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org/softmatter | Soft Matter

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 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.

1. Introduction thread core, and glue-like protein termed sericin that

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 self-
association of molecules into structurally well-defined archi-
tectures 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 struc-
tures 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: zzshao@fudan.edu.cn; Fax: +86 21 65640293;
Tel: +86 21 65642866
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 mem-
brane, which give silk fibroin a wide range of application.24–26
However, there is few reports about microparticles and nano-
particles 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

910 | Soft Matter, 2007, 3, 910–915 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 concen-
trated 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.
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.
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
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, anneal-
ing 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 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.

This journal is ß The Royal Society of Chemistry 2007 Soft Matter, 2007, 3, 910–915 | 911
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 well-
known 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 resolu-
tion.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.
Conformation of silk fibroin in microspheres
The conformational transition of silk fibroin in the micro-
spheres 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.
912 | Soft Matter, 2007, 3, 910–915
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 micro-
spheres 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 deter-
mination 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
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 measure-
ment 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 transi-
tion 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 cross-
linked 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 concen-
trations 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 micro-
spheres 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.
Fig. 6 The scheme of silk fibroin self-assembly to microspheres with
the presence of alcohol and freezing.
914 | Soft Matter, 2007, 3, 910–915
The glass transition temperature (Tg) of protein is con-
sidered to be one of the major determinants of protein self-
assembly. 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.
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