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Powder Technology 215-216 (2012) 85–90

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Self-assembled silk fibroin 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 fibroin had various applications especially outstanding for drug delivery due to its protein component, biocompatibility and biodegradability. In this paper, silk fibroin particles were prepared via self-assembly. Their sizes and appearances could be modified by adjusting of volume ratios among poly vinyl alcohol (PVA), silk fibroin 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 fibroin solution, 2) blending the silk fibroin/ethanol solution with PVA, 3) freezing the ternary solution for 48 h and collection of silk fibroin 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 fibroin Particles Self-assembly

1. Introduction Silk fibroin has widespread applications in biomedical research, such as controlled release and enzyme immobilization, due to its splendid biocompatibility and biodegradability [1–5]. Therefore, design and manufacture of devices composed by silk fibroin have been reported, including scaffolds and particles [4–7]. Most importantly, silk fibroin 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. [8–9]. The advantages of silk fibroin fulfill most of necessary requirements for a successful material on biomedical purpose. Techniques for fabrication of particles are vital. Sophisticated methods include emulsification, spray drying, lipid templating and self assembly. Silk fibroin particles can be developed by these methods. Further, current methods of preparing silk fibroin particles can be improved to avoid aggregation and deformation [2,10–12]. In this article, a proposal for preparation of silk fibroin particles has been investigated. Briefly, the particles are produced via self-assembly of regenerated silk fibroin under gentle condition, and PVA hydrogel is applied to improve quality of silk fibroin particles. The PVA molecular can give silk particles independent space to assemble, and simultaneously prevent particle aggregation. The size and appearance of regular silk fibroin particles

are controllable, and particle morphology also can be modified 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, 85,000–124,000 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 purification Bombyx mori silk was degummed in 0.02 M NaHCO3 solution at 90°C for 1.5 h to remove sericin completely. Then the silk solution was obtained by dissolving sericin-free silk fibers 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 fibroin and ethanol. Several parallel tests were preceded to define the best volume ratio among PVA, ethanol and silk fibroin. Briefly the volume ratio of ethanol to silk fibroin 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: (J.C.H. Goh). 0032-5910/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2011.09.012

USA). SEM images of b) silk precipitation formed without addition of PVA.5 (e).4. f) silk sedimentation formed with addition of PVA. a) Influences of volume ratio of ethanol to silk solution on formation silk particles. supernatant was discarded.009.4.86 P. 1. 2. Particle size analysis Silk particles fabricated under various conditions were washed for 3 times to eliminate all liquid chemicals. The samples were sputtered with platinum. Then. freeze-dried and analyzed by FTIR (Varian FT-IR 3100 Excalibur Series. 2. 2. Scanning electron microscopy (SEM) Silk particles after lyophilization were added directly on top of conductive tapes mounted on SEM sample stubs. c–e) silk particles fabricated with addition of PVA (VPVA = 2Vsilk + ethanol ). And then. 30 min).5.4. Vsilk/Vethanol = 5. and subsequently washed by milli-Q water for 3 times.4.3. from 400 to 4000 cm −1. Vsilk/Vethanol = 20 (c). Shi.H. The Netherlands). and pellets were collected and washed 3 times by ultrapure water prior to lyophilization.C. the volume ratio of ethanol to silk fibroin was modified from 1:20 to 20:1 under constant total addition of PVA. After centrifugation. they were resuspended . Goh / Powder Technology 215-216 (2012) 85–90 Fig. Vsilk/Vethanol = 0. Then pellets were collected. J.05. 5 (d) and 2. Fourier transform infrared (FTIR) spectroscopy Silk particles were centrifugated at 4000 g for half an hour. Software: Resolution 4. The morphologies of silk particles were investigated using a FEI XL30FEG scanning electron microscopy (FEI Company of USA. constant at first. to investigate the best proportion of PVA in total solution. Characterization 2. and then the volume ratio of PVA to silk/ethanol mixed solution was adjusted from 1:10 to 10:1.1.2. The whole liquid solution was frozen for 48 h before thawing and centrifugation (4000 g.

during the freezing process. [14]. or there would be massive aggregation instead of particle formation (Fig. UK). which was free from PVA molecular network and stuck together to form unpredictable conformation. when volume ratio of silk solution to ethanol ranged from 2 to 1. which would restrain silk nucleation's movement as well as aggregation. Then silk/ethanol solution was mixed with double volume (volume: V and VPVA = 2Vsilk + ethanol) of 2% (w/v) PVA solution thoroughly. Finally. typically when Vsilk:Vethanol b 1 (Fig. d and e.5 and VPVA = 2Vsilk + ethanol. Further. Mechanism of silk particles formation: a) regular formation under influences of PVA. the addition of ethanol to silk solution would induce silk molecular to form ß-sheet structure and self-assemble to form nucleation (micellar like structure). due to the ethanol concentration going up as water was slowly solidified. Silk precipitation efficiency ð%Þ ¼ silk pellet after lyophilization ×100 initial silk addition ð1Þ In the line with the conclusion reported by Cao et al. (1). then froze for 48 h. intermediate status existed. Results and discussions 3. 3a Fig. in Milli-Q water and analyzed by high performance particle size analyzer (HPPS. Then. the total addition of ethanol could not be increased excessively. SEM images of silk particles formed at a) Vsilk/Vethanol = 1. more silk molecular would change to ß-sheet structure. Along with volume ratio of silk to ethanol decreasing. This situation was probably caused by too many and tiny silk nucleation that was induced by excessive ethanol added. 1b. 1f). silk particles were collected by centrifugation. 1c. However. the sizes of silk particles were changed from micro to nano. .P.1. as can be seen in Fig.C. Moreover. 1a). Malvern Instruments Ltd. and attached to the nucleation. b) unpredictable silk particles conformation free of PVA. The silk precipitation efficiency was calculated using Eq. Goh / Powder Technology 215-216 (2012) 85–90 87 Fig. PVA had vital influences on the formation of silk particles. 3. Silk precipitation efficiency The influence of ethanol on silk sedimentation was discovered by addition of pure ethanol to an aqueous silk fibroin solution of c = 20 mg/ml. The diameters of these silk particles were controllable. 2. the nucleation would form silk particles. PVA would form hydrogel during freezing process. b) Vsilk/Vethanol = 5 and VPVA b 2Vsilk + ethanol. Shi. well formed silk particles were obtained (Fig. namely both regular and irregular particles were observed. Therefore. 2). 2). helped silk pellets to show regular conformation. confirmed by FTIR results. 3.H. as can be seen in Fig. and their molecular realigned to form crosslink network. as well as VPVA b 2Vsilk + ethanol (Fig. Therefore. appropriate amount of silk nucleation could connect to each other. J. Finally. 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.

and PVA molecular would crosslink to form network where silk nucleation was restrained and growth to regular shape. Obviously. once the particles were prepared. for amide I. . Further. 14]. In the mean time. J. 1540 cm −1 as well as 1230 cm -1 representing random coil. their molecular rearranged and took over the position of silk nucleation which would aggregate with each other after been pushed out [Fig. 1d). 1531 and 1237 cm −1 correspondently in Fig. In Fig. the manufacture process reported in this article included 3 major steps. Vsilk/Vethanol = 5 and VPVA = 2Vsilk + ethanol. 5a1) moved to 1629. 4a and b. after PVA hydrogel formed by 4% solution. compared with that of the particles fabricated in 2% PVA (Fig. In Fig. such as silk particles induced by ethanol. The proposed theoretical explanation of this phenomenon (Fig. the characteristics vibration bands of silk particles were obviously shift to other positions where indicated ß-sheet structure. 3. compared with a2. 4c (1)]. the silk nucleation was surrounded completely and compressed by PVA molecular during hydrogel formation [Fig. they could be stored in a freezer protecting any cargo inside. Goh / Powder Technology 215-216 (2012) 85–90 and b). 4. Particle morphology As abovementioned. the shape of particles formed in 4 and 6% PVA was changed obviously. FTIR analysis Silk fibroin had characteristics vibration bands in their FTIR spectra. the bands' positions also indicated the conformations of the protein material. 3. modification of PVA concentration also influenced the particle morphology significantly. 1541 and 1238 cm −1 (Fig. 4a and b. Therefore. II and III separately. Most importantly. aggregated and tabular silk particles were observed.88 P. beta sheet peek appeared (1610–1635 cm −1) and random coil peak shifted (1635–1645 cm −1) in silk particle group (Fig. 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)[15–16]. and some of the particles were even crushed. for example). Moreover.2. as can be seen in Fig. stabilized by PVA and preserved in low temperature (− 25 °C. that silk fibroin particles manufactured by ethanol or PVA separately. and 1630 cm −1. including 1630–1650 cm −1 for amide I (C_O stretching). This conclusion was in the line with the results reported by Wang et al. Therefore. Probable explanation for the intermediate status was that some of excessive silk nucleation produced by ethanol escaped from control of PVA network. 5a1. 5c) compared with that of raw silk group (Fig. 5a2. after PVA concentration went up to 6%.C. Moreover. 4c (2)]. and aggregated. Shi. 4c) was that PVA solution would turn into hydrogel during freezing process. 1520 cm −1 as well as 1270 cm −1 representing ß-sheet structure. c) possible mechanism of PVA induced silk microspheres' transformation. Further. mostly composed by ß-sheet silk molecular [11. such as absorption peaks of 1651. addition of PVA was necessary for formation of regular silk particles and reduction of aggregation. SEM images of silk particles formed after addition of a) 4% PVA and b) 6% PVA. which could be described as follows: 1650 cm −1. as can be seen in Fig. and Cao et al.3. the secondary structure composition of raw silk and particles produced by ethanol and PVA were determined by Fourier self deconvolution (FSD). 5b and c.H. However. the silk particles were mostly composed by ß-sheet silk molecular. which was the major advantage compared with that of the a b c Fig. This method avoided the particle aggregation compared with that of using ethanol only. 5b).

H. as can be seen in Fig. Malvern Instruments Ltd. there would be a maximum limitation for addition of ethanol. and 20. a) size distributions of silk particles as a function of volume ratio of silk to ethanol. However. The size distribution and a b 1600 1500 Average size (nm) 1400 1300 1200 1100 1000 30 00 25 00 0 20 0 15 00 10 00 0 50 0 5 1 10 5 20 25 volum e ratio (%) Fig. particles prepared by PVA alone which required dry and dissolving process to be collected.4.62 nm. (a1) and silk particles prepared by ethanol induced self-assembly (a2). The peaks are marked with abbreviations that stand for: beta turns (Bt).5. the average size went down from 1525. otherwise massive aggregation would happen.C. Conclusions In this article. Error bars indicate the width of size distribution. Cytokines. d and e). Shi.3 nm to 983. UK). 6a and b. red. 4. VEGF and FGF were easily deactivated due to environmental changes. (Volume ratio of silk to ethanol: 2. the mean diameter of silk particles decreased obviously. 5.and intramolecular beta sheets (Bs). 6. a method of fabrication of silk fibroin particles via selfassembly using ethanol and PVA was reported. 3.P. 1c. inter.5. Fourier self deconvolution (FSD) of FTIR spectra for raw silk (b) and particles (c). random coil (R). Moreover. alpha helix (A). yellow. Size control During manufacture process of silk particles. FTIR spectra of raw silk (Bombyx mori. 5. like BPM-2. J. b) average size of silk particles as a function volume ratio of silk to ethanol. their size was controllable by modification of the volume ratio of silk to ethanol. Goh / Powder Technology 215-216 (2012) 85–90 89 a b %T 82 80 78 80 c %T 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. When the volume ratio of silk to ethanol decreased from 20 to 2. Size analysis of silk particles produced from several volume ratio of silk to ethanol. probably caused by more and smaller nucleation (ß-sheet structure) induced by additional ethanol. blue. especially when the volume ratio of silk to ethanol b 1 (Fig. 1f).) Di et am er 900 5 10 15 20 (n m ) Volume ratio (silk/ethanol) . confirmed by high performance particle size analyzer (HPPS. better to be processed and encapsulated as described in this article. also endorsed by SEM observation (Fig.

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