Effect of Solvent on the Characteristics of Electrospun Regenerated Silk Fibroin Nanofibers

Lim Jeong1, Kuen Yong Lee2,a and Won Ho Park1,b 1Department of Textile Engineering, College of Engineering, Chungnam National University,

Daejeon 305-764, Republic of Korea

2Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea

aleeky@hanyang.ac.kr, bparkwh@cnu.ac.kr

Keywords: silk fibroin, nanofiber, electrospinning, secondary structure, β-sheet

Abstract. Nonwoven nanofiber matrices were prepared by electrospinning a solution of silk fibroin

(SF) dissolved either in formic acid or in 1,1,1,3,3,3-hexafluoro-2-isopropyl alcohol (HFIP). The

mean diameter of the electrospun nanofibers prepared from SF dissolved in formic acid was 80 nm

with a unimodal size distribution, which was smaller than those prepared from HFIP (380 nm). SF

nanofibers were then treated with an aqueous methanol solution, and structural changes due to

solvent-induced crystallization of SF were investigated using IR and 13

C solid-state CP/MAS NMR

spectroscopy. SF nanofibers prepared from formic acid were found to have a higher proportion of

β-sheet conformations than those prepared from HFIP. Methanol treatment provided a fast and

effective means to alter the secondary structure of both types of SF nanofibers from a random coil

form to a β-sheet form. As demonstrated in the present study, this approach to controlling the

dimensions and secondary structure of proteins using various solvents may be useful for the design

and tailoring of materials for biomedical applications, especially for tissue engineering applications.

Introduction

Silk fibroin (SF) has found useful applications in the areas of biomedical science and engineering

due to its distinctive biological properties [1,2]. Potential biomedical applications of SF include

scaffolds for tissue engineering and wound dressings. SF has been reported to be useful for the

formation of various types of scaffolds, which have been successfully used to culture various cell

types including stem cells [3-5]. We have previously reported that nonwoven matrices of

electrospun nanofibers can be prepared from a regenerated SF solution, and that the matrices were

effective for the cell attachment and spreading of normal human keratinocytes and fibroblasts [6].

However, electrospun SF nanofibers, as well as other types of SF matrices (e.g., film), should be

typically followed by chemical treatment with an aqueous alcohol solution in order to increase their

stability and mechanical properties in the presence of water. Methanol has been frequently used for

this purpose, and SF matrices that are not treated with an aqueous methanol solution can easily

dissolve or swell in water. Thus, the solvent-induced structural changes of SF have been widely

investigated and utilized to form stable SF matrices [7]. We also reported a novel approach to

stabilizing electrospun SF nanofibers by treatment with water vapor instead of alcohol solutions.

The solvent-induced conformational changes of SF treated with water vapor were investigated and

compared with methanol-treated SF nanofiber matrices [8].

Characteristics of electrospun SF nanofibers can be controlled either by chemical treatment (e.g.,

methanol treatment) after electrospinning, as mentioned above, or by changing the electrospinning

conditions, including the solvent types. In this study, we investigated the effect of an organic

solvent used to prepare a SF solution on the resultant characteristics of electrospun nanofiber

matrices. Formic acid and HFIP, typical organic solvents used to dissolve SF, were used to prepare

an SF solution for electrospinning, and the dimensional and structural changes of the SF nanofibers

were studied using SEM, IR, and NMR spectroscopy.

Key Engineering Materials Vols. 342-343 (2007) pp 813-816Online available since 2007/Jul/15 at www.scientific.net© (2007) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/KEM.342-343.813

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Materials & Methods

Degummed silk yarn was dissolved in a ternary solvent system composed of calcium chloride,

ethanol, and water (1:2:8 molar ratio) at 70oC for 6 h, followed by dialysis with cellulose tubular

membranes (molecular weight cut-off, 12,000; Sigma) against distilled water for three days. The

resultant solution was filtered and lyophilized. The SF powder was then dissolved in HFIP or

formic acid to prepare a regenerated SF solution. The SF nanofibers were prepared by

electrospinning a 7% (w/v) regenerated silk fibroin solution, and were collected on a target drum

that was placed at a distance of 8 cm from the syringe tip (inner diameter 0.0838 mm). A voltage of

16 kV was applied to the collecting target, and the flow rate of the solution was 2 mL/min. The

electrospun SF nanofiber matrices were treated with an aqueous methanol solution to achieve the

solvent-induced crystallization. In brief, nanofiber matrices were treated with an aqueous methanol

solution (50%), rinsed with distilled water, and dried in a vacuum at room temperature for 24 h to

prepare the methanol-treated SF nanofiber matrices. A scanning electron microscope (Hitachi S-

2350) was used to investigate the morphology of gold-coated SF nanofibers. IR spectra of the SF

nanofiber matrices were taken with a Magma 560 spectrometer (Nicolet) at a resolution of 2 cm-1.

13C solid-state CP/MAS NMR spectra of the SF nanofibers were obtained on a DSX 400 NMR

spectrometer (Bruker), using a cross-polarization pulse sequence and magic-angle spinning at 6.5

kHz.

Results & Discussion

Regenerated silk fibroin was dissolved either in formic acid or in HFIP, and nonwoven matrices

composed of randomly arranged nanofibers were prepared by electrospinning the SF solution. The

morphology and size of the SF nanofibers were determined by image analysis of scanning electron

microscopic pictures (Fig. 1). The mean diameter of nanofibers prepared from SF dissolved in

formic acid was 80 nm, with a unimodal size distribution in the range of 30-120 nm. The mean

diameter of SF nanofibers prepared from HFIP was 380 nm, with a range of 250-530 nm.

Fig. 1. SEM images of electrospun nanofibers prepared from regenerated silk fibroin

dissolved in (A) formic acid and (B) HFIP.

Major conformations of SF include random coil, α-helix, and β-sheet. The crystallization of SF

can be easily induced by simple physical (e.g., thermal) or chemical (e.g., methanol) treatments [9].

The most common method to convert a random coil form of SF into a more stable β-sheet form is

treatment with an organic solvent. It is well accepted that methanol is highly effective in the

crystallization of SF from a random coil conformation to a β-sheet conformation. The IR

spectroscopic method has been frequently used to investigate the conformational changes of silk

fibroin. The characteristic absorption bands of SF are found in the regions of 1625 (amide I), 1528

(amide II), 1260 (amide III), and 700 (amide V) cm-1. The characteristic absorption bands of SF

nanofibers prepared with formic acid were observed at 1642 (amide I) and 1517 (amide II) cm-1,

which can be attributed to the random coil conformation and β-sheet conformation, respectively

(Fig. 2A). These peaks were shifted to 1622 and 1514 cm-1, assigned to β-sheet conformation, after

(A) (B)

814 Advanced Biomaterials VII

methanol treatment. Interestingly, the characteristic absorption peaks of SF nanofibers prepared

with HFIP were observed at 1647 (amide I) and 1541 (amide II) cm-1, which can be attributed to the

random coil conformation (Fig. 2B). These peaks were also shifted to 1620 and 1516 cm-1 after

methanol treatment, which is indicative of structural changes in SF from a random coil

conformation to a β-sheet conformation by methanol treatment. It is likely that SF nanofibers

prepared from formic acid contain larger amounts of β-sheet structures than those prepared from

HFIP.

Fig. 2. Infrared spectra of (a) non-treated and (b) methanol-treated electrospun nanofibers

prepared from regenerated silk fibroin dissolved in (A) formic acid and (B) HFIP.

Fig. 3. Extended 13

C NMR spectra of (a) non-treated and (b) methanol-treated electrospun

nanofibers prepared from regenerated silk fibroin dissolved in (A) formic acid and (B)

HFIP.

Solid-state 13

C NMR also provides a useful means to confirm the structural changes of silk

proteins from a random coil conformation to a β-sheet conformation, as the chemical shifts of

carbon atoms in silk proteins are strongly related to the secondary structure of β-sheet

conformations. The β-sheet form can be identified by the 13

C chemical shifts of Gly (glycine), Ser

(serine), and Ala (alanine). Ala is a major constituent of SF, and the chemical shifts of Ala,

particularly of the methyl groups of Ala residues (Ala Cβ), are representative of the conformational

status of the protein. Expanded views of the chemical shifts of the Ala Cβ region of the SF

nanofibers are shown in Fig. 3. The chemical shifts of Ala Cβ and Ala C=O in non-treated SF

nanofibers prepared from formic acid were 17.1 and 172.2 ppm, respectively, indicating that the SF

Key Engineering Materials Vols. 342-343 815

nanofibers contained substantial amounts of the β-sheet conformation. The methanol-treated SF

nanofibers showed a strong peak at 20.5 ppm, which can be assigned to Ala Cβ in the β-sheet

conformation. In contrast, the chemical shifts of Ala Cβ and Ala C=O in non-treated SF nanofibers

prepared from HFIP were 16.3 and 173.1 ppm, respectively, and assigned to a random coil

conformation of SF. These peaks were shifted to 20.4 and 170.3, respectively, after methanol

treatment. This finding was consistent with the results obtained from the IR spectroscopic method.

The difference in the dimensions and secondary structure of electrospun SF nanofibers prepared

from different organic solvents was considered to be dependent on the volatility of the solvent used.

For example, the slow removal of formic acid during the electrospinning and drying process may

have caused the slow crystallization of SF and resulted in the formation of nanofibers with higher β-

sheet content than those prepared with HFIP.

Conclusions

Both the dimensions and secondary structure of regenerated SF nanofibers were greatly affected by

the organic solvent used for the electrospinning process. Formic acid was useful in forming

nanofibers with smaller diameters and higher β-sheet content than those prepared with HFIP, as

confirmed by SEM, IR, and solid-state CP-MAS 13

C NMR spectroscopy. This approach to

controlling the dimension and structure of regenerated SF nanofibers using different solvents may

be useful in the design and fabrication of electrospun SF nanofiber matrices for wound dressings

and tissue engineering applications.

Acknowledgement

This work was supported by the NanoBio R&D Program (Grant No. 2005-00009 and 2005-00115)

of the Korea Science & Engineering Foundation.

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Advanced Biomaterials VII 10.4028/www.scientific.net/KEM.342-343 Effect of Solvent on the Characteristics of Electrospun Regenerated Silk Fibroin Nanofibers 10.4028/www.scientific.net/KEM.342-343.813