The preparation of regenerated silk fibroin

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Zhengbing Cao, Xin Chen, Jinrong Yao, Lei Huang and Zhengzhong Shao*. Received 1st .... stirring (100 rpm) at 25 uC over 2 min, each of the RSF solutions ...

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The preparation of regenerated silk fibroin microspheres Zhengbing Cao, Xin Chen, Jinrong Yao, Lei Huang and Zhengzhong Shao* Received 1st March 2007, Accepted 8th May 2007 First published as an Advance Article on the web 22nd May 2007 DOI: 10.1039/b703139d The objective of the present study is to investigate the possibility of preparing pure protein microspheres from regenerated silk fibroin (RSF). It is found that RSF microspheres, with predictable and controllable sizes ranging from 0.2 to 1.5 mm, can be prepared via mild selfassembling of silk fibroin molecular chains. The merits of this novel method include a rather simple production apparatus and no potentially toxic agents, such as surfactants, initiators, crosslinking agents, etc. The results show that the particle size and size distribution of RSF microspheres are greatly affected by the amount of ethanol additive, the freezing temperature and the concentration of silk fibroin. Finally, the mechanism of RSF microspheres formation is also discussed based on our experimental results.

1. Introduction Recently, nanoparticles with sizes between 100 to 1000 nm based on synthetic polymers as well as natural materials have been widely investigated for various applications, such as carriers for drug delivery.1–4 Nanoparticles have attracted great research interest in the field of drug delivery because they have the ability to deliver many kinds of drugs to targeted areas of the body for sustained periods of time. Compared to conventional dosage forms, these delivery systems provide numerous advantages, which include improved efficacy, reduced toxicity, and improved patient compliance and convenience.5,6 Among these colloidal systems, the one based on proteins may be rather promising, since it is biodegradable, non-antigenic and relatively easy to prepare. Furthermore, special amino acid sequences of protein may provide such protein-based nanoparticles with various possibilities for further surface modification and covalent drug attachment.7 In nature, we can find many examples of spontaneous selfassociation of molecules into structurally well-defined architectures on mesoscopic to macroscopic length scales.8,9 In particular, the b-sheet structure has received considerable attention for its self-assembling properties. It can form amyloid fibril, or as a building block in the formation of supramolecular materials such as peptide filament, film, nanotube, and crystal, and also plays a crucial role in pathogenesis.10,11 Therefore, understanding and controlling the self-assembly process of b-sheet in proteins may provide unique opportunities in the design of supramolecular structures for advanced material applications.12,13 Silks have been used by human beings for centuries mainly because of their unique luster, fineness, and mechanical properties. The most extensively characterized silks are from silkworms and spiders.14 The domestic silkworm (Bombyx mori) silk contains protein termed fibroin, which forms a Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai, 200433, People’s Republic of China. E-mail: [email protected]; Fax: +86 21 65640293; Tel: +86 21 65642866

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thread core, and glue-like protein termed sericin that surrounds the fibroin threads to cement them together.15 It is also found that silk fibroin is a native fibrous protein and consists chiefly of the repeated polypeptide sequence of Gly– Ala–Gly–Ala–Gly–Ser.16,17 In recent years, silks and the silk fibroins have been widely used as biomaterials, e.g. surgical suture, wound covering material, soft contact lens, scaffold for tissue engineering and controlled release carrier for their impressive biocompatibility and biodegradability.18–20 Previous research has indicated that the immune response mounted against silk in practical applications was mostly attributable to the glue-like sericin protein, not the core fibroin fibre.21 Also, there was evidence clearly showing that the silk was susceptible to proteolytic degradation in vivo and over longer periods it would be absorbed.14 Therefore, extensive studies have been carried out on the application of silk fibroin in non-textile fields regarding it as a kind of protein rather than a fibre.22–24 It is well known that silk fibroin can be processed into various forms, such as gel, powder, porous scaffold, nanofibre and membrane, which give silk fibroin a wide range of application.24–26 However, there is few reports about microparticles and nanoparticles which have potential application in such fields as controlled release and gene engineering.27,28 In this paper, we present a new approach to prepare a protein microsphere based on the self-assembling of regenerated silk fibroin (RSF) under very mild conditions, with rather simple production apparatus, and without any surfactant, initiator, cross-linking agent, or toxic organic reagent that would adversely affect the living body.

2. Experimental Materials To remove the sericin, the silkworm (Bombyx mori) silk was degummed in 0.5% (w/w) aqueous Na2CO3 solution at 95 uC for 1 h, washed with copious distilled water and then dried. The degummed silk was dissolved in 9.5 mol L21 aqueous LiBr solution. After being filtered, the fibroin solution was dialyzed against de-ionized water for 4 days at room temperature with 12–14 kDa cutoff semi-permeable membrane to remove the This journal is ß The Royal Society of Chemistry 2007

salt. The dialyzed silk fibroin solution was centrifuged at 6000 rpm for 10 min. Then, the supernatant was concentrated according to the method reported previously.29 The concentrated silk fibroin was diluted to a series of RSF solutions with concentration ranging from 1.0 to 10% (w/w) (determined by weighting method), and stored at 4 uC for further use. All other reagents were analytical grade and used as received.

were suspended in a series of phosphate buffers and measured by a zeta potential analyzer (zetaplus, Brookhaven, US) at 25 uC. The instrument was routinely calibrated with a 250 mV latex standard.

3. Results and discussion The shape and surface morphology of microspheres

Preparation of RSF microspheres After a certain amount of ethanol was added under gentle stirring (100 rpm) at 25 uC over 2 min, each of the RSF solutions with concentration from 1.0 to 10% (w/w) was incubated in a refrigerator at freezing temperature (25 to 240 uC) for 24 h. Then the frozen sample was defrosted at room temperature. It was found that the original transparent solution was turned into a milky suspension. Eventually, dry particles of RSF could be obtained by lyophilizing with a freezing dryer after stable RSF microspheres were collected by centrifugation (12 000 rpm, 30 min). Determination of particle size and its distribution The milky suspension was diluted 100 fold with de-ionized water, then the size and distribution of RSF microspheres was measured by photon-correlation spectroscopy (PCS) using a Malvern autosizer 4700 (Malvern Instruments Ltd., Malvern, UK) with a scattering angle of 90u at 25 uC. In order to verify the reproducibility of the preparation RSF microspheres assembled under the typical process described above, the procedure was carried out at least three times for each of the individual RSF microspheres.

Fig. 1 shows SEM photographs of RSF microparticles obtained by self-assembling with different amounts of ethanol additive under the typical process, in which the freezing temperature was 220 uC, RSF concentration was 3% (w/w) and the volume ratio of ethanol to RSF solution was 1 : 20 to 9 : 20. It can be seen that the particles with a coarse surface are spherical ones without apparent aggregation or adhesion. This was possibly caused by the amphiphilic property of silk fibroin in an aqueous environment. The silk fibroin is composed of hydrophilic side chains and hydrophobic segments. In the hydrophobic microcrystalline region, the distal end of the hydrophilic side chain might be extended into the aqueous solution, anchoring the hydrophilic block to the substrate surface through the hydrophobic segment. The intrusion of hydrophilic side chains into the solution resulted in the coarse sphere surface. It is worth noticing that Daamen et al. have recently prepared a new class of nano-/microcapsules using elastin as a model system, by three phases including fast-freezing, annealing and lyophilization.30 Although the freezing process was employed as well, we think the formation of RSF microspheres

Determination of morphology with scanning electron microscopy (SEM) and atomic force microscopy (AFM) For SEM, the diluted microspheres suspension was dropped on a clean graphite surface. After air-drying, the sample was coated with gold for 3 min using ion sputter. The shape and morphology of the RSF microspheres was observed using a Philips XL30 scanning electron microscope (Netherlands). For AFM, the diluted microspheres suspension was dropped on a freshly cleaved mica substrate and dried at room temperature, and was observed with Nanoscope IV Digital Instruments (Veeco Metrology Group, USA) in tapping mode, using a Si3N4 cantilever with a spring constant of 50 N m21 and a resonance frequency of 340 kHz. The scanning was performed at a scan speed of 0.8 Hz with a resolution of 512 6 512 pixels. FTIR spectroscopy A drop of RSF microspheres suspension was cast onto the aluminium foil and dried at room temperature. The FTIR spectra of RSF microspheres were obtained on a Magna-550 FTIR spectrometer (Nicolet, USA) in reflection mode with a resolution of 2 cm21. Zeta potential measurement To determine their surface charges using the technique of electrophoretic laser doppler anemometry, the microspheres This journal is ß The Royal Society of Chemistry 2007

Fig. 1 SEM images of RSF microspheres prepared with different volume ratios of ethanol added to SF solution. SF concentration was 3% (w/w), freezing temperature was 220 uC. VEtOH/VSF (A) 1 : 20, (B) 3 : 20, (C) 6 : 20, (D) 8 : 20 and (E) 9 : 20.

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might differ from those of ‘‘lyophilisomes’’ because silk fibroin is a particular fibrils protein for which the conformational transition is very sensitive to the ethanol additive and strength induced during the icing of aqueous solution (see discussion below). Also, there was no evidence to show that the produced RSF microspheres were hollow particles. Particle size analysis showed that the RSF microspheres were quite homogeneous in size, which could be varied from 0.2 to 1.5 mm in a dry state by controlling the amount of ethanol added. Larger spheres with a wider size distribution was formed by adding less ethanol. When the ratio of ethanol to RSF increase to 9 : 20 (v/v) or higher, an interesting phenomena can be found. There were a number of irregular aggregates formed in addition to the small microspheres (Fig. 1E). These aggregates indicated the formation of silk fibroin gel at higher ethanol concentration. We guess that the formation of the aggregates is an intermediate step in the wellknown sol–gel transition of RSF solution induced by a variety of factors.31 The AFM technique has been widely applied to obtain surface-dependent information in three dimensions on the nanometre scale. It can resolve surface details down to the atomic level and give morphological image in high resolution.32,33 The image of the shape and surface characteristic of the microspheres produced by RSF self-assembly was obtained successfully by tapping mode (Fig. 2), showing a uniform structure as in SEM photographs above. The 3D image of Fig. 2B further confirmed the fine spherical shape of RSF microspheres . The zeta potential is an important feature for the particle because a more pronounced zeta potential value, either positive or negative, favors the particle-suspension stability.34 The measurement on a zeta potential analyzer showed that the RSF microspheres were negatively charged. Their zeta potential values were slightly varied around 229 mV at the suspension of pH range 7–11. It suggested that the suspension of microspheres would be quite stable. Furthermore, the microspheres suspension could be stored for at least 3 months without visible coagulation occurring.

prepared with a high ethanol–RSF ratio was predominantly the b-sheet structure, with peaks at 1623 cm21 (amide I) and 1526 cm21 (amide II), whereas silk I structure was observed in those microspheres obtained with lower ethanol–RSF ratios, with peaks at 1648 cm21 (amide I) and 1539 cm21 (amide II).35,36 In other words, the higher the ethanol–RSF ratio, the more b-sheet structures in the microspheres, which is in accordance with the well-known conformational transition of silk fibroin from random coil and/or helical conformation to b-sheet induced by low dielectric organic solvents, such as methanol and ethanol.37,38 This means the increase of ethanol concentration favors the b-sheet structure, and results in higher crystallinity in the microspheres at the same freezing temperature and RSF concentration.37 Effect of ethanol addition and freezing temperature With the addition of ethanol, the conformational transition of silk fibroin from random coil to b-sheet is a spontaneous process, however, the transition rate is dependent on ethanol concentration.39 Therefore, we may prepare the RSF microspheres with different particle sizes by freezing the ethanol– RSF mixture with different ethanol concentration and temperature. In our experiments, we found RSF microspheres only formed within a narrow range of ethanol–RSF ratio, i.e. from 2 : 20 to 10 : 20. The silk fibroin precipitated when the ratio was less than 2 : 20 (we could find the particles at 1 : 20 occasionally, as showed in Fig. 1A) and gelated when the ratio was more than 10 : 20 under the typical conditions (RSF concentration was 3% (w/w) and freezing temperature was 220 uC). From the particle size and size distribution determination by photon-correlation spectroscopy (PCS), we found that the ethanol concentration had great effect on the size of microspheres. Both the size and the polydispersity index (PI) of the microspheres decreased with the increase of ethanol–RSF ratio from 2 : 20 to 8 : 20 (Fig. 4). It should be noted that the particle sizes presented in Fig. 4 were much larger than those of the counterparts shown in SEM images (Fig. 1). We

Conformation of silk fibroin in microspheres The conformational transition of silk fibroin in the microspheres was monitored by FTIR. As shown in Fig. 3, the secondary structure of the silk fibroin in microspheres

Fig. 2 AFM images of RSF microspheres: (A) High image, (B) 3D image. SF concentration was 5.0% (w/w), freezing temperature was 220 uC, VEtOH/VSF was 8 : 20.

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Fig. 3 FTIR spectra of RSF microspheres prepared with different volume ratios of ethanol to RSF solution. SF concentration was 3.0% (w/w), freezing temperature was 220 uC. VEtOH/VRSF (A) 1 : 20, (B) 4 : 20, (C) 6 : 20 and (D) 8 : 20, respectively.

This journal is ß The Royal Society of Chemistry 2007

Fig. 4 Particle size of RSF microspheres in the PCS measurement in correlation to the volume ratio of ethanol added to the SF solution at different freezing temperature (mean ¡ S.D., n ¢ 3). SF concentration was 3% (w/w).

supposed that this was mainly due to swelling of the RSF microspheres in the aqueous solution, regarding the measurement of PCS. The b-sheet structure of silk fibroin induced by ethanol could exist stably in the form of microcrystals in the ethanol– RSF mixture solution. The silk fibroin microcrystals could grow and aggregate together with the conformational transition process due to the ‘‘free’’ silk fibroin chains in the solution. The silk fibroin molecular chains may also entangle with one another, thus eventually forming a physically crosslinked gel. When the mixture solution was frozen, the gel particle was squeezed into a solid and prevented further aggregation. Therefore, the size of RSF microspheres was attributed to the kinetics of silk fibroin crystallization (conformation transition), which depended on the amount of ethanol added. The conformation transition was slow with low ethanol concentration and could be completed in a long period of up to several days.40 The RSF could not form microspheres when the freezing temperature was above 25 uC or below 245 uC. Between 25 and 245 uC, the size of microspheres decreased with the decrease of freezing temperature, as shown in Fig. 4. This phenomenon may be attributed to the kinetics of the freezing process. When the mixture solution was quenched at a lower temperature, the molecule chain movement of silk fibroin was restricted and the microcrystal was isolated within a very short time, which prevented the development of b-sheet structure and the aggregation of silk fibroin. Therefore, the size of RSF microspheres was small. Effect of RSF concentration Fig. 5 shows the PCS results of the size and size distribution of RSF microspheres prepared with various silk fibroin concentrations ranging from 1.0 to 10.0% (w/w) at 220 uC and 8 : 20 of ethanol–RSF ratio. The size of RSF microspheres increased from about 200 to 700 nm when RSF concentration increased. In the meantime, the PI of the size of microspheres also increased. It means the higher the RSF concentration, the This journal is ß The Royal Society of Chemistry 2007

Fig. 5 Particle size and polydispersity index (PI) of RSF microspheres prepared at different concentration SF solution (mean ¡ S.D., n ¢ 3). VEtOH/VRSF was 8 : 20, freezing temperature was 220 uC.

larger size with the wider size distribution of the microspheres formed In our previous work, we found aggregation of silk fibroin became faster when the microcrystal served as a seed.41 On the other hand, with the increase of silk fibroin concentration, the chance of interaction between microcrystals and ‘‘free’’ silk fibroin molecules, and the interactions among microspheres also increased. Thus in a limited space, microspheres were easier to aggregate and form larger congeries. This could be the reason that the size distribution of microspheres became wider with the increase of silk fibroin concentration. In particular, the PI increased significantly when the silk fibroin concentration was larger than 7.0% (w/w). In addition, we found it was very difficult to obtain uniform microspheres from high RSF concentration solution, which was very easy to form gel. The mechanism of RSF microspheres formation Polymers that contain both hydrophobic and hydrophilic segments could self-assemble in aqueous solution to form distinct structures, such as micelles, vesicles and tubules.42–44 This is largely due to the hydrophobic effect, which drives the non-polar region of each polymer molecule away from water and towards one another. The dimensions and shapes of the supermolecular structure formed from such assemblies depend on a variety factors, such as geometry of the polar head group and the shape of each molecule. Silk fibroin contains hydrophobic and hydrophilic segments, in which 73% of the amino acid residues are hydrophobic,25,45 but there are still a lot of amino acids with polar side groups, such as Ser, Tyr, Glu, and Asp that have strong affinity for water. It is generally accepted that silk fibroin is a fibrous protein characterized by the high content of Gly–Ala repeating units giving rise to some crystalline features.46,47 Furthermore, silk fibre is composed of amorphous regions and crystalline regions. The amorphous regions that are composed of amino acids with bulk side groups have poor orientation, but the repeated amino acid sequence (Gly–Ala–Gly–Ala–Gly–Ser)n tends to form the well-oriented anti-parallel b-sheet crystalline regions.48 Soft Matter, 2007, 3, 910–915 | 913

In a fresh RSF aqueous solution, the hydrophobic segments and hydrophilic segments are supposed to disperse randomly. Therefore, the silk fibroin molecules are easy to aggregate by physical or chemical stimuli, such as vibration, agitation, freezing and addition of organic solvents, to form the b-sheet structure and to be insolubilized.23,49,50 It is well known that ethanol is miscible with water in any ratio, but is a poor solvent for silk fibroin.51 In our study, when ethanol was added into the RSF aqueous solution, the molecular chains of silk fibroin interacted quickly and strongly with each other, and then the chains were rearranged in a regular array to some extent, resulting in a conformational transition from random and/or helical structure to b-sheet.28 As shown in Fig. 6, silk fibroin molecules firstly formed b-sheet microcrystals after the ethanol addition with gentle stirring. It has been reported that silk fibroin can exist as the b-sheet structure without precipitation from aqueous ethanol solution,52 for example, Yamada and co-workers have successfully found the microcrystals in an alcohol–silk fibroin mixture.33,53 After the formation of silk fibroin microcrystals, these b-sheet microcrystals could act as a seed for the growth of silk fibroin aggregation with continuous stirring.41 Subsequently, the shearing force produced during the freezing procedure of aqueous solution (kept in a refrigerator with a slow decrease of temperature) would induce silk fibroin conformational transition more completely. During the above process, the RSF microspheres could be maturated from the microcrystals by self-assembling. Such a microsphere may be composed of two ‘‘phases’’, e.g. the crystalline phase is the well-ordered core, and amorphous phase is the poor-ordered shell. Another possibility is that the amorphous phase entraps and attaches the crystalline phase to form a fine spherical shape. This solidification process of silk fibroin could be controlled by manipulating parameters, such as freezing temperature, concentration of silk fibroin and alcohol added that influence the hydrogen-bond formation to control the silk fibroin crystallization.54–56 It should be pointed out that none of those processes involves covalent cross-linking.

The glass transition temperature (Tg) of protein is considered to be one of the major determinants of protein selfassembly. Li et al. reported the effect of freezing temperature on the silk fibroin conformation and crystalline structure. They found that there exists a Tg of silk fibroin ranging from 234 to 220 uC, and the initial melting temperature of ice in frozen silk fibroin solution is about 28.5 uC.57 In our case, we think that the amount of ethanol added and the process of freezing are crucial for the formation of RSF microspheres. The assembly of silk fibroin initiated with the b-sheet formation was induced by ethanol, and then the RSF microcrystals grew and solidified into a well-defined structure by isolation of frozen water (ice), and the shearing force produced by the freezing process.

4. Conclusion The present study shows that RSF microspheres with predictable and reproducible sizes ranging from 0.2 mm to 1.5 mm could be prepared by a mild self-assembling of RSF solution by adding a small amount of ethanol and quenching below the freezing point. The obtained microspheres all have fine spherical shapes with coarse surfaces without apparent adhesion. The particle size and size distribution could be controlled by the conditions of preparation, such as the amount of ethanol added and the freezing temperature. Moreover, after lyophilization, these RSF microspheres did not aggregate and could be easily dispersed in distilled water again.

Acknowledgements This work is supported by the National Natural Science Foundation of China (NSFC, 20434010 and 20525414), the Science and Technology Development Foundation of Shanghai (05JC14009), the Program for New Century Excellent Talents in University of China (NCET, 00085901) and the Program for Changjiang Scholars and Innovative Research Team in University.

References

Fig. 6 The scheme of silk fibroin self-assembly to microspheres with the presence of alcohol and freezing.

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