effect of surfactants on sol–gel transition of silk fibroin

8
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 Ó 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 Á Gelation time Á Sol–gel transition Á 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 [25]. 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 [610]. 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 [1114]. 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 [1518]. 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 Á M. H. Kim Á L. Jeong Á W. H. Park (&) Department of Textile Engineering, College of Engineering, Chungnam National University, Daejeon 305-764, South Korea e-mail: [email protected] D. Cho Á 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

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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).

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