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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
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predictable and controllable sizes ranging from 0.2 to 1.5 mm, can be prepared via mild self-
assembling 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, cross-
linking 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.
910 | Soft Matter, 2007, 3, 910915 This journal is The Royal Society of Chemistry 2007
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salt. The dialyzed silk fibroin solution was centrifuged at were suspended in a series of phosphate buffers and measured
6000 rpm for 10 min. Then, the supernatant was concentrated by a zeta potential analyzer (zetaplus, Brookhaven, US) at
according to the method reported previously.29 The concen- 25 uC. The instrument was routinely calibrated with a 250 mV
trated silk fibroin was diluted to a series of RSF solutions with latex standard.
concentration ranging from 1.0 to 10% (w/w) (determined by
weighting method), and stored at 4 uC for further use. All 3. Results and discussion
other reagents were analytical grade and used as received.
The shape and surface morphology of microspheres
Preparation of RSF microspheres Fig. 1 shows SEM photographs of RSF microparticles
After a certain amount of ethanol was added under gentle obtained by self-assembling with different amounts of ethanol
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stirring (100 rpm) at 25 uC over 2 min, each of the RSF additive under the typical process, in which the freezing
solutions with concentration from 1.0 to 10% (w/w) was temperature was 220 uC, RSF concentration was 3% (w/w)
incubated in a refrigerator at freezing temperature (25 to and the volume ratio of ethanol to RSF solution was 1 : 20 to
240 uC) for 24 h. Then the frozen sample was defrosted at 9 : 20. It can be seen that the particles with a coarse surface are
room temperature. It was found that the original transparent spherical ones without apparent aggregation or adhesion. This
solution was turned into a milky suspension. Eventually, dry was possibly caused by the amphiphilic property of silk fibroin
in an aqueous environment. The silk fibroin is composed
particles of RSF could be obtained by lyophilizing with a
of hydrophilic side chains and hydrophobic segments. In
freezing dryer after stable RSF microspheres were collected by
the hydrophobic microcrystalline region, the distal end of the
centrifugation (12 000 rpm, 30 min).
hydrophilic side chain might be extended into the aqueous
solution, anchoring the hydrophilic block to the substrate
Determination of particle size and its distribution
surface through the hydrophobic segment. The intrusion of
The milky suspension was diluted 100 fold with de-ionized hydrophilic side chains into the solution resulted in the coarse
water, then the size and distribution of RSF microspheres was sphere surface.
measured by photon-correlation spectroscopy (PCS) using a It is worth noticing that Daamen et al. have recently
Malvern autosizer 4700 (Malvern Instruments Ltd., Malvern, prepared a new class of nano-/microcapsules using elastin as a
UK) with a scattering angle of 90u at 25 uC. In order to verify model system, by three phases including fast-freezing, anneal-
the reproducibility of the preparation RSF microspheres ing and lyophilization.30 Although the freezing process was
assembled under the typical process described above, the employed as well, we think the formation of RSF microspheres
procedure was carried out at least three times for each of the
individual RSF microspheres.
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 Fig. 1 SEM images of RSF microspheres prepared with different
volume ratios of ethanol added to SF solution. SF concentration was
To determine their surface charges using the technique of 3% (w/w), freezing temperature was 220 uC. VEtOH/VSF (A) 1 : 20, (B)
electrophoretic laser doppler anemometry, the microspheres 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 prepared with a high ethanolRSF ratio was predominantly
is a particular fibrils protein for which the conformational the b-sheet structure, with peaks at 1623 cm21 (amide I) and
transition is very sensitive to the ethanol additive and strength 1526 cm21 (amide II), whereas silk I structure was observed in
induced during the icing of aqueous solution (see discussion those microspheres obtained with lower ethanolRSF ratios,
below). Also, there was no evidence to show that the produced with peaks at 1648 cm21 (amide I) and 1539 cm21 (amide
RSF microspheres were hollow particles. II).35,36 In other words, the higher the ethanolRSF ratio, the
Particle size analysis showed that the RSF microspheres more b-sheet structures in the microspheres, which is in
were quite homogeneous in size, which could be varied from accordance with the well-known conformational transition of
0.2 to 1.5 mm in a dry state by controlling the amount of silk fibroin from random coil and/or helical conformation to
ethanol added. Larger spheres with a wider size distribution b-sheet induced by low dielectric organic solvents, such as
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was formed by adding less ethanol. When the ratio of ethanol methanol and ethanol.37,38 This means the increase of ethanol
to RSF increase to 9 : 20 (v/v) or higher, an interesting concentration favors the b-sheet structure, and results in
phenomena can be found. There were a number of irregular higher crystallinity in the microspheres at the same freezing
aggregates formed in addition to the small microspheres temperature and RSF concentration.37
(Fig. 1E). These aggregates indicated the formation of silk
fibroin gel at higher ethanol concentration. We guess that the Effect of ethanol addition and freezing temperature
formation of the aggregates is an intermediate step in the well-
With the addition of ethanol, the conformational transition of
known solgel transition of RSF solution induced by a variety
silk fibroin from random coil to b-sheet is a spontaneous
of factors.31
process, however, the transition rate is dependent on ethanol
The AFM technique has been widely applied to obtain
concentration.39 Therefore, we may prepare the RSF micro-
surface-dependent information in three dimensions on the
spheres with different particle sizes by freezing the ethanol
nanometre scale. It can resolve surface details down to the
RSF mixture with different ethanol concentration and
atomic level and give morphological image in high resolu-
temperature. In our experiments, we found RSF microspheres
tion.32,33 The image of the shape and surface characteristic of
the microspheres produced by RSF self-assembly was obtained only formed within a narrow range of ethanolRSF ratio, i.e.
successfully by tapping mode (Fig. 2), showing a uniform from 2 : 20 to 10 : 20. The silk fibroin precipitated when the
structure as in SEM photographs above. The 3D image of ratio was less than 2 : 20 (we could find the particles at 1 : 20
Fig. 2B further confirmed the fine spherical shape of RSF occasionally, as showed in Fig. 1A) and gelated when the ratio
microspheres . was more than 10 : 20 under the typical conditions (RSF
The zeta potential is an important feature for the particle concentration was 3% (w/w) and freezing temperature was
because a more pronounced zeta potential value, either 220 uC). From the particle size and size distribution deter-
positive or negative, favors the particle-suspension stability.34 mination by photon-correlation spectroscopy (PCS), we found
The measurement on a zeta potential analyzer showed that that the ethanol concentration had great effect on the size of
the RSF microspheres were negatively charged. Their zeta microspheres. Both the size and the polydispersity index (PI) of
potential values were slightly varied around 229 mV at the the microspheres decreased with the increase of ethanolRSF
suspension of pH range 711. It suggested that the suspension ratio from 2 : 20 to 8 : 20 (Fig. 4). It should be noted that the
of microspheres would be quite stable. Furthermore, the particle sizes presented in Fig. 4 were much larger than those
microspheres suspension could be stored for at least 3 months of the counterparts shown in SEM images (Fig. 1). We
without visible coagulation occurring.
912 | Soft Matter, 2007, 3, 910915 This journal is The Royal Society of Chemistry 2007
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In a fresh RSF aqueous solution, the hydrophobic segments The glass transition temperature (Tg) of protein is con-
and hydrophilic segments are supposed to disperse randomly. sidered to be one of the major determinants of protein self-
Therefore, the silk fibroin molecules are easy to aggregate by assembly. Li et al. reported the effect of freezing temperature
physical or chemical stimuli, such as vibration, agitation, on the silk fibroin conformation and crystalline structure.
freezing and addition of organic solvents, to form the b-sheet They found that there exists a Tg of silk fibroin ranging from
structure and to be insolubilized.23,49,50 It is well known that 234 to 220 uC, and the initial melting temperature of ice in
ethanol is miscible with water in any ratio, but is a poor frozen silk fibroin solution is about 28.5 uC.57 In our case, we
solvent for silk fibroin.51 In our study, when ethanol was think that the amount of ethanol added and the process of
added into the RSF aqueous solution, the molecular chains of freezing are crucial for the formation of RSF microspheres.
silk fibroin interacted quickly and strongly with each other, The assembly of silk fibroin initiated with the b-sheet
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and then the chains were rearranged in a regular array to some formation was induced by ethanol, and then the RSF
extent, resulting in a conformational transition from random microcrystals grew and solidified into a well-defined structure
and/or helical structure to b-sheet.28 by isolation of frozen water (ice), and the shearing force
As shown in Fig. 6, silk fibroin molecules firstly formed produced by the freezing process.
b-sheet microcrystals after the ethanol addition with gentle
stirring. It has been reported that silk fibroin can exist as the 4. Conclusion
b-sheet structure without precipitation from aqueous ethanol
solution,52 for example, Yamada and co-workers have The present study shows that RSF microspheres with
successfully found the microcrystals in an alcoholsilk fibroin predictable and reproducible sizes ranging from 0.2 mm to
mixture.33,53 After the formation of silk fibroin microcrystals, 1.5 mm could be prepared by a mild self-assembling of RSF
these b-sheet microcrystals could act as a seed for the growth solution by adding a small amount of ethanol and quenching
of silk fibroin aggregation with continuous stirring.41 below the freezing point. The obtained microspheres all have
Subsequently, the shearing force produced during the freezing fine spherical shapes with coarse surfaces without apparent
procedure of aqueous solution (kept in a refrigerator with a adhesion. The particle size and size distribution could be
slow decrease of temperature) would induce silk fibroin controlled by the conditions of preparation, such as the
conformational transition more completely. During the above amount of ethanol added and the freezing temperature.
process, the RSF microspheres could be maturated from the Moreover, after lyophilization, these RSF microspheres
microcrystals by self-assembling. Such a microsphere may be did not aggregate and could be easily dispersed in distilled
composed of two phases, e.g. the crystalline phase is the water again.
well-ordered core, and amorphous phase is the poor-ordered
shell. Another possibility is that the amorphous phase entraps Acknowledgements
and attaches the crystalline phase to form a fine spherical
This work is supported by the National Natural Science
shape.
Foundation of China (NSFC, 20434010 and 20525414),
This solidification process of silk fibroin could be controlled
the Science and Technology Development Foundation of
by manipulating parameters, such as freezing temperature,
Shanghai (05JC14009), the Program for New Century
concentration of silk fibroin and alcohol added that influence
Excellent Talents in University of China (NCET, 00085901)
the hydrogen-bond formation to control the silk fibroin
and the Program for Changjiang Scholars and Innovative
crystallization.5456 It should be pointed out that none of
Research Team in University.
those processes involves covalent cross-linking.
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