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

⁎ Corresponding author at: Division of Bioengineeringpore, 9 Engineering Drive 1, Singapore 117575, Singapor65 68723069.

E-mail address: biegohj@nus.edu.sg (J.C.H. Goh).

0032-5910/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.powtec.2011.09.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 May 2011Received in revised form 27 August 2011Accepted 9 September 2011Available online 16 September 2011

Keywords:Silk fibroinParticlesSelf-assembly

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 ratioof silk to ethanol ranged from 2 to 20. Preparation pathways could be concluded as 1) mixing ethanol withsilk fibroin solution, 2) blending the silk fibroin/ethanol solution with PVA, 3) freezing the ternary solutionfor 48 h and collection of silk fibroin particles via thaw and centrifugation. Silk particles with various appear-ances were also obtained by addition of concentrated PVA solution. Silk particles reported have potential asdrug delivery carriers in a variety of biomedical applications.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Silk fibroin has widespread applications in biomedical research,such as controlled release and enzyme immobilization, due to its splen-did biocompatibility and biodegradability [1–5]. Therefore, design andmanufacture of devices composed by silk fibroin have been reported,including scaffolds and particles [4–7]. Most importantly, silk fibroinhas great potential applications in controlled release system and targetdrug delivery, due to its unique protein component, strong mechanicalstability and slow degradability, compared with that of the other natu-ral and synthetic polymers, such as chitosan, alginate, starch, ethyl cellu-lose, polyanhydrides, polyorthoesters and polyesters etc. [8–9]. Theadvantages of silk fibroin fulfill most of necessary requirements for asuccessful material on biomedical purpose.

Techniques for fabrication of particles are vital. Sophisticatedmethods include emulsification, spray drying, lipid templating and selfassembly. Silk fibroin particles can be developed by thesemethods. Fur-ther, current methods of preparing silk fibroin particles can be im-proved 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 silkfibroin under gentle condition, and PVA hydrogel is applied to improvequality of silk fibroin particles. The PVAmolecular can give silk particlesindependent space to assemble, and simultaneously prevent particleaggregation. The size and appearance of regular silk fibroin particles

are controllable, and particle morphology also can be modified byadjusting the amount of ethanol and PVA applied in the reaction.

2. Materials and methods

2.1. Material

Bombyxmori silkwas supplied by Silk Innovation Center, Thailand.Calcium chloride, poly vinyl alcohol (PVA, 85,000–124,000 Da), andother chemicals used in this study were purchased from Sigma-Aldrich. 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 at90°C for 1.5 h to remove sericin completely. Then the silk solutionwas obtained by dissolving sericin-free silk fibers in to a ternary sol-vent system of CaCl2/CH3CH2OH/H2O (1:2:8 in molar ratio) followedby dialysis [13]. Finally, 6% (w/v) silk solution was prepared andstored at 4°C for further application.

2.3. Preparation of silk particles

The concentration of silk and PVA solution used here were adjust-ed to the same concentration (w/v), typically 2% silk and PVA solutionwere used here. Certain amount of silk solution and ethanol wasmixed, and then PVA was added into the mixture, and fully blendedwith silk fibroin and ethanol. Several parallel tests were preceded todefine the best volume ratio among PVA, ethanol and silk fibroin.Briefly the volume ratio of ethanol to silk fibroin solution was kept

Fig. 1. a) Influences 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; c–e)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.

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constant at first, and then the volume ratio of PVA to silk/ethanolmixed solution was adjusted from 1:10 to 10:1, to investigate thebest proportion of PVA in total solution. Then, the volume ratio of eth-anol to silk fibroin was modified from 1:20 to 20:1 under constanttotal addition of PVA. The whole liquid solution was frozen for 48 hbefore thawing and centrifugation (4000 g, 30 min). After centrifuga-tion, supernatant was discarded, and pellets were collected andwashed 3 times by ultrapure water prior to lyophilization.

2.4. Characterization

2.4.1. Fourier transform infrared (FTIR) spectroscopySilk 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 Excal-ibur Series, Software: Resolution 4.05.009, USA), from 400 to4000 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 weresputtered with platinum. The morphologies of silk particles were in-vestigated using a FEI XL30FEG scanning electron microscopy (FEICompany of USA, The Netherlands).

2.4.3. Particle size analysisSilk particles fabricated under various conditions were washed for 3

times to eliminate all liquid chemicals. And then, theywere resuspended

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

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inMilli-Qwater and analyzed by high performance particle size analyzer(HPPS, Malvern Instruments Ltd, UK).

3. Results and discussions

3.1. Silk precipitation efficiency

The influence of ethanol on silk sedimentation was discovered byaddition of pure ethanol to an aqueous silk fibroin solution ofc=20 mg/ml. Then silk/ethanol solution was mixed with double vol-ume (volume: V and VPVA=2Vsilk+ethanol) of 2% (w/v) PVA solutionthoroughly, then froze for 48 h. Finally, silk particles were collectedby centrifugation. The silk precipitation efficiency was calculatedusing Eq. (1).

Silk precipitation efficiency %ð Þ ¼ silk pellet after lyophilizationinitial silk addition

×100

ð1Þ

In the line with the conclusion reported by Cao et al. [14], the ad-dition of ethanol to silk solution would induce silk molecular to formß-sheet structure and self-assemble to form nucleation (micellar likestructure), confirmed by FTIR results. Then, during the freezing pro-cess, more silk molecular would change to ß-sheet structure, and

Fig. 3. SEM images of silk particles formed at a) Vsilk/Vethanol=1.5 and

attached to the nucleation, due to the ethanol concentration goingup as water was slowly solidified. Finally, the nucleation wouldform silk particles. Moreover, PVA had vital influences on the forma-tion of silk particles, helped silk pellets to show regular conformation,as can be seen in Fig. 1b. PVA would form hydrogel during freezingprocess, and their molecular realigned to form crosslink network,which would restrain silk nucleation's movement as well as aggrega-tion, appropriate amount of silk nucleation could connect to eachother. Therefore, well formed silk particles were obtained (Fig. 2).The diameters of these silk particles were controllable, as can beseen in Fig. 1c, d and e. Along with volume ratio of silk to ethanol de-creasing, the sizes of silk particles were changed from micro to nano.However, the total addition of ethanol could not be increased exces-sively, or there would be massive aggregation instead of particle for-mation (Fig. 1f), typically when Vsilk:Vethanol b1 (Fig. 1a). Thissituation was probably caused by too many and tiny silk nucleationthat was induced by excessive ethanol added, which was free fromPVA molecular network and stuck together to form unpredictableconformation. Therefore, massive silk aggregation without uniformappearance would be obtained when no PVA or too much ethanolwas added into the reaction of silk particles fabrication (Fig. 2).

Further, intermediate status existed, namely both regular and ir-regular particles were observed, when volume ratio of silk solutionto ethanol ranged from 2 to 1, as well as VPVAb2Vsilk+ethanol (Fig. 3a

VPVA=2Vsilk+ethanol; b) Vsilk/Vethanol=5 and VPVAb 2Vsilk+ethanol.

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and b). Probable explanation for the intermediate status was thatsome of excessive silk nucleation produced by ethanol escaped fromcontrol of PVA network, and aggregated.

3.2. Particle morphology

As abovementioned, addition of PVA was necessary for formationof regular silk particles and reduction of aggregation. Moreover, mod-ification of PVA concentration also influenced the particle morpholo-gy significantly. In Fig. 4a and b, the shape of particles formed in 4 and6% PVA was changed obviously, compared with that of the particlesfabricated in 2% PVA (Fig. 1d). The proposed theoretical explanationof this phenomenon (Fig. 4c) was that PVA solution would turn intohydrogel during freezing process, and PVA molecular would crosslinkto form network where silk nucleation was restrained and growth toregular shape. However, after PVA hydrogel formed by 4% solution,their molecular rearranged and took over the position of silk nucle-ation which would aggregate with each other after been pushed out[Fig. 4c (1)]. Further, after PVA concentration went up to 6%, the silknucleation was surrounded completely and compressed by PVA mo-lecular during hydrogel formation [Fig. 4c (2)]. Therefore, aggregatedand tabular silk particles were observed, and some of the particleswere even crushed, as can be seen in Fig. 4a and b.

3.3. FTIR analysis

Silk fibroin had characteristics vibration bands in their FTIR spec-tra, including 1630–1650 cm−1 for amide I (C_O stretching), 1540–

a

c

Fig. 4. SEM images of silk particles formed after addition of a) 4% PVA and b) 6% PVA, Vsilk/Vspheres' transformation.

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]. Fur-ther, the bands' positions also indicated the conformations of the pro-tein material, which could be described as follows: 1650 cm−1,1540 cm−1 as well as 1230 cm-1 representing random coil, and1630 cm−1, 1520 cm−1 as well as 1270 cm−1 representing ß-sheetstructure, for amide I, II and III separately. In Fig. 5a1, comparedwith a2, the characteristics vibration bands of silk particles were ob-viously shift to other positions where indicated ß-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 parti-cles produced by ethanol and PVA were determined by Fourier selfdeconvolution (FSD), as can be seen in Fig. 5b and c. Obviously, betasheet peek appeared (1610–1635 cm−1) and random coil peakshifted (1635–1645 cm−1) in silk particle group (Fig. 5c) comparedwith that of raw silk group (Fig. 5b). Therefore, the silk particleswere mostly composed by ß-sheet silk molecular.

This conclusion was in the line with the results reported by Wanget al. and Cao et al. that silk fibroin particles manufactured by ethanolor PVA separately, mostly composed by ß-sheet silk molecular [11,14]. Most importantly, the manufacture process reported in this arti-cle included 3 major steps, such as silk particles induced by ethanol,stabilized by PVA and preserved in low temperature (−25 °C, for ex-ample). This method avoided the particle aggregation compared withthat of using ethanol only. In the mean time, once the particles wereprepared, they could be stored in a freezer protecting any cargo in-side, which was the major advantage compared with that of the

b

ethanol=5 and VPVA=2Vsilk+ethanol; c) possible mechanism of PVA induced silk micro-

1600 1620 1640 1660 1680 170066

68

70

72

74

76

78

80

82

%T

Bs

R

A

BtBs

1600 1620 1640 1660 1680 170071

72

73

74

75

76

77

78

79

80

Wave Number (cm )Wave Number (cm )

Bs

Bs

A

BtR

%T

cb

a

Fig. 5. FTIR spectra of raw silk (Bombyxmori, (a1) and silk particles prepared by ethanol induced self-assembly (a2); Fourier self deconvolution (FSD) of FTIR spectra for raw silk (b) andparticles (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).

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particles prepared by PVA alone which required dry and dissolvingprocess to be collected. Cytokines, like BPM-2, VEGF and FGF wereeasily deactivated due to environmental changes, better to be pro-cessed and encapsulated as described in this article.

3.4. Size control

Duringmanufacture process of silk particles, their sizewas control-lable by modification of the volume ratio of silk to ethanol, confirmedby high performance particle size analyzer (HPPS, Malvern Instru-ments Ltd, UK), as can be seen in Fig. 6a and b. When the volumeratio of silk to ethanol decreased from 20 to 2.5, the mean diameter

500

1000

1500

2000

2500

3000

05

1015

20

25

volume ratio (%)

Diameter (nm

)

a

Fig. 6. Size analysis of silk particles produced from several volume ratio of silk to ethanol, a) sizeof silk particles as a function volume ratio of silk to ethanol. Error bars indicate the width

of silk particles decreased obviously, also endorsed by SEM observa-tion (Fig. 1c, d and e). Moreover, the average size went down from1525.3 nm to 983.62 nm, probably caused by more and smaller nucle-ation (ß-sheet structure) induced by additional ethanol. However,there would be a maximum limitation for addition of ethanol, other-wise massive aggregation would happen, especially when the volumeratio of silk to ethanol b1 (Fig. 1f).

4. Conclusions

In this article, amethod of fabrication of silk fibroin particles via self-assembly using ethanol and PVAwas reported. The size distribution and

5 10 15 20

900

1000

1100

1200

1300

1400

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1600

Ave

rage

siz

e (n

m)

Volume ratio (silk/ethanol)

b

distributions of silk particles as a function of volume ratio of silk to ethanol; b) average sizeof size distribution. (Volume ratio of silk to ethanol: 2.5, red; 5, yellow; and 20, blue.)

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appearance of regular spheres can be controlled by varying the amountof ethanol and PVA used in the fabrication process, moreover particlemorphology could be modified by increasing concentration of PVA.The preparation method of silk particles reported in this article wasmild, convenient, and compromised to loading requirements of medi-cines and cytokines which were easy to denature or degrade, henceshould be helpful for silk-based drug delivery systems.

Acknowledgment

This research was supported by a grant from the Biomedical Re-search Council, Singapore. The authors also wish to thank colleaguesin the National University of Singapore Tissue Engineering Programfor their help.

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