effect of surfactants on sol–gel transition of silk fibroin
TRANSCRIPT
ORIGINAL PAPER
Effect of surfactants on sol–gel transition of silk fibroin
Ji Hun Park • Min Hee Kim • Lim Jeong •
Donghwan Cho • Oh Hyeong Kwon •
Won Ho Park
Received: 3 December 2013 / Accepted: 28 April 2014
� Springer Science+Business Media New York 2014
Abstract In this study, various surfactants were added to
control the gelation time of silk fibroin (SF) aqueous
solution. The gelation behaviors of SF aqueous solution in
the presence of surfactant were investigated with attenu-
ated total reflectance infrared, SEM, and a viscometer.
When surfactants other than chitooligosaccharide were
added into an SF aqueous solution, the gelation time of the
solution was decreased under the fixed conditions. Partic-
ularly, anionic surfactant was found to be more effective
than non-ionic and cationic surfactants in accelerating the
gelation of SF. In addition, the conformational changes of
SF hydrogel with or without surfactant were investigated in
a time-resolved manner using infrared spectroscopy. Con-
formational transitions of SF nanofibers from random coil
to b-sheet forms were strongly dependent on the inherent
properties of surfactant, and on the different interactions
between surfactant and SF molecules in aqueous solution.
This approach to controlling the gelation of SF aqueous
solution by the surfactant, and to monitoring their confor-
mational changes on a real-time scale, may be critical in
the design and tailoring of SF hydrogels useful for bio-
medical applications.
Keywords Silk fibroin � Gelation time � Sol–gel
transition � Surfactant
1 Introduction
Hydrogel is an attractive physical form for tissue engi-
neering and regenerative medicine, because of its excellent
biocompatibility due to high water content, as well as the
nature of polymer [1]. Hydrogels are widely used in vari-
ous biomedical applications, such as drug delivery systems,
contact lenses, encapsulation materials for gene or cell
therapeutics, wound dressings, and scaffolds for soft tissue
engineering [2–5].
Silk is a natural fibrous protein that is spun into fiber by
silkworms and spiders. Silkworm silk primarily consists of
two protein components: fibroin and sericin. Pure fibroin
component can be obtained by removing the sericin using
various degumming conditions. Silk fibroin (SF) can also
be regenerated by dissolving with an appropriate solvent,
and subsequently fabricated into various forms, such as
gels, powders, fibers, and films [6–10]. SF has been widely
used for cosmetics and food additives, and has recently
been found to have potential in the areas of biomedical
science and engineering due to its distinctive biological
properties including biocompatibility, oxygen and water
vapor permeability, biodegradability, and minimal inflam-
matory responses in vivo [11–14]. One of the promising
applications of SF in biomedical engineering is as a scaf-
folding material for tissue regeneration. It was reported that
SF can be useful for the culture of fibroblasts and osteo-
blasts, and can enhance the adhesion, growth, and differ-
entiation of cells with benefits quite similar to those of
collagen matrices [15–18]. In addition, SF hydrogel pre-
pared by physical crosslinking (i.e., crystallization) is very
attractive in biomedical fields because it has an excellent
biocompatibility. The gelation mechanism of SF was
studied in terms of the formation of b-sheet structure in an
SF aqueous solution. The formation of SF hydrogel can be
J. H. Park � M. H. Kim � L. Jeong � W. H. Park (&)
Department of Textile Engineering, College of Engineering,
Chungnam National University, Daejeon 305-764, South Korea
e-mail: [email protected]
D. Cho � O. H. Kwon
Department of Polymer Science and Engineering, Kumoh
Institute of Technology, Kumi city, South Korea
123
J Sol-Gel Sci Technol
DOI 10.1007/s10971-014-3379-4
adjusted by changing physical or chemical conditions, such
as concentration of SF aqueous solution, temperature, pH
and additives [3, 19–23]. The results indicated that the
gelation of SF is closely associated with the formation of
the b-sheet structure. The presence of a large amount of
amino acids with a short side chain such as Gly and Ala in
SF molecular chains favors the formation of b-sheet
structure, not a-helical structure. This transition in the
secondary structure of SF from random coil structure to b-
sheet structure is irreversible, unlike with other
biopolymers.
In this study, non-ionic, cationic, and anionic surfactants
were used to change the gelation rate of SF aqueous solu-
tion. A custom-made chamber was used to obtain time-
resolved infrared spectra of an SF solution during hydrog-
elation. The effect of ionic group and alkyl group of sur-
factants on the hydrogelation of SF was also investigated.
2 Experimental methods
2.1 Materials
Triton X-100, octyltrimethyl ammonium bromide (OTAB),
dodecyltrimethyl ammonium bromide (DTAB), hexade-
cyltrimethyl ammonium bromide (HTAB) and sodium
dodecyl benzene sulfonate (SDBS) were purchased from
Sigma-Aldrich. Chitooligosaccharide (CHI) (DD = 87 %)
was supplied by Hyosung Co. (Korea) and its compositions
are as follows: dimer 2.31, trimer 12.53, tetramer 15.11,
pentamer 13.59, hexamer 8.86, heptamer 6.46, octamer
8.87, nonamer or higher 32.27 mol%.
2.2 Regenerated SF solution
Degummed silk yarn was dissolved in a ternary solvent
system composed of calcium chloride, ethanol and water
(1:2:8 molar ratio) at 70 �C for 6 h, followed by dialysis
with cellulose tubular membranes (250-7 l, Sigma) against
distilled water for 3 days [17]. The dialyzed SF solution
was centrifuged to remove the insoluble residues, and the
final concentration of resultant SF solution was approxi-
mately 2.5 wt%.
2.3 Gelation of SF/surfactant solution
In order to investigate the effect of various surfactants on
the gelation of SF aqueous solution, the surfactant solutions
with different concentrations were prepared by dissolving
and defoaming in water for 12 h. The structure of surfac-
tants used is shown in Fig. 1. The surfactant solution was
added slowly into the SF aqueous solution in the range of
0–100 mM. 1.0 mL of SF aqueous solution containing
surfactants was placed in 10 mL flat-bottomed vials kept at
37 �C. The gelation time was determined when the sample
appeared in an opaque white color and did not flow from an
inverted vial within 30 s [20].
2.4 Characterization
A custom-made chamber with a temperature controller was
used for the time-resolved measurements of IR spectra of
the samples (Fig. 2). The measurement was started once
the sample was placed in the chamber. IR spectra were
taken at a resolution of 2 cm-1 using a Magma 560 spec-
trometer (Nicolet). A scanning electron microscope (Hit-
achi S-2350) was used to investigate the macroscopic
morphology of the freeze-dried SF gel. A viscometer (SV-
10, A&D Company) was used to determine the viscosity
change in SF solution until the gelation was completed.
3 Results and discussion
3.1 Effect of non-ionic surfactant
Hydrophobic association and hydrogen bonding are the
main interactions involved in the aggregation of polymer/
non-ionic surfactant systems [24]. Figure 3 shows the
gelation time of SF solution with different Triton X-100
contents at 37 �C. By adding Triton X-100, a non-ionic
surfactant, the gelation time of SF was abruptly reduced to
about 15 h until a concentration of 50 mM, and slowed
down thereafter to 12 h at 100 mM with increasing Triton
X-100 concentration. This seems to have been induced by
dehydration caused by the surfactants (‘‘salt out’’ effect).
This supports that the hydrophilic group of surfactant took
away the hydrated water molecules from SF molecular
chains and was combined into SF hydrophobic chains,
because they have a tendency to combine polymer chains
rather than to generate micelles in polymer solution. Sub-
sequently, the gelation of SF aqueous solution was pro-
moted due to the enhanced hydrophobic interaction
between SF molecular chains.
The gelation process is accompanied by a transition of the
SF conformation from a predominantly random coil state in
the sol to a predominantly b-sheet state in the gel. Attenuated
total reflectance infrared (ATR-IR) spectroscopy and Fourier
transform infrared (FT-IR) spectroscopy have been often used
to investigate the conformational changes during SF gelation
reaction [25, 26]. The time-resolved changes of the IR
absorption bands of SF during gelation were investigated
using a custom-made chamber (Fig. 2). The structural change
of the SF aqueous solution at 1,725–1,525 cm-1 (amide I
band region) and 3,900–2,700 cm-1 was closely examined by
time-resolved measurements using FT-IR. Figure 4 shows the
J Sol-Gel Sci Technol
123
changes in expanded FT-IR spectra of the SF/Triton-X solu-
tion in the spectral range of 1,725–1,525 cm-1. The absorp-
tion peak at 1,639 cm-1 (amide I) were observed in the initial
SF/Triton-X solution, indicating that the SF solution mainly
consisted of random coil conformation (Fig. 4a). The peaks at
1,639 cm-1 started to be shifted after a gelation time of
630 min, and then a peak at 1,631 cm-1 appeared after
900 min (Fig. 4c, d). This shift in the peak position supports
that the conformational structure of SF/Triton X-100 solution
was changed from random coil structure to b-sheet structure.
The formation of an absorption peak at 1,631 cm-1 after a
gelation time of 900 min (15 h) is closely correlated with the
gelation time described previously in Fig. 3. From these
results, the gelation in the SF/Triton X-100 mixture started to
occur at about 630 min, and was completed at 900 min,
indicating that the conformational change of SF/Triton X-100
solution, from random coil structure to b-sheet structure,
occurred faster than that of pure SF solution. The sol–gel
Fig. 1 Chemical structures of
various surfactants. a Triton
X-100, b sodium
dodecylbenzene sulfonate
(SDBS),
c octyltrimethylammonium
bromide (OTAB),
d dodecyltrimethyl ammonium
bromide (DTAB) and
e hexadecyltrimethyl
ammonium bromide (HTAB)
Fig. 2 Schematic diagram of
custom-made IR-chamber for
time-resolved FT-IR
measurement
J Sol-Gel Sci Technol
123
transition of SF arises from a combination of hydrophobic
interaction and hydrogen bonding leading tob-sheet structure.
In the solution state, the SF molecular chains are hydrated by
many water molecules. The transition of random coil structure
to b-sheet structure was caused mainly by the change of the
water state in SF aqueous solution [27]. The dehydration (‘‘salt
out’’) process in the presence of Triton X-100 results in the
formation of b-sheet structure. The addition of Triton-X
promotes this dehydration process to combine with SF mol-
ecules. As a result, the hydrophobic interactions between SF
molecules were facilitated by removing water molecules
which inhibit hydrophobic interactions of the SF molecules.
3.2 Effect of ionic surfactant
We next investigated the effect of ionic surfactant on the
gelation of SF aqueous solution. To investigate the effect
of ionic group and alkyl group of the surfactant on the
gelation of SF, the ionic surfactants, sodium dodecyl ben-
zene sulfonate (SDBS), octyltrimethyl ammonium bromide
(OTAB), dodecyltrimethyl ammonium bromide (DTAB)
and hexadecyltrimethyl ammonium bromide (HTAB) were
used.
The gelation behaviors of SF containing ionic surfactants
were summarized in Table 1. In the anionic surfactant
SDBS, SF hydrogel could be formed at concentrations up to
100 mM. In cationic surfactants, the solubility and alkyl
group of the surfactant affect the gelation of the SF aqueous
solution. OTAB and DTAB were soluble in water up to a
concentration of 100 mM. HTAB, which as the longest alkyl
chain, showed limited solubility up to 20 mM in water.
However, a lower concentration of HTAB (B20 mM) and a
higher concentration of DTAB (C25 mM) induce aggre-
gates immediately after mixing with SF aqueous solution. In
addition, the aggregates, together with phase separation,
were generated during the gelation process at a lower con-
centration of DTAB (B20 mM) and higher concentration of
OTAB ([25 mM). The formation of aggregates seems to be
related to the length of alkyl groups and cationic character in
surfactants. On the whole, a fibroin molecule has an overall
negative charge in neutral pH because it has an isoelectric
point at pH = 3.8–3.9 [10, 17, 18]. The strong electrostatic
interactions between SF chains with negative charge and
cationic surfactant with positive charge promote the
Fig. 4 FT-IR spectral change of SF/Triton X-100 during gelation (spectral range 1,800–1,000 cm-1)
Fig. 3 Change in gelation time of SF/Triton X-100 solution at 37 �C
J Sol-Gel Sci Technol
123
generation of aggregates, not gelation. In this process, the
hydrophobic interaction with SF aqueous solution also
increased with the length of the alkyl group, and thus,
aggregates were generated more easily, while the anionic
surfactant SDBS did not generate the aggregates during the
gelation process.
Figure 5 shows the gelation time of the SF solution with
SDBS or OTAB. The gelation time slightly decreased in the
SF/OTAB solution at up to 20 mM (OTAB induces aggre-
gates at C25 mM during the gelation process). In the SF/
SDBS solution, the gelation was promoted considerably, and
was completed within 1 h at 100 mM SDBS, which is 10
times faster than when the non-ionic surfactant Triton X-100
was used. This result can be explained by an increased
dehydration (‘‘salt out’’) effect by the anionic group in
SDBS. The sulfonate groups in SDBS exist mainly outside
the SF molecules with an overall negative charge because of
electrostatic repulsion. The sulfonate groups took away
water molecules around the SF molecular chains, and thus
promoted hydrophobic interactions not only between SF
molecules but also between SF molecules and long alkyl
chains of SDBS. Subsequently, the conformational change
Table 1 Gelation behavior of SF/surfactant solutions
Fig. 5 Change in gelation time of SF aqueous solutions containing
SDBS (filled square) and OTAB (filled circle) surfactants
Fig. 6 Viscosity data of SF gel containing SDBS; a 25 mM,
b 75 mM, c 100 mM and d 150 mM
J Sol-Gel Sci Technol
123
of SF from a random coil to a stable b-sheet structure was
facilitated by increased hydrophobic interactions between
SF molecular chains. The observed faster gelation in SF/
SDBS solution may be attributable to an increased dehy-
dration effect, compared with nonionic surfactant Triton
X-100. Wu et al. [28] reported that SF gelation was accel-
erated using an anionic surfactant, sodium dodecyl sulfate
(SDS) as a gelling agent. The gelation time of SF solution
(4 wt%) was completed within 20 min at 8 mM SDS. This
difference in the gelation time between SF/SDBS and SF/
SDS solutions may be associated with the concentration of
SF solution (SF solution of 2.5 wt% was used in this study).
Figure 6 depicts the changes in viscosity of the SF/
SDBS solution over time. The abrupt increase in viscosity
indicates a transition from a sol state to a gel state. The
viscosity was 2–3 cP in the early solution state, and
reached 10,000–12,000 cP after gelation. The gelation
times determined from changes in viscosity were coinci-
dent with the results in Fig. 5.
3.3 Effect of chitooligosaccharide
Chitooligosaccharide (CHI), which has excellent physio-
logical functions, is a weak cationic oligosaccharide pre-
pared from chitin or chitosan by chemical or enzymatic
Fig. 7 Change in gelation time of SF/chitooligosaccharide solution at
37 �C
Fig. 8 SEM images of a pure SF gel, b SF gel with 75 mM Triton X-100, and c SF gel with 75 mM SDBS (950)
J Sol-Gel Sci Technol
123
degradation. The color of SF aqueous solutions containing
CHI was changed to a deeper yellow with the CHI concen-
tration. Unexpectedly, the gelation time of the SF solution
increased linearly with increasing CHI concentration,
although the CHI contained cationic amino groups (Fig. 7).
It seems that the electrostatic interactions between SF chains
with negative charge and CHI molecules with partly positive
charge (amino groups) exist in the SF/CHI aqueous solution.
Therefore, the CHI molecules can go between the SF mol-
ecules. The existence of hydrophilic CHI molecules
increased the locational hindrance between SF molecular
chains, and thus the transition from random coil structure to
b-sheet structure of the SF aqueous solution was inhibited.
This result indicates that hydrophobic interactions play an
important role in the gelation of SF. Therefore, the sol–gel
transition of SF arises from a combination of intermolecular
hydrophobic interaction and polar interaction (ionic inter-
action and hydrogen bonds).
3.4 Morphology of SF hydrogels
SEM pictures were taken to investigate the morphological
changes of SF hydrogels containing surfactants after
freeze-drying at -80 �C. Figure 8 shows SEM images of
freeze-dried SF and SF/surfactant hydrogels. Freeze-dried
SF hydrogel showed leaf-like morphologies and intercon-
nected pores (Fig. 8a). The pore sizes of freeze-dried SF
hydrogels with Triton X-100 or SDBS were larger than that
of SF hydrogel prepared from aqueous solution. Interest-
ingly, no significant changes in the SF hydrogel with Triton
X-100 or SDBS were observed (Fig. 8b, c).
4 Conclusion
The effect of surfactants on the gelation time of SF was
varied due to their inherent properties and different
interactions with SF molecules in aqueous solution. The
transition rate of SF from sol to gel was mainly based on the
extent of hydrophobic interaction and electrostatic interac-
tion (repulsion) in the presence of surfactant. Particularly,
the gelation time of an SF aqueous solution seems to be
dependent on the dehydration rate by the surfactant. When
non-ionic or anionic surfactant was added into an SF aqueous
solution, the gelation of SF with anionic surfactant was 10
times faster than that with non-ionic surfactant. In the case of
cationic surfactant, SF aggregates and phase separation were
generated because of stronger electrostatic interaction. With
a proper surfactant, the gelation time of SF could be regu-
lated to a desired period of time (Fig. 9). This approach to
controlling the gelation of regenerated SF solution may find
useful applications in areas of biomedical engineering,
including wound dressings and tissue regeneration.
Acknowledgments This work was supported by the National
Research Foundation of Korea (NRF-2012M2A2A6035747).
References
1. Numata K, Katashima T, Sakai T (2011) State of water, molec-
ular structure, and cytotoxicity of silk hydrogels. Biomacromol-
ecules 12:2137–2144
2. West JL, Hubbell JA (1995) Photopolymerized hydrogel mate-
rials for drug-delivery applications. React Polym 25:139–147
3. Wang X, Kluge JA, Leisk GG, Kaplan DL (2008) Sonication-
induced gelation of silk fibroin for cell encapsulation. Biomate-
rials 29:1054–1064
4. de Vos P, Bucko M, Gemeiner P, Navratil M, Svitel J, Faas M,
Strand BL, Skjak-Braek G, Morch YA, Vikartovska A, Lacik I,
Kollarikova G, Orive G, Poncelet D, Pedraz JL, Ansorge-Schum-
acher MB (2009) Multiscale requirements for bioencapsulation in
medicine and biotechnology. Biomaterials 30:2559–2570
5. Kundu J, Poole-Warren LA, Martens P, Kundu SC (2012) Silk
fibroin/poly(vinyl alcohol) photocrosslinked hydrogels for deliv-
ery of macromolecular drugs. Acta Biomater 8:1720–1729
6. Li M, Lu S, Wu Z, Tan K, Minoura N, Kuga S (2002) Structure
and properties of silk fibroin-poly(vinyl alcohol) gel. Int J Biol
Macromol 30:89–94
Fig. 9 Expectation on gelation
time of SF solution using
various surfactants
J Sol-Gel Sci Technol
123
7. Takeshita H, Ishida K, Kamiishi Y, Yoshii F, Kume T (2000)
Production of fine powder from silk by radiation. Macromol
Mater Eng 283:126–131
8. Yao J, Masuda H, Zhao C, Asakura T (2002) Artificial spinning
and characterization of silk fiber from Bombyx mori silk fibroin
in hexafluoroacetone hydrate. Macromolecules 35:6–9
9. Putthanarat S, Zarkoob S, Magoshi J, Chen JA, Eby RK, Stone
M, Adams WW (2002) Effect of processing temperature on the
morphology of silk membranes. Polymer 43:3405–3413
10. Lee KY, Kong SJ, Park WH, Ha WS, Kwon IC (1998) Effect of
surface properties on the antithrombogenicity of silk fibroin/S-
carboxymethyl kerateine blend films. J Biomater Sci Polym Ed
9:905–914
11. Draelos ZD (2000) Novel topical therapies in cosmetic derma-
tology. Curr Probl Dermatol 12:235–239
12. Zhang YF, Wu CT, Luo T, Li S, Cheng XR, Miron RJ (2012)
Synthesis and inflammatory responses of a novel silk fibroin
scaffold containing BMP7 adenovirus for bone regeneration.
Bone 51:704–713
13. Vepari C, Kaplan DL (2007) Silk as a biomaterial. Prog Polym
Sci 32:991–1007
14. Park WH, Jeong L, Yoo DI, Hudson S (2004) Effect of chitosan
on morphology and conformation of electrospun silk fibroin
nanofibers. Polymer 45:7151–7157
15. Cai K, Yao K, Cui Y, Yang Z, Li X, Xie H, Qing T, Gao L (2002)
Influence of different surface modification treatments on poly(D,
L-lactic acid) with silk fibroin and their effects on the culture of
osteoblast in vitro. Biomaterials 23:1603–1611
16. Cai K, Yao K, Lin S, Yang Z, Li X, Xie H, Qing T, Gao L (2002)
Poly(D, L-lactic acid) surfaces modified by silk fibroin: effects on
the culture of osteoblast in vitro. Biomaterials 23:1153–1160
17. Min BM, Lee G, Kim SH, Nam YS, Lee TS, Park WH (2004)
Electrospinning of silk fibroin nanofibers and its effect on the
adhesion and spreading of normal human keratinocytes and
fibroblasts in vitro. Biomaterials 25:1289–1297
18. Karageorgiou V, Meinel L, Hofmann S, Malhotra A, Volloch V,
Kaplan DL (2004) Bone morphogenetic protein-2 decorated silk
fibroin films induce osteogenic differentiation of human bone
marrow stromal cells. J Biomed Mater Res 71A:528–537
19. Matsumoto A, Chen J, Collette AL, Kim UJ, Altman GH, Cebe P,
Kaplan DL (2006) Mechanism of silk fibroin sol-gel transition.
J Phys Chem B 110:21630–21638
20. Kim UJ, Park J, Li C, Jin HJ, Valluzzi R, Kaplan DL (2004) Structure
and properties of silk hydrogels. Biomacromolecules 5:786–792
21. Nagarkar S, Nicolai T, Chassenieux C, Lele A (2010) Structure
and gelation mechanism of silk hydrogels. Phys Chem Chem
Phys 12:3834–3844
22. Yucel T, Cebe P, Kaplan DL (2009) Vortex-induced injectable
silk fibroin hydrogels. Biophys J 97:2044–2050
23. Li XG, Wu LY, Huang MR, Shao HL, Hu XC (2008) Confor-
mational transition and liquid crystalline state of regenerated silk
fibroin in water. Biopolymers 89:497–505
24. Anghel DF, Winnik FM, Galatanu N (1999) Effect of the sur-
factant head group length on the interactions between polyeth-
ylene glycol monononylphenyl ethers and poly(acrylic acid).
Colloids Surf A Physicochem Eng Asp 149:339–345
25. Chen X, Knight DP, Shao Z, Vollrath F (2001) Regenerated
Bombyx silk solutions studied with rheometry and FTIR. Poly-
mer 42:9969–9974
26. Mathur AB, Tonelli A, Rathke T, Hudson S (1997) The disso-
lution and characterization of Bombyx mori silk fibroin in cal-
cium nitrate methanol solution and the regeneration of films.
Biopolymers 42:61–74
27. Hu X, Kaplan DL, Cebe P (2008) Dynamic protein–water relation-
ships during beta-sheet formation. Macromolecules 41:3939–3948
28. Wu X, Hou J, Li M, Wang J, Kaplan DL, Lu S (2012) Sodium
dodecyl sulfate-induced rapid gelation of silk fibroin. Acta Bio-
mater 8:2185–2192
J Sol-Gel Sci Technol
123