytotoxic potential of antibiotic impregnated nanosilver expected to be released from nanosilver phosphate particles.
phosphate-doped hydroxyapatite sample were determined Minimum Inhibitory Concentration (MIC) of silver nitrate
and compared to antibiotics impregnated only and nanosilver and each antibiotic used against two bacterial strains (gram
phosphate loaded only samples. negative P. aeruginosa ATCC 27853 and gram positive S.
aureus ATCC 25923) was determined by the broth macrodi-
2. Materials and Method lution method. Bacterial inoculum was prepared by transfer-
ring 3–5 colonies of bacterial isolate from a fresh (18–24 h)
2.1. Materials. Raw materials used in this study were calcium nutrient agar plate to 5 mL of sterile nutrient broth (NB). It
sulfate hemihydrate (Lafarge Prestia Co., Ltd, Thailand) and was incubated at 35∘ C ± 2∘ C for 4–6 h (become visibly turbid)
pregelatinized starch (Thaiwah Co., Ltd, Thailand). These and the culture was adjusted to a 0.5 McFarland standard
materials were supplied in the form of powders and used (≈1.5 × 108 CFU/mL). Tenfold serial dilution of the inoculum
without further sieving. Antibiotics used were gentam- suspension was made by adding 1.0 mL of the inoculum
icin sulfate (T.P Drug Laboratories (1969) Co., Ltd) and suspension to 9.0 mL of NB to achieve 1.5 × 106 CFU/mL and
vancomycin hydrochloride (CJ CheilJedang Corporation, gently mixing by the vortex mixture. Serial 2-fold dilution of
Korea), abbreviated as CN and VC, respectively. the silver nitrate or antibiotic solution was made in the series
of tubes and 0.5 mL of the prepared inoculum suspension was
2.2. Sample Preparation. Calcium sulfate hemihydrate pow- added to each tube and incubated at 35∘ C ± 2∘ C for 16–18 h.
ders was mixed with pregelatinized starch powders using The growth of bacteria was shown as the visual turbidity and
a mechanical blender and loaded into a three dimensional the MIC value was determined from the final silver nitrate
printing machine (Z400, Z Corporation) to print 7 mm in or antibiotic concentration of the lowest concentration of
diameter spherical specimens. Solutions containing 1 M of nonturbid tube.
disodium hydrogen phosphate (Fluka) and two concentra-
tion of silver nitrate (BDH) (0.001 and 0.005 M) were pre- 2.3.4. Antimicrobial Activity. All the samples were sterilized
pared, designated HA 001 and HA 005, respectively. Ammo- by ethylene oxide gas prior to the tests. Antimicrobial perfor-
nium (BDH) was then added dropwise to the solution until mance tests were carried out by modified agar diffusion assay
clear solutions were obtained. The fabricated 3DP beads were against two bacterial strains (gram negative P. aeruginosa
then immersed in the solution and kept at 80∘ C for 24 hours ATCC 27853 and gram positive S. aureus ATCC 25923).
to transform to nanosilver phosphate-doped hydroxyapatite These two strains were selected to represent gram negative
by low temperature phosphorization reaction. Samples were and gram positive strains that are commonly found in bone
then taken out, rinsed by distilled water, and oven-dried. They infection areas [18, 19]. Bacterial strains were inoculated
were then loaded with two types of antibiotics, using vacuum- on each agar plate. The sample beads were submerged in
assisted method similarly to previous studies [1, 2]. simulated body fluid (SBF) at 37∘ C for 15 days and the beads
were withdrawn and placed in a new SBF at every 24 hours.
Each eluate was placed in the bored holes in the agar plates
2.3. Characterization
and incubated at 37∘ C. Antibiotic assay was performed by
2.3.1. Microstructure, Phase Composition and Total Silver measuring inhibition zone by a vernier caliper.
Content. XRD characterization was carried out using X-
ray diffractometer (JDX 3530, JEOL, Japan) with Co K- 2.3.5. Cytotoxic Potential. All the samples were sterilized
alpha radiation in the range of 10–80∘ 2𝜃 with step angle of by ethylene oxide gas and incubated in 1 mL of DMEM
0.02 degree. JCPDS files were used to identify the peaks of (Biowhittaker) completed medium at 37∘ C. The eluates were
main compositions in sample. Microstructures of nanosilver drawn at 24, 48, and 72 hours with replenishment of a
phosphate loaded hydroxyapatite samples were examined new medium after each eluate aspiration. This was devised
using a transmission electron microscope (JEOL JEM-2010) to study and compare the effect of the releasing behavior
(a) (b)
Figure 1: TEM micrographs showing microstructure of nanosilver phosphate-doped hydroxyapatite. (a) HA001; (b) HA005. Scale bar =
20 nm.
of each sample on the cytotoxic potential at each period comprised the nanosized crystals of hydroxyapatite with
by the replenishment of a new medium after each eluate the distribution of spherical-shaped silver phosphate
aspiration similar to the antimicrobial activity test. By this nanoparticles within the cluster. Particle sizes of silver
way, the media which contained released antibiotics and phosphate particles in both samples were estimated from
silver ions from the previous extraction period was discarded the images to be less than 5 nm. SEM images showed that
and the concentration of antibiotics or silver ions in the solu- as-fabricated nanosilver phosphate-doped hydroxyapatite
tion would be changed accordingly with extraction periods sample was porous comprising numerous pores (Figures 2(a)
depending on the releasing characteristic. The elutes were and 2(b)). No significant difference in the microstructure
then added to each tissue culture dish which contained 1 between samples could be detected. Figure 3 shows the
× 105 human osteoblast cells per one milliliter of DMEM XRD pattern of sample fabricated by low temperature
medium and incubated for 24 hours. After incubation, 100 𝜇L coconversion technique. HA 005 showed the characteristic
of 0.5 mg mL−1 MTT (Sigma-Aldrich) was added in each peaks of hydroxyapatite and silver phosphate. HA 001
well and incubated for another 2 hours. Dimethyl sulfoxide also showed the characteristic peaks of hydroxyapatite,
was then added and transferred to a 96-well plate. Optical but silver phosphate peaks were not clearly observed. In
density was measured at the wavelength of 570 nm using all samples, hydroxyapatite peaks were similarly broad
a microplate reader (Easys Model UVM 340) to quantify while the silver phosphates peaks were sharp. Total silver
the cell viability. Thermanox (Nunc) cover slip was used as content in each sample was determined by an atomic
negative control (NC) while polyurethane film containing absorption technique and observed to be 0.09 and 0.42% for
0.1% Zinc diethyldithiocarbamate (ZDEC): RM-A for ISO HA 001 and HA 005, respectively. After impregnation by
10993 cytotoxicity testing (Hatano) was used as positive antibiotic, the microstructure of the gentamicin impregnated
control (PC). Reagent control (RC) was the well which samples appeared relatively unchanged (Figures 2(c) and
contained no samples. Hydroxyapatite sample (HA) which 2(d)). In contrast, the surface of vancomycin impregnated
was fabricated by similar process, but without antibiotics or samples was extensively coated by vancomycin (Figures
nanosilver phosphate loading, was also tested as a control 2(e) and 2(f)). This was owing to the greater gelation
sample. ability of vancomycin at high concentration compared to
gentamicin.
2.3.6. Statistical Analysis. The differences in properties
amongst samples were analyzed using an analysis of variance 3.2. Total Antibiotics Loading. Figure 4 shows the total antibi-
(ANOVA) and Tukey post hoc testing. A value of 𝑃 < 0.05 otics loading in the nanosilver phosphate-doped hydroxya-
was considered significant. patite at different silver content. It could be seen that drug
loading in pure hydroxyapatite samples was greater than
3. Results those of nanosilver phosphate-doped samples for both type
of antibiotics. In the case of gentamicin, the drug content
3.1. Phase Composition and Microstructure. Figure 1 shows in HA CN 001 was not significantly different to HA CN,
the typical microstructures of fabricated nanosilver but approximately twice greater than that of HA CN 005.
phosphate-doped hydroxyapatite samples, HA 001 and In contrast, the drug content in HA VC 001 was lower than
HA 005. It was observed that both samples similarly those of HA VC and HA VC 005.
600 𝜇m 600 𝜇m
(a) (b)
600 𝜇m 600 𝜇m
(c) (d)
600 𝜇m 600 𝜇m
(e) (f)
Figure 2: SEM images showing microstructure of nanosilver phosphate-doped hydroxyapatite and antibiotic impregnated nanosilver
phosphate-doped hydroxyapatite samples: (a) HA 001; (b) HA 005; (c) HA CN 001; (d) HA CN 005; (e) HA VC 001; (f) HA VC 005. Scale
bar = 600 𝜇m.
3.3. Minimum Inhibition Concentration. Table 1 shows the P. aeruginosa. In the case of vancomycin impregnated
MIC values for each antibiotic and silver nitrate (in the samples (Figure 5(a)), no inhibition zone was seen for
form of silver ions) against two bacterial strains. Gentam- HA VC sample since vancomycin was known to be inactive
icin displayed the lowest MIC values, whereas vancomycin against gram negative strains [20]. In contrast, vancomycin
showed the highest values against S. aureus. In the case of impregnated nanosilver phosphate samples (HA VC 001 and
P. aeruginosa, MIC value was not obtained for vancomycin HA VC 005) showed inhibition zone. However, the sizes
since it is not active against gram negative bacteria. Silver ions of the inhibition zone at the same period were observed
showed lower MIC value than that of gentamicin. to be similar to those of the hydroxyapatite samples con-
taining similar content of nanosilver phosphate such as
HA VC 001 versus HA 001 or HA VC 005 versus HA 005.
3.4. Antibacterial Performance against P. aeruginosa. Figure 5 In the case of gentamicin impregnated samples (Figure 5(b)),
shows the antimicrobial performance of the samples against all gentamicin impregnated samples showed inhibition zone.
Table 1: Minimum inhibition concentration of the employed bacterial strains against each antibiotics and silver nitrate solution.
Minimum inhibition concentration (g/mL)

Antibiotics
P. aeruginosa ATCC 27853 S. aureus ATCC 25923
Vancomycin (VC) No inhibition (>125) 0.975
Gentamicin (CN) 1.5625 0.0977
Ag+ (AgNO3 ) 0.3125 0.3125
HA VC 005 and HA CN 005 samples which contained large

amount of nanosilver phosphate displayed longer antibacte-
rial duration for about 15 days.
∗∗
∗ ∗□ HA005
□ ∗ □ ∗ ∗∗ ∗ 3.5. Antibacterial Performance against S. aureus. In the case
∗∗ of S. aureus, inhibition zones were seen for all samples.
∗ ∗ HA001
∗ ∗ ∗ ∗ ∗ HA 001 showed limited antibacterial activity at day 2 extrac-
tion only while HA 005 displayed inhibition zone for only 5
20 25 30 35 40 45 50
days. The sizes of inhibition zone were also significantly lower
2𝜃 (deg) than those of antibiotics impregnated nanosilver phosphate-
□ Silver phosphate doped samples. In the case of vancomycin impregnated sam-
∗ Hydroxyapatite ples (Figure 6(a)), HA VC 005 showed the longest antibac-
terial duration followed by HA VC and HA VC 001, respec-
Figure 3: Phase composition of fabricated nanosilver phosphate-
doped hydroxyapatite by low temperature phosphorization tech-
tively. In contrast, both HA CN 001 and HA CN 005 showed
nique. about two times longer antibacterial duration than HA CN
sample (Figure 6(b)). The inhibition zones of samples con-
taining both antibiotics and nanosilver phosphate displayed
12 decreasing trends with extraction periods, whereas the inhi-
∗ bition zone size of nanosilver phosphate-doped samples was
∗
∗ relatively constant throughout the extraction periods.
10 ∗ ∗
8 3.6. Cytotoxic Potential. Cytotoxic potential test (Figure 7)

Drug loading (mg)
indicated that no cytotoxic potential at all extraction periods

was observed for HA, HA VC, and HA VC 001 samples.
6 However, HA VC 005 sample showed cytotoxic potential
(cell viability lower than 70%) even at day 3 extraction. In
4 the case of gentamicin impregnated samples, HA CN and
HA CN 005 samples displayed cytotoxic potential at day 1
extraction, whereas cytotoxic potential was observed until
2
day 2 extraction for HA CN 001 sample. In the case of
nanosilver phosphate-doped hydroxyapatite alone, HA 001
0 sample displayed cytotoxic potential only at day 1 extrac-
HA VC
HA VC 001
HA VC 005
HA CN
HA CN 001
HA CN 005
tion, but no cytotoxic potential was observed at day 2 and

longer extraction periods. HA 005 sample showed cytotoxic
potential at all extraction periods, but cell viability tended
to increase at longer extraction periods. The cell viability of
Figure 4: Total antibiotics content in the hydroxyapatite and the positive control sample remained low (less than 4%) at all
nanosilver phosphate-doped hydroxyapatite samples (error bars = extraction periods.
standard deviation, 𝑛 = 2). ∗ 𝑃 < 0.05.
4. Discussion
The inhibition zones of samples containing both gentamicin A combination of a three dimensional printing technique
and nanosilver phosphate were significantly greater than and low temperature phosphorization process was previously
those of samples containing nanosilver phosphate alone and shown to provide a simple mean to direct fabricate low
exhibited decreasing trends with extraction periods, whereas crystalline nanostructure hydroxyapatite which is close to
the inhibition zone size of nanosilver phosphate-doped those of bone [21, 22]. This manufacturing route was subse-
samples was relatively constant throughout the extraction quently developed to possess the antibacterial performance
periods. HA 001, HA VC 001 and HA CN 001 samples dis- by employing either antibiotics impregnation or nanosilver
played antibacterial duration for about 7 days while HA 005, compound incorporation [1, 2, 12, 23]. In this study, the
15
15
Inhibition zone (mm)

10
10
5 5
0 0
0 5 10 15 0 5 10 15
Days Days
HA VC HA 001 HA CN HA 001
HA VC 001 HA 005 HA CN 001 HA 005
HA VC 005 HA CN 005
(a) (b)
Figure 5: Antibacterial profile of VC (a) and CN (b) impregnated hydroxyapatites and nanosilver phosphate-doped hydroxyapatites against
P. aeruginosa (𝑛 = 2).
15 15
10 10
5 5
0 0
0 5 10 15 0 5 10 15
Days Days
HA VC HA 001 HA CN HA 001
HA VC 001 HA 005 HA CN 001 HA 005
HA VC 005 HA CN 005
(a) (b)
Figure 6: Antibacterial profile of VC (a) and CN (b) impregnated hydroxyapatites and nanosilver phosphate-doped hydroxyapatites against
S. aureus (𝑛 = 2).
impregnation of antibiotics in nanosilver phosphate-doped comprised the distribution of nanosized silver phosphate
hydroxyapatite was further developed to further provide the particles in the structure of nanosized hydroxyapatite crys-
improvement in antibacterial performance of hydroxyapatite. tals. After impregnation by antibiotics, difference in loading
From XRD analysis, the characteristic peaks of nanosil- efficiency was observed. This difference in drug loading effi-
ver phosphate-doped hydroxyapatite were found to be ciency for both antibiotics could be related to the microstruc-
broad and overlapped indicating the low crystallinity and ture of the samples as a result of the fabrication process. The
nanosized crystals of the materials similar to those of increase in degree of nanosilver phosphate incorporation in
bone mineral [24, 25]. Only silver phosphate peaks were the sample tended to decrease the adsorption sites for the
observed without any other metallic silver bands. SEM and drugs in the samples due to the greater number and size
TEM analysis showed that the fabricated 3DP nanosilver of formed silver phosphate particles which could obstruct
phosphate-doped hydroxyapatite was highly porous and the diffusion of antibiotics solution into the samples. In
180 increased from 7 days for HA CN to 14 days for HA CN 001.

160 Therefore, the synergic effect resulting from the combination
of gentamicin and nanosilver phosphate in HA CN 001 was
140
observed against S. aureus. The absence in enhancement of
Cell viability (%)
120 HA CN 005 was thought to be due to its low gentamicin

100 loading (Figure 4) which might be not sufficient to produce
80
an improvement when combined with nanosilver phosphate.
The antibacterial activity of nanosilver particles was generally
60 attributed to its small size that could attach or penetrate
40 the cell membrane of bacteria and disrupt the integrity of
20 bacterial membrane [27, 28]. The possible mechanism of
synergic antibacterial effect of nanosilver particles and antibi-
0
D1 D2 D3
otics combination was hypothesized to be due to the binding
Extraction periods
of drug molecules onto the nanosilver which increasing the
destruction and penetration into the cell wall of bacteria
HA HA VC 005 [16, 17]. However, in this study, nanosilver phosphate particles
HA 001 HA CN were incorporated into hydroxyapatite structure and could
HA 005 HA CN 001
HA VC
not attach and penetrate the cell membrane by themselves
HA CN 005
HA VC 001 but only release silver ions to produce the antibacterial
activity [27]. Therefore, the degree of bactericidal perfor-
Figure 7: Cell viability of antibiotics impregnated nanosilver mance of nanosilver phosphate-doped hydroxyapatite was
phosphate-doped hydroxyapatite samples using serial extraction expected to be different from that of nanosilver phosphate
technique (error bars = standard deviation, 𝑛 = 3). particles. In this case, silver ions which were released from
nanosilver phosphate-doped hydroxyapatite would prevent
the DNA of bacteria from unwinding [17]. This would
result in the difference in antibacterial activity enhancement
the case of vancomycin impregnated samples, the reincrease caused by antibiotics impregnated nanosilver phosphate-
in antibiotics content in HA VC 005 was noted. It was doped hydroxyapatite as prepared in this study and the direct
thought that this might be related to the greater gelling combination of nanoparticles and antibiotics as reported
tendency of vancomycin on the sample surface than that previously [13–17].
of gentamicin. However, further investigation is needed to Cytotoxic potential of antibiotics impregnated samples
clarify this observation. and nanosilver phosphate-doped samples alone could be
In term of antibacterial activity, no enhancement was related to the amount of released silver and type of antibi-
observed from the combination of nanosilver phosphate and otics. It was reported previously that vancomycin was less
vancomycin against P. aeruginosa. However, some improve- toxic to osteoblasts than gentamicin and required greater
ment in antibacterial duration for HA VC 005 (7 days) concentration to induce cytotoxicity [29, 30]. Therefore,
when testing against S. aureus compared to HA VC (6 HA VC was less cytotoxic than HA CN and HA 001 was
days) or HA 005 (5 days) was noted. The combination of less cytotoxic than HA 005. When the extraction periods
nanosilver phosphate and gentamicin seemed to give greater increased, lower concentrations of both antibiotics and silver
enhancement in antibacterial activity than vancomycin. This were released from the samples so the level of cytotoxic of the
is possible due to the greater spectrum of activity, lower samples diminished with increasing numbers of extraction
molar mass and lower minimum inhibitory concentration periods [31–33]. In the case of samples containing both
(MIC) of gentamicin [26]. MIC determination in Table 1 nanosilver phosphate and antibiotics, HA VC 005 samples
also confirmed that vancomycin has higher MIC values than showed cytotoxic potential even at day 3, whereas HA VC
both gentamicin and silver ions. In the case of P. aeruginosa, and HA VC 001 did not showed cytotoxic at all periods.
21% and 814% increase in inhibition zone size at day 1 of This could be related to the cytotoxic potential of silver ions
both gentamicin impregnated nanosilver phosphate-doped that was released from HA 005 samples which also showed
samples (9.5–9.87 mm) compared to HA CN (8 mm) and cytotoxic potential at all periods. In the case of HA VC 001,
nanosilver phosphate-doped samples alone (0.87–1.25 mm), the noncytotoxic potential was possibly due to the lower
respectively, was observed. However, no improvement in level of released silver ions and the non-cytotoxic potential of
antibacterial duration was observed. This improvement was vancomycin. In the case of gentamicin impregnated nanosil-
thought to be the additional effect since the increase in the ver phosphate-doped hydroxyapatite, HA CN 001 showed
inhibition zone size was resulted from the sum of those of the lower cell viability than HA CN and HA CN 005. One
HA CN and HA 001 or HA 005 samples. In the case of S. might expect that HA CN 005 would show the greatest
aureus, the enhancement in both initial inhibition zone size at cytotoxic potential since it contained greater amount of
day 1 and the antibacterial duration was seen for HA CN 001, silver phosphate similarly to that of HA VC 005 sample.
but not for HA CN 005. A 22% increase in inhibition Unlike vancomycin, gentamicin was observed to be cyto-
zone size at day 1 of HA CN 001 (11.25 mm) compared to toxic to osteoblasts. Thus, the content of gentamicin in the
HA CN (9.25 mm) was observed. The antibacterial duration samples that could be released and resulted in cytotoxic
potential should also be taken into account. It was found [5] A. Ślósarczyk, J. Szymura-Oleksiak, and B. Mycek, “The kinet-
that the gentamicin loading in HA CN 001 was similar to ics of pentoxifylline release from drug-loaded hydroxyapatite
that of HA CN while that of HA CN 005 was about twice implants,” Biomaterials, vol. 21, no. 12, pp. 1215–1221, 2000.
lower (Figure 4). Therefore, the greater cytotoxic potential of [6] F. Chai, J.-C. Hornez, N. Blanchemain, C. Neut, M. Descamps,
HA CN 001 was thought to be due to the combination of high and H. F. Hildebrand, “Antibacterial activation of hydroxya-
content of gentamicin and nanosilver phosphate compared patite (HA) with controlled porosity by different antibiotics,”
to the cytotoxic potential of gentamicin only in HA CN Biomolecular Engineering, vol. 24, no. 5, pp. 510–514, 2007.
and lower gentamicin content and higher silver phosphate [7] C. Marambio-Jones and E. M. V. Hoek, “A review of the antibac-
content in HA CN 005. terial effects of silver nanomaterials and potential implications
for human health and the environment,” Journal of Nanoparticle
5. Conclusion
[8] S. Sarkar, A. D. Jana, S. K. Samanta, and G. Mostafa, “Facile
The combined use of nanosilver phosphate and antibiotics synthesis of silver nano particles with highly efficient anti-
could enhance the antibacterial performance of the hydrox- microbial property,” Polyhedron, vol. 26, no. 15, pp. 4419–4426,
yapatite samples. Synergic enhancement was found for gen- 2007.
tamicin impregnated nanosilver phosphate-doped hydrox- [9] Y. Li, L. Li, J. Li et al., “Antibacterial properties of nanosilver
yapatite against S. aureus, but additive effect was observed PLLA fibrous membranes,” Journal of Nanomaterials, vol. 2009,
against P. aeruginosa. Limited enhancement was observed Article ID 168041, 5 pages, 2009.
for vancomycin impregnated nanosilver phosphate-doped [10] C.-T. Dinh, T.-D. Nguyen, F. Kleitz, and T.-O. Do, “Large-
hydroxyapatite against S. aureus, but no improvement was scale synthesis of uniform silver orthophosphate colloidal
seen against P. aeruginosa. These behaviors were related to the nanocrystals exhibiting high visible light photocatalytic activ-
antibiotics and nanosilver phosphate content in the samples, ity,” Chemical Communications, vol. 47, no. 27, pp. 7797–7799,
type of antibiotics used, and bacterial strains tested. 2011.
[11] J. J. Buckley, A. F. Lee, L. Olivi, and K. Wilson, “Hydroxyap-
atite supported antibacterial Ag3 PO4 nanoparticles,” Journal of
Conflict of Interests Materials Chemistry, vol. 20, no. 37, pp. 8056–8063, 2010.
The authors declare that there is no conflict of interests [12] J. Suwanprateeb, F. Thammarakcharoen, K. Wasoontararat, W.
Chokevivat, and P. Phanphiriya, “Preparation and character-
ization of nanosized silver phosphate loaded hydroxyapatite
by single step co-conversion process,” Materials Science and
Acknowledgments Engineering C, vol. 32, pp. 2122–2128, 2012.
[13] K. I. Wolska, K. Grzes, and A. Kurek, “Synergy between novel
Lafarge Prestia Co., Ltd. is thanked for the supply of cal- antimicrobials and conventional antibiotics or bacteriocins,”
cium sulfate and Thaiwah Co., Ltd, Thailand, for the sup- Polish Journal of Microbiology, vol. 61, pp. 95–104, 2012.
ply of pregelatinized starch. Cluster and Program Manage- [14] S. Ruden, K. Hilpert, M. Berditsch, P. Wadhwani, and A.
ment Office, National Science and Technology Development S. Ulrich, “Synergistic interaction between silver nanoparti-
Agency is acknowledged for financial support. W. Chokevivat cles and membrane-permeabilizing antimicrobial peptides,”
(MTEC) was thanked for cytotoxic potential study. Antimicrobial Agents and Chemotherapy, vol. 53, no. 8, pp. 3538–
3540, 2009.
References [15] G. Geoprincy, P. Saravanan, N. Nagendra Gandhi, and S.

Renganathan, “A novel approach for studying the combined
[1] F. Thammarakcharoen and J. Suwanprateeb, “The effect of bead antimicrobial effects of silver nanoparticles and antibiotics
size on drug loading and releasing characteristic of antibiotic through agar over layer method and disk diffusion method,”
loaded 3DP calcium phosphate bead,” in Proceedings of the Pure Digest Journal of Nanomaterials and Biostructures, vol. 6, no. 4,
and Applied Chemistry International Conference, pp. 473–476, pp. 1557–1565, 2011.
2011. [16] A. M. Fayaz, K. Balaji, M. Girilal, R. Yadav, P. T. Kalaichelvan,
[2] P. Phanphiriya, F. Thammarakcharoen, W. Chokevivat, and and R. Venketesan, “Biogenic synthesis of silver nanoparticles
J. Suwanprateeb, “Antimicrobial performance and cytotoxicity and their synergistic effect with antibiotics: a study against
of antibiotics impregnated hydroxyapatite for osteomyelitis gram-positive and gram-negative bacteria,” Nanomedicine:
treatment,” Advanced Materials Research, vol. 506, pp. 513–516, Nanotechnology, Biology, and Medicine, vol. 6, no. 1, pp. e103–
2012. e109, 2010.
[3] Y. Shinto, A. Uchida, F. Korkusuz, N. Araki, and K. Ono, [17] P. Li, J. Li, C. Wu, Q. Wu, and J. Li, “Synergistic antibacterial
“Calcium hydroxyapatite ceramic used as a delivery system for effects of 𝛽-lactam antibiotic combined with silver nanoparti-
antibiotics,” Journal of Bone and Joint Surgery B, vol. 74, no. 4, cles,” Nanotechnology, vol. 16, no. 9, pp. 1912–1917, 2005.
pp. 600–604, 1992. [18] P. J. Carek, L. M. Dickerson, and J. L. Sack, “Diagnosis and
[4] Y. Yamashita, A. Uchida, T. Yamakawa, Y. Shinto, N. Araki, management of osteomyelitis,” American Family Physician, vol.
and K. Kato, “Treatment of chronic osteomyelitis using calcium 63, no. 12, pp. 2413–2420, 2001.
hydroxyapatite ceramic implants impregnated with antibiotic,” [19] P. D. P. Lew and P. F. A. Waldvogel, “Osteomyelitis,” The Lancet,
International Orthopaedics, vol. 22, no. 4, pp. 247–251, 1998. vol. 364, no. 9431, pp. 369–379, 2004.
[20] O. S. Kluin, H. C. van der Mei, H. J. Busscher, and D. Neut,

“A surface-eroding antibiotic delivery system based on poly-
(trimethylene carbonate),” Biomaterials, vol. 30, no. 27, pp.
4738–4742, 2009.
[21] J. Suwanprateeb, W. Suvannapruk, and K. Wasoontararat, “Low
temperature preparation of calcium phosphate structure via
phosphorization of 3D-printed calcium sulfate hemihydrate
based material,” Journal of Materials Science: Materials in
Medicine, vol. 21, no. 2, pp. 419–429, 2010.
[22] J. Suwanprateeb, F. Thammarakcharoen, K. Wasoontararat, and
W. Suvannapruk, “Influence of printing parameters on the
transformation efficiency of 3D-printed plaster of paris to
hydroxyapatite and its properties,” Rapid Prototyping Journal,
vol. 18, pp. 490–499, 2012.
[23] J. Suwanprateeb, F. Thammarakcharoen, K. Wasoontararat, W.
Chokevivat, and P. Phanphiriya, “Single step preparation of
nanosilver loaded calcium phosphate by low temperature co-
conversion process,” Journal of Materials Science: Materials in
Medicine, vol. 23, pp. 2091–2100, 2012.
[24] R.-J. Chung, “Study of hydroxyapatite nano composites with
photoluminescence properties,” Biomedical Engineering, vol. 23,
no. 2, pp. 107–112, 2011.
[25] F. Peters, K. Schwarz, and M. Epple, “The structure of bone
studied with synchrotron X-ray diffraction, X-ray absorption
spectroscopy and thermal analysis,” Thermochimica Acta, vol.
361, no. 1-2, pp. 131–138, 2000.
[26] S. Alvarez, M. Jones, and S. L. Berk, “In vitro activity of
fosfomycin, alone and in combination, against methicillin-
resistant Staphylococcus aureus,” Antimicrobial Agents and
Chemotherapy, vol. 28, no. 5, pp. 689–690, 1985.
[27] J. R. Morones, J. L. Elechiguerra, A. Camacho et al., “The
bactericidal effect of silver nanoparticles,” Nanotechnology, vol.
16, no. 10, pp. 2346–2353, 2005.
[28] M. J. Hajipour, K. M. Fromm, A. A. Ashkarran et al., “Antibac-
terial properties of nanoparticles,” Trends in Biotechnology, vol.
30, pp. 499–511, 2012.
[29] V. Antoci Jr., C. S. Adams, N. J. Hickok, I. M. Shapiro, and J.
Parvizi, “Antibiotics for local delivery systems cause skeletal cell
toxicity in vitro,” Clinical Orthopaedics and Related Research, no.
462, pp. 200–206, 2007.
[30] C. R. Rathbone, J. D. Cross, K. V. Brown, C. K. Murray, and J.
C. Wenke, “Effect of various concentrations of antibiotics on
osteogenic cell viability and activity,” Journal of Orthopaedic
[31] W. Chen, Y. Liu, H. S. Courtney et al., “In vitro anti-bacterial
and biological properties of magnetron co-sputtered silver-
containing hydroxyapatite coating,” Biomaterials, vol. 27, no. 32,
pp. 5512–5517, 2006.
[32] B. Singh, A. K. Dubey, S. Kumar, N. Saha, B. Basu, and R.
Gupta, “In vitro biocompatibility and antimicrobial activity of
wet chemically prepared Ca10−𝑥 Ag𝑥 (PO4 )6 (OH)2 (0.0 ≤ x ≤ 0.5)
hydroxyapatites,” Materials Science and Engineering C, vol. 31,
no. 7, pp. 1320–1329, 2011.
[33] G. Gosheger, J. Hardes, H. Ahrens et al., “Silver-coated megaen-
doprostheses in a rabbit model—an analysis of the infection rate
and toxicological side effects,” Biomaterials, vol. 25, no. 24, pp.
5547–5556, 2004.
http://dx.doi.org/10.1155/2013/587021
Research Article
Iron Oxide Magnetic Nanoparticles: Characterization and
Toxicity Evaluation by In Vitro and In Vivo Assays
Alina Mihaela Prodan,1,2 Simona Liliana Iconaru,3 Carmen Steluta Ciobanu,3

Mariana Carmen Chifiriuc,4 Mihai Stoicea,2 and Daniela Predoi3
1
Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari, Sector 5, 050474 Bucharest, Romania
2
Emergency Hospital Floreasca, Bucharest 5, Calea Floreasca Nr 8, Sector 1, 014461 Bucarest, Romania
3
National Institute of Materials Physics, 105 bis Atomistilor, P.O. Box MG 07, 077125 Bucuresti-Magurele, Romania
4
Microbiology Department, Faculty of Biology, University of Bucharest, Aleea Portocalelor 1-3, 60101 Bucharest, Romania
Correspondence should be addressed to Daniela Predoi; dpredoi@gmail.com
Received 21 August 2013; Revised 4 October 2013; Accepted 5 October 2013
Copyright © 2013 Alina Mihaela Prodan et al. This is an open access article distributed under the Creative Commons Attribution
cited.
The aim of this study was to evaluate the biological properties of iron oxide nanoparticles (IO-NPs) obtained in the aqueous
suspension. The iron oxide nanoparticles were characterized by scanning electron microscopy (SEM) and transmission electron
microscopy (TEM). The biocompatibility of the iron oxide was demonstrated by the in vitro quantification of HeLa cells viability
using propidium iodide (PI) and fluorescein diacetate (FdA) and the MTT colorimetric assay. The toxicity of small size iron
oxide nanoparticles was also evaluated by means of histological examination on male Brown Norway rats after intraperitoneal
injection. At the tested concentrations, the nanoparticles proved to be not cytotoxic on HeLa cells. The rat’s behavior, as well as
the histopathological aspect of liver, kidney, lung, and spleen tissues at 48 h after intraperitoneal injection did not present any
modifications. The in vivo and in vitro assays suggested that the IO-NPs could be further used for developing new in vivo medical
applications.
1. Introduction the use of these nanomaterials in biomedical applications, for

example, as contrast agents for magnetic resonance imaging
Nowadays, finding new approaches for solving pressing (MRI) [6, 7], tissue-specific release of therapeutic agents,
problems in the field of medical science is the focus of targeted drug delivery in tumor therapy [8], hyperthermia,
research institutes everywhere. The most studied materials cell labeling [9], magnetic cell sorting [10], and magnetic field
with promising potential in the field of biomedical applica- assisted radionuclide therapy [11].
tions are those with magnetic properties. Magnetic materials,
especially iron oxides nanoparticles, are known since ancient In the past few years, superparamagnetic iron oxide
times to have many spectacular properties, but in the last nanoparticles with controlled and enhanced surface chem-
decade the properties that they possess at nanometric scale istry properties have been used successfully as contrast
have been the starting point of great potential applications agents for magnetic resonance imaging in vivo [12, 13]. The
such as drug delivery, magnetic cell separation, tumor label- new direction of research aims to develop new compounds
ing and cell labeling. The most common forms of iron oxides, based on iron oxide nanoparticles for in vivo biomedical
magnetite and maghemite (Fe3 O4, 𝛾-Fe2 O3 ), are studied due applications. Recent studies in the field of malignant tumors
to the outstanding properties they exhibit at nanometric scale are focused on developing a new drug delivery systems based
(high specific surface area, superparamagnetism, etc.) [1–5]. on iron oxide nanoparticles in order to avoid damaging
The nanometric dimensions of these materials makes them the healthy cells around the tumor mass in the process of
ideal candidates for surface engineering and functionaliza- cancerous cell destruction. These types of nanosystems based
tion. Surface enhancement and functionalization facilitate on iron oxide nanoparticles have the ability to heat up,
delivering toxic amounts of thermal energy to tumors, or as from EDAX. The operating conditions were an accelerating
chemotherapy and radiotherapy enhancement agents, where voltage between 2 and 25 keV (depending of the signal/noise
a controlled degree of tissue warming leads to an effective cell ratio) for samples tilted at 25∘ in order to get the optimal
destruction [14, 15]. take off angle (30∘ ) allowing a dead time around 20–30%
In agreement with Pisanic II et al. [16], magnetic nanopar- and a collecting time of 90–120 s. Transmission electron
ticles could be used as tools in a wide variety of biomedical microscopy (TEM) images for these samples were recorded
applications. On the other hand, Pisanic II et al. showed using a FEI Tecnai 12 equipped with a low dose digital camera
that failure to fully and properly evaluate nanostructures from Gatan. The specimen for TEM imaging was prepared
on an individual case-by-case basis may lead to lack of by ultramicrotomy in order to obtain a thin section of about
parameter control in in vitro experiments, as well as incorrect 60 nm. The powder was embedded in an epoxy resin (polaron
assumptions concerning their biocompatibility and biosafety 612) before microtomy.
of their in vivo use [16]. In order to improve the knowledge
on cytotoxicity of iron oxide nanoparticles, we performed an 2.4. Cytotoxicity Assay. Quantification of cell viability was
in vivo toxicity study (48 h) by administration by intraperi- performed using propidium iodide (PI) and fluorescein diac-
toneal injection of 𝛾-Fe2 O3 dispersion at concentrations of etate (FdA). Briefly, 5×104 HeLa cells were seeded in each well
0.7 mL/kg, 1.7 mL/kg, and 3.7 mL/kg. of a 24-well plate and after 24 h, the monolayers were treated
The aim of this study was to develop iron oxide nanopar- with a suspension of 𝛾-Fe2 O3 (200 𝜇L) nanoparticles diluted
ticles by an adapted coprecipitation method [17–22] with 100 times. The effects on cellular viability were evaluated
controllable parameters and enhanced biocompatible prop- after 48 h by adding 100 𝜇L PI (0.1 mg/mL) and 100 𝜇L FdA
erties for in vivo applications. Scanning electron microscopy (0.1 mg/mL) and fluorescence studies have been performed
(SEM) and transmission electron microscopy (TEM) studies using Observer D1 Carl Zeiss microscope. The cell viability
have been conducted to obtain information about the size, was established by the ratio between viable (green) and dead
structure, and morphology of IO-NPs. The biocompatibility cells (red) counted on several microscopic fields [24].
of the iron oxide was evaluated using in vitro and in vivo The cell viability was determined by MTT colorimetric
assays, consisting in the quantification of HeLa cells viability assay developed by Mosmann for in vitro cytotoxicity and
and the histological evaluation of the nanoparticles effects on cell proliferation measurements [25]. It was reported that
the male Brown Norway rat’s tissues. the mitochondrial enzyme succinate-dehydrogenase within
viable cells is able to cleave the MTT salt into formazan, a blue
2. Experimental Section colored product. The amount of formazan produced, read
on scanning multiwell spectrophotometer, is proportional to
2.1. Materials. Ferrous chloride tetrahydrate (FeCl2 ⋅4H2 O), the number of viable cells present [25–27]. The cells were
ferric chloride hexahydrate (FeCl3 ⋅6H2 O), natrium hydrox- cultured in the medium (2.5 × 105 cells/mL) containing iron
ide (NaOH), and chlorhidric acid (HCl) were purchased oxide nanoparticles for 12, 24, and 72 hours periods. Culture
from Merck. Deionized water was used in the synthesis of medium without iron oxide nanoparticles served as control
nanoparticles and for rinsing the clusters. in each experiment. The different final concentrations of
the suspension of iron oxide nanoparticles were prepared
2.2. Synthesis of Iron Oxide Ferrofluid. Iron oxide nanopar- in cell growth medium. Concentration ranges were 10, 20,
ticles were prepared by coprecipitation [17–22]. Ferrous and 30 𝜇g/mL. The medium from each well was removed
chloride tetrahydrate (FeCl2 ⋅4H2 O) in 2 M HCl and ferric by aspiration, the cells were washed with 200 𝜇L phosphate
chloride hexahydrate (FeCl3 ⋅6H2 O) were mixed at 100∘ C buffer saline solution (PBS)/well, and then 50 𝜇L of 1 mg/mL
MTT solution was added on each well. After 2 h of incu-
(Fe2+ /Fe3+ = 1/2). The mixture was dropped into 200 mL
bation, the MTT solution from each well was removed by
of NaOH (2 mol⋅L−1 ) solution under vigorous stirring for
aspiration. A volume of 50 𝜇L isopropanol was added and
about 30 min. The precipitate of magnetite (black precipitate
the plate was shaken to dissolve formazan crystals. The
immediately formed) was converted into 𝛾-Fe2 O3 particles
optical density at 595 nm, for each well, was then determined
by repeated treatment with HNO3 (2 mol⋅L−1 ) and FeNO3 using a Tecan multiplate reader (Tecan GENios, Grödic,
(0.3 mol⋅L−1 ) solutions [23]. The acidic precipitate was iso- Germany). The percent of viable cells cultured on the iron
lated by decantation on a magnet, separated by centrifuga- oxide nanoparticles was calculated in comparison with a
tion (6000 rpm), then washed in acetone, and dispersed in control sample; the cells cultured on uncoated culture plastic
deionized water at pH = 2.5. The final ion concentration vessels, being considered to have a viability of 100%.
was 0.38 mol⋅L−1 . For biological investigations, the pH was
adjusted to 7 using aqueous ammonia. The iron content of 2.5. Animals. Male Brown Norway rats (weighing ∼300 ±
the suspensions was determined by redox-titration [23]. 10 g) were purchased from the National Institute of Research
and Development for Microbiology and Immunology “Can-
2.3. Characterization of Nanoparticles. The morphology of tacuzino,” Bucharest. The rats were housed in an environment
the obtained material was studied using a Quanta Inspect controlled for temperature (22 ± 2∘ C), light (12 h light/dark
F scanning electron microscope (SEM), operating at 25 kV cycles), and humidity (60 ± 10%). The animals were main-
in vacuum. The elemental local analysis was performed tained under specific pathogen free-conditions in accordance
using an energy dispersive spectroscopy (EDS) detector with NIH Guide for the Care and Use of laboratory Animals.
established that the optimum size of magnetic nanoparticles

to promote an effective biodistribution is ranging from
10 to 100 nm [32]. From this point of view, the obtained
nanoparticles meet this criterium, with a diameter of 10 nm.
The results have shown that the obtained nanoparticles were
not cytotoxic on the HeLa cells after 48 h exposure to a
suspension of 𝛾-Fe2 O3 (200 𝜇L) nanoparticles diluted 100
times (Figure 3(b)), as revealed by the absence of dead,
red cells stained with propidium iodide (Figure 3). The low
cytotoxicity of iron nanoparticles has been reported also by
other authors and has been explained by the fact that the
nanoparticles are not degraded within the timescale of the
cellular assay (48 h) [33].
To examine the cytotoxicity of the iron oxide nanopar-
ticles, the MTT assay was used. The HeLa cells were treated
Figure 1: Scanning electron microscopy image of the synthesized
iron oxide sample. on/in a medium containing different concentrations (10, 20,
and 30 𝜇g/mL) of the suspension of iron oxide nanoparticles.
Cell viability was determined at 12 h, 24 h, and 72 h after
treatment and the test results are shown in Figure 4. We can
2.6. Histological Examination. For the analysis of iron oxide see that the cell viability decreased when the concentration
toxicity in vivo, the rats (𝑛 = 4 per group) were treated with and time period increased. These results are in agreement
normal saline and iron oxide (at concentrations of 0.7 mL/kg, with previous studies presented by Kouchesfehani et al.
1.7 mL/kg, and 3.7 mL/kg) via intraperitoneal injection. The [34]. The toxic effect was taken into consideration when the
final ion concentration in iron oxide solution prepared survival rate was below 80%. The graph shows the mean +/−
by coprecipitations was 0.38 mol⋅L−1 . For histopathological s.d. of normalized values on three independent experiments.
examinations, selected organs (liver, kidney, lung and spleen) The in vivo toxicity study (48 h) was performed with 𝛾-
were removed from the rats and fixed in 10% formalin. Fe2 O3 dispersion administered by intraperitoneal injection at
The organs were prepared as paraffin-embedded glass slides concentrations of 0.7 mL/kg, 1.7 mL/kg, and 3.7 mL/kg. The
stained with hematoxylin and eosin. The morphological rats were observed after 48 h from each administration and
changes were observed by microscopic examination [28]. their behavior was evaluated. All animals survived the admin-
istration of 𝛾-Fe2 O3 on all tested concentrations and did not
3. Results and Discussion show any sign of discomfort (lethargy, nausea, vomiting or
diarrhea) during the whole duration of the experiment. The
SEM analysis was used to confirm the morphology of the histopathological assessment of the selected tissues including
synthesized iron oxide sample (Figure 1). The obtained results liver, kidney, lung, and spleen was conducted.
using scanning electron microscopy analysis clearly show At 48 h after the intraperitoneal injection no significant
that the IO-NPs have spherical shape. Detailed structural macroscopic histopathological changes were observed in the
information and the growth direction of the maghemite, 𝛾- case of liver and kidney for all tested concentrations in the
Fe2 O3 , were obtained from TEM and HRTEM micrographs. treated group compared with the control.
Figure 2(a) shows TEM picture of iron oxide nanopar- In Figure 5, the microscopic observations of the rat liver
ticles (IO-NPs), clearly showing that the product is entirely injected with different 𝛾-Fe2 O3 concentrations after 48 h
composed of crystals with a relatively uniform, spherical are shown. The microscopic observations of the rat kidney
morphology. Grain size distribution was determined by injected with different 𝛾-Fe2 O3 concentrations after 48 h are
measuring the mean diameter, 𝐷, of about 500 particles presented in Figure 6.
on the micrographs (Figure 2(b)). TEM images indicate a Pathological sections of liver after injection with a
very uniform size distribution of iron oxide nanoparticles. 0.7 mL/kg dose of iron oxide nanoparticles (Figure 5(b))
The average grain size of the monodisperse nanoparticles show that the architecture of the liver was not affected by IO-
is 10 ± 0.3 nm. Figure 2(c) shows the selected area electron NPs (0.7 mL/kg). Hepatocytes with discreet anisokaryosis,
diffraction (SAED) pattern recorded from an area containing formation of chromocenters and nucleoli, and focal intra-
a large number of nanoparticles and the high-resolution TEM hepatocyte cholestasis (HE, 600x) were found in the liver
picture. The rings in the SAED pattern can be indexed as the of both the IO-NPs (0.7 mL/kg) treated and control groups
(220), (311), (400), (422), (511), and (440) reflections of the (Figure 5(a)) with no significant difference between them.
cubic maghemite in agreement with the XRD results [29]. Greaves in histopathology of preclinical toxicity studies [35]
Despite the great potential of iron nanoparticles to be showed that laboratory animals under conventional housing
used for different industrial and medical applications, data can undergo liver changes. On the other hand, he showed
about their toxicity are still scarce [30]. Bearing in mind that granulomas are common spontaneous lesions in the liver.
that in vitro tests represent a first step of biomedical appli- The liver changes may occur due to multiple causes such as
cation investigation [31], we have studied the toxicity of the drugs, bacterial, fungal, parasitic or viral infections, and liver
obtained nanoparticles on HeLa cells. It has been previously or systemic disorders and are usually asymptomatic [36].
100 nm 18
15
12
Frequency (%)
9
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Diameter (nm)
(a) (b)
2 mm
(c)
Figure 2: Bright field TEM picture showing a homogeneous distribution of iron oxide nanoparticles (a), size distribution of IO-NPs (b), and
SAED pattern from a region including a large number of nanoparticles (c).
(a) (b)
Figure 3: Inverted microscope image of HeLa cells after 48 h exposure to a suspension of 𝛾-Fe2 O3 (200 𝜇L) nanoparticles diluted 100 times
(b). Control cells cultured in free medium were run in parallel to the treated groups (a) (×200).
100
90
Viability (%)
80
70
60
50
0 10 20 30 40 50 60 70 80
Incubation time (h)
Control 20 𝜇g/mL
10 𝜇g/mL 30 𝜇g/mL
Figure 4: Effect of different concentrations of iron oxide nanoparticles on HeLa cells viability.
(a) (b)
(c) (d)
Figure 5: Light optical image of the liver after 48 h exposure to 𝛾-Fe2 O3 nanoparticles at concentrations of 0.7 mL/kg (b), 1.7 mL/kg (c), and
3.7 mL/kg (d). The reference sample is also presented (a).
The liver examination after the injection of 1.7 mL/kg injection with 1.7 mL/kg dose of iron oxide nanoparticles
dose of iron oxide nanoparticles (Figure 5(c)) indicates hep- (Figure 5(c)). After injection with 3.7 mL/kg dose of iron
atocytes with moderate anisokaryosis, formation of chro- oxide, nanoparticles (Figure 5(d)) were noticed hepatocytes
mocenters and nucleoli, and moderate granular cytoplasmic with moderate anisokaryosis, formation of chromocenters
degeneration. The microgranular brown pigment deposits in and nucleoli. The granulovacuolar cytoplasmic degenera-
Kupffer cells (HE, 600x) were also observed in the liver after tion and microgranular brown pigment deposits in Kupffer
(a) (b)
(c) (d)
Figure 6: Light optical image of the kidney after 48 h exposure to 𝛾-Fe2 O3 nanoparticles at concentrations of 0.7 mL/kg (b), 1.7 mL/kg (c),
and 3.7 mL/kg (d). The reference sample is also presented (a).
cells and hepatocytes (HE, 600x) were also distinguished On the other hand, the microscopic observations of
(Figure 5(d)). the rat lung and spleen injected with different 𝛾-Fe2 O3
The pathological micrographs of kidneys in rats after concentrations after 48 h are presented in Figures 7 and 8.
injection with 0.7 mL/kg dose of iron oxide nanoparticles In Figure 7, the pathological micrographs of lungs in
(Figure 6(b)) show that the kidney has preserved the architec- rats after the injection with doses containing 0.7 mL/kg,
ture of the control specimen (Figure 6(a)) with no significant 1.7 mL/kg, and 3.7 mL/kg of iron oxide nanoparticles and
differences. The tubular cells with moderate anisokaryosis the pathological micrographs of the control specimen
and anisochromia with formation of chromocenters and (Figure 7(a)) are presented. After injection with 0.7 mL/kg
minimal granular cytoplasmic degeneration (HE, 600x) are (Figure 6(b)) dose of iron oxide nanoparticles, the lung
also presented in Figure 6(b). parenchyma of the rats shows preserved alveolar architec-
The specimen injected with a solution of 1.7 mL/kg iron ture with rare macrophages in the alveolar septa, discreet
oxide (Figure 6(c)) preserves the architecture, tubular cells anisokaryosis, and anisochromia of type II pneumocytes with
with moderate anisokaryosis, and anisochromia with for- rare nucleoli. The focal ectatic capillaries in the alveolar
mation of chromocenters, moderate granulovacuolar cyto- septa are also presented. We can see that the pathological
plasmic degeneration with focal clear cells, and moderate micrographs of lung in rats after injection with 0.7 mL/kg
vascular congestion (HE, 400x). For the specimen injected dose of iron oxide nanoparticles (Figure 7(b)) show that the
with a solution containing 3.7 mL/kg iron oxide (Figure 6(d)) lung has preserved the architecture of the control specimen
were observed tubular cells with pronounced architectural (Figure 7(a)) with no significant differences. For the speci-
distortions, enlarged nuclei with irregular contours, for- men injected with a solution containing 1.7 mL/kg iron oxide
mation of prominent nucleoli, marked granular cytoplas- (Figure 7(c)), we observed that the lung parenchyma shows
mic degeneration and discreet deposition of microgranular preserved alveolar architecture with rare macrophages in the
brown pigment in the renal interstitium (HE, 600x). alveolar septa, discreet anisokaryosis, and anisochromia of
(a) (b)
(c) (d)
Figure 7: Light optical image of the lung after 48 h exposure to 𝛾-Fe2 O3 nanoparticles at concentrations of 0.7 mL/kg (b), 1.7 mL/kg (c), and
3.7 mL/kg (d). The reference sample is also presented (a).
type II pneumocytes, with rare chromocenters and nucleoli. In the present study, we have established that the tested
The focal ectatic capillaries in the alveolar septa are also IO-NPs did not induce any morphological alterations such as
presented. Lung parenchyma of the specimen injected with an increase of granulomas or tissue damage to the liver and
a solution of 3.7 mL/kg iron oxide (Figure 7(d)) shows pre- kidneys. The lack of morphological modifications to the liver
served alveolar architecture with rare macrophages in the and kidneys could be explained by the low amount of IO-
alveolar septa, discreet anisokaryosis, and anisochromia of NPs. For low concentrations of IO-NPs, the histopathological
type II pneumocytes, with rare chromocenters and nucleoli. investigations performed after injection showed that the
In the lung parenchyma it is also observed focal ectatic architecture of the liver and kidneys was not affected and
capillaries in the alveolar septa. no significant differences between the control groups and
Pathological sections of spleen after injection with a injected groups were observed. These results are in agreement
0.7 mL/kg and 1.7 mL/kg dose of iron oxide nanoparticles with previous studies conducted by Wang et al. [32–37]
(Figures 8(b)-8(c)) show that the architecture of the spleen which ascertained that the toxicity apparently depends on the
was not affected by IO-NPs compared with the architecture type of nanoparticules and their concentration. Furthermore,
of the control specimen (Figure 8(a)). After injection with Wang et al. showed that some metal nanoparticles as well
0.7 and 1.7 mL/kg dose of iron oxide nanoparticles, there as Zn nanoparticles are highly toxic in acute assessments.
were noticed splenic red pulp with discreet nuclear contour Previous studies realized by Dekkers et al. [38] showed that
irregularities, discreet anisochromia with focal chromocenter metal oxide nanoparticles, like some forms of silica (SiO2 ),
formation, and rare nucleoli. After injection with a 3.7 mL/kg induce toxicity after subacute assessments.
dose of iron oxide nanoparticles (Figure 8(d)), we observed Hillyer and Albrecht, in gastrointestinal persorption and
the splenic red pulp with increased number of monocytes, tissue distribution of differently sized colloidal gold nanopar-
with nuclear contour irregularities. The discreet anisochro- ticles studies [39], show that the acute and subacute in vivo
mia with focal chromocenter formation were also remarked intraperitoneal administration studies are very important,
in the splenic pulp after injection with a 3.7 mL/kg dose of allowing us to find the potential toxicological effects that iron
iron oxide nanoparticles. oxide nanoparticles may have in key organs (gastrointestinal
(a) (b)
(c) (d)
Figure 8: Light optical image of the spleen after 48 h exposure to 𝛾-Fe2 O3 nanoparticles at concentrations of 0.7 mL/kg (b), 1.7 mL/kg (c),
and 3.7 mL/kg (d). The reference sample is also presented (a).
tract, liver, kidneys, and spleen) and the cardiovascular proved to be not cytotoxic on HeLa cells and did not modify
system. On the other hand, Kim et al. [40] showed that the rat’s behavior or the histopathological aspect of liver,
the study of the possibility of iron oxide nanoparticles to kidney, lung, and spleen tissues. Intraperitoneal injection
cross the intestinal barrier as well as their effects in blood of 𝛾-Fe2 O3 nanoparticles at several concentrations showed
serum, and the possible alteration to urinary parameters a normal macroscopic histopathological behavior of liver,
(potassium, sodium, and osmolality) is very important in kidney, lung, and spleen after 48 h for each concentration in
order to understand the toxicity effects of these particles. the treated group compared with the control. Therefore, the
Understanding the potential risks associated with exposure preserved architecture of the control or slightly pathological
to iron oxide nanoparticles used for a great variety of changes of liver, kidney, lung, and spleen joint were induced
medical applications is crucial. It is very important to design by the low-dose of IO-NPs. The results of the present study
functionalize iron oxide nanoparticles that can be effectively suggested that the Fe2 O3 nanoparticles could be used for
internalized and which can meet the demands of a particular future therapeutic alternative treatment strategies.
application without compromising on cellular toxicity.
Acknowledgments
4. Conclusions The financial and encouragement support provided by the
Ministry of Educations of the Romania, Project no. C2-06
The toxicity of the uniform, spherical obtained nanoparticles under program CEA-IFA, POSDRU/107/1.5/S/76813 (DocIn-
with 10 ± 0.3 nm in size has been investigated by in vitro and vest), and by the scholarship of the French government via
in vivo assays. At the tested concentrations, the nanoparticles the cultural section of the French Embassy.
References [17] R. Massart, “Magnetic fluids and process for obtaining them,”
US Patent 4329241, 1982.
[1] S. Laurent, D. Forge, M. Port et al., “Magnetic iron oxide nano- [18] R. Massart, “Preparation of aqueous magnetic liquids in alkaline
particles: synthesis, stabilization, vectorization, physic chem- and acidic media,” IEEE Transactions on Magnetics, vol. 17, pp.
ical characterizations and biological applications,” Chemical 1247–1248, 1981.
Reviews, vol. 108, no. 6, pp. 2064–2110, 2008.
[19] R. Massart, J. Roger, and V. Cabuil, “New trends in chemistry
[2] S. Mornet, S. Vasseur, F. Grasset, and E. Duguet, “Magnetic of magnetic colloids: polar and non polar magnetic fluids,
nanoparticle design for medical diagnosis and therapy,” Journal emulsions, capsules and vesicles,” Brazilian Journal of Physics,
of Materials Chemistry, vol. 14, no. 14, pp. 2161–2175, 2004. vol. 25, no. 2, pp. 135–141, 1995.
[3] A. M. Prodan, S. L. Iconaru, C. M. Chifiriuc et al., “Magnetic [20] D. Predoi and C. Valsangiacom, “Thermal studies of magnetic
properties and biological activity evaluation of iron oxide spinel iron oxide in solution,” Journal of Optoelectronics and
nanoparticles,” Journal of Nanomaterials, vol. 2013, Article ID Advanced Materials, vol. 9, no. 6, pp. 1797–1799, 2007.
893970, 7 pages, 2013. [21] D. Zins, V. Cabuil, and R. Massart, “New aqueous magnetic
[4] E. Katz and I. Willner, “Integrated nanoparticle-biomolecule fluids,” Journal of Molecular Liquids, vol. 83, no. 1–3, pp. 217–232,
hybrid systems: synthesis, properties, and applications,” Ange- 1999.
wandte Chemie, vol. 43, no. 45, pp. 6042–6108, 2004. [22] D. Predoi, “A study on iron oxide nanoparticles coated with
[5] S. Laurent, S. Dutz, U. O. Häfeli, and M. Mahmoudi, “Magnetic dextrin obtained by coprecipitation,” Digest Journal of Nanoma-
fluid hyperthermia: focus on superparamagnetic iron oxide terials and Biostructures, vol. 2, no. 1, pp. 169–173, 2007.
nanoparticles,” Advances in Colloid and Interface Science, vol. [23] S. Mornet, F. Grasset, J. Portier, and E. Duguet, “Maghemite@
166, no. 1-2, pp. 8–23, 2011. silica nanoparticles for biological applications,” European Cells
[6] C. H. Cunningham, T. Arai, P. C. Yang, M. V. McConnell, J. M. and Materials, vol. 3S2, article 110, 2002.
Pauly, and S. M. Conolly, “Positive contrast magnetic resonance [24] A. M. Grumezescu, E. Andronescu, A. Ficai, C. Bleotu, and M.
imaging of cells labeled with magnetic nanoparticles,” Magnetic C. Chifiriuc, “Chitin based biomaterial for antimicrobial ther-
Resonance in Medicine, vol. 53, no. 5, pp. 999–1005, 2005. apy: fabrication, characterzation and in vitro profile based
[7] S. A. Anderson, R. K. Rader, W. F. Westlin et al., “Mag- intercation with eukaryotic and prokaeryotic cells,” Biointerface
netic resonance contrast enhancement of neovasculature with Research in Applied Chemistry, vol. 2, p. 446, 2012.
alpha(v)beta(3)-targeted nanoparticles,” Magnetic Resonance in [25] T. Mosmann, “Rapid colorimetric assay for cellular growth and
Medicine, vol. 44, no. 3, pp. 433–439, 2000. survival: application to proliferation and cytotoxicity assays,”
[8] B. Polyak and G. Friedman, “Magnetic targeting for site-specific Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55–63,
drug delivery: applications and clinical potential,” Expert Opin- 1983.
ion on Drug Delivery, vol. 6, no. 1, pp. 53–70, 2009. [26] F. Denizot and R. Lang, “Rapid colorimetric assay for cell gro-
[9] R. Weissleder, H.-C. Cheng, A. Bogdanova, and A. Bogdanov wth and survival: modifications to the tetrazolium dye pro-
Jr., “Magnetically labeled cells can be detected by MR imaging,” cedure giving improved sensitivity and reliability,” Journal of
Journal of Magnetic Resonance Imaging, vol. 7, no. 1, pp. 258–263, Immunological Methods, vol. 89, no. 2, pp. 271–277, 1986.
1997. [27] H. Wan, R. L. Williams, P. J. Doherty, and D. F. Williams, “The
cytotoxicity evaluation of Kevlar and silicon carbide by MTT
[10] E. A. Schellenberger, F. Reynolds, R. Weissleder, and L. Joseph-
assay,” Journal of Materials Science, vol. 5, no. 6-7, pp. 441–445,
son, “Surface-functionalized nanoparticle library yields probes
1994.
for apoptotic cells,” ChemBioChem, vol. 5, no. 3, pp. 275–279,
2004. [28] B. Su, S. L. Xiang, J. Su et al., “Diallyl disulfide increases histone
acetylation and P21WAF1 expression in human gastric cancer
[11] A. R. Jalilian, A. Panahifar, M. Mahmoudi, M. Akhlaghi, and A.
cells in vivo and in vitro,” Biochemical Pharmacology, vol. 1, no.
Simchi, “Preparation and biological evaluation of [67Ga]-
7, pp. 1–10, 2012.
labeled- superparamagnetic nanoparticles in normal rats,”
[29] C. S. Ciobanu, S. L. Iconaru, E. Gyorgy et al., “Biomedical prop-
Radiochimica Acta, vol. 97, no. 1, pp. 51–56, 2009.
erties and preparation of iron oxide-dextran nanostructures by
[12] C. Corot, P. Robert, J.-M. Idée, and M. Port, “Recent advances MAPLE technique,” Chemistry Central Journal, vol. 6, article 17,
in iron oxide nanocrystal technology for medical imaging,” 2012.
Advanced Drug Delivery Reviews, vol. 58, no. 14, pp. 1471–1504,
[30] O. Krystofova, J. Sochor, O. Zitka et al., “Effect of magnetic nan-
2006.
oparticles on tobacco BY-2 cell suspension culture,” Interna-
[13] C. Fan, W. Gao, Z. Chen et al., “Tumor selectivity of stealth tional Journal of Environmental Research and Public Health, vol.
multi-functionalized superparamagnetic iron oxide nanoparti- 10, no. 1, pp. 47–71, 2013.
cles,” International Journal of Pharmaceutics, vol. 404, no. 1-2, [31] L. L. C. Estevanato, J. R. Da Silva, A. M. Falqueiro et al.,
pp. 180–190, 2011. “Co-nanoencapsulation of magnetic nanoparticles and selol
[14] Q. A. Pankhurst, J. Connolly, S. K. Jones, and J. Dobson, “Appli- for breast tumor treatment: in vitro evaluation of cytotoxicity
cations of magnetic nanoparticles in biomedicine,” Journal of and magnetohyperthermia efficacy,” International Journal of
Physics D, vol. 36, no. 13, pp. R167–R181, 2003. Nanomedicine, vol. 7, pp. 5287–5299, 2012.
[15] R. K. Gilchrist, W. D. Shorey, R. C. Hanselman, J. C. Parrott, and [32] M. L. B. Carneiro, E. S. Nunes, R. C. A. Peixoto et al., “Free Rho-
C. B. Taylor, “Selective inductive heating of lymph,” Annals of dium (II) citrate and rhodium (II) citrate magnetic carriers
Surgery, vol. 146, pp. 596–606, 1957. as potential strategies for breast cancer therapy,” Journal of
[16] T. R. Pisanic II, J. D. Blackwell, V. I. Shubayev, R. R. Fiñones, and Nanobiotechnology, vol. 9, article 11, 2011.
S. Jin, “Nanotoxicity of iron oxide nanoparticle internalization [33] L. Gu, R. H. Fang, M. J. Sailor, and J.-H. Park, “In vivo clearance
in growing neurons,” Biomaterials, vol. 28, no. 16, pp. 2572–2581, and toxicity of monodisperse iron oxide nanocrystals,” ACS
2007. Nano, vol. 6, no. 6, pp. 4947–4954, 2012.
[34] H. M. Kouchesfehani, S. rKiani, A. A. Rostami, and R. Fakheri,

“Cytotoxic effect of iron oxide nanoparticles on mouse embry-
onic stem cells by MTT assay,” Iranian Journal of Toxicology, vol.
7, no. 21, pp. 849–853, 2013.
[35] P. Greaves, “Liver and pancreas,” in Histopathology of Preclinical
Toxicity Studies, pp. 457–503, Academic Press, Elsevier, New
York, NY, USA, 2007.
[36] A. Shiga, Y. Ota, Y. Ueda et al., “Study on the pathogenesis of
foreign body granulomatous inflammation in the livers of
sprague-dawley rats,” Journal of Toxicologic Pathology, vol. 23,
no. 4, pp. 253–260, 2010.
[37] B. Wang, W. Feng, M. Wang et al., “Acute toxicological impact of
nano- and submicro-scaled zinc oxide powder on healthy adult
mice,” Journal of Nanoparticle Research, vol. 10, no. 2, pp. 263–
276, 2008.
[38] S. Dekkers, P. Krystek, R. J. B. Peters et al., “Presence and risks
of nanosilica in food products,” Nanotoxicology, vol. 5, no. 3, pp.
393–405, 2011.
[39] J. F. Hillyer and R. M. Albrecht, “Gastrointestinal persorption
and tissue distribution of differently sized colloidal gold
nanoparticles,” Journal of Pharmaceutical Sciences, vol. 90, no.
12, pp. 1927–1936, 2001.
[40] Y. S. Kim, J. S. Kim, H. S. Cho et al., “Twenty-eight-day oral toxi-
city, genotoxicity, and gender-related tissue distribution of silver
nanoparticles in Sprague-Dawley rats,” Inhalation Toxicology,
vol. 20, no. 6, pp. 575–583, 2008.
http://dx.doi.org/10.1155/2013/613638
Research Article
Electrospun Hyaluronan-Gelatin Nanofibrous Matrix for
Nerve Tissue Engineering
Hau-Min Liou,1 Lih-Rou Rau,1 Chun-Chiang Huang,1 Meng-Ru Lu,1

and Fu-Yin Hsu1,2
1
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan
2
Department of Life Sciences, National Taiwan Ocean University, Keelung 20224, Taiwan
Correspondence should be addressed to Fu-Yin Hsu; fyhsu5565@gmail.com
Received 23 July 2013; Revised 25 September 2013; Accepted 26 September 2013
Copyright © 2013 Hau-Min Liou et al. This is an open access article distributed under the Creative Commons Attribution License,
Schwann cells play a critical role in the repair of the peripheral nerve. The goal of this study was to fabricate electrospun gelatin
(Gel) and hyaluronan-gelatin (HA-Gel) composite nanofibers to provide a suitable growth environment for Schwann cells. The
fiber diameters of Gel, 0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm,
respectively. The biological performance of Gel and HA-Gel was evaluated using an in vitro culture of RT4-D6P2T rat Schwann
cells. We found that the cell attachment and proliferation rates were not significantly different on these matrices. However, the
Schwann cells displayed better organized F-actin on HA-Gel than on Gel. Moreover, the expression levels of several genes, including
Nrg1, GFAP, and P0, were significantly higher on HA-Gel than on Gel. In addition, the levels of Nrg1 and P0 protein expression
were also higher on the HA-Gel than on Gel. These results indicate that the hyaluronan-gelatin composite nanofibrous matrix could
potentially be used in peripheral nerve repair.
1. Introduction made of natural and synthetic materials is a promising alter-

native for promoting successful nerve regeneration.
Regeneration of the nervous system is a major challenge. Gelatin is obtained by the partial hydrolysis of native col-
However, the peripheral nervous system has a better intrinsic lagen in an alkaline or acidic environment. Gelatin exhibits
ability to repair and regenerate axons after axonal damage due excellent biocompatibility and biodegradability properties;
to the ability of Schwann cells to enhance the environment for thus, it has been widely used for nerve repair [4]. Hyaluronan
regeneration [1]. For the treatment of peripheral nerve injury, is a naturally biopolymer composed of repeating disaccharide
nerve autografts or direct end-to-end surgical reconnection is units of glucuronic acid and N-acetylglucosamine. It is the
used to repair nerve injury. Unfortunately, direct end-to-end major constituent of the extracellular matrix (ECM) of con-
surgical reconnection only can repair small gaps in the nerve, nective tissues and has many important biological functions.
because the tension introduced into the nerve cable would Because of its excellent biocompatibility, nonimmunogenic-
inhibit nerve regeneration for longer nerve gaps [2]. A nerve ity, and specific biological functions, hyaluronan is used in
autograft harvested from another site in the body is used in a variety of clinical therapies, including supplementing joint
these cases and is considered the “gold standard” for repair fluid in arthritis and facilitating wound healing and regen-
of the longer nerve damage gaps without immunological eration [5, 6]. Furthermore, hyaluronan is known to prevent
rejection problems. However, a nerve autograft has several perineural scar formation, which improves peripheral nerve
disadvantages, including neuroma, hypofunction at the regeneration [7].
donor nerve graft site, and nerve site mismatch. Allografts Tissue engineered scaffolds are another choice for im-
and xenografts are possible replacements for autografts. Nev- plantation to facilitate neural repair. Fibrous scaffolds have
ertheless, the major clinical problems are the risk of immune become very popular in tissue engineering because they
repulsion and disease transmission [3]. Thus, a nerve graft mimic the physical architecture of the ECM. The architecture
Table 1: Oligonucleotide primer for real-time PCR amplification. 2.2. Preparation of Hyaluronan-Gelatin Composite Nanofibr-
ous Matrix (HA-Gel). To allow complete evaporation of the
Gene Primer sequence: sense/antisense solvent, the solvent used in this study was a mixture of formic
5 -GGTGTGGAGTGCCTTCGTAT-3󸀠
󸀠
acid and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a vol-
GFAP
5󸀠 -TACGATGTCCTGGGAAAAGG-3󸀠 ume/volume ratio of 3/7. The gelatin powder was dissolved in
5 -CCAAGCACTGGAACTCATACTGC-3󸀠
󸀠
the solvent mixture to a concentration of 12% (w/v). Various
NGF
5󸀠 -CTGCTGAGCACACACACGCAG-3󸀠 concentrations of sodium hyaluronan (0%, 0.5%, 1%, and 1.5%
5󸀠 -GGCAGTCAGCCCCTTTGTG-3󸀠 w/v) were added to the gelatin solution and dissolved by
Nrg1
5 -TGCAGGGTTGTGATGAAAGGA-3󸀠
󸀠 vortexing until the solution was clear. For the electrospinning
5󸀠 -CATCTCTGTGGACAGCCAGA-3󸀠 process, the polymer solution was placed in a plastic syringe
p75 fitted with an 18 G needle and attached to a syringe pump that
5󸀠 -CTCTACCTCCTCACGCTTGG-3󸀠
provided a steady flow rate of 8.5 𝜇L/min. The electrospinning
5󸀠 -CTGCACTGCTCCTTCTGGT-3󸀠
P0 voltage of 2.3 kV/cm was applied using a high-voltage power
5󸀠 -CCTTGGCATAGTGGAAGATTG-3󸀠 supply (Glassman High Voltage, Inc.).
5󸀠 -ATAGCACCTCCGTTGGACAG-3󸀠
S100
5󸀠 -TCGTTTGCACAGAGGACAAG-3󸀠
2.3. Characterization of the Electrospun Nanofibers. The fiber
5󸀠 -GGCCCGAAGCGTTTACTT-3󸀠
18S rRNA morphology and diameter of the HA-Gels were determined
5󸀠 -CGGCCGTCCCTCTTAATC-3󸀠 by a field emission scanning electron microscope (SEM;
model S-4800, Hitachi, Tokyo, Japan). Briefly, the electrospun
matrices were sputter-coated with gold and visualized by
SEM at an accelerating voltage of 5 kV. The fiber diameter was
of the ECM plays an important role in regulating cell behavior analyzed using image analysis (ImageJ software 1.42, National
with biochemical signals and topographical cues [8, 9]. Institutes of Health, USA). The average fiber diameter and
Electrospinning is an easy method to fabricate ultrafine standard deviation were calculated from 100 random mea-
fibers with submicrometer to nanometer diameters. Electro- surements.
spun fibrous matrices exhibit certain special characteristics,
such as a high specific surface area, a high aspect ratio, and a
high porosity surface area. Most importantly, the topological 2.4. Cell Culture on HA-Gel. Because the HA-Gels readily
structure of electrospun matrices mimics the architecture of dissolved in aqueous media, it was necessary to cross-link
the ECM and could enhance cell attachment, migration, pro- them before using them for cell culture. The electrospun
liferation, and differentiation [10]. matrices were cross-linked by treatment with N-(3-dimethyl-
aminopropyl)-N-ethylcarbodiimide hydrochloride (EDC).
Schwann cells are a major component of the peripheral The matrix was immersed in 1% EDC solution in 95% ethanol
nerve system and play an important role in Wallerian degen- for 2 hrs and then washed repeatedly with 0.01 M PBS to
eration and the subsequent axon regeneration in peripheral remove the residual EDC. Finally, the electrospun matrices
nerve injury [11]. After Wallerian degeneration, the Schwann were sterilized by exposure to ultraviolet light in a sterile hood
cells begin to proliferate and migrate to the lesion, forming overnight. The electrospun matrices were placed in 24-well
longitudinally oriented cell strands called the bands of Büng- tissue culture plates containing a suspension of RT4-D6P2T
ner; thereafter, Schwann cells secrete a number of neurotro-
rat Schwann cells (BCRC no. 60508) (5 × 104 cells/well) in
phic factors such as nerve growth factor and cytokines that
DMEM supplemented with 10% (v/v) FBS, 100 U/mL of peni-
support the survival of injured neurons [12], secrete extracel-
cillin, and 100 𝜇g/mL of streptomycin. The cultures of cell-
lular matrix components, such as laminin and collagen type
seeded electrospun matrices were harvested at 1, 2, 3, and
IV, for axonal growth, and produce neurite promoting factors
4 hrs for cell attachment assays and on days 1, 3, and 6 days for
to guide the growing axon [13, 14]. Thus, the aim of the present
cell proliferation assays. Cell viability was determined using
study was to investigate whether a hyaluronan-gelatin elec-
the MTT assay. At each time point, three samples were used to
trospun matrix has potential as a neurograft for peripheral
measure the number of cells attached to the electrospun
nerve repair.
matrices. The experiments were performed in triplicate.
2. Materials and Methods 2.5. Fluorescent Staining of the Cytoskeleton. The morphology
of cells with their F-actin cytoskeleton fluorescently stained
2.1. Reagents. Dulbecco’s Modified Eagle Medium (DMEM), with fluorescein isothiocyanate (FITC)-conjugated phalloi-
fetal bovine serum (FBS), and trypsin were purchased from din and nuclei stained with DAPI was examined. After 1 or
GIBCO (Grand Island, NY, USA). Gelatin type A (Gel) was 4 hrs of cell culture, cells were fixed with 3.7% (v/v) para-
purchased from the Sigma-Aldrich Chemical Company (St. formaldehyde in PBS for 10 min, followed by 2 times washing
Louis, MO, USA). Sodium hyaluronan was purchased from with PBS and permeabilization with 0.1% (v/v) Triton X-100
NovaMatrix (Drammen, Norway). The RT4-D6P2T rat in PBS for 5 min. After washing with PBS, the samples were
Schwann cells were purchased from BCRC (Bioresource Col- blocked with 1% bovine serum albumin in PBS for 1 hr.
lection and Research Center, Taiwan). The other chemicals After blocking, the samples were stained with 6.4 𝜇M FITC-
used were of reagent grade unless otherwise stated. conjugated phalloidin for 20 min. Cell nuclei were stained
Table 2: Sample notation, average diameter of hyaluronan-gelatin composite nanofibers.
Sample Gelatin concentration Hyaluronan Average diameter 1st quartile; 3rd quartile
notation (mg/mL) concentration (mg/mL) Standard deviation (nm) (nm)
Gel 12 0 130 ± 30 109; 144
0.5 HA-Gel 12 0.5 294 ± 87 230; 352
1 HA-Gel 12 1 362 ± 129 282; 422
1.5 HA-Gel 12 1.5 224 ± 54 179; 256
with 4󸀠 ,6-diamidino-2-phenylindole (DAPI). After staining, immunoblotting were analyzed with the nonparametric
the samples were washed with PBS and observed under a flu- Mann-Whitney U test. Differences at 𝑃 < 0.05 were consid-
orescence microscope (TS100, Nikon) and a laser scanning ered statistically significant.
confocal microscope (LSCM, Zeiss LSM 510 META).
3. Results
2.6. Quantitative Real-Time PCR. Real-time PCR was used to
determine the levels of neuregulin 1 (Nrg1), S100, nerve 3.1. Fabrication of Hyaluronan-Gelatin Electrospun Nano-
growth factor (NGF), low-affinity nerve growth factor recep- fibers. Formic acid is a good solvent for hyaluronan and gela-
tor (p75), glial fibrillary acidic protein (GFAP), and myelin tin. However, gelatin and hyaluronan/gelatin which dissolved
protein zero (P0) mRNA and 18S ribosomal RNA (as the in a formic acid solution cannot be fabricated into nanofibr-
internal control). Table 1 shows the sequences of the oligonu- ous matrices by electrospinning. In a previous study, hexaflu-
cleotides that were used as PCR primers. Briefly, the reaction oropropanol was found to be a suitable solvent to dis-
is followed by the manufacture protocol of SYBR Green PCR solve hyaluronan/collagen [15]. To obtain hyaluronan-gelatin
Master Mix Kit (Protech SA-SQGLR-V2). The real-time PCR nanofibers (HA-Gel), we used the cosolvent method by mix-
conditions for the promoter region were 15 mins at 94∘ C, ing formic acid and HFIP.
1 min at 62.5∘ C, and 15 s at 94∘ C. Real-time PCR reactions Figure 1 shows that the synthetic product was a three-di-
were performed using an iQ5 Gradient Real-Time PCR sys- mensional nonwoven nanofibrous hyaluronan-gelatin matrix
tem (Bio-Rad). The levels of RNA expression were calculated with interconnected pores. The fiber diameters were 130 ±
using the 2−ΔΔCt method. 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm for Gel,
0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel, respectively. These
dimensions are similar to those of the native fibrous proteins
2.7. Immunoblotting Analysis. Cells were seeded on substrates in the ECM. We found that the diameters of the fibers in the
at 3 × 104 cells/cm2 in medium. Immunoblotting to detect the HA-Gel matrix were significantly different from those in the
Schwann cell-specific proteins neuregulin 1 (Nrg1), myelin Gel matrix (𝑃 < 0.05, one-way ANOVA with Tukey’s post hoc
protein zero (P0), and CD44 was performed after day 3 and test) (Table 2). In addition, the diameters of the fibers grad-
day 6 of culturing. Cells were collected and lysed in lysis ually increased and then decreased as the concentration of
buffer. The supernatants were obtained by centrifugation at hyaluronan was gradually increased.
4∘ C, 15,000 ×g for 10 min. The concentration of protein was The Gel and HA-Gel fibers cross-linked with EDC are
analyzed using a Bradford Coomassie assay. The proteins shown in Figure 2. The Gel and HA-Gel fibers still maintained
(30 𝜇g/𝜇L) were fractionated by electrophoresis and electro- their morphology. However, the fibers appeared to be more
transferred to polyvinylidene difluoride film (PVDF). Block- rubbery and were fused at fiber junctions.
ing was performed using 5% (w/v) nonfat milk, and then the
primary antibody was applied to the membrane overnight at 3.2. Cell Attachment and Proliferation. Cellular functions, in-
4∘ C. Antibodies specific to CD44 (Santa Cruz Biotechnology- cluding spreading, proliferation, and differentiation, are sen-
INC. sc-9960), Nrg1 (Santa Cruz Biotechnology-INC. sc- sitive to the composition and surface topography of the
28916), P0 (Santa Cruz Biotechnology-INC. sc-18531), and matrix. To test the effects of the hyaluronan/gelatin weight
nucleophosmin B23 (Invitrogen 325200) were used. After ratios of the nanofibrous matrix on cell morphology, we incu-
incubation with a HRP-conjugated secondary antibody, the bated the RT4-D6P2T rat Schwann cells on hyaluronan and
immunoreactive bands were detected using enhanced chemi- gelatin matrices with different weight ratios. The morpholo-
luminescence detection (Millipore WBKLS0500). The immu- gies of the Schwann cells that adhered to the different matri-
noreactive bands were analyzed by ImageJ software (National ces were investigated by staining the actin cytoskeleton with
Institutes of Health, USA). Nucleophosmin B23 was used as FITC-phalloidin and visualizing the cells with a fluorescence
the internal control. microscope (shown in Figure 3) and a laser scanning confocal
microscope (shown in Figure 4). We found that most of the
2.8. Statistical Analyses. Statistical analyses were performed Schwann cells cultured on the HA-Gel matrix appeared to
using SPSS v.11 software. For each condition, the diameters of have spread during the 4 hr attachment period. However, it
at least 100 randomly chosen fibers were measured. The fiber was obvious that the cells cultured on the Gel matrix re-
diameters were analyzed using a one-way ANOVA with mained round after 4 h (Figure 3(e) and Figure 4(e)). The
Tukey’s post hoc test. The cell viability, gene expression, and attachment and proliferation of the Schwann cells on the Gel
(a) (b)
(c) (d)
Figure 1: Scanning electron microscopy images of as-electrospun HA-Gel matrix at a magnification of 5000x. (a) Gel, (b) 0.5 HA-Gel, (c) 1 HA-
Gel, and (d) 1.5 HA-Gel. The scale bar represents 10 𝜇m.
(a) (b)
(c) (d)
Figure 2: Scanning electron microscopy images of electrospun HA-Gel matrix after EDC treatment at a magnification of 10000x. (a) Gel,
(b) 0.5 HA-Gel, (c) 1 HA-Gel, and (d) 1.5 HA-Gel. The scale bar represents 5 𝜇m.
(a) (b) (c) (d)
20 𝜇m
(e) (f) (g) (h)
Figure 3: Fluorescence micrographs of F-actin stained RT4-D6P2T Schwann cell grown on an electrospun matrix of Gel ((a), (e)), 0.5 HA-Gel
((b), (f)), 1 HA-Gel ((c), (g)), and 1.5 HA-Gel ((d), (h)) for 1 hr ((a), (b), (c), and (d)) or 4 hrs ((e), (f), (g), and (h)). Cytoskeletal F-actin was
stained green with FITC, and cell nuclei were stained blue with DAPI. (Scale bar = 20 𝜇m.)
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 4: Confocal micrographs of F-actin stained RT4-D6P2T Schwann cell on an electrospun matrix of Gel ((a), (e)), 0.5 HA-Gel ((b),
(f)), 1 HA-Gel ((c), (g)), and 1.5 HA-Gel ((d), (h)) for 1 hour ((a), (b), (c), and (d)) and 4 hours ((e), (f), (g), and (h)). Cytoskeletal F-actin was
stained green with FITC, and cell nuclei were stained blue with DAPI. (Scale bar = 10 𝜇m.)
and HA-Gel matrices were quantified using the MTT assay. PCR for Nrg1, S100, NGF, p75, GFAP, and P0 and 18S riboso-
There were no significant differences in attachment or prolif- mal RNA (as shown in Figure 6). On day 3, the Schwann cells
eration among the cells cultured on 0.5 HA-Gel, 1 HA-Gel, or displayed insignificantly increased gene expression levels of
1.5 HA-Gel (𝑃 > 0.05) (as shown in Figure 5). Nrg1, S100, NGF, p75, GFAP, and P0 when grown on 0.5 HA-
Gel, 1 HA-Gel, or 1.5 HA-Gel relative to those obtained from
3.3. Real-Time PCR Analysis. The expression levels of cells grown on Gel (𝑃 > 0.05). However, on day 6, the
Schwann cell-specific genes were analyzed using real-time Schwann cells showed significantly higher gene expression
0.5 1.6
1.4
0.4
MTT assay (OD at 570 nm)
MTT assay (OD at 570 nm)

1.2
0.3 1.0
0.8
0.2 0.6
0.4
0.1
0.2
0.0 0.0
1 2 3 4 1 3 5
Incubation time (hours) Incubation time (days)
Gel 1 HA-gel Gel 1 HA-gel

0.5 HA-gel 1.5 HA-gel 0.5 HA-gel 1.5 HA-gel
(a) (b)
Figure 5: Quantification of RT4-D6P2T Schwann cells on Gel and HA-Gel matrices. The MTT assay was used to quantify cell attachment
and proliferation on Gel and HA-Gel. (a) The attachment of RT4-D6P2T Schwann cells on various matrices after culturing for up to 4 hrs.
(b) The viability of RT4-D6P2T Schwann cells on various matrices after culturing for up to 6 days. The data are presented as the means ± SD,
𝑛 = 4. No statistically significant differences in cell attachment or proliferation on the HA-Gel and Gel were found.
4 4
Relative gene expression
3 3
∗
2 2 ∗
∗ ∗
∗ ∗
1 1 ∗∗
0 0
Nrg1 S100 NGF p75 GFAP P0 Nrg1 S100 NGF p75 GFAP P0

(a) (b)
Figure 6: Real-time PCR analyses of the nerve-associated genes expressed by RT4-D6P2T Schwann cells on various matrices after culturing
for (a) 3 days or (b) 6 days. The data are shown as the fold change relative to those obtained from cells grown on the culture dish surface. The
data are presented as the means ± SD (𝑛 = 4). (∗ ) denotes significant difference between HA-Gel and Gel (𝑃 < 0.05).
levels of Nrg1, GFAP, and P0 when grown on 0.5 HA-Gel, However, the levels of Nrg1 and P0 on day 3 and day 6 were
1 HA-Gel, or 1.5 HA-Gel compared to cells grown on Gel not significantly different among the cells grown on 0.5 HA-
(𝑃 < 0.05) (as shown in Figure 6). Gel, 1 HA-Gel, or 1.5 HA-Gel (𝑃 > 0.05).
3.4. Western Blot Analysis. The CD44 levels expressed on day 4. Discussion
3 and day 6 by the RT4-D6P2T rat Schwann cells grown on
the different matrices were not significantly different (𝑃 > Sponge forms of gelatin, hyaluronan, and their composites
0.05). The levels of Nrg1 and P0 were higher in the cells grown have been widely used to fabricate scaffolds for tissue engi-
on 0.5 HA-Gel, 1 HA-Gel, or 1.5 HA-Gel than in the cells neering [16–18]. However, the physical structure of the
grown on Gel on day 6 (𝑃 < 0.05) (as shown in Figure 7). sponge form of these matrices is not acceptable for the tissue
(1) (2) (3) (4) (5) (1) (2) (3) (4) (5)
CD44 CD44
Nrg1
Nrg1
P0
P0
B23
B23
5 5
4 4

3 3 ∗
∗
2 2
∗
∗ ∗ ∗
1 1
0 0
CD44 Nrg1 P0 CD44 Nrg1 P0

(a) (b)
Figure 7: Western blot analysis of CD44, Nrg1, and P0 proteins in RT4-D6P2T Schwann cells cultured for (a) 3 or (b) 6 days. Densitometric
quantification of the protein bands was performed by normalizing with B23. The data are shown as the fold change relative to those obtained
from cells grown on the culture dish surface. The data are presented as the means ± SD (𝑛 = 3). (∗ ) denotes significant difference between
HA-Gel and Gel (𝑃 < 0.05). (1) Gel, (2) 0.5 HA-Gel, (3) 1 HA-Gel, (4) 1.5 HA-Gel, and (5) culture dish.
regeneration. A porous and nanofibrous biodegradable ma- factors might explain why the mean fiber diameters increased
trix that mimics a natural ECM is the optimal scaffold for at lower hyaluronan concentrations and decreased at higher
tissue engineering [19]. It is widely accepted that cell adhesion hyaluronan concentrations.
and most cellular activities, including spreading, migration, Hyaluronan is found in all vertebrate tissues and is impor-
proliferation, gene expression, and cytoskeletal function, are tant for the organization of pericellular and extracellular
sensitive to the nanotopography [20] and molecular compos- matrices [22]. Hyaluronan plays a crucial role in tissue hydr-
ition of the matrix [15]. Xu et al. demonstrated that there were ation and lubrication and in the regulation of cell functions,-
stronger interactions between the cells and the nanofibers including cell attachment, cell mitosis, cell migration, tumor
[21]. Consequently, most researchers believe that a nanofibr- development, metastasis, cell apoptosis, and inflammation
ous matrix may promote cell growth more effectively than a [23, 24]. The function of hyaluronan depends upon its inter-
sponge substrate. action with the cell surface receptors, including the receptor
Several methods have been used to fabricate the fibrous for hyaluronan-mediated motility (RHAMM), cluster deter-
matrix, but the electrospinning is a simple and effective fabri- minant 44 (CD44), lymphatic vessel endothelial hyaluronan
cation technique to produce nano-/microfibers. The mor- receptor (LYVE-1), hyaluronan receptor for endocytosis
phology and diameter of electrospun fibers are dependent on (HARE), liver endothelial cell clearance receptor (LEC recep-
many processing parameters, including the molecular weight tor), and toll-like receptor 4 (TLR4) [25, 26]. Hyaluronan has
of the polymer, the concentration (or viscosity), the conduc- been shown to influence the interaction of glial cells with
tivity, the applied voltage, and the feeding rate of the polymer axons, to reduce the formation of scar tissue and to provide
solution. In our previous study, we found that increasing the a suitable environment for tissue repair [27–29]. Wang et al.
concentration of hyaluronan would increase the charge den- found that hyaluronan played a vital role in the biological pro-
sity on the surface of the electrospinning jet, which would in- cesses involved in the deposition and remodeling of the fibrin
duce higher electrostatic forces and result in the formation of extracellular matrix to increase the level of myelination of
smaller diameter fibers [15]. On the other hand, the diameter axons after 4 weeks in vivo [30]. Sherman noted that the CD44
of the electrospun fibers increases with increasing concentra- transmembrane glycoprotein plays a key role in Schwann cell-
tions of a polymer solution. A combination of the aforesaid neuron interaction. The reduction of CD44 expression
in vitro decreased Schwann cell-neurite adhesion and caused Schwann cells and dorsal root ganglia,” Biomaterials, vol. 33, no.
the apoptosis of Schwann cells [31]. 33, pp. 8529–8539, 2012.
The peripheral nerve repair process depends on Schwann [5] J. M. Medina, A. Thomas, and C. R. Denegar, “Knee osteoarthri-
cells, which synthesize and secrete important substances such tis: should your patient opt for hyaluronic acid injection?” Jour-
as neuregulin-1 (Nrg1), GFAP, and P0. Nave proposed that nal of Family Practice, vol. 55, no. 8, pp. 669–675, 2006.
Nrg1 is essential for every developmental stage of Schwann [6] E. Nyman, F. Huss, T. Nyman, J. Junker, and G. Kratz, “Hyalu-
cells and for the myelination of axons [32]. Nrg1 is an epi- ronic acid, an important factor in the wound healing properties
dermal growth factor-like ligand that interacts with the tyro- of amniotic fluid: in vitro studies of re-epithelialisation in hu-
sine kinase ErbB receptor to regulate many aspects of neural man skin wounds,” Journal of Plastic Surgery and Hand Surgery,
development. Moreover, Ghatak et al. demonstrated that vol. 47, no. 2, pp. 89–92, 2013.
ErbB2 can be activated by HA through CD44-mediated [7] G. Y. Özgenel, “Effects of hyaluronic acid on peripheral nerve
mechanisms to promote the proliferation and differentiation scarring and regeneration in rats,” Microsurgery, vol. 23, no. 6,
pp. 575–581, 2003.
of Schwann cells [33].
GFAP is an intermediate filament protein that functions [8] J. Taipale and J. Keski-Oja, “Growth factors in the extracellular
as a scaffold for cytoskeletal assembly and maintenance [34]. matrix,” FASEB Journal, vol. 11, no. 1, pp. 51–59, 1997.
GFAP also plays a vital role in Schwann cell proliferation and [9] F. Berthiaume, P. V. Moghe, M. Toner, and M. L. Yarmush,
the upregulation of Schwann cell-specific cytoskeletal con- “Effect of extracellular matrix topology on cell structure, func-
tion, and physiological responsiveness: hepatocytes cultured in
stituents after nerve damage [35]. Myelin protein zero (P0) is
a sandwich configuration,” FASEB Journal, vol. 10, no. 13, pp.
the major adhesive and structural protein of the myelin 1471–1484, 1996.
sheath of peripheral nerves [36]. P0 is expressed by myelinat-
[10] C. Li, C. Vepari, H.-J. Jin, H. J. Kim, and D. L. Kaplan, “Elec-
ing Schwann cells and is necessary for normal myelin struc-
trospun silk-BMP-2 scaffolds for bone tissue engineering,” Bio-
ture and function [37]. Some studies have demonstrated that materials, vol. 27, no. 16, pp. 3115–3124, 2006.
P0 promoted the regeneration of injured axons [38]. From the
[11] M. G. Burnett and E. L. Zager, “Pathophysiology of peripheral
results of real-time Q-PCR and western blotting, we found nerve injury: a brief review,” Neurosurgical Focus, vol. 16, no. 5,
that Nrg1, GFAP, and P0 gene expression and Nrg1 and P0 article E1, 2004.
protein expression were enhanced at day 6 when Schwann [12] S. S. Scherer and J. Salzer, “Axon-Schwann cell interaction dur-
cells were cultured on HA-Gel. We speculate that the HA-Gel ing peripheral nerve degeneration and regeneration,” in Glial
enhanced myelination via the interaction of the surface CD44 Cell Development, pp. 299–330, Oxford University Press, Lon-
receptors of Schwann cells with hyaluronan. don, UK, 2003.
[13] J. Hu, J. Zhou, X. Li, F. Wang, and L. Hezuo, “Schwann cells pro-
5. Conclusion mote neurite outgrowth of dorsal root ganglion neurons
through secretion of nerve growth factor,” Indian Journal of
Schwann cells grown on hyaluronan-gelatin nanofibrous ma- Experimental Biology, vol. 49, no. 3, pp. 177–182, 2011.
trices showed better organized F-actin stress fibers, higher [14] A. I. Gravvanis, A. A. Lavdas, A. Papalois, D. A. Tsoutsos, and
levels of Nrg1, GFAP, and P0 mRNA, and higher levels of R. Matsas, “The beneficial effect of genetically engineered
Nrg1 and P0 protein compared to those grown on the gela- Schwann cells with enhanced motility in peripheral nerve re-
generation: review,” Acta Neurochirurgica, vol. 100, pp. 51–56,
tin nanofibrous matrix. These findings suggest that the hyalu-
2007.
ronan-gelatin nanofibrous matrix could potentially be used in
the repair of injured peripheral nerves. [15] F.-Y. Hsu, Y.-S. Hung, H.-M. Liou, and C.-H. Shen, “Electrospun
hyaluronate-collagen nanofibrous matrix and the effects of
varying the concentration of hyaluronate on the characteristics
Conflict of Interests of foreskin fibroblast cells,” Acta Biomaterialia, vol. 6, no. 6, pp.
2140–2147, 2010.
The authors declare that there is no conflict of interests re- [16] L. Hong, I. Peptan, P. Clark, and J. J. Mao, “Ex vivo adipose
garding the publication of this paper. tissue engineering by human marrow stromal cell seeded gelatin
sponge,” Annals of Biomedical Engineering, vol. 33, no. 4, pp. 511–
517, 2005.
References [17] Y. C. Chen, W. Y. Su, S. H. Yang, A. Gefen, and F. H. Lin, “In situ
[1] T. A. Ferguson and Y. J. Son, “Extrinsic and intrinsic determi- forming hydrogels composed of oxidized high molecular weight
nants of nerve regeneration,” Journal of Tissue Engineering, vol. hyaluronic acid and gelatin for nucleus pulposus regeneration,”
2, no. 1, 2011. Acta Biomaterialia, vol. 9, no. 2, pp. 5181–5193, 2013.
[18] Y. Liu, X. Z. Shu, S. D. Gray, and G. D. Prestwich, “Disulfide-
[2] C. E. Schmidt and J. B. Leach, “Neural tissue engineering: strate-
crosslinked hyaluronan-gelatin sponge: growth of fibrous tissue
gies for repair and regeneration,” Annual Review of Biomedical
in vivo,” Journal of Biomedical Materials Research A, vol. 68, no.
Engineering, vol. 5, pp. 293–347, 2003.
1, pp. 142–149, 2004.
[3] T. E. Trumble and F. G. Shon, “The physiology of nerve trans- [19] H. M. Lin, Y. H. Lin, and F. Y. Hsu, “Preparation and character-
plantation,” Hand Clinics, vol. 16, no. 1, pp. 105–122, 2000. ization of mesoporous bioactive glass/polycaprolactone nanofi-
[4] R. E. Gámez Sazo, K. Maenaka, W. Gu, P. M. Wood, and M. B. brous matrix for bone tissues engineering,” Journal of Materials
Bunge, “Fabrication of growth factor- and extracellular matrix- Science: Materials in Medicine, vol. 23, no. 11, pp. 2619–2630,
loaded, gelatin-based scaffolds and their biocompatibility with 2012.
[20] K. S. Park, K. J. Cha, I. B. Han et al., “Mass-producible nano-fea- formation in Schwann cells,” Molecular and Cellular Neurosci-
tured polystyrene surfaces for regulating the differentiation of ence, vol. 18, no. 6, pp. 606–618, 2001.
human adipose-derived stem cells,” Macromolecular Bioscience, [38] L. B. Spiryda, “Myelin protein zero and membrane adhesion,”
vol. 12, no. 11, pp. 1480–1489, 2012. Journal of Neuroscience Research, vol. 54, no. 2, pp. 137–146, 1998.
[21] C. Xu, R. Inai, M. Kotaki, and S. Ramakrishna, “Electrospun
nanofiber fabrication as synthetic extracellular matrix and its
potential for vascular tissue engineering,” Tissue Engineering,
vol. 10, no. 7-8, pp. 1160–1168, 2004.
[22] T. Laurent, “The biology of hyaluronan. Introduction,” Ciba
Foundation Symposium, vol. 143, pp. 1–20, 1989.
[23] B. P. Toole and M. G. Slomiany, “Hyaluronan: a constitutive reg-
ulator of chemoresistance and malignancy in cancer cells,” Sem-
inars in Cancer Biology, vol. 18, no. 4, pp. 244–250, 2008.
[24] C. Cencetti, D. Bellini, C. Longinotti, A. Martinelli, and P. Mat-
ricardi, “Preparation and characterization of a new gellan gum
and sulphated hyaluronic acid hydrogel designed for epidural
scar prevention,” Journal of Materials Science: Materials in Med-
icine, vol. 22, no. 2, pp. 263–271, 2011.
[25] J. B. Park, H.-J. Kwak, and S.-H. Lee, “Role of hyaluronan in gli-
oma invasion,” Cell Adhesion & Migration, vol. 2, no. 3, pp. 202–
207, 2008.
[26] M. A. Solis, Y.-H. Chen, T. Y. Wong, V. Z. Bittencourt, Y.-C. Lin,
and L. L. H. Huang, “Hyaluronan regulates cell behavior: a
potential niche matrix for stem cells,” Biochemistry Research
International, vol. 2012, Article ID 346972, 11 pages, 2012.
[27] L. S. Sherman, T. A. Rizvi, S. Karyala, and N. Ratner, “CD44 en-
hances neuregulin signaling by Schwann cells,” Journal of Cell
Biology, vol. 150, no. 5, pp. 1071–1083, 2000.
[28] Y. T. Wei, W. M. Tian, X. Yu et al., “Hyaluronic acid hydrogels
with IKVAV peptides for tissue repair and axonal regeneration
in an injured rat brain,” Biomedical Materials, vol. 2, no. 3, pp.
S142–S146, 2007.
[29] Z. Z. Khaing and C. E. Schmidt, “Advances in natural biomate-
rials for nerve tissue repair,” Neuroscience Letters, vol. 519, no. 2,
pp. 103–114, 2012.
[30] K. K. Wang, I. R. Nemeth, and B. R. Seckel, “Hyaluronic acid en-
hances peripheral nerve regeneration in vivo,” Microsurgery,
vol. 18, no. 4, pp. 270–275, 1998.
[31] L. S. Sherman, T. A. Rizvi, S. Karyala, and N. Ratner, “CD44 en-
hances neuregulin signaling by Schwann cells,” Journal of Cell
Biology, vol. 150, no. 5, pp. 1071–1083, 2000.
[32] K.-A. Nave and J. L. Salzer, “Axonal regulation of myelination by
neuregulin 1,” Current Opinion in Neurobiology, vol. 16, no. 5, pp.
492–500, 2006.
[33] S. Ghatak, S. Misra, and B. P. Toole, “Hyaluronan constitutively
regulates ErbB2 phosphorylation and signaling complex forma-
tion in carcinoma cells,” Journal of Biological Chemistry, vol.
280, no. 10, pp. 8875–8883, 2005.
[34] P. A. Coulombe and P. Wong, “Cytoplasmic intermediate fila-
ments revealed as dynamic and multipurpose scaffolds,” Nature
Cell Biology, vol. 6, no. 8, pp. 699–706, 2004.
[35] D. Triolo, G. Dina, I. Lorenzetti et al., “Loss of glial fibrillary
acidic protein (GFAP) impairs Schwann cell proliferation and
delays nerve regeneration after damage,” Journal of Cell Science,
vol. 119, no. 19, pp. 3981–3993, 2006.
[36] D. D’Urso, P. J. Brophy, S. M. Staugaitis et al., “Protein zero of
peripheral nerve myelin: biosynthesis, membrane insertion,
and evidence for homotypic interaction,” Neuron, vol. 4, no. 3,
pp. 449–460, 1990.
[37] D. M. Menichella, E. J. Arroyo, R. Awatramani et al., “Protein
Zero is necessary for E-cadherin-mediated adherens junction
http://dx.doi.org/10.1155/2013/515741
Research Article
Investigation of the In Vitro Degradation of a Novel
Polylactide/Nanohydroxyapatite Composite for Artificial Bone
Jianghong Huang,1,2 Jianyi Xiong,1,2 Jianquan Liu,1,2 Weimin Zhu,1,2 and Daping Wang1,2
1
State Key Department of Orthopedics, The Second People’s Hospital of Shenzhen, Shenzhen, Guangdong 518035, China
2
Key Laboratory of Tissue Engineering, Shenzhen, Guangdong 518035, China
Correspondence should be addressed to Daping Wang; dapingwang1972@163.com
Received 22 April 2013; Revised 7 August 2013; Accepted 20 September 2013
Copyright © 2013 Jianghong Huang et al. This is an open access article distributed under the Creative Commons Attribution
cited.
We prepared the poly-L-lactic acid (PLLA)/nanohydroxyapatite (n-HA) composite and investigated the in vitro degradation of
pure PLLA material and PLLA/n-HA composites in order to identify a suitable and ideal artificial bone tissue repair material. The
water uptake, weight loss, and changes in the PBS pH value and in the mechanical properties of material were measured during
the processes that PLLA and PLLA/n-HA biological composites were degraded in PBS. We also performed electron microscopic
scanning of the material fracture surface and observed the microscopic morphologies of materials during the degradation process.
We found that the degradation rate of the PLLA/n-HA material was slower than the PLLA material, and there was a little degradation
of the PLLA/n-HA material at early stages. The PLLA/n-HA material also maintained the initial mechanical strength better than
the pure PLLA material. The PLLA/n-HA material is thus a better material for artificial bone than the pure PLLA material.
1. Introduction are weak and cannot match the mechanical strength of

human bone. In addition, the degradation of the n-HA is
The quest for an ideal artificial bone tissue repair material is a slow [4–6]. PLLA/n-HA composites can be generated using
hot topic in the bone tissue engineering research field. Poly- the poly-L-lactic acid (PLLA) as a base material and n-HA
lactic acid is biomedical synthetic material that is most com- particles as reinforcement substances, thus fully utilizing the
monly used in bone tissue repair, and it has good biocompat- advantages of the two materials as biomedical engineering
ibility, degradability, and processing controllability. However,
materials [7, 8]. The paper focused on the in vitro degradation
polylactic acid still has defects such as poor hydrophilicity,
characteristics of pure PLLA and PLLA/n-HA composites in
generation of acidic degradation products, and insufficient
order to provide the ideal bone tissue repair material for bone
retention time for its mechanical strength [1–3]. Nanohydrox-
yapatite (n-HA) is a type of bone graft substitute material tissue engineering.
that has similar physicochemical and biological properties as
the human skeleton and can be absorbed by the body and 2. Materials and Methods
gradually transformed into autologous bone component. n-
HA is strongly hydrophilic and is a mildly basic material. 2.1. Preparation of Composites
Moreover, it can be degraded via the solution-mediated
process (dissolved in physiological solutions) and the cell- 2.1.1. Preparation of PLLA. L-lactide (LLA) with high purity
mediated process (phagocytosis). The calcium and phosphate (99.95%) was prepared by a combination of distillation
ions released after degradation participate in the local bone technology and washing-recrystallization with lactic acid.
tissue calcification or enter the calcium and phosphorus PLLA was synthesized via ring-opening polymerization using
pools of the body, and they can be subsequently utilized or prepared L-lactide at 140∘ C for 24 hours. The solution spin-
discharged physiologically. However, as a scaffold material ning method was used to obtain sheets of the PLLA material
for tissue engineering, the mechanic characteristics of n-HA (Figure 1).
2 mm
Figure 1: Sheets of the PLLA material.

5 mm
(a)
2 mm
Figure 2: n-HA powder.

5 mm
(b)
2.1.2. Preparation of n-HA Powder. n-HA was generated from
Ca(NO3 )2 and NH4 PO3 via sol flocculation. During the Figure 3: Sample strips after being mold-pressed: (a) PLLA; (b)
chemosynthesis of n-HA, aqua ammonia was used to adjust PLLA/n-HA.
the PH value to 8∼13. With dispersant agent and appropriate
agitator, the n-HA deposit was separated out of the solution.
With series of processes including scouring, filtering, drying, (1) prepare 1/15 mol/L KH2 PO4 , that is, 9.078 g KH2 PO4
sinter-roasting, and milling, the n-HA powder has been pre- per liter of water;
pared (Figure 2). (2) prepare 1/15 mol/L Na2 HPO4 ⋅2H2 O, that is, 11.876 g
Na2 HPO4 ⋅2H2 O per liter of water;
2.1.3. Preparation of PLLA/n-HA Artificial Bone. The PLLA/ (3) mix 18.2% (volume fraction) of the KH2 PO4 solution
n-HA composite was jointly developed by the Tissue Engi- and 81.8% of the Na2 HPO4 ⋅2H2 O solution;
neering Laboratory of the Second People’s Hospital of Shen- (4) adjust the pH of the PBS solution to be about 7.4 by
zhen and the Powder Metallurgy Research Institute of Central using acid/base solution.
South University. The PLLA/n-HA composite was prepared
via melt blending method, which includes 2 steps. Firstly,
the PLLA was heated into viscous flow state at 160∘ C. Then 2.3. In Vitro Degradation Experiments. We used the compres-
n-HA was mixed with PLLA. The PLLA/n-HA artificial sion molding method to prepare strips of PLLA and PLLA/n-
bone material was processed into cylinders (with a length of HA composite materials (10 mm × 5 mm × 5 mm with a n-
10 mm, diameter of 5 mm, and height of 5 mm) (Figure 3) and HA content of 20 wt%) (Figure 3). The prepared biological
preserved in vacuum packaging. material was cleaned with deionized water and placed in a
40∘ C vacuum oven. After being sufficiently dried, the sample
stripes were weighed, and the weight was recorded as 𝑚0 .
2.2. Preparation of the In Vitro Degradation Medium, Phos- The weighted and dry strips were divided into two categories
phate Buffered Saline (PBS). The preparation of PBS solution based on the material composition (PLLA and PLLA/n-HA).
was carried out according to the following steps: Each category of material was further divided into 10 groups
8000
3500
7000
3000
6000
Intensity (cps)
2500
Intensity (cps)
5000
2000
4000
3000 1500
2000 1000
1000 500
0 0
5 10 15 20 25 30 35 40 45 50 55 60 5 10 15 20 25 30 35 40 45 50 55 60
2𝜃 (deg) 2𝜃 (deg)
(a) (b)
5000
4500
4000
Intensity (cps)
3500
3000
2500
2000
1500
1000
500
0
5 10 15 20 25 30 35 40 45 50 55 60
2𝜃 (deg)
(c)
Figure 4: (a) XRD curves of PLLA; (b) XRD curves of HA powder; (c) XRD curves of PLLA/n-HA composite.
based on the degradation time (2 weeks, 4 weeks, 6 weeks, 8 grids, and stained with 4% uranyl acetate and Reynolds, lead
weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, and citrate. Sections were observed under a transmission electron
20 weeks), and each group contained three samples. microscope.
All the samples were placed in clean glass bottles con-
taining PBS, which were then sealed and placed in an electric 2.4.3. Calculation of the Biological Material Water Uptake
thermostatic shaker. The shaker temperature was 37 ± 0.5∘ C, Ratio and Weight Loss Ratio. One group was taken for each
and the vibration speed was 100 rpm. type of material at various degradation time points (2 weeks,
4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks,
16 weeks, 18 weeks, and 20 weeks). The sample weight was
2.4. Evaluation Indicators measured in accordance with the following steps.
2.4.1. Assessing the PLLA, n-HA Powder, and PLLA/n-HA The weight of the sample before degradation is called the
Composite Using X-Ray Diffraction (XRD). The crystal struc- initial weight (𝑚0 ). After a certain period of degradation, the
ture of PLLA, n-HA, and PLLA/n-HA composite was mea- samples were removed, blotted gently with paper to remove
sured by X-ray diffraction system (D/max 2550, Japan). The the water on the surface, and weighed to get the wet weight
scanning range is from 0 to 80 degree and the scanning speed (𝑚1 ). The degraded experimental samples were vacuum-
dried for 24 h at 40∘ C, and the residual weight (𝑚2 ) was then
is 8.0∘ /min. Data were acquired using a CuK𝛼1 source at 40 kV
obtained. The sample water uptake ratio (𝑚𝐴 ) and the weight
and 300 mA.
loss ratio (𝑚𝐿 ) were calculated as follows [9]:
2.4.2. Assessing the PLLA/n-HA Composite Using Transmis- 100 (𝑚1 − 𝑚2 )
𝑚𝐴 (%) = (1)
sion Electron Microscope (TEM). The PLLA/n-HA composite 𝑚2
was cut into blocks measuring about 1 mm wide, 1 mm long,
and 1 mm thick. The blocks and n-HA powder were post- 100 (𝑚0 − 𝑚2 )
𝑚𝐿 (%) = . (2)
fixed in 1% osmium tetroxide for 2 h at 4∘ C. They were rinsed 𝑚0
in distilled water for several times, dehydrated in graded
series (20∼100%) of ethanol and then in propylene oxide, 2.4.4. pH of the PBS Solution. A digital pH meter (pHS-25 pH
infiltrated with Epon 812, and finally polymerized in pure meter with digital display, Shanghai REX Instrument Factory)
Epon 812 for 48 h at 65∘ C. Ultrathin sections were cut on an was used to measure the pH value of the PBS solution at
ultramicrotome using diamond knives, collected on copper different degradation stages.
(a) (b)
Figure 5: (a) TEM micrograph of the n-HA powder; (b) TEM micrograph of PLLA/n-HA composites.
2.4.5. Measurement of Flexural Strength and Modulus. The 10

flexural strength and modulus of each dried sample strip
9
were measured using the three-point bending method on a
universal testing machine (Instron-1121, UK). 8
2.4.6. Scanning Electron Microscopy (SEM) Morphological 7

Features of the Fracture Surface. After measuring the flexural
Water uptake (%)
6
strength, the fracture surface of the sample strip was sprayed
with gold, and SEM (field emission scanning electron micro- 5
scope, Tescan, Czech) was used to observe the changes in the
bending fracture microscopic morphology of samples in the 4
degradation process.
3
3. Results 2
3.1. XRD Analysis of the PLLA, n-HA Powder, and PLLA/n- 1

HA Composite. Figure 4 shows the XRD curves of PLLA, n- 0
HA, and PLLA/n-HA composite. From Figure 4(a), we can 0 2 4 6 8 10 12 14 16 18 20
see that PLLA shows its most intense diffraction peaks at 2𝜃 Degradation time (week)
values of 16.6 and 18.96, which is in agreement with 2013
PLLA
international center for diffraction data (PDF no. 54-1917). PLLA/n-HA
Figure 4(b) shows the XRD curve of n-HA. n-HA shows its
most intense diffraction peaks at 2𝜃 values of 31.74, 32.12, Figure 6: Variation of water uptake of biomaterials with degrada-
and 32.9, which coincides with 2013 international center for tion time.
diffraction data (PDF no. 9-432). Figure 4(c) shows XRD
curve of PLLA/n-HA composite. PLLA/n-HA composite
3.3. Changes in the Material Water Uptake Ratio and Weight
shows its most intense diffraction peaks at 2𝜃 values of 16.6
Loss Ratio. Figure 6 showed that with the extension of the
and 18.96, which is in agreement with the PLLA data in PDF
degradation time, the water uptake ratios of the two samples
no. 54-1917. In addition, PLLA/n-HA shows its most intense
were gradually increased, but the rate of increase in the water
diffraction peaks at 2𝜃 values of 31.74, 32.12, and 32.9, which
uptake ratio became slower. During the entire degradation
coincides with the n-HA data in PDF no. 9-432. period, the water uptake ratios of the PLLA/n-HA samples
were significantly higher than those of the PLLA samples.
3.2. TEM Analysis of the n-HA Morphology in the PLLA/n- As seen from the curves in Figure 7, the weight loss ratios
HA Composite. Results of TEM showed that n-HA presents of the PLLA and PLLA/n-HA materials were relatively low
needle-like structure, which is similar to human bone compo- before the 6th week of degradation, and they were 1.038% and
nents (Figure 5(a)). In addition, n-HA was distributed evenly 0.698%, respectively. After 6 weeks of degradation, with the
in n-HA/PLLA composite (Figure 5(b)). rapid increase of the water uptake ratio, the weight loss ratio
30 8
25 7
20 6
Weight loss (%)
pH value
15 5
10 4
5 3
0 2
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Degradation time (week) Degradation time (week)
PLLA PLLA
PLLA/n-HA PLLA/n-HA
Figure 7: Change of sample weight loss as a function of degradation Figure 8: Effect of degradation time on pH value of PBS.
time.
of the pure PLLA material was also increased sharply and seen from Figure 9, in the early stages of degradation (0–6
showed a linear upward trend with the degradation time. In weeks), the strengths of all materials showed little reduction.
contrast, the weight loss ratio of PLLA/n-HA composite did The strength of the pure PLLA material decreased abruptly
not increase with the degradation time until the degradation between 6∼12 weeks, and the decrease of strength was
time reached 10 weeks, and the extent of increase was smaller alleviated after 12 weeks. The strength of the PLLA/n-HA
than the pure PLLA material. composite started to show significant decrease after eight
weeks and lasted until the end of the degradation experiment.
The curves in Figure 10 that depicted the changes of material
3.4. pH Changes of the PBS Solution. Figure 8 showed the
flexural over the degradation time revealed similar changes
curve that the pH value of the PBS solution changed with as the curves in Figure 9.
the degradation time. It demonstrated that prior to 4 weeks
of degradation, the decrease in the pH value was small
for all PBS solution. After 4 weeks, the pH value of the 3.6. Analysis of the Fracture Morphology of Biological Mate-
PBS solutions containing the PLLA samples began to show rials in the Degradation Process. Figure 11 shows the SEM
the phenomenon of accelerated decrease. This phenomenon images of the morphologies of the bending fracture of the
became even more significant after the pH value dropped pure PLLA biological material in the degradation process
(×8000). Figure 12 shows the SEM images of the morpholo-
below 6. At the 10th week, the pH value was reduced to
gies of the bending fracture of the PLLA/n-HA composite
about 3. After 10 weeks, the decrease in the pH value became
biomaterial in the degradation process (×8000).
slow and basically stopped decreasing. Before the degradation
Before degradation, the fractures of each biological
time reached six weeks, the pH value of the PBS solution con- material sample showed dense structures. After 4 weeks of
taining the PLLA/n-HA sample almost showed no changes. degradation, the fracture surface of the pure PLLA material
And between 6∼12 weeks, the pH value of the buffer was did not show significant changes, and the fracture surface
slightly decreased with the extension of degradation time. was smooth without cracks (Figure 11(b)). After 6 weeks of
After 12 weeks, the decrease in the buffer pH was intensified. degradation, a small amount of cracks could be observed
The pH value at 16 weeks was about 4.2, and after 16 weeks, in the middle of the fracture, although the fracture edge
the decrease in the pH value started to slow down. still showed compact structures (Figure 11(c)). It might be
related to that fact that the polymeric material first underwent
3.5. Changes in the Material Mechanical Properties in the autocatalytic degradation. After 8 weeks of degradation, the
Degradation Process. Figures 9 and 10, respectively, demon- cracks extended to peripheral areas and a large number of
strate the curves by which the bending strength and bending the small holes could be observed in the fracture surface
modulus of each sample of biological material changes with (Figure 11(d)), which might be caused by the degradation of
the degradation time in the process of degradation. As the interior of the material. This suggested that the PLLA
200 the material fracture surface (Figure 11(e)). When the degra-
dation time was further extended, the slits continued to
expand, and 18 weeks later, the PLLA material had collapsed,
180
and the material was destructed as a whole (Figure 11(f)).
Figure 12 shows the SEM images of the fracture surfaces
160 of the PLLA/n-HA material. Compared with the morphology
of the fracture surface of the pure PLLA material, the fracture
Flexural strength
surface of the PLLA/n-HA material showed dense structures

140
with only a small amount of cracks prior to 10 weeks of
degradation. After 14 weeks, the fracture surface showed
120 a large number of mesh-like holes, accompanied by the
generation of large cracks. In addition, exposed HA particles
could also be clearly seen, suggesting the occurrence of the
100
degradation and absorption of the PLLA/n-HA materials.
With the extension of the degradation time, the PLLA/n-
80 HA material underwent accelerated degradation, and criss-
cross cracks appeared on the fracture surface, although
exposed HA particles were not found. It suggested that with
60
0 2 4 6 8 10 12 14 16 18 20 the progress of the degradation, HA particles were already
Degradation time (week) dissolved in the PBS solution. After 20 weeks, the fracture
surface presented more depression, widened and deepened
PLLA
cracks, and loose structures. Large amounts of degradation
PLLA/n-HA
fragments could not be decomposed in a timely manner and
Figure 9: Variations of bending strength of biomaterials with degra- were thus filled into the cracks in the interior of the materials.
dation time. The composite materials showed significant degradation.
5
4. Discussion
The biggest advantage of biomedical polymeric materials is its
4.5
biodegradability and bioresorbabilty. Biocomposite materials
4 not only keep the features of the component materials, but
also can acquire new properties that individual compo-
3.5 nent materials do not have. Therefore, the comprehensive
properties of the composite materials are better than those
Flexural modulus
3 of the original component materials [10]. Biodegradability

refers to the process that the polymeric chains of solid-
2.5
state polymeric materials or devices are broken by the
2 complex physiological environment in vivo, leading to the
generation of large quantities of small molecule fragments
1.5 and the complete destruction of the polymeric materials
or devices. Bioresorbabilty refers to the process that solid-
1 state polymeric materials or devices are biodegraded in vivo
and that the resulting degradation products are absorbed or
0.5
excreted via metabolism.
0 Besides the general characteristics such as good bio-
0 2 4 6 8 10 12 14 16 18 20 compatibility and the lack of antigenicity, rejection reaction,
Degradation time (week) teratogenicity, carcinogenicity, and toxicity and side effects,
biodegradable polymeric composites that can be used as an
PLLA
PLLA/n-HA artificial bone tissue repair materials also need to have other
features as follows: (1) moderate degradation rate, that is, the
Figure 10: Effects of the degradation time on the bending modulus degradation rate of the artificial bone tissue repair material
of biomaterials. must match the rate of bone healing, (2) appropriate biome-
chanical properties, that is, the biomechanical properties of
the implanted artificial bone tissue repair material have to
materials had undergone partial degradation. After 12 weeks match the mechanical properties of the implant site [11].
of degradation, the cracks of the material expanded to form Hydroxyapatite (HA), whose chemical formula is
slits, providing passage for the water molecules, thus accel- Ca10 (PO4 )6 (OH)2 , is the major inorganic component of
erating the degradation of the PLLA materials. Many holes human bone. Sixty percent of HA in bone is needle-like
generated by the degradation process could be observed in and less than 100 nm in size [12]. TEM results demonstrated
(a) (b) (c)
(d) (e) (f)
Figure 11: SEM images of fracture surfaces of degraded PLLA biomaterials (×8000) ((a)-0 w; (b)-4 w; (c)-6 w; (d)-8 w; (e)-12 w; (f)-18 w). (a)
and (b) smooth fracture surface without substantial cracks; (c) Surfaces showed a small amount of cracks as red arrows indicated, and the
fracture edges still had compact structure; (d) the cracks in material surface extended to the peripheral area, and a large number of small
holes appeared in the fracture surface as red arrows indicated; (e) the cracks of the material expanded to form slits, and the fracture surface
was covered with holes as red arrows indicated; (f) materials had collapsed, and the material was destructed as a whole.
that the n-HA in this study was 50∼200 nm in size and studies, PLLA/n-HA was put into PBS to observe the water
showed needle-like appearance, which was close to that uptake ratio, weight loss ratio, and other biological properties.
of human bone. Results of XRD showed that PLLA and There are multiple interfaces between the respective
n-HA manufactured in this study are of good degree of components of the PLLA/n-HA composite material. The
crystallization. Furthermore, both PLLA and n-HA keep the interfaces exposed in the medium present the “suction effect”
integrated structure of crystal in the PLLA/n-HA composite. due to the capillary action. As a result, water molecules
Several methods have been used to prepare the PLLA/n- are prone to diffuse along the interfaces and enter into the
HA composite, such as coating method [13], solvent cast- inside of the material. Therefore, the water uptake ratio of
ing/particulate leaching [14], supercritical CO2 foaming [15], the PLLA/n-HA composite material is higher than that of the
solution blending method [16], and melt blending method. non-interfaced and closely combined pure PLLA material. In
Organic solvent is often used in the preparation of composites addition, the hydrophilic n-HA particles are more conducive
via the above methods except melt blending method. Besides for absorption and lead to the increase in the water uptake
its unfavorable effect on grafted tissues, organic solvent may ratio of the composite material.
inactivate biological growth factors and negatively influence As compared to the pure polymeric materials, composite
cell adherence and proliferation. In order to avoid the materials have a slower degradation rate. The specific reasons
negative effect of organic solvent, melt blending method was might include the following aspects. First, micropores among
used in this study. the pure PLLA material molecules provide channels for the
Varila et al. compared in vitro the reactivity of the penetration of water molecules, so that water molecules can
composites in simulated body fluid, Tris-buffered solution, easily get access to the interior of the material and accel-
and phosphate buffered saline (PBS). They concluded that erate the material degradation. Second, as to the PLLA/n-
degradation of the composites containing the bioactive HA composite material, although the water uptake of the
glasses was faster in PBS than in the two other solutions composite material is greater than that of the pure PLLA
[17]. Furthermore, PBS was often used as in vitro degradation material, the basic ions released by HA into the PBS buffer
system in other studies [9, 18–26]. In consistent with previous can neutralize the acidic substances generated in the PLLA
(a) (b) (c)
(d) (e) (f)
Figure 12: SEM images of fracture surfaces of degraded PLLA/n-HA biomaterials (×8000) ((a)-0 w; (b)-6 w; (c)-10 w; (d)-14 w; (e)-18 w; (f)-
20 w). (a) and (b) No significant changes on the fracture surface, which had no substantial cracks; (c) The fracture showed compact structure
with only a small number of cracks as red arrows indicated; (d) Fracture surface showed a large number of mesh-like holes, accompanied
by the generation of large cracks as red arrows indicated; (e) Criss-cross cracks on the fracture surface as red arrows indicated; (f) Fracture
surface showed more depression, widened and deepened cracks as red arrows indicated, loose structure, and significant degradation.
degradation process, slowing the autocatalytically accelerated polymeric PLLA/n-HA composite material has wide applica-
degradation of the polymeric material in acidic environment tion prospect and practical value.
and inhibiting the degradation of the polymeric material. The ions released from the dissolution of n-HA in the
As compared to the pure PLLA biological material, the PLLA/n-HA composite material are basic and can neutralize
degradation rate of the PLLA/n-HA composite material in the acidic degradable substances generated during the PLLA
the initial stages of degradation is slower, thus delaying the degradation process. The autocatalytic degradation effect
degradation of the PLLA/n-HA composite. Therefore, the of acids on the polymeric material can be alleviated, thus
weight loss of the composite material is smaller than that of slowing down the degradation rate. In the early phases
the pure polymer materials in the same period. of degradation, the composite material can still maintain
The incorporation of n-HA particles successfully reduces relatively high strength. After 6 weeks of degradation, the
the acidity caused by the acidic products of polylactic acid decrease rate in the composite material flexural strength and
degradation generated in the early phase of degradation. It modulus accelerated. After 10 weeks of degradation, the com-
thus can reduce or avoid the aseptic inflammation caused by posite material had a flexural strength of 151 MPa, equivalent
the acidic substance, and it can improve the biocompatibility to that of the fresh adult bone (160 Mpa), and it could still
of the polymeric biomaterial. In addition, in the early phase provide a good supporting role for the newly generated bone
of degradation, due to the slowing down of the degradation tissue. Thus, the PLLA/n-HA composite material prepared by
rate, the PLLA/n-HA composite material can maintain high the addition of n-HA can maintain relatively high flexural
initial mechanical strength and provide sufficient supporting strength and modulus in the early and middle phases of
effect for new bone tissue. In the late phase of degradation, the degradation. After 16 weeks of degradation, it was still able
degradation of the PLLA/n-HA composite material acceler- to maintain a flexural strength of 100 MPa, higher than the
ates, which is conducive to new bone tissue ingrowth in order minimum flexural strength of the human bones (96 Mpa),
to achieve good therapeutic effect. Therefore, the prepared thus providing a reliable support for the regeneration of new
bone tissue. In addition, the improvement in the acidic envi- rabbits,” Journal of Biomedical Materials Research B, vol. 66, no.
ronment can reduce or avoid the occurrence of inflammatory 2, pp. 539–547, 2003.
reaction caused by the acidic degradation products. In the late [5] L. MacArini, P. Milillo, A. Mocci, R. Vinci, and G. C. Ettorre,
phase of degradation, due to the degradation of the polymeric “Poly-L-lactic acid—hydroxyapatite (PLLA-HA) bioabsorbable
substance in the composite material, the amount of composite interference screws for tibial graft fixation in anterior cruciate
material is gradually reduced, leaving space for new generated ligament (ACL) reconstruction surgery: MR evaluation of
bone tissue. osteointegration and degradation features,” Radiologia Medica,
Changes in the material fracture surface morphology vol. 113, no. 8, pp. 1185–1197, 2008.
revealed that the degradation rate of the PLLA/n-HA material [6] M. Ngiam, S. Liao, A. J. Patil et al., “Fabrication of mineralized
was slower than that of the PLLA material. Most importantly, polymeric nanofibrous composites for bone graft materials,”
Tissue Engineering A, vol. 15, no. 3, pp. 535–546, 2009.
before the degradation time reached 14 weeks, there was little
degradation of the PLLA/n-HA material, and its mechanical [7] F. Peng, X. Yu, and M. Wei, “In vitro cell performance on
hydroxyapatite particles/poly(L-lactic acid) nanofibrous scaf-
strength loss was also small. It thus is able to maintain a
folds with an excellent particle along nanofiber orientation,”
high mechanical strength and provide a good supporting role
Acta Biomaterialia, vol. 7, no. 6, pp. 2585–2592, 2011.
for new bone tissue. This is mainly related to the following
[8] F. Peng, J. R. Olson, M. T. Shaw, and M. Wei, “Influence
reasons. (1) The addition of n-HA particles neutralizes the
of pretreatment on the surface characteristics of PLLA fibers
acidic products generated during the degradation process of and subsequent hydroxyapatite coating,” Journal of Biomedical
polymeric materials and slows down the PLLA autocatalytic Materials Research B, vol. 88, no. 1, pp. 220–229, 2009.
degradation. (2) It also effectively reduces the inflammatory [9] H. Zhao, L. Ma, Y. Gong, C. Gao, and J. Shen, “A polylac-
response caused by the acidic products and improves the tide/fibrin gel composite scaffold for cartilage tissue engineer-
biocompatibility of the composite materials. ing: fabrication and an in vitro evaluation,” Journal of Materials
The in vitro degradation characteristics of our PLLA/n- Science, vol. 20, no. 1, pp. 135–143, 2009.
HA composite material as mentioned above might be benefi- [10] D. D. Wright-Charlesworth, J. A. King, D. M. Miller, and C.
cial for its application in bone tissue repair. However, since the H. Lim, “In vitro flexural properties of hydroxyapatite and self-
body’s osteogenic process is very complicated, investigation reinforced poly(L-lactic acid),” Journal of Biomedical Materials
of the physicochemical properties of in vitro degradation Research A, vol. 78, no. 3, pp. 541–549, 2006.
alone cannot confirm its value in application. It is thus neces- [11] G. Sui, X. Yang, F. Mei et al., “Poly-L-lactic acid/hydroxyapatite
sary to carry out a series of follow-up experiments focusing hybrid membrane for bone tissue regeneration,” Journal of
on the in vivo bone tissue repair and construction, coupling Biomedical Materials Research A, vol. 82, no. 2, pp. 445–454,
with the bone growth factors and co-culture with osteoblasts 2007.
in vitro. [12] M. A. Malik, D. A. Puleo, R. Bizios, and R. H. Doremus,
“Osteoblasts on hydroxyapatite, alumina and bone surfaces in
vitro: morphology during the first 2 h of attachment,” Biomate-
Acknowledgments rials, vol. 13, no. 2, pp. 123–128, 1992.
The authors acknowledge the Shenzhen Key Laboratory [13] T. Tian, D. Jiang, J. Zhang, and Q. Lin, “Fabrication of bioactive
composite by developing PLLA onto the framework of sintered
Enhancement Projects (CXB201104220049A), Shenzhen-
HA scaffold,” Materials Science and Engineering C, vol. 28, no. 1,
Hong Kong Innovation Circle Project Fund pp. 51–56, 2008.
(JSE201007190007A), Shenzhen Second People’s Hospital
[14] T. Kasuga, Y. Ota, M. Nogami, and Y. Abe, “Preparation and
Seeding Plan for providing the funding source for this work. mechanical properties of polylactic acid composites containing
hydroxyapatite fibers,” Biomaterials, vol. 22, no. 1, pp. 19–23,
References 2001.
[15] C. Delabarde, C. J. G. Plummer, P.-E. Bourban, and J.-A. E.
[1] C. Zhang, Q. Feng, T. Zhang, J. Chen, C. Lu, and H. Wu, “In vitro Månson, “Biodegradable polylactide/hydroxyapatite nanocom-
biologic evaluation on nano-hydroxyapatite/poly (L-lactic acid) posite foamscaffolds for bone tissue engineering applications,”
biocomposites fabricated using in-situ growth method,” Sheng Journal of Materials Science, vol. 23, no. 6, pp. 1371–1385, 2012.
Wu Yi Xue Gong Cheng Xue Za Zhi, vol. 29, no. 2, pp. 307–310, [16] X. Deng, J. Hao, and C. Wang, “Preparation and mechanical
2012. properties of nanocomposites of poly(D,L-lactide) with Ca-
[2] H. K. Moon, Y. S. Choi, J.-K. Lee, C.-S. Ha, W.-K. Lee, deficient hydroxyapatite nanocrystals,” Biomaterials, vol. 22, no.
and J. A. Gardella Jr., “Miscibility and hydrolytic behavior of 21, pp. 2867–2873, 2001.
poly(trimethylene carbonate) and poly(Ll-lactide) and their [17] L. Varila, T. Lehtonen, and J. Tuominen, “In vitro behaviour of
blends in monolayers at the air/water interface,” Langmuir, vol. three biocompatible glasses in composite implants,” Journal of
25, no. 8, pp. 4478–4483, 2009. Materials Science, vol. 23, no. 10, pp. 2425–2435, 2012.
[3] X. Niu, Q. Feng, M. Wang, X. Guo, and Q. Zheng, “Porous nano- [18] S. Zhou, B. Song, and X. Li, “In vitro degradation and release
HA/collagen/PLLA scaffold containing chitosan microspheres profiles for poly-dl-lactide film containing paracetamol,” Jour-
for controlled delivery of synthetic peptide derived from BMP- nal of Materials Science, vol. 18, no. 8, pp. 1623–1626, 2007.
2,” Journal of Controlled Release, vol. 134, no. 2, pp. 111–117, 2009. [19] S. Kobayashi and K. Sakamoto, “Effect of hydrolysis on mechan-
[4] S. Ishii, J. Tamura, T. Furukawa et al., “Long-term study of high- ical properties of tricalcium phosphate/poly-L-lactide compos-
strength hydroxyapatite/poly(L-lactide) composite rods for the ites,” Journal of Materials Science, vol. 20, no. 1, pp. 379–386,
internal fixation of bone fractures: a 2-4-year follow-up study in 2009.
[20] M. Mehdikhani-Nahrkhalaji, M. H. Fathi, V. Mortazavi, S. B.

Mousavi, B. Hashemi-Beni, and S. M. Razavi, “Novel nanocom-
posite coating for dental implant applications in vitro and in vivo
evaluation,” Journal of Materials Science, vol. 23, no. 2, pp. 485–
495, 2012.
[21] T. Niemelä, H. Niiranen, and M. Kellomäki, “Self-reinforced
composites of bioabsorbable polymer and bioactive glass with
different bioactive glass contents. Part II: in vitro degradation,”
Acta Biomaterialia, vol. 4, no. 1, pp. 156–164, 2008.
[22] N. A. Weir, F. J. Buchanan, J. F. Orr, and G. R. Dickson,
“Degradation of poly-L-lactide. Part 1: in vitro and in vivo physi-
ological temperature degradation,” Proceedings of the Institution
of Mechanical Engineers H, vol. 218, no. 5, pp. 307–319, 2004.
[23] S. Chen, Y. Hao, and W. Cui, “Biodegradable electrospun PLLA/
chitosan membrane as guided tissue regeneration membrane
for treating periodontitis,” Journal of Materials Science, vol. 48,
no. 19, pp. 6567–6577, 2013.
[24] J. Russias, E. Saiz, R. K. Nalla, and A. P. Tomsia, “Microspheres
as building blocks for hydroxyapatite/polylactide biodegradable
composites,” Journal of Materials Science, vol. 41, no. 16, pp.
5127–5133, 2006.
[25] L. M. Ehrenfried, M. H. Patel, and R. E. Cameron, “The effect
of tri-calcium phosphate (TCP) addition on the degradation of
polylactide-co-glycolide (PLGA),” Journal of Materials Science,
vol. 19, no. 1, pp. 459–466, 2008.
[26] Z. Zhou, Q. Yi, X. Liu, L. Liu, and Q. Liu, “In vitro degradation
behaviors of poly-L-lactide/bioactive glass composite materials
in phosphate-buffered solution,” Polymer Bulletin, vol. 63, no. 4,
pp. 575–586, 2009.
http://dx.doi.org/10.1155/2013/138083
Research Article
A Study on the Effect of Plasma Treatment for
Waste Wood Biocomposites
MiMi Kim, Heung Soo Kim, and Joong Yeon Lim

Department of Mechanical, Robotics and Energy Engineering, Dongguk University-Seoul, 26 Pil-dong 3-ga, Jung-gu,
Seoul 100-715, Republic of Korea
Correspondence should be addressed to Joong Yeon Lim; jylim@dongguk.edu
Received 12 June 2013; Accepted 3 July 2013
Copyright © 2013 MiMi Kim et al. This is an open access article distributed under the Creative Commons Attribution License,
The surface modification of wood powder by atmospheric pressure plasma treatment was investigated. The composites were
manufactured using wood powder and polypropylene (wood powder: polypropylene = 55 wt% : 45 wt%). Atmospheric pressure
plasma treatment was applied under the condition of 3 KV, 17±1 KHz, 2 g/min. Helium was used as the carrier gas and hexamethyl-
disiloxane (HMDSO) as the monomer to modify the surface property of the waste wood biocomposites by plasma polymerization.
The tensile strengths of untreated waste wood powder (W3) and single species wood powder (S3) were about 18.5 MPa and 21.5 MPa
while those of plasma treated waste wood powder (PW3) and plasma treated single species wood powder (PS3) were about 21.2 MPa
and 23.4 MPa, respectively. Tensile strengths of W3 and S3 were improved by 14.6% and 8.8%, respectively. From the analyses
of mechanical properties and morphology, we conclude that the interfacial bonding of polypropylene and wood powder can be
improved by atmospheric pressure plasma treatment.
1. Introduction the productivity of WPCs. Moreover, coupling agents are

chemical materials that are not environment friendly. There-
Governmental regulations and growing environmental fore, new technologies have been developed to produce
awareness worldwide have triggered a paradigm shift towards environment friendly materials from biomass resources. The
designing materials compatible with the environment [1]. interfacial adhesion in biocomposites can be enhanced by two
Biocomposites are most actively investigated as environment types of methods: one type modifies the base polymers and
friendly materials [2]. Biofibers or natural fibers have been another type treats the biodegradable fillers to increase their
generally used as reinforcements for biocomposites. The hydrophobic properties. The latter type includes methods
matrices of biocomposites are biodegradable or nonbiode- such as corona treatment, plasma treatment, heating, and so
gradable polymers that can be extracted from natural forth [5]. Plasma treatment changes the chemical structure
resources instead of existing oil resources [3]. and surface properties of hydrophilic natural fibers to give
Wood plastics composites (WPCs) are made of wood them hydrophobic properties, which would enhance their
powder. Composites are made by combining two materials interfacial adhesion with the base polymer [6, 7].
to obtain better performance than either of the original ones For mass production of biocomposites, atmospheric
while the original phases of these materials are retained pressure plasma was used in this study instead of vacuum
[4]. Strong interfacial adhesion between fibers and matrices, plasma. Biocomposites production in an atmospheric pres-
however, is very important in making composites with sure plasma is a dry process that has many advantages
high mechanical properties. For production of WPCs, the such as low contamination, energy savings, and enhanced
two materials that are to be combined must be treated by interfacial adhesion [8, 9]. It also removes foreign matters
coupling agents because of the incompatibility between the by making chemical reactions only on surfaces of polymer
hydrophilic wood powder and the hydrophobic polymer. while preserving the basic physical properties of the polymer
However, the high prices of coupling agents have reduced [10]. Plasma processes, which have been usually carried out
in vacuum, are now carried out at atmospheric pressure, and A film type of polyethylene terephthalate (PET) was produced
these atmospheric pressure plasma processes have become using a press. The plasma treatment was conducted using a
very popular because of their reduced production cost and vacuum plasma. The monomer used in the experiment was
shortened process time. Recently, the application of atmo- evaporated and then used to modify the PET film surface. Ten
spheric pressure plasma processes has been extended. Plasma microliters of water and glycerol droplets was dropped on the
processes in vacuum, on the other hand, take a long time processed film using the sessile drop method, and then the
because of the less numbers of electrons and ions in vacuum contact angle was measured. All contact angle measurements
plasma than in atmospheric pressure plasma, and they also were repeated three times to obtain the mean value and the
have difficulty in providing stable and uniform modification. deviation.
In this study, we tried to make biocomposites with
increased interfacial adhesion between the wood power and
2.3. Atmospheric Pressure Plasma Treatment. In the atmo-
the polypropylene (PP) of hydrophobic thermoplastic poly-
spheric pressure plasma treatment for the modification of the
olefin polymers by modifying the surfaces of the hydrophilic
wood powder surface, helium was used to make the plasma
wood powder particles. To achieve this objective, a lab scale
generation atmosphere and as the carrier gas. Wood powder,
chamber was made. A screw was inserted in the chamber
injected through a hopper, was moved by a screw inserted at
to move the wood powder. Electrodes were installed outside
the chamber. The plasma generated between the two types
the chamber so that the wood powder could move and
of electrodes surrounding the chamber treated the wood
plasma processes could be carried out by rotating the screw.
powder being moved by the screw. At this time, the plasma
Continuity and good productivity were achieved.
treatment condition was 3 kV at 17 ± 1 kHz. Figure 1 shows
The surfaces of the wood powder particles are modified
schematic of the atmospheric pressure plasma treatment.
by plasma processes as follows. First, electrons and gas
molecules collide in the plasma. When an electron collides
with a molecule XY, an electron escapes from the molecule 2.4. Composite Processing. For the mechanical mixing of
XY and the molecule becomes ionized. Sometimes, the bond wood powder and polypropylene, pellets were produced at
of the molecule XY is broken, and the molecule divides 210∘ C at 180 rpm using a twin screw extruder. The wood
into X and Y. The divided molecules X and Y are then powder content in the biocomposites was fixed at 55 wt%.
likely to interact with the many unbounded electrons. These The dispersion of the wood powder in PP was increased by
molecules are called radicals or chemically-active species. carrying out the extrusion process three times. The pellets
Through the chemical reactions of the ionized electrons in produced were dried for 24 hours at 60∘ C, and then, the
collisions in the plasma, wood powder becomes coated and specimens whose mechanical properties were to be evaluated
the dispersion components on the surfaces are increased. were injected by the injection molding machine.
Then, the surfaces of the wood power particles are modified
to have hydrophobicity. To investigate such effects of surface
modification, experiments were carried out for waste wood 2.5. Mechanical Properties. The tensile test was conducted at
powder and single species wood powder. If the interfa- 5 mm/min at room temperature using a 100 kN load accord-
cial adhesion of waste wood powder with polypropylene ing to ASTM D638, while the flexural test was conducted
is enhanced by surface modification, the strength of the at 3.4 mm/min at room temperature using a 100 kN load
waste wood biocomposites would increase and the waste according to ASTM D790. Five specimens were tested for
wood powder could be used as a recycled raw material of each experiment. The average value of the measurements was
biocomposites. calculated and used for analysis.
2. Experimental Procedure 3. Results and Discussion
2.1. Materials. Two types of wood powder were used: one 3.1. Surface Free Energy. The intermolecular forces acting
was a single species powder of spruce and the other was a on the surface of the polymer can be divided into three
waste wood powder of crushed, construction waste wood categories: primary (chemical) force, quasi-chemical (hydro-
chips. Wood powder was extracted from a 60–80 mesh sieve. gen bond) force, and secondary (Van der Waals) force. The
Before wood powder was treated by plasma, it was dried in secondary force is again subdivided into Keesom force, Debye
vacuum at moisture content of less than 1%. Hexamethyl- force, and London dispersion force, which is much larger
disiloxane (HMDSO) was used in the plasma treatment to than the other two Van der Waals forces. Surface tension
modify the surface properties of the wood powder to make is surface free energy per unit area, thermodynamically.
it hydrophobic. We used H1501 Polypropylene (LG Chem.) Interfacial tension is created by cohesion and adhesion, which
are the results of intermolecular forces at the interface of two
of less than 3 mm in diameter, 0.9 g/cm3 in density, and
different molecules. Since the free energy of a solid surface
17 g/10 min in melting index.
cannot be measured directly, unlike that of a liquid, it is
calculated indirectly using a liquid whose surface tension is
2.2. Contact Angle. A series of experiments were conducted known.
to select the monomer that would be used to modify the Surface energy can be expressed in Young-Dupre’ equa-
hydrophilic wood powder surface into a hydrophobic surface. tion as follows.
Wood powder
input
Chamber
Electrode
He + HMDSO
He
He
Wood powder
output
A.C. power supply
HMDSO
Bubbler
Figure 1: Schematic of atmospheric pressure plasma treatment.
Young equation: Table 1: Contact angle and surface energy of PET film.
𝛾SV = 𝛾SL + 𝛾LV cos 𝜃. (1) Contact angle (∘ ) Surface energy (dyne/cm)
Monomer
Water Glycerol Total Dispersion Polar
Dupre equation: Oxygen/PET 8 9 73.73 12.78 60.96
𝑊𝐴 = 𝛾LV + 𝛾SV − 𝛾SL . (2) Benzene/PET 58.8 65 49.17 1.08 47.37
CH4 /PET 55.7 47.6 59.56 2.14 57.42
Young-Dupre equation: Acrylic acid/PET 35.7 47.7 71.94 1.72 70.22
HMDSO/O2
𝑊𝐴 = 𝛾LV (1 + cos 𝜃) . (3) 28 41 75.74 2.54 73.21
(1 : 9)/PET
In addition, it can be expressed as the sum of polar compo- HMDSO/O2 56.5 60 47.25 4.32 42.93
nents and dispersion components. Consider the following: (2 : 8)/PET
HMDSO/O2 66 68.8 38.03 4.39 33.64
1/2 𝑝 1/2 (3 : 7)/PET
𝛾SL = 𝛾SV + 𝛾LV − 2(𝛾𝑆𝑑 𝛾LV
𝑑
) 𝑃
− 2(𝛾𝑆 𝛾LV ) . (4)
HMDSO/O2 72 73 32.39 4.57 27.81
(4 : 6)/PET
Contact angle is a measure that represents the wettability
of a solid surface. It can be calculated by measuring the HMDSO/O2 73.5 73.5 30.64 5.43 25.21
(5 : 5)/PET
contact angle of a water droplet on the PET film surface-
modified by plasma treatment. In general, a low contact HMDSO/PET 105 76 74.77 71.68 3.09
angle indicates high wettability (hydrophilic property), and
a high contact angle represents low wettability (hydrophobic
property). Figure 2 shows equilibrium contact angle of a
solid/liquid surface. component among the surface energy of the film after
Table 1 provides the measured contact angle and surface HMDSO plasma treatment was very low, which was also
energy of the PET film. Experimental results showed that verified by measurement of the contact angle. However,
the plasma-treated film had a low contact angle and a oxygen mixing ratio also plays an important role in plasma
high surface energy but that the polar components were treatments using HMDSO. The values of the plasma-treated
much higher than the dispersion components. These results polar component and dispersion component obtained by
confirm the modification of the film surface into that having mixing HMDSO and oxygen at a mixing ratio of 1 : 9 showed
the hydrophilic property. the opposite of those when treated using HMDSO alone. As
This experiment aims to modify the hydrophilic surface a result, it was found that HMDSO was most effective when
of wood powder into a hydrophobic surface. The suitable modifying a surface to have hydrophobic property, but the
monomer is hexamethyl-disiloxane (HMDSO). The polar results depend on the amount of oxygen input.
Vapor
5
Liquid 𝛾LV
Tensile modulus (GPa)

4
𝜃
3
𝛾SL 𝛾SV
2
Solid
1
Figure 2: Equilibrium contact angle of solid/liquid surface.
0
W3 PW3 S3 PS3
25
W3-waste wood powder
PW3-waste wood powder by plasma treatment
20
S3-single species wood powder
Tensile strength (MPa)
PS3-single species wood powder by plasma treatment

15 Figure 4: Tensile modulus of composites.
10 Table 2: Flexural strength and flexural modulus of composites.
W3 PW3 S3 PS3
5
Flexural strength (MPa) 36.0 39.5 41.1 42.6
Flexural modulus (GPa) 3.8 4.0 4.3 4.5
0
W3 PW3 S3 PS3
nontreated waste wood powder. The plasma-treated single
W3-waste wood powder species wood powder also showed an increase in tensile
PW3-waste wood powder by plasma treatment strength. These results confirm that atmospheric pressure
S3-single species wood powder plasma treatment is effective in the surface modification of
PS3-single species wood powder by plasma treatment
wood powder. The tensile modulus of the single species wood
Figure 3: Tensile strength of composites. powder was higher than that of the waste wood powder. They
were also increased by plasma treatment.
3.2. Tensile Strength. Figures 3 and 4 show the effect of plasma 3.3. Flexural Strength. The flexural strength and flexural
treatment on the tensile strength and tensile modulus of modulus of the composites according to the type of wood
the composites according to the type of wood powder. The powder are summarized in Table 2. The flexural strength of
tensile strength of the composites formed from the single the composite made from the single species wood powder was
species wood powder was 21.5 MPa, which is 16.2% higher 41.1 MPa, which is 14.2% higher than that of the composite
than that of the composites formed from the waste wood made from the waste wood powder (36.0 MPa). It was also
powder (18.5 MPa). The reason why the tensile strength of observed that the flexural strength of the composite made
the composites made of the waste wood powder was lower from the waste wood powder with surface modification
than that of the composites made of the single species wood by atmospheric pressure plasma treatment was 39.5 MPa,
powder was due to the foreign matters in the waste wood which is an increase of 9.7% from that made from the
powder. These foreign matters come from the surface overlay, nontreated waste wood powder. In addition, flexural moduli
coating and adhesives of living wood waste and construction also increased in the single species wood powder.
waste woods, which are the raw materials of waste wood
powder. The foreign matters make recycling waste wood 3.4. SEM Images. Figures 5 and 6 present the cross-sections of
difficult. The strength of waste wood powder should be the composites observed by SEM. In the case of the composite
improved to broaden its application. The surface of waste made of the nontreated wood powder, voids were observed
wood powder can be modified to increase the strength of at the interface between the wood powder and polymer.
waste wood composites. The experimental results showed However, voids were not observed at the interface in the case
that the tensile strength of waste wood powder after surface of the composite made of the plasma-treated wood powder.
modification by the atmospheric pressure plasma treatment These results showed that the hydrophilic surface of the wood
was 21.2 MPa, which is an increase of 14.6% from that of the powder was modified into a hydrophobic surface, which
Weak interfacial adhesion the surface energy calculated by measurement of the

contact angle.
(3) Tensile strengths of the composites made of the
waste wood powder and single species wood powder
were 18.5 MPa and 21.5 MPa, respectively. The tensile
strength of the single species wood powder compos-
ites was 16.2% higher than that of the waste wood
powder composites. Foreign matters on the wood
powder surface decreased the tensile strength of the
waste wood powder composites. This is why plasma
treatment is required for recycling waste wood.
100 𝜇m (4) When the surface of the waste wood powder was
modified by atmospheric pressure plasma treatment,
the tensile strength increased by 14.6% from 18.5 MPa
Figure 5: SEM micrograph of nontreated composites’ cross-section. to 21.2 MPa and the flexural strength also increased
by 9.7%, from 36.0 MPa to 39.5 MPa. In the case of
the single species wood powder, the tensile strength
Strong interfacial adhesion
increased by 8.8% and flexural strength increased by
3.6%. These results confirm that surface modification
by atmospheric pressure plasma treatment is effective
for increasing the strength of both single species wood
powder and waste wood powder.
(5) Through surfaces’ modification by plasma treatment,
the mechanical properties of the waste wood pow-
der became similar to those of the single species
wood powder without modification. Therefore, it is
expected that waste wood powder can be recycled as
raw biomaterials by various treatment processes.
100 𝜇m
Acknowledgments
Figure 6: SEM micrograph of plasma-treated composites’ cross- This research was supported by the Ministry of Land,
section. Infrastructure and Transport and the Korea Agency for
Infrastructure Technology Advancement.
helped to improve the interfacial adhesion with polymer,

which is hydrophobic. The experimental results of tensile and
References
flexural strengths also provided the same tendency. [1] A. K. Mohanty, M. A. Khan, and G. Hinrichsen, “Surface
modification of jute and its influence on performance of
biodegradable jute-fabric/Biopol composites,” Composites Sci-
4. Conclusion ence and Technology, vol. 60, no. 7, pp. 1115–1124, 2000.
The strengths of waste wood biocomposites were investigated [2] S.-Y. Park, G.-S. Han, H.-S. Kim, H.-S. Yang, and H.-J. Kim,
for recycling waste wood. The surfaces of the wood powders “Evaluation of the impact on manufacturing temperature and
were modified to enhance their interfacial adhesion with time in the production process of bio-composites,” Mokchae
polypropylene by atmospheric pressure plasma treatment. Konghak, vol. 33, no. 1, pp. 29–37, 2005.
Several important observations were made from the tensile [3] T. Takayama, K. Komabayasi, M. Itou, and Y. Miyake, “Devel-
and flexural strength tests as follows. opment of Bio-based plastics for injection molding,” SAE
International Journal of Materials and Manufacturing, vol. 2, no.
(1) Although the total energy of the surfaces was 1, pp. 12–17, 2009.
high by the atmospheric pressure plasma treatment [4] C.-H. Kim, K.-J. Kim, and T.-J. Eom, “Properties of WPC
using monomers, the surfaces were modified into prepared with various size and amount of wood particle,” Palpu
hydrophobic or hydrophilic surfaces depending on Chongi Gisul/Journal of Korea Technical Association of the Pulp
polar or dispersed components. and Paper Industry, vol. 40, no. 3, pp. 59–64, 2008.
[5] B.-H. Lee, H.-S. Kim, S.-W. Choi, and H.-J. Kim, “Improvement
(2) The most efficient monomer for the modification of of interfacial adhesion for surface treated rice husk flour-filled
the hydrophilic wood powders was HMDSO, which polypropylene bio-composites,” Mokchae Konghak, vol. 34, no.
had the lowest polar component (3.09 dyne/cm) in 3, pp. 38–45, 2006.
[6] T. S. Lee, S. G. Lee, and K. H. Song, “Natural fiber reinforced

biocomposites and biodegradation,” Fashion Information and
Technology, vol. 7, pp. 10–21, 2010.
[7] S. Singh, D. Y. Sasaki, J. Cesarano III, and A. J. Hurd, “Nanome-
ter pores in ultrathin silica films prepared by self-assembly of
organic spacers in an alkylsiloxane monolayer,” Thin Solid Films,
vol. 339, no. 1-2, pp. 209–215, 1999.
[8] J. Lai, B. Sunderland, J. Xue et al., “Study on hydrophilicity
of polymer surfaces improved by plasma treatment,” Applied
Surface Science, vol. 252, no. 10, pp. 3375–3379, 2006.
[9] V. Kotal, V. Svorcık, P. Slepicka et al., “Gold coating of
poly(ethyleneterephthalate) modified by argon plasma,” Plasma
Process and Polymers, vol. 4, no. 1, pp. 69–76, 2007.
[10] S.-H. Seo, S. Chang, Y.-E. Yoo, and J. D. Chung, “Surface char-
acteristics of polymer material treated by atmospheric pressure
plasma,” Korean Journal of Air-Conditioning and Refrigeration
Engineering, vol. 22, no. 5, pp. 282–288, 2010.
http://dx.doi.org/10.1155/2013/479109
Research Article
Water Absorption and Diffusion Characteristics of
Nanohydroxyapatite (nHA) and
Poly(hydroxybutyrate-co-hydroxyvalerate-) Based Composite
Tissue Engineering Scaffolds and Nonporous Thin Films
Naznin Sultana1,2 and Tareef Hayat Khan3

1
Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong
2
IJN-UTM Cardiovascular Engineering Centre (CEC), Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia,
81310 UTM Johor Bahru, Johor, Malaysia
3
KALAM, Faculty of Built Environment, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
Correspondence should be addressed to Naznin Sultana; naznin@biomedical.utm.my
Received 15 March 2013; Revised 10 April 2013; Accepted 10 April 2013
Copyright © 2013 N. Sultana and T. H. Khan. This is an open access article distributed under the Creative Commons Attribution
cited.
Water uptake characteristics of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV-) based composite tissue engineering (TE)
scaffolds incorporating nanosized hydroxyapatite (nHA) have been investigated. The water absorption of these composite scaffolds
obeyed the classical diffusion theory for the initial period of time. The diffusion coefficients of the composite scaffolds during the
water absorption were much faster than those for the nonporous thin films, suggesting that the water uptake process depends on the
presence of porosity and porous microstructure of the composite scaffolds. The incorporation of nHA increased the water uptake
of both the composite scaffolds and thin films. It was also observed that the equilibrium uptake increased with the incorporation
of nHA. This increase in the water uptake was largely due to the nHA particle aggregates in the microstructure of both composite
scaffolds and thin films. The activation energy for diffusion was also determined using the Arrhenius equation for both porous
scaffolds and thin films and the results suggested that the activation energy for scaffolds was lower than that for thin films.
1. Introduction Both PLLA and PHBV possess good biocompatibility with

tissue and blood and are regarded as good candidates as
Through the combination of cell biology, biomaterials, and biodegradable polymer matrix of composite scaffolds [6].
engineering, tissue engineering (TE) seeks to provide a Blending of PHBV with PLLA can shorten the degradation
solution to replace, repair, or regenerate tissues/organs [1, time and rate of PHBV which has much longer degradation
2]. To overcome the limitations of the autogenous and time [7, 8]. Several biodegradable polymer-hydroxyapatite
allogenic bone graft, development of new synthetic scaffolds (HA) composites have been developed as bone substitute
has received increasing interest. In TE, the biomaterial based material or bone TE scaffolds [9–11]. As HA is a natural
scaffolds are the most important aspects [3, 4]. A number of component of bones and possesses the osteoconductive prop-
approaches such as phase separation, selective laser sintering, erties, polymer-HA composite scaffold is a promising system
and electrospinning are being used for fabricating scaffolds for bone tissue regeneration as it can mimic the composition
for bone regeneration. Biodegradable polymers, including and structure with the mineral phase of the bone.
poly(caprolactone), poly(l-lactic acid) (PLLA), and poly(hy- By guiding new tissue growth in vivo and in vitro,
droxybutyrate-co-hydroxyvalerate) (PHBV), have been pro- scaffolds play an important role in TE. The scaffolds pos-
cessed into 3D scaffolds with the development of TE [5]. sess 3D macroporous and interconnected network with
the macropore diameter of a few hundreds of microme-

ters to allow cell invasion. The water uptake properties
of composite scaffolds for tissue TE are of importance as
the primary mechanism of the polymer degradation inside
body is hydrolysis. The ingress of water into polymer-based
scaffolds can have both adverse and beneficial effects on
the properties of the scaffolds. Hydrolysis and microcrack
can be formed due to water exposure. On the other hand,
breakdown of three-dimensional scaffolds can occur due to
excessive water uptake. Several studies have been performed
for dental application to study the characteristics of polymeric
systems and composites [12, 13]. However, the water uptake
and diffusion characteristics of TE scaffolds have been rarely
assessed and reported. Figure 1: SEM micrograph of nanosized HA particles synthesized
Previously, we reported the fabrication, characteriza- in-house through a nanoemulsion process.
tion, and in vitro biological evaluation of PHBV/PLLA-
based scaffolds [14]. The aim of the present study was to
determine the water uptake and diffusion characteristics
of 100/0 PHBV/PLLA, 50/50 PHBV/PLLA blend, and 10%
nHA incorporated 50/50 PHBV/PLLA composite scaffolds. 2.3. Preparation of Polymer and Composite Specimens. Rect-
For comparison, solvent-cast thin films were also used in angular shape specimens (20 mm × 5 mm × 0.5 mm) were
the study. The effect of incorporation of HA, porosity, and cut with sharp razor blade from three types of polymer
microstructures of the composite scaffolds on the diffusion and composite scaffolds, namely, PHBV scaffolds (10% w/v),
coefficient, equilibrium uptake, and temperature dependence 50/50 PHBV/PLLA (10% w/v) blend scaffolds, and 10% nHA
was also investigated. in PHBV/PLLA (10% w/v) composite scaffolds. Rectangular
specimens of (20 mm × 5 mm × 0.5 mm) were also pre-
pared from solvent-cast films of the same compositions. The
2. Experimental porous microstructure, morphology, and pore sizes of the
scaffolds specimens were studied using a scanning electron
2.1. Materials and General Methods. PHBV with 6% of 3- microscopy (SEM; Stereoscan 440, UK). In order to deter-
hydroxyvalerate was purchased from Sigma-Aldrich. In order mine the presence and distribution of nHA nanoparticles in
to blend with PHBV, PLLA with viscosity 1.6 dL g−1 (Medis- composite scaffolds, energy dispersive X-ray spectrometry
orb 100L 1A) was purchased from Lakeshore Biomaterials (EDX, INCA 300, UK) was performed. The skeletal density
(Birmingham, AL). The nHA nanoparticles were produced and the porosity of the scaffolds were measured using
in-house which were used for producing composite scaffolds equations described elsewhere [16]. The wettability of the
and composite thin films [15]. Figure 1 shows the SEM scaffolds was measured by measuring contact angles using a
micrograph of nanosized HA. The freeze-dried HA powders contact angle measuring machine (SL200B, Shanghai Salon
used in this investigation consisted of tiny agglomerates of Tech Inc., Ltd., China). With distilled water as liquid, the
HA nanocrystallites. The particle size of the HA powders was sessile drop method was employed. The contact angle of
found to be in the range of 20–30 nm. Chloroform and acetic the water drop on the scaffold specimen was determined at
acid were analytical grade. room temperature using proprietary software. At least three
measurements were conducted at different locations of the
scaffold specimen. As the mean ± standard deviation, the
2.2. Fabrication of Scaffolds and Solvent Cast Films. Pure contact angle was expressed.
100/0 PHBV/PLLA, 50/50 PHBV/PLLA, and 10% nHA in
50/50 PHBV/PLLA tissue engineering scaffolds with 10%
(w/v) polymer concentration were fabricated using the emul- 2.4. Water Absorption. The specimens were placed in an oven
sion freezing/freeze-drying technique described elsewhere at 37∘ C for 48 hours and then weighed to an accuracy of
[16]. Briefly, to produce polymer scaffolds, 1 g polymer or ±0.0001 g on a balance. The specimens were then placed into
polymer blend was dissolved in 5 mL chloroform and then 100 mL of distilled water at 37∘ C. During the first day of
5 mL acetic acid was added. The emulsion was then homog- immersion, the specimens were removed at intervals of 2,
enized and kept frozen at −18∘ C in a freezer for overnight 5, 10, 15 min, and so forth, blotted dry on filter paper to
to induce phase separation. Then the frozen emulsion was remove excess water, weighed, and returned to the water.
freeze-dried using a freeze-drying vessel (Labconco, USA). Following the first day, the samples were weighed daily, until
Similarly, to produce 10% nHA in 50/50 PHBV/PLLA com- the uptake slowed. Until there was no significant change in
posite scaffolds, 0.1 g nHA was added in the emulsion and weight, the uptake of water was recorded. When equilibrium
homogenized prior to the phase-separation process. Non- was attained, the samples were then transferred to a drying
porous thin films with the same compositions were fabricated oven at 37∘ C. Similarly, the experiment was repeated at 25∘ C
using solvent casting technique. and at 50∘ C.
2.5. Diffusion Coefficients. For the earlier stages of uptake Table 1: Average pore diameter, porosity, and contact angle of the
(usually where 𝑀𝑡 /𝑀∞ ≤ 0.5), classical diffusion theory scaffold.
predicts [1, 17] the following: Average pore Contact
Scaffold Porosity %
diameter (𝜇m) angle
𝑀𝑡 𝐷𝑡 1/2 75 ± 2.0 103 ± 1.6
= 2( 2 ) , (1) 100/0 PHBV/PLLA 290
𝑀∞ 𝜋𝑙 50/50 PHBV/PLLA 271 77 ± 1.5 98 ± 2.5
10% nHA in 50/50
where 𝑀𝑡 is the mass uptake at time 𝑡, 𝑀∞ is the mass 250 72 ± 3.0 83 ± 1.5
PHBV/PLLA
uptake at equilibrium, 2𝑙 is the thickness of the specimen, 𝐷
is the diffusion coefficient, and 𝑡 is the water uptake time. A
plot of 𝑀𝑡 /𝑀∞ against 𝑡1/2 should provide a straight line for 3.2. Diffusion Study of Solvent-Cast Thin Films. Figure 5
earlier stages of water uptake. Diffusion coefficients can be shows the water uptake curve of 100/0 PHBV/PLLA thin
calculated from the slope of the straight line. film at 37∘ C as 𝑀𝑡 /𝑀∞ versus the square root of immersion
The temperature dependence of the diffusion coefficient time. The specimens were immersed in distilled water. The
is given by the Arrhenius equation: maximum uptake at equilibrium was 1.4%. The diffusion
coefficients were calculated from the initial linear region of
𝑄 1
ln 𝐷 = ln 𝐷0 − ( ), (2) the curves.
𝑅 𝑇 In order to observe the temperature dependence of the
diffusion coefficients of 100/0 PHBV/PLLA thin film, the
where 𝑇 is the absolute temperature (∘ K), 𝑄 is the activation
experiment was performed at three different temperatures,
energy for diffusion (J/mol or eV/atom), and 𝐷𝑜 is a temper-
namely, at 25∘ C, 37∘ C, and 50∘ C. Water uptake study was also
ature independent constant (preexponential) (m2 /s), 𝑅 is the
performed for 50/50 PHBV/PLLA thin film and 10% HA in
gas constant (8.314 J/mol-K or 8.62 × 10−5 eV/atom).
50/50 PHBV/PLLA thin film following the same procedure
mentioned above. The calculated diffusion coefficients are
3. Results and Discussion given in Table 2. It can be seen that diffusion coefficient is
significantly higher at high temperature of 50∘ C than that of
Several assumptions were used for this study. They were as 25∘ C, 37∘ C.
follows. In order to study the temperature dependence of diffusion
coefficients, Arrhenius plot was constructed for 10% HA in
(i) Distilled water was in infinite supply. 50/50 PHBV/PLLA thin films as shown in Figure 6. From
(ii) Polymer and composite films and scaffolds were the slope of the curve, activation energy can be calculated
surrounded by water. which is referred to as the energy required to produce the
diffusive motion of one mole of atoms. The activation energy
3.1. Morphological Observation. Figure 2 shows the morphol- calculated from the slope of the best fit linear curve was
ogies of thin films of 100/0 PHBV/PLLA, 50/50 PHBV/PLLA, 76 ± 20 KJ/mole with the regression coefficient, 𝑟2 value of
and 10% nHA incorporated 50/50 PHBV/PLLA. Some micro- 0.94. The activation energy of 100/0 PHBV/PLLA and 50/50
pores were observed in 50/50 PHBV/PLLA thin film due PHBV/PLLA thin film was 99.8 ± 21.9 KJ/mole and 112 ±
to the immiscibility of PHBV and PLLA. Figure 3 shows 45 KJ/mole, respectively.
the morphologies of 100/0 PHBV/PLLA, 50/50 PHBV/PLLA,
and 10% nHA in 50/50 PHBV/PLLA scaffolds. It was 3.3. Diffusion Study of Scaffolds. Figure 7 is the plot of
observed that highly porous scaffold specimens consisted of water uptake as 𝑀𝑡 /𝑀∞ versus square root of immersion
many micropores. Pore structure and size depended on the time at 37∘ C for 100/0 PHBV/PLLA scaffolds. The diffusion
phase separation process used in the fabrication technique. coefficients were calculated from the initial linear regions of
Scanning electron microscopy (SEM) and energy dispersive the curves. In order to study the temperature dependence
X-ray spectroscopy showed the distribution of HA nanopar- of diffusion coefficients of the scaffolds, the experiment was
ticles in the pore walls of the scaffolds, and some particle performed at three different temperatures, namely, at 25∘ C,
agglomeration was also observed (Figure 4). The average pore 37∘ C, and 50∘ C.
diameter of the 100/0 PHBV/PLLA scaffold was higher than Table 3 shows the diffusion coefficients of 100/0 PHBV/
that of 10% nHA incorporated 50/50 PHBV/PLLA scaffold as PLLA scaffolds at 25∘ C, 37∘ C, and 50∘ C, which were 51.75 ×
incorporation of nHA perturbed the phase separation process 10−12 , 67.60 × 10−12 , and 165 × 10−12 m2 /s, respectively. It was
slightly (Table 1). As the scaffolds were prepared from same observed that the initial water uptake of HA incorporated
polymer emulsion concentration, 10% (w/v), the porosity composite scaffold was much higher than that of polymer
did not change significantly in the scaffolds. The contact blend scaffolds, and the diffusion coefficients were also
angle was much higher in 100/0 PHBV/PLLA scaffold than higher. The value of diffusion coefficient was lower at low
50/50 PHBV/PLLA and 10% nHA in PHBV/PLLA scaffolds temperature and relatively high at high temperature. Figure 8
as PHBV polymer is well known for its hydrophobic nature. shows the Arrhenius plot which represents the tempera-
Blending with PLLA and incorporporation of nHA increased ture dependence of the diffusion coefficients of 10% HA in
the hydrophilicity of the composite scaffolds. 50/50 PHBV/PLLA scaffolds. From the slope of the best
(a) (b)
(c)
Figure 2: SEM micrographs of thin films of different compositions. (a) 100/0 PHBV/PLLA thin film; (b) 50/50 PHBV/PLLA thin film; (c) 10%
nHA in 50/50 PHBV/PLLA blend film.
(a) (b)
(c)
Figure 3: SEM micrographs of scaffolds of different compositions. (a) 100/0 PHBV/PLLA scaffold; (b) 50/50 PHBV/PLLA scaffold; (c) 10%
nHA in 50/50 PHBV/PLLA scaffold.
(a) (b)
Ca
P
Ca Ka1 P Ka1
(c)
Figure 4: SEM micrographs of (a) 10% nHA in 50/50 PHBV/PLLA composite scaffold; (b) EDX spectrum; (c) Mapping of Ca and P.
fit linear line, the activation energy was calculated, which 1.4
was 100.9 ± 5.3 KJ/mole. The activation energies were found
120.3 ± 45 KJ/mole for 50/50 PHBV/PLLA scaffold and 83.5 ± 1.2
53 KJ/mole for 100/0 PHBV/PLLA scaffolds, respectively.
Table 4 shows the comparison between the amounts 1
of water uptake (g/g) at equilibrium between the different
0.8
𝑀𝑡 /𝑀∞
compositions of thin films and scaffolds. Among the dif-

ferent compositions of thin films and scaffolds, the amount
0.6
(g/g) of equilibrium water uptake of 10% nHA in 50/50
PHBV/PLLA films and scaffolds was higher than that of
0.4
100/0 PHBV/PLLA and 50/50 PHBV/PLLA thin films and
scaffolds. This result was opposite to other studies where
0.2
the uptake of HA/polymer composite films was slower than
the pure polymeric film as the polymers were very much 0
hydrophilic and water uptake was high for the polymer itself 0 100 200 300 400 500 600 700 800 900 1000
[12]. In the present study, it was also observed that water Square root of immersion time (s)
uptake increased significantly with the increasing porosity.
The scaffolds which were more than 70% porous, absorbed Figure 5: Water uptake curve for 100/0 PHBV/PLLA thin film at
more water at the same condition than that of dense films. 37∘ C in the form of 𝑀𝑡 /𝑀∞ versus square root of immersion time.
Besides, the incorporation of HA increased the water uptake
in both composite thin film and composite scaffold.
Water uptake can occur by the materials in terms of porosity and the amount of available liquid water at the
“absorbed water” that means the amount of water absorbed surface of the material. For this reason, porous material can
from media which mainly depends on the hydrophilicity uptake and store more water whereas the nonporous (dense)
of material. Capillary water is termed as liquid water that material can store a limited amount of water (Table 4). It can
is “drawn in” through pores or capillaries of the materials. be seen from the plots of 𝑀𝑡 /𝑀∞ versus square root of time
Moreover, the amount of water absorbed is related to the (Figures 5 and 7) that the plots are almost linear for both the
Table 2: Diffusion coefficients of thin films at 25∘ C, 37∘ C, and 50∘ C.
Temperature (∘ C) 100/0 PHBV/PLLA thin films 50/50 PHBV/PLLA thin films 10% nHA in 50/50 PHBV/PLLA thin films
(m2 /s) (m2 /s) (m2 /s)
25 3.8 × 10−13 ± 1.5 × 10−13 2.12 × 10−13 ± 0.65 × 10−13 2.3 × 10−13 ± 0.7 × 10−13
37 11.3 × 10−13 ± 1.0 × 10−13 3.215 × 10−13 ± 0.47 × 10−13 11.6 × 10−13 ± 0.9 × 10−13
50 59.3 × 10−13 ± 12.0 × 10−13 59.8 × 10−13 ± 3.53 × 10−13 22.9 × 10−13 ± 0.8 × 10−13
Table 3: Diffusion coefficients of scaffolds at 25∘ C, 37∘ C, and 50∘ C.
Temperature (∘ C) 100/0 PHBV/PLLA scaffolds 50/50 PHBV/PLLA scaffolds 10% nHA in 50/50 PHBV/PLLA
(m2 /s) (m2 /s) scaffolds (m2 /s)
25 51.7 × 10−12 ± 1.1 × 10−12 0.16 × 10−11 ± 0.06 × 10−11 0.2 × 10−11 ± 0.05 × 10−11
37 67.6 × 10−12 ± 3.2 × 10−12 3.58 × 10−11 ± 0.7 × 10−11 1.3 × 10−11 ± 0.5 × 10−11
50 165 × 10−12 ± 9.1 × 10−12 6.92 × 10−11 ± 2 × 10−11 5.4 × 10−11 ± 1.35 × 10−11
−20 Table 4: Comparison of equilibrium water uptake between thin

films and scaffolds at 37∘ C.
−21
Compositions of films Equilibrium water Equilibrium water
−22 and scaffold uptake for thin films uptake for scaffolds
specimens (g/g) (g/g)
−23
100/0 PHBV/PLLA 0.01 1.24
ln 𝐷
−24 50/50 PHBV/PLLA 0.03 1.35

−25 10% nHA in 50/50
0.09 2.97
PHBV/PLLA
−26
−27 −20
−28 −21
0.0031 0.00315 0.0032 0.00325 0.0033 0.00335 0.0034 −22
1/𝑇
−23
Figure 6: Arrhenius plot of 10% HA in 50/50 PHBV/PLLA thin −24
films (the graph is plotted as logarithm of diffusion coefficients (𝐷)
ln 𝐷
versus the reciprocal of absolute temperature, 𝑇). −25

−26
−27
−28
1.4 −29
−30
1.2 0.0031 0.00315 0.0032 0.00325 0.0033 0.00335 0.0034
1/𝑇
1
Figure 8: Arrhenius plot of 10% HA in 50/50 PHBV/PLLA scaffolds
(the graph is plotted as logarithm of diffusion coefficients (𝐷) versus
𝑀𝑡 /𝑀∞
0.8
the reciprocal of absolute temperature, 𝑇).
0.6
0.4
thin films and scaffolds in the initial period of time. As Fick’s
second law is applicable for both the thin films and scaffolds
0.2
for the initial period of time, it can be stated that the initial
stage is diffusion controlled. Similar results were obtained by
0
0 200 400 600 800 1000 1200 1400
several studies of polymeric composite materials for dental
Square root of immersion time (s)
applications [12, 18, 19].
It was also found that the water uptake of PHBV/PLLA
Figure 7: Water uptake curve for 100/0 PHBV/PLLA scaffolds at scaffold was lower than 10 wt% nHA incorporated PHBV/
37∘ C in the form of 𝑀𝑡 /𝑀∞ versus square root of immersion time. PLLA scaffold. For the scaffold specimens, when all the
pores were assumed to be filled with water, the equilibrium showed decrease in water uptake after saturation
water uptake of polymer blend scaffold and composite scaf- due to dissolution of HA and also degradation of
fold specimens were found to be 1.35 g/g and 2.97 (g/g), amorphous part of PLLA/PHBV polymer.
respectively (Table 4). Moreover, water uptake of composite (4) The diffusion coefficients were higher in porous scaf-
scaffold specimen decreased after few days. The reason might folds than thin films as the rate of diffusion increased
be the starting of dissolution of nHA within the composite with decreasing density of atomic packing. Porosity
scaffold. The initial increase of water uptake of the composite and surface to volume ratio had a profound effect on
films and scaffolds may be due to hydrophilic nature of the the water uptake of scaffolds.
nanosized nHA particles or the inclusions of HA nanoparticle
aggregates. The nHA nano-particles may appear as loosely
embedded aggregates in the polymer matrix. As a result, Conflict of Interests
additional amount of water at the interface between the The authors, hereby, declare that they do not have any conflict
agglomerates and the matrix can be accommodated. of interests.
The calculated diffusion coefficients expressed are given
in Tables 2 and 3 during water absorption. The values of
diffusion coefficients are available in the literature for other Acknowledgments
conventional composites [18, 20]. The diffusion coefficients
N. Sultana thanks The University of Hong Kong (HKU)
were higher in scaffolds than thin films. This behaviour can
for providing her with a research studentship. This work
occur during the initial uptake of water through the pores of
was supported by a GRF Grant (HKU 7182/05E) from the
the scaffolds which can cause the formation of water clusters.
Research Grants Council of Hong Kong. Assistance provided
Surface area to volume ratio and thickness can also have
by technical staff in the Department of Mechanical Engi-
effects on this.
neering, Faculty of Engineering, is acknowledged. Authors
Temperature and composition are the two most impor-
also thank Universiti Teknologi Malaysia, Ministry of Higher
tant factors which can affect the diffusion coefficient [21].
Education (MOHE), RMC, IJN-UTM Cardiovascular Engi-
Between these two factors, temperature has the most pro-
neering Centre, and FRGS Grant (vot: 4F126), GUP Tier 1 (03
found influence on the coefficients and diffusion rates. The
H13, 05H07) for financial supports.
diffusion coefficients are related to temperature according to
Arrhenius equation. From Arrhenius plots of thin film and
scaffold (Figures 6 and 8), it can be seen that the plot is almost References
linear. These results can further confirm that water uptake of
[1] E. Bell, Tissue Engineering: Current Perspectives, Birkhäuser,
thin films is controlled by diffusion process. From the plots Boston, Mass, USA, 1993.
of 𝑀𝑡 /𝑀∞ versus square root of immersion time, it is also
[2] R. P. Lanza, R. S. Langer, and J. Vacanti, Principles of Tissue
possible to estimate when a plateau region can be established Engineering, Elsevier/Academic Press, Amsterdam, The Nether-
and how long the diffusion phenomena can be observed. lands, 2007.
It was also found by other studies that after saturation, [3] Z. Li, Y. Su, B. Xie et al., “A tough hydrogel-hydroxyapatite
many other properties should be taken into account such as bone-like composite fabricated in situ by the electrophoresis
structure and surface chemistry. approach,” Journal of Materials Chemistry B, vol. 1, no. 12, pp.
1755–1764, 2013.
4. Conclusions [4] J. Lacroix, J. Lao, and E. Jallot, “Green and safe in situ templating
of bioactive glass scaffolds for bone tissue engineering,” Journal
(1) The amount of water absorption or uptake depends of Materials Chemistry B, vol. 1, no. 13, pp. 1782–1785, 2013.
mainly on the hydrophilicity and porosity of the [5] S. Lalan, I. Pomerantseva, and J. P. Vacanti, “Tissue engineering
materials. For this reason, PHBV/PLLA and 10% HA and its potential impact on surgery,” World Journal of Surgery,
vol. 25, no. 11, pp. 1458–1466, 2001.
in PHBV/PLLA scaffolds and thin films absorbed
more water than that of 100/0 PHBV/PLLA scaffolds [6] C. Bastioli, Handbook of Biodegradable Polymers, Rapra Tech-
nology, Shrewsbury, UK, 2005.
and thin films as PHBV is more hydrophobic than
[7] E. Blümm and A. J. Owen, “Miscibility, crystallization and melt-
PLLA and HA. The water uptake of the scaffolds
ing of poly(3-hydroxybutyrate)/poly(l-lactide) blends,” Poly-
was much higher than that of thin films because the mer, vol. 36, no. 21, pp. 4077–4081, 1995.
scaffolds had high porosity. [8] N. Sultana and M. Wang, “PHBV/PLLA-based composite scaf-
(2) For thin films and scaffold, Arrhenius plot was almost folds containing nano-sized hydroxyapatite particles for bone
linear. For the initial period of time, water uptake of tissue engineering,” Journal of Experimental Nanoscience, vol. 3,
the thin films was controlled by diffusion process. It no. 2, pp. 121–132, 2008.
was possible to estimate when a plateau region could [9] P. X. Ma, R. Zhang, G. Xiao, and R. Franceschi, “Engineering
be established and how long the diffusion phenomena new bone tissue in vitro on highly porous poly(𝛼-hydroxyl
could be observed. acids)/hydroxyapatite composite scaffolds,” Journal of Biomedi-
cal Materials Research, vol. 54, pp. 284–293, 2001.
(3) After saturation, many other properties should be [10] B. D. Ratner, BioMaterials Science: An Introduction to Materials
taken into account such as structure and surface in Medicine, Elsevier Academic Press, San Diego, Calif, USA,
chemistry. 10% HA in 50/50 PHBV/PLLA scaffolds 2004.
[11] N. Sultana and T. H. Khan, “Factorial study of compressive

mechanical properties and primary in vitro osteoblast response
of PHBV/PLLA scaffolds,” Journal of Nanomaterials, vol. 2012,
Article ID 656914, 8 pages, 2012.
[12] M. Braden, “Water absorption characteristics of dental micro-
fine composite filling materials. II. Experimental materials,”
Biomaterials, vol. 5, no. 6, pp. 373–375, 1984.
[13] S. Deb, M. Braden, and W. Bonfield, “Water absorption charac-
teristics of modified hydroxyapatite bone cements,” Biomateri-
als, vol. 16, no. 14, pp. 1095–1100, 1995.
[14] N. Sultana and M. Wang, “PHBV/PLLA-based composite
scaffolds fabricated using an emulsion freezing/freeze-drying
technique for bone tissue engineering: surface modification
and in vitro biological evaluation,” Biofabrication, vol. 4, no. 1,
Article ID 015003, 2012.
[15] W. Y. Zhou, M. Wang, W. L. Cheung, B. C. Guo, and D.
M. Jia, “Synthesis of carbonated hydroxyapatite nanospheres
through nanoemulsion,” Journal of Materials Science: Materials
in Medicine, vol. 19, no. 1, pp. 103–110, 2008.
[16] N. Sultana and M. Wang, “Fabrication of HA/PHBV composite
scaffolds through the emulsion freezing/freeze-drying process
and characterisation of the scaffolds,” Journal of Materials
Science: Materials in Medicine, vol. 19, no. 7, pp. 2555–2561, 2008.
[17] J. Crank and G. S. Park, Eds., Diffusion in Polymers, Academic
Press, New York, NY, USA, 1977.
[18] M. Braden and R. L. Clarke, “Water absorption characteristics
of dental microfine composite filling materials. I. Proprietary
materials,” Biomaterials, vol. 5, no. 6, pp. 369–372, 1984.
[19] C. Santos, R. L. Clarke, M. Braden, F. Guitian, and K. W. M.
Davy, “Water absorption characteristics of dental composites
incorporating hydroxyapatite filler,” Biomaterials, vol. 23, no. 8,
pp. 1897–1904, 2002.
[20] J. Crank and G. S. Park, Diffusion in Polymers, Academic Press,
London, UK, 1968.
[21] R. E. Smallman and A. H. W. Ngan, Physical Metallurgy and
Advanced Materials, Butterworth-Heinemann; Elsevier, Ams-
terdam, The Netherlands, 2007.

Nano For Biomimetics

Uploaded by

Copyright:

Available Formats

Nano For Biomimetics

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nano For Biomimetics

Uploaded by

Copyright:

Available Formats

Journal of Nanomaterials

Nano for Biomimetics

Nano for Biomimetics and Biomaterials

Guest Editors: Il-Kwon Oh, Anchal Srivastava, In-Kyu Park,

A Facile Nanodelivery Platform Based on Functionalized Hyperbranched Poly(ether-ester) for

Nanostructural Colouration in Malaysian Plants: Lessons for Biomimetics and Biomaterials,

A Dual-Functional [SBA-15/Fe3 O4 /P(N-iPAAm)] Hybrid System as a Potential Nanoplatform for

Characterization of the Casein/Keratin Self-Assembly Nanomicelles, Su Xiao-Zhou, Wang Hong-Ru,

Development of Antibiotics Impregnated Nanosized Silver Phosphate-Doped Hydroxyapatite Bone

Investigation of the In Vitro Degradation of a Novel Polylactide/Nanohydroxyapatite Composite for

Water Absorption and Diffusion Characteristics of Nanohydroxyapatite (nHA) and

Il-Kwon Oh,1 Anchal Srivastava,2 In-Kyu Park,3 and Michael Z. Hu4

Correspondence should be addressed to Il-Kwon Oh; ikoh@kaist.ac.kr

Received 3 April 2014; Accepted 3 April 2014; Published 29 June 2014

This special issue has been considered as a necessary Acknowledgment

Correspondence should be addressed to Li-min Mao; hrbmlm@126.com

Academic Editor: In-Kyu Park

1. Introduction methacrylate, polyphenylene, poly(ether ketone), polyester,

Scheme 1: End-functionalization between HPEE and amino group/carbonyl group.

Table 1: Correlation between surface potential and end-functionalized polymers.

Delivery vehicle (PEE) Potential Comparable analogue (PE) Potential

2000 1500 1000 2000 1500 1000

Cell viability (% control)

3T3 cell viability for 96 h incubation

Concentration 0.001 mg/mL Concentration 1 mg/mL

Scheme 2: Structure of COOH-HPEE (a) and pingyangmycin (b).

Size distribution by intensity Size distribution by intensity

Cell viability (% control)

S. Zaleha M. Diah,1 Salmah B. Karman,1,2 and Ille C. Gebeshuber1,3

Correspondence should be addressed to Ille C. Gebeshuber; gebeshuber@iap.tuwien.ac.at

Academic Editor: Il-Kwon Oh

1. Introduction deter herbivores, and help in light management (e.g., UV

Spongy Vascular bundle

2.2.4. Functions and Potential Applications. Colours serve a

Figure 5: The principle of Snell’s law.

Figure 6: Thin-film interference (modified from [33]).

the two waves will produce constructive interference. In

Physical mechanism Device structure Engineering application References

[67] J. A. Rego, J. A. A. Harvey, A. L. MacKinnon, and E. Gatdula,

Andreza de Sousa,1,2 Karynne Cristina de Souza,1 Paula Maria da Silva Leite,1,2

Correspondence should be addressed to Edésia Martins Barros de Sousa; sousaem@cdtn.br

Academic Editor: Anchal Srivastava

1. Introduction The delivery of these molecules was once considered

1200 than 700∘ C that are characteristic of phase transformation of

4000 (220) XRD peaks of SBA-15/Fe3 O4 can be indexed to a hexagonal

perpendicular to the channel axis (Figure 4(b)). The Fe3 O4

1500 (100) 150 (200) 1500 (100) 150 (200)

(a) (b) (c)

1.2 1.2 SBA-15/Fe3 O4 /P(N-iPAAm) after washing process

0.06 SBA-15/Fe3 O4 /P(N-iPAAm)

Antochshuk and coauthors [26] attributed this stepwise

42 field for 30 min, the temperature ranged from 24 to 39∘ C and

pension after sonication and under 126 Oe AC magnetic

[24] D. Zhao, Q. Huo, J. Feng, B. F. Chmelka, and G. D. Stucky,

Su Xiao-Zhou, Wang Hong-Ru, and Huang Mian

Correspondence should be addressed to Wang Hong-Ru; wanghr@sust.edu.cn

Academic Editor: Anchal Srivastava

1. Introduction Based on these viewpoints, our studies intended for structural

Figure 6: FT-IR spectra of the casein/keratin nanomicelles mixtures