Powder Technology 215-216 (2012) 8590

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Powder Technology
journal homepage: www.elsevier.com/locate/powtec

Self-assembled silk broin particles: Tunable size and appearance

Pujiang Shi a, James C.H. Goh a, b,
a b

Department of Orthopedic Surgery, National University of Singapore, 10 Lower Kent Ridge Road, Singapore 119260, Singapore Division of Bioengineering, National University of Singapore, Singapore

a r t i c l e

i n f o

a b s t r a c t
Silk broin had various applications especially outstanding for drug delivery due to its protein component, biocompatibility and biodegradability. In this paper, silk broin particles were prepared via self-assembly. Their sizes and appearances could be modied by adjusting of volume ratios among poly vinyl alcohol (PVA), silk broin and ethanol. Regular silk particles were formed in PVA solution when the volume ratio of silk to ethanol ranged from 2 to 20. Preparation pathways could be concluded as 1) mixing ethanol with silk broin solution, 2) blending the silk broin/ethanol solution with PVA, 3) freezing the ternary solution for 48 h and collection of silk broin particles via thaw and centrifugation. Silk particles with various appearances were also obtained by addition of concentrated PVA solution. Silk particles reported have potential as drug delivery carriers in a variety of biomedical applications. 2011 Elsevier B.V. All rights reserved.

Article history: Received 16 May 2011 Received in revised form 27 August 2011 Accepted 9 September 2011 Available online 16 September 2011 Keywords: Silk broin Particles Self-assembly

1. Introduction Silk broin has widespread applications in biomedical research, such as controlled release and enzyme immobilization, due to its splendid biocompatibility and biodegradability [15]. Therefore, design and manufacture of devices composed by silk broin have been reported, including scaffolds and particles [47]. Most importantly, silk broin has great potential applications in controlled release system and target drug delivery, due to its unique protein component, strong mechanical stability and slow degradability, compared with that of the other natural and synthetic polymers, such as chitosan, alginate, starch, ethyl cellulose, polyanhydrides, polyorthoesters and polyesters etc. [89]. The advantages of silk broin fulll most of necessary requirements for a successful material on biomedical purpose. Techniques for fabrication of particles are vital. Sophisticated methods include emulsication, spray drying, lipid templating and self assembly. Silk broin particles can be developed by these methods. Further, current methods of preparing silk broin particles can be improved to avoid aggregation and deformation [2,1012]. In this article, a proposal for preparation of silk broin particles has been investigated. Briey, the particles are produced via self-assembly of regenerated silk broin under gentle condition, and PVA hydrogel is applied to improve quality of silk broin particles. The PVA molecular can give silk particles independent space to assemble, and simultaneously prevent particle aggregation. The size and appearance of regular silk broin particles

are controllable, and particle morphology also can be modied by adjusting the amount of ethanol and PVA applied in the reaction. 2. Materials and methods 2.1. Material Bombyx mori silk was supplied by Silk Innovation Center, Thailand. Calcium chloride, poly vinyl alcohol (PVA, 85,000124,000 Da), and other chemicals used in this study were purchased from SigmaAldrich. Ultrapure water from the Milli-Q system(Synthesis, A10) was used. 2.2. Silk purication Bombyx mori silk was degummed in 0.02 M NaHCO3 solution at 90C for 1.5 h to remove sericin completely. Then the silk solution was obtained by dissolving sericin-free silk bers in to a ternary solvent system of CaCl2/CH3CH2OH/H2O (1:2:8 in molar ratio) followed by dialysis [13]. Finally, 6% (w/v) silk solution was prepared and stored at 4 C for further application. 2.3. Preparation of silk particles The concentration of silk and PVA solution used here were adjusted to the same concentration (w/v), typically 2% silk and PVA solution were used here. Certain amount of silk solution and ethanol was mixed, and then PVA was added into the mixture, and fully blended with silk broin and ethanol. Several parallel tests were preceded to dene the best volume ratio among PVA, ethanol and silk broin. Briey the volume ratio of ethanol to silk broin solution was kept

Corresponding author at: Division of Bioengineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117575, Singapore. Tel.: + 65 65161911; fax: + 65 68723069. E-mail address: biegohj@nus.edu.sg (J.C.H. Goh). 0032-5910/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2011.09.012

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Fig. 1. a) Inuences of volume ratio of ethanol to silk solution on formation silk particles; SEM images of b) silk precipitation formed without addition of PVA, Vsilk/Vethanol = 5; ce) silk particles fabricated with addition of PVA (VPVA = 2Vsilk + ethanol ), Vsilk/Vethanol = 20 (c), 5 (d) and 2.5 (e); f) silk sedimentation formed with addition of PVA, Vsilk/Vethanol = 0.5.

constant at rst, and then the volume ratio of PVA to silk/ethanol mixed solution was adjusted from 1:10 to 10:1, to investigate the best proportion of PVA in total solution. Then, the volume ratio of ethanol to silk broin was modied from 1:20 to 20:1 under constant total addition of PVA. The whole liquid solution was frozen for 48 h before thawing and centrifugation (4000 g, 30 min). After centrifugation, supernatant was discarded, and pellets were collected and washed 3 times by ultrapure water prior to lyophilization. 2.4. Characterization 2.4.1. Fourier transform infrared (FTIR) spectroscopy Silk particles were centrifugated at 4000 g for half an hour, and subsequently washed by milli-Q water for 3 times. Then pellets were

collected, freeze-dried and analyzed by FTIR (Varian FT-IR 3100 Excalibur Series, Software: Resolution 4.05.009, USA), from 400 to 4000 cm 1. 2.4.2. Scanning electron microscopy (SEM) Silk particles after lyophilization were added directly on top of conductive tapes mounted on SEM sample stubs. The samples were sputtered with platinum. The morphologies of silk particles were investigated using a FEI XL30FEG scanning electron microscopy (FEI Company of USA, The Netherlands). 2.4.3. Particle size analysis Silk particles fabricated under various conditions were washed for 3 times to eliminate all liquid chemicals. And then, they were resuspended

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Fig. 2. Mechanism of silk particles formation: a) regular formation under inuences of PVA; b) unpredictable silk particles conformation free of PVA.

in Milli-Q water and analyzed by high performance particle size analyzer (HPPS, Malvern Instruments Ltd, UK). 3. Results and discussions 3.1. Silk precipitation efciency The inuence of ethanol on silk sedimentation was discovered by addition of pure ethanol to an aqueous silk broin solution of c = 20 mg/ml. Then silk/ethanol solution was mixed with double volume (volume: V and VPVA = 2Vsilk + ethanol) of 2% (w/v) PVA solution thoroughly, then froze for 48 h. Finally, silk particles were collected by centrifugation. The silk precipitation efciency was calculated using Eq. (1). Silk precipitation efficiency % silk pellet after lyophilization 100 initial silk addition 1

In the line with the conclusion reported by Cao et al. [14], the addition of ethanol to silk solution would induce silk molecular to form -sheet structure and self-assemble to form nucleation (micellar like structure), conrmed by FTIR results. Then, during the freezing process, more silk molecular would change to -sheet structure, and

attached to the nucleation, due to the ethanol concentration going up as water was slowly solidied. Finally, the nucleation would form silk particles. Moreover, PVA had vital inuences on the formation of silk particles, helped silk pellets to show regular conformation, as can be seen in Fig. 1b. PVA would form hydrogel during freezing process, and their molecular realigned to form crosslink network, which would restrain silk nucleation's movement as well as aggregation, appropriate amount of silk nucleation could connect to each other. Therefore, well formed silk particles were obtained (Fig. 2). The diameters of these silk particles were controllable, as can be seen in Fig. 1c, d and e. Along with volume ratio of silk to ethanol decreasing, the sizes of silk particles were changed from micro to nano. However, the total addition of ethanol could not be increased excessively, or there would be massive aggregation instead of particle formation (Fig. 1f), typically when Vsilk:Vethanol b 1 (Fig. 1a). This situation was probably caused by too many and tiny silk nucleation that was induced by excessive ethanol added, which was free from PVA molecular network and stuck together to form unpredictable conformation. Therefore, massive silk aggregation without uniform appearance would be obtained when no PVA or too much ethanol was added into the reaction of silk particles fabrication (Fig. 2). Further, intermediate status existed, namely both regular and irregular particles were observed, when volume ratio of silk solution to ethanol ranged from 2 to 1, as well as VPVA b 2Vsilk + ethanol (Fig. 3a

Fig. 3. SEM images of silk particles formed at a) Vsilk/Vethanol = 1.5 and VPVA = 2Vsilk + ethanol; b) Vsilk/Vethanol = 5 and VPVA b 2Vsilk + ethanol.

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and b). Probable explanation for the intermediate status was that some of excessive silk nucleation produced by ethanol escaped from control of PVA network, and aggregated. 3.2. Particle morphology As abovementioned, addition of PVA was necessary for formation of regular silk particles and reduction of aggregation. Moreover, modication of PVA concentration also inuenced the particle morphology signicantly. In Fig. 4a and b, the shape of particles formed in 4 and 6% PVA was changed obviously, compared with that of the particles fabricated in 2% PVA (Fig. 1d). The proposed theoretical explanation of this phenomenon (Fig. 4c) was that PVA solution would turn into hydrogel during freezing process, and PVA molecular would crosslink to form network where silk nucleation was restrained and growth to regular shape. However, after PVA hydrogel formed by 4% solution, their molecular rearranged and took over the position of silk nucleation which would aggregate with each other after been pushed out [Fig. 4c (1)]. Further, after PVA concentration went up to 6%, the silk nucleation was surrounded completely and compressed by PVA molecular during hydrogel formation [Fig. 4c (2)]. Therefore, aggregated and tabular silk particles were observed, and some of the particles were even crushed, as can be seen in Fig. 4a and b. 3.3. FTIR analysis Silk broin had characteristics vibration bands in their FTIR spectra, including 16301650 cm 1 for amide I (C_O stretching), 1540

1520 cm 1 for amide II (secondary N\H bending) and 1270 1230 cm 1 for amide III (C\N and N\H functionalities)[1516]. Further, the bands' positions also indicated the conformations of the protein material, which could be described as follows: 1650 cm 1, 1540 cm 1 as well as 1230 cm -1 representing random coil, and 1630 cm 1, 1520 cm 1 as well as 1270 cm 1 representing -sheet structure, for amide I, II and III separately. In Fig. 5a1, compared with a2, the characteristics vibration bands of silk particles were obviously shift to other positions where indicated -sheet structure, such as absorption peaks of 1651, 1541 and 1238 cm 1 (Fig. 5a1) moved to 1629, 1531 and 1237 cm 1 correspondently in Fig. 5a2. Moreover, the secondary structure composition of raw silk and particles produced by ethanol and PVA were determined by Fourier self deconvolution (FSD), as can be seen in Fig. 5b and c. Obviously, beta sheet peek appeared (16101635 cm 1) and random coil peak shifted (16351645 cm 1) in silk particle group (Fig. 5c) compared with that of raw silk group (Fig. 5b). Therefore, the silk particles were mostly composed by -sheet silk molecular. This conclusion was in the line with the results reported by Wang et al. and Cao et al. that silk broin particles manufactured by ethanol or PVA separately, mostly composed by -sheet silk molecular [11, 14]. Most importantly, the manufacture process reported in this article included 3 major steps, such as silk particles induced by ethanol, stabilized by PVA and preserved in low temperature ( 25 C, for example). This method avoided the particle aggregation compared with that of using ethanol only. In the mean time, once the particles were prepared, they could be stored in a freezer protecting any cargo inside, which was the major advantage compared with that of the

Fig. 4. SEM images of silk particles formed after addition of a) 4% PVA and b) 6% PVA, Vsilk/Vethanol = 5 and VPVA = 2Vsilk + ethanol; c) possible mechanism of PVA induced silk microspheres' transformation.

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b
%T

82 80 78

80

c
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79 78 77

76 74 72 70 68 A 66 1600 1620 1640 1660 1680 1700 Bs R

76 75 74 Bt Bs 73 72 71 1600 A Bs 1620 1640 1660 1680 1700 Bs R Bt

Wave Number (cm )

Wave Number (cm )

Fig. 5. FTIR spectra of raw silk (Bombyx mori, (a1) and silk particles prepared by ethanol induced self-assembly (a2); Fourier self deconvolution (FSD) of FTIR spectra for raw silk (b) and particles (c). The peaks are marked with abbreviations that stand for: beta turns (Bt), alpha helix (A), random coil (R), inter- and intramolecular beta sheets (Bs).

particles prepared by PVA alone which required dry and dissolving process to be collected. Cytokines, like BPM-2, VEGF and FGF were easily deactivated due to environmental changes, better to be processed and encapsulated as described in this article.

3.4. Size control During manufacture process of silk particles, their size was controllable by modication of the volume ratio of silk to ethanol, conrmed by high performance particle size analyzer (HPPS, Malvern Instruments Ltd, UK), as can be seen in Fig. 6a and b. When the volume ratio of silk to ethanol decreased from 20 to 2.5, the mean diameter

of silk particles decreased obviously, also endorsed by SEM observation (Fig. 1c, d and e). Moreover, the average size went down from 1525.3 nm to 983.62 nm, probably caused by more and smaller nucleation (-sheet structure) induced by additional ethanol. However, there would be a maximum limitation for addition of ethanol, otherwise massive aggregation would happen, especially when the volume ratio of silk to ethanol b 1 (Fig. 1f).

4. Conclusions In this article, a method of fabrication of silk broin particles via selfassembly using ethanol and PVA was reported. The size distribution and

b
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Fig. 6. Size analysis of silk particles produced from several volume ratio of silk to ethanol, a) size distributions of silk particles as a function of volume ratio of silk to ethanol; b) average size of silk particles as a function volume ratio of silk to ethanol. Error bars indicate the width of size distribution. (Volume ratio of silk to ethanol: 2.5, red; 5, yellow; and 20, blue.)

Di et am er

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Volume ratio (silk/ethanol)

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P. Shi, J.C.H. Goh / Powder Technology 215-216 (2012) 8590 [5] S. Sahoo, S.L. Toh, J.C.H. Goh, A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells, Biomaterials 31 (11) (2010) 29902998. [6] R.J. Xie, M.Z. Li, H.Y. Wu, Y.X. Feng, J.F. Yang, The preparation of silk broin microspheres, in: L. Bai (Ed.), Researches and Progresses of Modern Technology on Silk, Textile and Mechanicals I, CHEMICAL INDUSTRY PRESS, Beijing, 2007, pp. 341345. [7] R.J. Xie, H.Y. Wu, J.M. Xu, Q.M. Deng, The preparation of silk broin drug-loading microspheres, in: Y. Li, X.O. Luo, J.S. Li, A.Z. Chen (Eds.), Textile Bioengineering and Informatics Symposium Proceedings, VOLS. 1 AND 2, HONG KONG POLYTECHNIC UNIV, Hong Kong, 2008, pp. 603609. [8] P.J. Shi, Y. Zuo, Q. Zou, J. Shen, L. Zhang, Y.B. Li, et al., Improved properties of incorporated chitosan lm with ethyl cellulose microspheres for controlled release, International Journal of Pharmaceutics 375 (12) (2009) 6774. [9] E. Wenk, A.J. Wandrey, H.P. Merkle, L. Meinel, Silk broin spheres as a platform for controlled drug delivery, Journal of Controlled Release 132 (1) (2008) 2634. [10] J.H. Yeo, K.G. Lee, Y.W. Lee, S.Y. Kim, Simple preparation and characteristics of silk broin microsphere, European Polymer Journal 39 (6) (2003) 11951199. [11] X.Q. Wang, T. Yucel, Q. Lu, X. Hu, D.L. Kaplan, Silk nanospheres and microspheres from silk/pva blend lms for drug delivery, Biomaterials 31 (6) (2010) 10251035. [12] A.S. Lammel, X. Hu, S.H. Park, D.L. Kaplan, T.R. Scheibel, Controlling silk broin particle features for drug delivery, Biomaterials 31 (16) (2010) 45834591. [13] B.M. Min, G. Lee, S.H. Kim, Y.S. Nam, T.S. Lee, W.H. Park, Electrospinning of silk broin nanobers and its effect on the adhesion and spreading of normal human keratinocytes and broblasts in vitro, Biomaterials 25 (78) (2004) 12891297. [14] Z.B. Cao, X. Chen, J.R. Yao, L. Huang, Z.Z. Shao, The preparation of regenerated silk broin microspheres, Soft Matter 3 (7) (2007) 910915. [15] S. Hofmann, C. Foo, F. Rossetti, M. Textor, G. Vunjak-Novakovic, D.L. Kaplan, et al., Silk broin as an organic polymer for controlled drug delivery, Journal of Controlled Release 111 (12) (2006) 219227. [16] J. Kundu, Y.I. Chung, Y.H. Kim, G. Taeb, S.C. Kundu, Silk broin nanoparticles for cellular uptake and control release, International Journal of Pharmaceutics 388 (12) (2010) 242250.

appearance of regular spheres can be controlled by varying the amount of ethanol and PVA used in the fabrication process, moreover particle morphology could be modied by increasing concentration of PVA. The preparation method of silk particles reported in this article was mild, convenient, and compromised to loading requirements of medicines and cytokines which were easy to denature or degrade, hence should be helpful for silk-based drug delivery systems. Acknowledgment This research was supported by a grant from the Biomedical Research Council, Singapore. The authors also wish to thank colleagues in the National University of Singapore Tissue Engineering Program for their help. References
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