Preparation and characterization of PEGDE crosslinked silk fibroin film

Journal of Wuhan University of

Technology-Mater. Sci. Ed.

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DOI 10.1007/s11595-014-1047-8

Preparation and Characterization of PEGDE Crosslinked Silk Fibroin Film

WEI Yali, SUN Dan, YI Honggen, ZHAO Huanrong, WANG Jiannan* (National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China) Abstract: To obtain water-insoluble silk fibroin (SF) materials, polyethylene glycol diglycidyl ether (PEG-DE) was selected as a crosslinking agent to prepare SF films (blends). The reaction conditions were optimized for the crosslinking of the SF molecules. The hot water stability of the blends was measured using BCA protein assay and gravimetric analysis. The molecular conformation and crystalline structure of the blends were analyzed by FTIR and XRD, respectively. When the mass ratio of SF:PEG-DE was 1.0:0.8, the hot water loss rate of the SF blends was minimized. PEG-DE could induce SF molecules to form β-sheets during the gel reaction process, resulting in improved crystallinity and hot water dissolved resistance of the blend films. In order to demonstrate the cytotoxicity of the chemical reagents used to crosslink SF, L929 cells were seeded on the blend film (SF:PEG-DE = 1:1) and cultured for 3 days. Cells of L929 readily adhered and spread in the fusiform on the blend film resulting in high cell viability. The extracted liquid from the SF porous film did not inhibit cell proliferation, as estimated by the MTT assay. Key words: silkworm; silk fibroin; PEG-DE; FTIR; XRD; cell compatibility

1 Introduction The silkworm silk fibroin consists of 20 kinds of amino acids and has excellent tissue affinity, and can support a variety of cell interactions aiding cell adhesion, spread and proliferation[1]. The regenerated SF materials have been increasingly investigated as a drug carrier and in tissue engineering of the cornea[2], ligament[3], bone[4], vessel[5], connective tissues[6] and nerve tissues[7]. The natural silk fibers are water insoluble crystalline copolymers due to the formation of a

©Wuhan University of Technology and SpringerVerlag Berlin Heidelberg 2014 (Received: Aug. 19, 2013; Accepted: May 24, 2014) WEI Yali(魏雅丽): E-mail: [email protected] *Corresponding author: WANG Jiannan(王建南): Prof.; Ph D; E-mail:[email protected] Funded by National Natural Science Foundation of China (Nos.51173125 and 51473108), Natural Science Foundation of Jiangsu Province of China (Nos.BK2012633 and BK2041210), College Natural Science Research Project of Jiangsu Province of China (No.12KJA43004), the Science and Technology Development Foundation of Suzhou of China (Nos.SYG201001 and SS201341) and Priority Academic Program Development of Jiangsu Higher Education Institutions [PAPD]

β-sheet structure (Silk II). But the regenerated SF materials prepared from SF aqueous solution are noncrystalline with weak intermolecular forces, easily dissolved in water and cannot be used for cell culture in vitro and transplantation in vivo. The insolubility of regenerated SF material can be enhanced through the molecular conformation change from random coil to β-sheets (Silk II) structures by treating with organic solvents or dehydration reagents. Such as methanol and ethanol are used more often to treat the regenerated SF materials to achieve SF insolubility in water[8,9]. Methylglyoxal, glutaraldehyde and proanthocyanidin are also potentially useful protein crosslinking agents used in biomaterials. Methylglyoxal has been shown to crosslink proteins in vitro[10]. Glutaraldehyde is a potent protein crosslinker that has been commonly used in the field of tissue engineering, but its acute toxicity has always been a concern [11,12] . Another polyphenol agent, proanthocyanidin, which is particularly abundant in grape seed extracts has been shown to be over 120-times less toxic than glutaraldehyde[13]. The molecular conformation of SF can also be translated into the Silk II structure from the amorphous state or from the Silk I structure by

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high temperature treatment[14]. But the SF materials after the organic solvent or heat-moisture treatment are generally brittle and inflexible in the dry state, causing difficulty in practical application. Studies have shown that polyols (such as glycerine and polyethylene glycol) can not only induce SF material to undergo a moderate conformation transition to obtain low waterdissolubility, but also improve the stress and strain properties[15]. Chemical crosslinking is an important method to prepare insoluble SF materials. These processes have also been used to improve the properties of biomaterials [16]. For example, 1-ethyl-3-3-dimethyl aminopropyl-carbodiimide hydrochloride (EDC) reacts with the primary amine group on the side chain and the carboxyl group on the peptide chain to form peptide bonds [4,17-19] . Silk fibroin contains many aspartic, glutamic and lysine residues, so it can be crosslinked by EDC to prepare insoluble SF materials[20]. Genipin, a heterocyclic compound, extracted from gardenia fruit, is an effective natural crosslinking agent that can covalently bind to free amine groups and has increasingly been used in the preparation of tissue engineering scaffolds[21,22]. The molecular conformation of regenerated SF material can be translated from random coil structures into β-sheets (Silk II) or β-turns induced by genipin, to obtain water-insoluble SF materials[23]. The genipin crosslinked silk film displayed highest elongation at break and tensile strength[24]. In addition, metal ions such as cupric ion can induce the SF secondary structure transition from an amorphous to a β-sheet structure[25]. Water-soluble epoxy resin polyethylene glycol diglycidyl ether (PEG-DE), a type of flexible chain polymer, is an available crosslinker. Motta et al [24] used PEG-DE to cross-link fibroin and sericin and indicated that PEG-DE stabilized the random/α-helix structures in the fibroin and prevented phase separation between fibroin and sericin, and the cross-linked films exhibited good biocompatibility. Antheraea pernyi silk fibroin was reported to have no changes in molecular conformations caused by PEG-DE in the regenerated film at 40 ℃, and the crosslinked regenerated film showed remarkable compliance and tenacity[26]. The reports related to the application of PED-DE are very few, in order to recognize PEG-DE further and promote its applications in biomaterials, in the present paper we selected PEG-DE as the crosslinking agent to prepare water-insoluble SF films. A suitable crosslinking reaction condition for stable film preparation was

explored. The secondary structure, crystalline structure and cell compatibility of the crosslinked SF film were investigated.

2 Experimental 2.1 Preparation of SF solution and blend films B. mori SF solution was prepared as described previously[27]. B. mori raw silk was initially treated three times in 0.06 wt% Na2CO3 solution at 98-100 ℃ for 30 min to remove sericin. After drying, the degummed silk was dissolved in the ternary solvent CaCl2•CH3CH2OH•H2O (mole ratio = 1:2:8) at 70 ± 2 ℃ while stirring until complete dissolution. The regenerated SF solution was obtained by dialyzing against the deionized water at 4 ℃ for 4 days and filtering the mixed solution. The regenerated SF solution (SF concentration 4%) and PEG-DE (Sigma, USA) were mixed together using the weight ratios of 1.0/0.0 (1.0:0.0), 1.0/0.2, 1.0/0.5, 1.0/0.8, 1.0/1.0 and 1.0/1.2 while stirring. All the films were prepared by casting the same volume of SF aqueous solution onto the same area of polyethylene dish, which was then and dried at 60 ℃. The uncross-linked SF film was immersed in 80% v/v ethanol for 2 h and washed with deionized water. The mixture of regenerated SF and PEG-DE at weight ratios of 1.0:1.0 was freeze-dried at 20 ℃ to obtain the regenerated SF porous film. 2.2 Measurement of the water solubility and degree of crosslinking All the fresh films were weighed to obtain the original mass G1 (mg). Sampling each sample and oven drying gave the dry weight G 2 (mg). The moisture content w (%) was calculated according to Eq.(1), and the dry weight G2 (mg) of all the fresh films was calculated according to Eq.(2). The fresh films were immersed in deionized water (0.02 mg/mL) while shaking at 37 ℃ for 24 h. The remaining solids were oven dried and weighed to obtain the dry mass G3 (mg). The dissolved loss rate D (%) was calculated according to Eq.(3) as follows. The SF concentration in the residual liquid was determined using a protein assay kit (BCA, Beyotime Institute of Biotechnology, China). A gradient concentration (mg/mL) of bovine serum albumin (BSA) standard solution was designed and the absorbance recorded at 562 nm (A562) on a microplate reader (Synergy HT, BIO-TEK). A standard curve was automatically generated using the regression equation y = ax + b (R2 0.99). The absorbance values of the



unknown samples were recorded and substituted in the regression equation to calculate the SF concentrations (mg/mL). The crosslinking degree of silk fibroin was calculated according to Eq.(4). (1) (2) (3)

(4) 2.3 Structural analysis The blended films were cut into powders with a radius less than 40 μm, and the samples were prepared in dried KBr pellets and analyzed with a Nicolet AvatarIR360 Fourier transform infrared spectrophotometer (Thermo Fisher Nicolet, USA). The crystal structures of all the SF films were determined with an X-ray diffractometer (XRD, Mercury CCD, Japan) using a tube voltage of 40 kV, a tube current of 40 mA, and a scanning speed of 2 °/min. The diffraction intensity curve was recorded by scanning the 2θ range 5-50°. The XRD curves were decomposed and curve-fitted to estimate the crystallinity degree of the SF films. 2.4 Cell culture The ethanol treated SF film and the blend film of SF/PEG-DE = 1.0/1.0 were immersed in deionized water at room temperature for 3 days, cut and placed carefully at the bottom of 24 well plates and sterilized by gamma rays. L929 fibroblasts were cultivated in Dulbecco’s Modified Eagles medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, American), 100 U/mL penicillin and 100 μg/ mL streptomycin at 37 ℃/5% CO 2. The cells were trypsinized using 0.25% trypsin (Sigma, USA) in the logarithmic growth state and resuspended at 5×10 4 cells/mL in fresh DMEM with 10% FBS containing dual antibiotics. The cells were seeded onto the films to continue cultivation, 1 mL/well, in triplicate. The cell morphology of L929 on the SF film and the blend film was observed after cultivation for 24, 48 and 72 h by an inverted microscope (TH4-200, Olympus, Japan). 2.5 MTT test The SF porous film was first sterilized, and then immersed in fresh DMEM (0.2 g/mL) at 37 ℃ for 24 h. The L929 fibroblasts were trypsinized using

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0.25% trypsin in the logarithmic growth state and resuspended to a concentration of 2.5×104 cells/mL in fresh DMEM. The cells were seeded into 96 well plates (200 μL/well) and cultivated for 24 h. Removing the medium and adding 100 μL maceration extract and 100 μL fresh DMEM to continue cultivation for 3 d. DMEM containing 5% dimethyl sulfoxide (DMSO) was used as a positive control and fresh DMEM used as a negative control. A MTT solution (5 mg/mL) was added to the wells (100 μL/well) to replace the medium and cultivated at 37 ℃/5% CO2 for 4 h. After carefully removing the medium, the blue formazan reaction product was dissolved by adding HCl-dimethylcarbinol (1 mL/well). The absorbance of the completely dissolved solution was measured at 490 nm using a microplate reader (Synergy HT, BIO-TEK). The fresh DMEM contained 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, respectively.

3 Results and discussion 3.1 Water solubility and crosslinking degree of blend films When used as a biological material, the hot water stability of the SF/PEG-DE composite is an important property to prevent allogeneic rejection and inflammatory reaction caused by the dissolved foreign matter. Fig.1 shows the hot water loss rates of the blend films. The loss rate increased with increasing PEG-DE/ SF mass ratio.

Furthermore, the SF content in the residual liquid was measured as shown in Fig.2. Fig.2(a) is the standard curve and automatically generated a regression equation y = 1.192x + 0.099 8 (R 2 = 0.992 8). The independent variable x was the protein concentration, the dependent variable y was the determined absorbance value. PEG-DE can chemically react with –NH2, – COOH or –OH, which exist in the SF molecular chain. Fig.2 (b) shows that the dissolved SF content decreased with increasing PEG-DE/SF mass ratio. The dissolved

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SF concentration reached a minimum when the SF/ PEG-DE mass ratio was between 1.0:0.2 and 1.0:0.8, however, parts of the silk fibroin were not crosslinked. There were no obvious differences in the dissolved SF concentrations in the range of 1.0:0.8 to 1.0:1.2 of SF/ PEG-DE mass ratio. This means that most of the silk fibroin was crosslinked at the mass ratio 1.0:0.8 of SF:PEG-DE, and the crosslinking degree of silk fibroin was about 29.19%.

The lost content of PEG-DE could also be calculated according to the total dissolved content of the film and the dissolved content of the silk fibroin. However, we observed that PEG-DE did not fully participate in the crosslinking reaction and the efficiency was only about 30%. Therefore the total loss continuously increased with increasing PEG-DE content, in the range of 1.0:0.2 to 1.0:1.2 of SF/PEGDE mass ratio. According to these results, we used the mass ratio 1.0:0.8-1.0:1.0 of SF:PEG-DE to prepare water insolube SF material for steady cell culture or tissue engineering applications. 3.2 FTIR analysis The molecular conformation of the regenerated SF film prepared from aqueous solution was mainly the random coil (1 250 cm1 amide III and 665 cm1 amide V) and α-form (Silk I, 1 660 cm1 amide I and 1 525 cm1 amide II) (Fig.3), and the regenerated film dissolved in water. Fig.3 shows that the characteristic absorption bands assigned to β-sheets (Silk II) appeared

in the PEG-DE crosslinked films (Fig.3 (a-d)) that were the same as the ethanol treated SF film (Fig.3 (e)). FIRT spectra of crosslinked films showed few minor changes that the characteristic peak of amide III shifted from 1 250 to 1 229 cm 1 and the amide V shifted from 665 to 700 cm 1 compared with the ethanol



untreated SF film (Fig.3 (f)). This showed that PEG-DE induced molecular conformational transition of the silk fibroin during the crosslinking reaction, leading to the reduction in the solubility of the SF film. 3.3 XRD analysis The regenerated SF film from aqueous solution was mainly of the random coil structure with a low crystallinity in the Silk I crystalline form, part of the random coil or Silk I would translate to Silk II after ethanol treatment. In all the PEG-DE cross-linked SF films, the significant characteristic peaks appeared at about 9.0°, 20.3° and 24.1° assigned to Silk II (Fig. 4 (a-d)), similar to the ethanol treated SF film (Fig. 4 (e)). The Silk II content in the 16.7% PEG-DE (SF:PEG-

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DE = 1.0:0.2) crosslinked SF film increased from 4.9% to 11.5% compared with that of the SF film without ethanol treatment. The Silk II content and crystallinity increased with increasing PEG-DE content in the blend film. When SF:PEG-DE = 1.0:0.8, the Silk II content and crystallinity were 14% and 27.2%, significantly higher than that of the ethanol treated SF film (Table 1). As observed in Fig. 4, another significant characteristic peak appeared in all blend films at about 40.1° that was assigned to Silk I, but was absent in the SF film without ethanol treatment. Fig.4 and Table 1 show that not only the Silk II increased, translated from random coil or Silk I, but also part of Silk I newly formed from the random coil. PEG-DE could promote silk fibroin

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forming crystals. The crystallinity increase of SF film was a main factor to enhance its hot water stability. 3.4 Cytotoxicity of the blend film Chemical crosslinking is an effective method to prepare insoluble SF materials, but when it is used in the biomedical field, the cytotoxicity and cell compatibility must be given close attention. Fig.5 shows the growth and proliferation of L929 on the PEG-DE crosslinked SF film (SF:PEG-DE =1.0:1.0), uncrosslinked (ethanol treated) SF film and plastic cell plate. After 24 h of culture, the cells adhered well to both the SF film and the plastic cell plate. Almost all of the cells spread in the fusiform on the PEGDE crosslinked SF film and the condition of the cells was improved compared with the plastic cell plate. Cells divided, proliferated and spread over 80% of the crosslinked SF film area after 2 days of culture. The cells cultured on day 3 were completely covered with the PEG-DE crosslinked SF film, and many cells were observed to be round because there was not sufficient interface for cell adhesion on the film surface. There was no significant difference in the cell proliferative activity compared with the uncrosslinked SF film and the plastic cell plate. In addition, we used the extract of PEG-DE crosslinked SF porous film (SF: PEG-DE = 1.0:1.0) to culture L929 cells. The MTT test was performed on day 3. Data from Table 1 indicated the cell proliferative activity cultured in the extract liquid was the same as that in fresh DMEM and remarkably higher than that in DMEM containing 5% DMSO. The results demonstrated that the positive control of 5% DMSO had serious cytotoxicity, inhibiting cell growth, however the blend film crosslinked by PEG-DE had no obvious inhibitory effects on cell proliferative activity when the crosslinking ratio of SF:PEG-DE increased to 1.0:1.0. Therefore, we thought that the PEG-DE crosslinked SF films would not be toxic for cell growth and proliferation when the mass ratio of PEG-DE:SF was less than 1.0:1.0.

fibroin materials. The molecular conformation of the regenerated SF film, prepared from aqueous solution was a random coil, the crystallinity was low and the major structure observed was Silk I. PEG-DE not only crosslinked the silk fibroin to form a gel, but also can induce SF molecular conformation change to β-sheet from random coil or Silk I structures, leading to an increase in Silk II and crystallinity, enhancing the hot water dissolved resistance of the regenerated SF materials. Most of the silk fibroin was crosslinked and the reduction in the dissolved SF concentration stopped when the mass ratio of SF:PEG-DE was 1.0:0.81.0:1.2. The crosslinking degree of the silk fibroin was about 30%. The PEG-DE crosslinked SF films had no cytotoxicity or inhibitory effect on cell growth and proliferation of L929. The results of this research would promote the applications of PEG-DE in biomaterials, as well as provide a method to prepare stable SF biomaterials for tissue Engineering applications. References [1]

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