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Magnetic auto-fluorescent microspheres for a drug delivery system
Materials Letters 119 (2014) 143–145
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Magnetic auto-fluorescent microspheres for a drug delivery system Hui Zhang a, Jingdi Chen a,n, Yujue Zhang a, Panpan Pan a, Qiqing Zhang a,b a Institute of Biomedical and Pharmaceutical Technology & College of Chemistry and Chemical Engineering, Fuzhou University, No. 523, Gongye Road, Fuzhou 350002, China b Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 October 2013 Accepted 5 January 2014 Available online 10 January 2014
A drug delivery system was prepared for potential bone regeneration. The fabrication process involved electrostatic self-assembly on sodium alginate to form global composite with a core–shell structure. The core of microsphere consisted of sodium alginate and nano-Fe3O4, which was prepared by a microcapsule shaping device. The core then electrostatically self-assembled with chitosan and finally cross-linked by genipin to form the shell structure. Superparamagnetism behaviors of nano-Fe3O4 and weak ferromagnetic behaviors of microsphere were proved by physical property measurement system (PPMS) test. The structural schematic diagram showed the structure of composite microsphere. The morphology of the composite microspheres was evaluated by a field scanning electron microscope (FSEM). Osteoblasts adhesion with microspheres was analyzed by inverted fluorescence microscopy. Cytotoxicity established by confocal scanning laser microscope (CLSM) observation confirmed the developed composite materials with good biocompatibility. Results show potential applications for a bone repairing drug delivery system. & 2014 Elsevier B.V. All rights reserved.
Keywords: Sodium alginate Chitosan Fluorescence Biomaterials Composite materials
1. Introduction Microcapsule has received considerable attention on controlling release of drug and it has been widely used as injectable bone-filling materials for bone regeneration [1,2]. Chitosan (CS) has been widely investigated owing to its superior biocompatibility, appropriate biodegradability and excellent antibacterial property [3–5]. Alginate (Alg) is a linear polysaccharide copolymer of α-L-mannuronic acid (M) and β-D-guluronic acid (G), which forms gel when it reacts with divalent metal ion [6,7]. CS molecule is positively charged when it is dissolved in acetic acid, while sodium alginate molecule is negatively charged in aqueous solution [8,9]. They form polyelectrolyte semipermeable membrane through electrostatic interaction [10,11]. Due to the unique characteristics of oriented movement in magnetic field, magnetic nanoparticles have been widely used in biomedical aspects [12,13], for example, drug delivery vehicle and cancer hyperthermia treatment. Recently, many researchers have found that magnetic nanoparticles are effective for osteoinduction. Gu et al. [14] proved that the introduction of magnetic nanoparticles to CaP bioceramics could promote bone formation and growth. Wei et al. [15] reported that Fe3O4/CS/PVA nanofibrous membranes exhibited good cell adhesion and proliferation of MG-63 human osteoblast-like cells. Meng et al. [16] demonstrated that the addition
n Corresponding author at: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, No. 523 Gongye Road, Fuzhou 350002, China. Tel./fax: +86 591 83725260. E-mail address: [email protected] (J. Chen).
0167-577X/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2014.01.008
of magnetic nanoparticles in HA/PLA composite nanofibrous films could induce higher proliferation rate and faster differentiation of osteoblast. Thus, microspheres containing magnetic nanoparticles may have great potential in bone regenerative medicine. Here, a composite microsphere (Alg–CS–Gip) with the core–shell structure was prepared through the microcapsule shaping device. When the raw materials (Alg and nano-Fe3O4) were dripped from the pinhole, the drops overcame the surface tension under the impact of electric field force and then fell into the coagulation bath (Ca2 þ aqueous solution) to form core. The simple and efficient microsphere forming process, mild forming condition and high activity of biomass are regarded as the advantages of this method. The core was then submerged in chitosan acetic acid solution to form polyelectrolyte semipermeable membrane. Besides, genipin (Gip) was selected as cross-linking agent to cross link with chitosan to form shell, because of its outstanding biocompatibility. Genipin has been reported to bind with biological tissues and biopolymers such as chitosan or gelatin through covalent coupling [17–19]. It not only enhances the tightness of microspheres but also provides intrinsic fluorescence for the composite microspheres [20,21]. The property of fluorescence provides effective ways for imaging the microsphere– cell interface, tracing cell adhesion and proliferation with a confocal laser scanning microscope (CLSM).
2. Experimental Materials and method: The process of microspheres fabrication contains several steps. First, 1.5 wt% sodium alginate (C.P., Sinopharm
144
H. Zhang et al. / Materials Letters 119 (2014) 143–145
Chemical Reagent Co., Ltd., China) with 0.01 wt% nano-Fe3O4 (Monodisperse magnetic single-crystal ferrite microspheres) [22] was dissolved, and the core of spheres was produced by the microcapsule shaping device (University of Shanghai for Science and Technology). Second, the prepared core of spheres was then electrostatic selfassembled with 1.0 wt% chitosan aqueous solution (A.R., Shanghai Shenggong Biological Engineering Co., Ltd., China) at 37 1C for 8 h. Finally, the polyelectrolyte semipermeable membrane bonded with geinpin (A.R., Linchuan Zhixin Biological Technology Co., Ltd., China) at neutral pH for 36 h to form shell. Composite microspheres were rinsed with distilled water and freeze dried. Characterization: The phases of the products were analyzed by X-ray diffraction (XRD, Philips X’Pert Pro MPD, Co Kα radiation). Magnetic property of nano-Fe3O4 and microspheres was measured by a physical property measurement system (PPMS, PPMS-9T, Quantum Design, America). To observe morphology and topography of microsphere, a field scanning electron microscope (FSEM, Nova NanoSEM 230, FEI, America) was employed. Viability of neonatal rat osteoblasts with microsphere was evaluated after 1, 4, 7, and 10 days of incubation. Cytotoxicity was established by the confocal scanning laser microscope (CLSM, TY1318, Olympus, Japan). Osteoblasts adhesion and morphology were analyzed by inverted fluorescence microscopy (IX71, Olympus, Japan).
3. Results and discussion XRD spectra of nano-Fe3O4 and composite microspheres containing 1% Fe3O4 are shown in Fig. 1a. The crystallinity of microsphere is lower than that of pure Fe3O4 because of the existence of organic matrix. The crystal phases of as-achieved Fe3O4 can be indexed to standard diffraction powder card (JCPDS 19-629). Result indicates that the position and intensity of diffraction peaks fit well. Fig. 1b shows the magnetic hysteresis loops of Fe3O4 and composite microsphere containing 1% Fe3O4 measured by PPMS. The saturation magnetization and coercivity of nano-Fe3O4 display superparamagnetism. Obviously, the magnetic property is much lower than pure Fe3O4 owing to the existence of organic matrix. Fig. 2a shows rough surface and uniform particle size of Alg– CS–Gip microspheres. The average diameter is about 185 μm under dry condition. This demonstrated that cell favorably responses to the charged and hydrophilic surfaces in vitro studies corresponding to superior adsorption and bioactivity of adhesion proteins [23]. So, the positively charged CS and hydrophilic Alg composite microsphere may be favorable for cell adhesion and proliferation. The structural schematic diagram of composite microsphere is shown in Fig. 2b.
Fig. 1. (a) XRD spectra of (1) Fe3O4 and (2) composite microspheres. (b) Magnetic hysteresis loop of (1) composite microspheres and (2) Fe3O4.
Fig. 2. (a) SEM image of Alg–CS–Gip microspheres. (b) Structural schematic diagram of composite microsphere (Alg–CS–Gip).
H. Zhang et al. / Materials Letters 119 (2014) 143–145
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Fig. 3. Inverted fluorescence microscope images of composite microspheres cultured with osteoblast for (a) 1 day, (b) 4 days, (c) 7 days and (d) 10 days.
Fig. 4. CLSM fluorescent images: (a) Hoechest 33258 of microsphere–rat osteoblasts’ DNA staining, (b) intrinsic fluorescent image of microsphere, (c) bright field image of microsphere, (d) merged image of (a) and (b), and (e) merged image of (a)–(c). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 3 shows that the diameter of microsphere is about 450 μm under wet condition observed by an inverted fluorescence microscope. The nano-Fe3O4 disperses uniformly in microspheres even though some of them agglomerate. Besides, Fig. 3a–d shows the growing states of rat osteoblast cultured with microspheres. During the first 3 days, osteoblasts proliferate at the bottom of culture dish. On the 4th day, osteoblasts adhere to microsphere. Cells stretch, migrate and proliferate on microsphere gradually on the 7th day. On the 10th day, osteoblasts almost surround the whole microsphere. Thus, the composite microspheres show no cytotoxicity with superior cell adhesion and proliferation ability. In Fig. 4a, the bright blue dots representing the nucleus clearly show uniform, extensive distribution of osteoblast on composite microsphere. In addition, Fig. 4b indicates that composite microspheres possess intrinsic fluorescence displaying green circle, which is due to the reaction between chitosan and genipin. This property may provide an effective way to track the cell–microsphere interaction and effectively monitor the adhesion and proliferation on the surface of microsphere. Fig. 4c shows the bright field image of microspheres observed by CLSM. Nano-Fe3O4 is uniformly distributed inside the microsphere which shows black dots in image. Fig. 4d is obtained by merging Fig. 4a and b; Fig. 4e is obtained by merging Fig. 4a–c.
4. Conclusion Alg–CS–Gip composite microspheres were prepared by the microcapsule forming device. CLSM and inverted fluorescence microscopy images showed that osteoblasts adhered and proliferated actively surrounding the microspheres, which indicated advantageous biocompatibility of microspheres. This suggests future applications of the composite microsphere as a drug delivery system for bone regeneration.
Acknowledgment This research was supported by National Natural Science Foundation of China (31100677 and 31370958) and Scientific Research Foundation in Fuzhou University (2011-XQ-18 and XRC0950). References [1] Rahman N, Mathiowitz E. J Control Release 2004;94:163–75. [2] Lee JW, Kang KS, Lee SH, Kim JY, Lee BK, Cho DW. Biomaterials 2011;32:744–52. [3] Muzzarelli R, Mattioli-Belmonte M, Tietz C, Biagini R, Ferioli G, Brunelli M, et al. Biomaterials 1994;15:1075–81. [4] Tomihata K, Ikada Y. Biomaterials 1997;18:567–75. [5] VandeVord PJ, Matthew HW, DeSilva SP, Mayton L, Wu B, Wooley PH. J Biomed Mater Res 2002;59:585–90. [6] Gacesa P. Carbohydr Polym 1988;8:161–82. [7] Gombotz WR, Wee S. Adv Drug Deliv Rev 1998;31:267–85. [8] Chen JD, Yu QF, Zhang GD, Yang S, Wu JL, Zhang QQ. Colloid Surf B 2012;93:100–7. [9] Chen JD, Nan KH, Yin SH, Wang YJ, Wu T, Zhang QQ. Colloid Surf B 2010;8:640–7. [10] Anal AK, Stevens WF. Int J Pharm 2005;290:45–54. [11] Decher G, Hong J, Schmitt J. Thin Solid Films 1992;210:831–5. [12] Akiyama H, Ito A, Kawabe Y, Kamihira M. Biomaterials 2010;31:1251–9. [13] Attaluri A, Ma R, Qiu Y, Li W, Zhu L. Int J Hyperth 2011;27:491–502. [14] Wu Y, Jiang W, Wen X, He B, Zeng X, Wang G, et al. Biomed Mater 2010;5:015001. [15] Wei Y, Zhang X, Song Y, Han B, Hu X, Wang X, et al. Biomed Mater 2011;6:055008. [16] Meng J, Zhang Y, Qi X, Kong H, Wang C, Xu Z, et al. Nanoscale 2010;2:2565–9. [17] Sung HW, Liang IL, Chen CN, Huang RN, Liang HF. J Biomed Mater Res 2001;55:538–46. [18] Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K. Biomaterials 2002;23: 4827–32. [19] Jin J, Song M, Hourston D. Biomacromolecules 2004;5:162–8. [20] Chen H, Ouyang W, Lawuyi B, Martoni C, Prakash S. J Biomed Mater Res A 2005;75:917–27. [21] Chen H, Ouyang W, Lawuyi B, Prakash S. Biomacromolecules 2006;7:2091–8. [22] Deng H, Li X, Peng Q, Wang X, Chen J, Li Y. Angew Chem 2005;117:2842–5. [23] Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Tissue Eng 2005;11:1–18.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Magnetic auto-fluorescent microspheres for a drug delivery system Hui Zhang a, Jingdi Chen a,n, Yujue Zhang a, Panpan Pan a, Qiqing Zhang a,b a Institute of Biomedical and Pharmaceutical Technology & College of Chemistry and Chemical Engineering, Fuzhou University, No. 523, Gongye Road, Fuzhou 350002, China b Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China
art ic l e i nf o
a b s t r a c t
Article history: Received 9 October 2013 Accepted 5 January 2014 Available online 10 January 2014
A drug delivery system was prepared for potential bone regeneration. The fabrication process involved electrostatic self-assembly on sodium alginate to form global composite with a core–shell structure. The core of microsphere consisted of sodium alginate and nano-Fe3O4, which was prepared by a microcapsule shaping device. The core then electrostatically self-assembled with chitosan and finally cross-linked by genipin to form the shell structure. Superparamagnetism behaviors of nano-Fe3O4 and weak ferromagnetic behaviors of microsphere were proved by physical property measurement system (PPMS) test. The structural schematic diagram showed the structure of composite microsphere. The morphology of the composite microspheres was evaluated by a field scanning electron microscope (FSEM). Osteoblasts adhesion with microspheres was analyzed by inverted fluorescence microscopy. Cytotoxicity established by confocal scanning laser microscope (CLSM) observation confirmed the developed composite materials with good biocompatibility. Results show potential applications for a bone repairing drug delivery system. & 2014 Elsevier B.V. All rights reserved.
Keywords: Sodium alginate Chitosan Fluorescence Biomaterials Composite materials
1. Introduction Microcapsule has received considerable attention on controlling release of drug and it has been widely used as injectable bone-filling materials for bone regeneration [1,2]. Chitosan (CS) has been widely investigated owing to its superior biocompatibility, appropriate biodegradability and excellent antibacterial property [3–5]. Alginate (Alg) is a linear polysaccharide copolymer of α-L-mannuronic acid (M) and β-D-guluronic acid (G), which forms gel when it reacts with divalent metal ion [6,7]. CS molecule is positively charged when it is dissolved in acetic acid, while sodium alginate molecule is negatively charged in aqueous solution [8,9]. They form polyelectrolyte semipermeable membrane through electrostatic interaction [10,11]. Due to the unique characteristics of oriented movement in magnetic field, magnetic nanoparticles have been widely used in biomedical aspects [12,13], for example, drug delivery vehicle and cancer hyperthermia treatment. Recently, many researchers have found that magnetic nanoparticles are effective for osteoinduction. Gu et al. [14] proved that the introduction of magnetic nanoparticles to CaP bioceramics could promote bone formation and growth. Wei et al. [15] reported that Fe3O4/CS/PVA nanofibrous membranes exhibited good cell adhesion and proliferation of MG-63 human osteoblast-like cells. Meng et al. [16] demonstrated that the addition
n Corresponding author at: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, No. 523 Gongye Road, Fuzhou 350002, China. Tel./fax: +86 591 83725260. E-mail address: [email protected] (J. Chen).
0167-577X/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matlet.2014.01.008
of magnetic nanoparticles in HA/PLA composite nanofibrous films could induce higher proliferation rate and faster differentiation of osteoblast. Thus, microspheres containing magnetic nanoparticles may have great potential in bone regenerative medicine. Here, a composite microsphere (Alg–CS–Gip) with the core–shell structure was prepared through the microcapsule shaping device. When the raw materials (Alg and nano-Fe3O4) were dripped from the pinhole, the drops overcame the surface tension under the impact of electric field force and then fell into the coagulation bath (Ca2 þ aqueous solution) to form core. The simple and efficient microsphere forming process, mild forming condition and high activity of biomass are regarded as the advantages of this method. The core was then submerged in chitosan acetic acid solution to form polyelectrolyte semipermeable membrane. Besides, genipin (Gip) was selected as cross-linking agent to cross link with chitosan to form shell, because of its outstanding biocompatibility. Genipin has been reported to bind with biological tissues and biopolymers such as chitosan or gelatin through covalent coupling [17–19]. It not only enhances the tightness of microspheres but also provides intrinsic fluorescence for the composite microspheres [20,21]. The property of fluorescence provides effective ways for imaging the microsphere– cell interface, tracing cell adhesion and proliferation with a confocal laser scanning microscope (CLSM).
2. Experimental Materials and method: The process of microspheres fabrication contains several steps. First, 1.5 wt% sodium alginate (C.P., Sinopharm
144
H. Zhang et al. / Materials Letters 119 (2014) 143–145
Chemical Reagent Co., Ltd., China) with 0.01 wt% nano-Fe3O4 (Monodisperse magnetic single-crystal ferrite microspheres) [22] was dissolved, and the core of spheres was produced by the microcapsule shaping device (University of Shanghai for Science and Technology). Second, the prepared core of spheres was then electrostatic selfassembled with 1.0 wt% chitosan aqueous solution (A.R., Shanghai Shenggong Biological Engineering Co., Ltd., China) at 37 1C for 8 h. Finally, the polyelectrolyte semipermeable membrane bonded with geinpin (A.R., Linchuan Zhixin Biological Technology Co., Ltd., China) at neutral pH for 36 h to form shell. Composite microspheres were rinsed with distilled water and freeze dried. Characterization: The phases of the products were analyzed by X-ray diffraction (XRD, Philips X’Pert Pro MPD, Co Kα radiation). Magnetic property of nano-Fe3O4 and microspheres was measured by a physical property measurement system (PPMS, PPMS-9T, Quantum Design, America). To observe morphology and topography of microsphere, a field scanning electron microscope (FSEM, Nova NanoSEM 230, FEI, America) was employed. Viability of neonatal rat osteoblasts with microsphere was evaluated after 1, 4, 7, and 10 days of incubation. Cytotoxicity was established by the confocal scanning laser microscope (CLSM, TY1318, Olympus, Japan). Osteoblasts adhesion and morphology were analyzed by inverted fluorescence microscopy (IX71, Olympus, Japan).
3. Results and discussion XRD spectra of nano-Fe3O4 and composite microspheres containing 1% Fe3O4 are shown in Fig. 1a. The crystallinity of microsphere is lower than that of pure Fe3O4 because of the existence of organic matrix. The crystal phases of as-achieved Fe3O4 can be indexed to standard diffraction powder card (JCPDS 19-629). Result indicates that the position and intensity of diffraction peaks fit well. Fig. 1b shows the magnetic hysteresis loops of Fe3O4 and composite microsphere containing 1% Fe3O4 measured by PPMS. The saturation magnetization and coercivity of nano-Fe3O4 display superparamagnetism. Obviously, the magnetic property is much lower than pure Fe3O4 owing to the existence of organic matrix. Fig. 2a shows rough surface and uniform particle size of Alg– CS–Gip microspheres. The average diameter is about 185 μm under dry condition. This demonstrated that cell favorably responses to the charged and hydrophilic surfaces in vitro studies corresponding to superior adsorption and bioactivity of adhesion proteins [23]. So, the positively charged CS and hydrophilic Alg composite microsphere may be favorable for cell adhesion and proliferation. The structural schematic diagram of composite microsphere is shown in Fig. 2b.
Fig. 1. (a) XRD spectra of (1) Fe3O4 and (2) composite microspheres. (b) Magnetic hysteresis loop of (1) composite microspheres and (2) Fe3O4.
Fig. 2. (a) SEM image of Alg–CS–Gip microspheres. (b) Structural schematic diagram of composite microsphere (Alg–CS–Gip).
H. Zhang et al. / Materials Letters 119 (2014) 143–145
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Fig. 3. Inverted fluorescence microscope images of composite microspheres cultured with osteoblast for (a) 1 day, (b) 4 days, (c) 7 days and (d) 10 days.
Fig. 4. CLSM fluorescent images: (a) Hoechest 33258 of microsphere–rat osteoblasts’ DNA staining, (b) intrinsic fluorescent image of microsphere, (c) bright field image of microsphere, (d) merged image of (a) and (b), and (e) merged image of (a)–(c). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 3 shows that the diameter of microsphere is about 450 μm under wet condition observed by an inverted fluorescence microscope. The nano-Fe3O4 disperses uniformly in microspheres even though some of them agglomerate. Besides, Fig. 3a–d shows the growing states of rat osteoblast cultured with microspheres. During the first 3 days, osteoblasts proliferate at the bottom of culture dish. On the 4th day, osteoblasts adhere to microsphere. Cells stretch, migrate and proliferate on microsphere gradually on the 7th day. On the 10th day, osteoblasts almost surround the whole microsphere. Thus, the composite microspheres show no cytotoxicity with superior cell adhesion and proliferation ability. In Fig. 4a, the bright blue dots representing the nucleus clearly show uniform, extensive distribution of osteoblast on composite microsphere. In addition, Fig. 4b indicates that composite microspheres possess intrinsic fluorescence displaying green circle, which is due to the reaction between chitosan and genipin. This property may provide an effective way to track the cell–microsphere interaction and effectively monitor the adhesion and proliferation on the surface of microsphere. Fig. 4c shows the bright field image of microspheres observed by CLSM. Nano-Fe3O4 is uniformly distributed inside the microsphere which shows black dots in image. Fig. 4d is obtained by merging Fig. 4a and b; Fig. 4e is obtained by merging Fig. 4a–c.
4. Conclusion Alg–CS–Gip composite microspheres were prepared by the microcapsule forming device. CLSM and inverted fluorescence microscopy images showed that osteoblasts adhered and proliferated actively surrounding the microspheres, which indicated advantageous biocompatibility of microspheres. This suggests future applications of the composite microsphere as a drug delivery system for bone regeneration.
Acknowledgment This research was supported by National Natural Science Foundation of China (31100677 and 31370958) and Scientific Research Foundation in Fuzhou University (2011-XQ-18 and XRC0950). References [1] Rahman N, Mathiowitz E. J Control Release 2004;94:163–75. [2] Lee JW, Kang KS, Lee SH, Kim JY, Lee BK, Cho DW. Biomaterials 2011;32:744–52. [3] Muzzarelli R, Mattioli-Belmonte M, Tietz C, Biagini R, Ferioli G, Brunelli M, et al. Biomaterials 1994;15:1075–81. [4] Tomihata K, Ikada Y. Biomaterials 1997;18:567–75. [5] VandeVord PJ, Matthew HW, DeSilva SP, Mayton L, Wu B, Wooley PH. J Biomed Mater Res 2002;59:585–90. [6] Gacesa P. Carbohydr Polym 1988;8:161–82. [7] Gombotz WR, Wee S. Adv Drug Deliv Rev 1998;31:267–85. [8] Chen JD, Yu QF, Zhang GD, Yang S, Wu JL, Zhang QQ. Colloid Surf B 2012;93:100–7. [9] Chen JD, Nan KH, Yin SH, Wang YJ, Wu T, Zhang QQ. Colloid Surf B 2010;8:640–7. [10] Anal AK, Stevens WF. Int J Pharm 2005;290:45–54. [11] Decher G, Hong J, Schmitt J. Thin Solid Films 1992;210:831–5. [12] Akiyama H, Ito A, Kawabe Y, Kamihira M. Biomaterials 2010;31:1251–9. [13] Attaluri A, Ma R, Qiu Y, Li W, Zhu L. Int J Hyperth 2011;27:491–502. [14] Wu Y, Jiang W, Wen X, He B, Zeng X, Wang G, et al. Biomed Mater 2010;5:015001. [15] Wei Y, Zhang X, Song Y, Han B, Hu X, Wang X, et al. Biomed Mater 2011;6:055008. [16] Meng J, Zhang Y, Qi X, Kong H, Wang C, Xu Z, et al. Nanoscale 2010;2:2565–9. [17] Sung HW, Liang IL, Chen CN, Huang RN, Liang HF. J Biomed Mater Res 2001;55:538–46. [18] Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K. Biomaterials 2002;23: 4827–32. [19] Jin J, Song M, Hourston D. Biomacromolecules 2004;5:162–8. [20] Chen H, Ouyang W, Lawuyi B, Martoni C, Prakash S. J Biomed Mater Res A 2005;75:917–27. [21] Chen H, Ouyang W, Lawuyi B, Prakash S. Biomacromolecules 2006;7:2091–8. [22] Deng H, Li X, Peng Q, Wang X, Chen J, Li Y. Angew Chem 2005;117:2842–5. [23] Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Tissue Eng 2005;11:1–18.