1. Introduction
It is well known that qualified biomaterials shall possess good mechanical properties and biological compatibility [
1]. Silk fibroin (SF), the major protein in the silk produced by
Bombyx mori, can meet above criteria. Silk fibroin is biodegradable [
2] with minimal inflammatory reaction [
3]. Therefore, silk fibroin has been widely used in the biomedical and clinical fields [
4,
5,
6].
Hydrogels are three-dimensional hydrophilic networks, which are crosslinked via chemical and physical bonds, with the ability to absorb and retain large amounts of water without dissolution [
7]. Many hydrogels, including natural and synthetic ones, were used as materials for drug delivery [
8] and tissue engineering [
9], as both drugs and cells could be encapsulated in the hydrogel matrices. Based on needs, silk fibroin can be made into different forms [
10,
11], and hydrogel is one of them. Although silk fibroin-based hydrogel has been widely investigated, its application is limited due to its poor mechanical properties [
12,
13], as well as its uncontrolled and long period of gelation time [
14,
15].
Poly(ethylene glycol) diacrylate (PEGDA) is a biologically inert material with particular use in tissue engineering and drug delivery [
16,
17]. PEGDA gels rapidly in the presence of a photoinitiator and UV irradiation at 365 nm at room temperature [
18], which would be extremely useful for bringing them to an industrial scale or approaching them with a wide variety of applications [
19], although many photoinitiators used are identified as hazards [
20,
21,
22,
23]. The initiator-free photopolymerization for PEGDA hydrogel formation also has been reported but is less common [
24]. Although demonstrating excellent mechanical properties, PEGDA-only gels do not support cell attachment, as the gel surface inhibits the adsorption of adhesion protein, such as fibronectin; therefore, there is a lack of cellular attachment sites [
25]. In addition, gelation at 365 nm UV light or near-UV radiation (with wavelengths of 200 to 400 nm) can cause pyrimidine dimer formation and other chemical changes in the DNA of cells [
26].
Although it is reported that norbornene-functionalized SF can be incorporated in norbornene-functionalized 4-arm poly(ethylene glycol) (PEG4NB) through thiol–ene photocrosslinking [
27], carbic anhydride used for norbornene functionalization is dangerous [
28]. It would be desirable to prepare hydrogels in the absence of toxic ingredients, with a short and controlled gelation time, while keeping their good mechanical properties and biocompatibility. Therefore, we adopted a different strategy in which thiol groups were first installed onto silk fibroin molecules through carbodiimide chemistry. Since PEGDA contains two alkene groups attached to each end of the molecule, they can graft themselves onto SF molecules. Photoinitiators for thiol–ene reactions have been extensively used [
29]. The usage of a photoinitiator could cause undesirable side reactions, such as surface oxidation or the removal of bound thiols [
30]. In addition, it increases the cost of manufacture with tedious procedures. According to safety data sheets [
31,
32,
33,
34], most of the photoinitiators used for thiol-ene reactions are harmful. Through the developed approach, the gelation of PEGDA/SF aqueous solution could be completed within three minutes without the appearance of a photoinitiator. Furthermore, the light used to mediate the thiol–ene reaction has a wavelength of 405 nm. Therefore, we present here a mild and green strategy for SF-based hydrogel fabrication. The release kinetics of the prepared hydrogel were studied using rhodamine B as a model molecule in order to evaluate its potential use in drug delivery application.
2. Materials and Methods
2.1. Materials
Cocoons of silkworm Bombyx mori (a Chinese strain demoted as 872) were provided by the College of Biotechnology, Southwest University. Tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), 2-Morpholinoethanesulfonic acid (MES), and sodium chloride were purchased from Aladdin Agent Co., Ltd. (Shanghai, China). Sodium carbonate, rhodamine B (RB), and sodium biphosphate dihydrate were purchased from KeLong Chemical Reagent Co., Ltd. (Chengdu, China). Sodium dihydrogen phosphate was purchased from Fangzheng reagent Co., Ltd. (Tianjin, China). N-hydroxysuccinimide (NHS) and reduced glutathione (GSH) were purchased from Solarbio Co., Ltd. (Beijing, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Adamas-beta (Shanghai, China). Calcium chloride was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ethanol was purchased from Chuandong Chemical Co., Ltd. (Chongqing, China). Poly(ethylene glycol) diacrylate (PEGDA, n = approximately 4) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Potassium bromide was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagles medium (DMEM, high glucose) and phosphate-buffered saline (PBS, 1×) were purchased from Hyclone (Logan, UT, USA). 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from BioFroxx (Einhausen, Germany). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Zhejiang, China). Hematoxylin and eosin (H&E) were purchased from Nanchang Yulu Experimental Equipment Co., Ltd. (Nanchang, China). All chemicals are of analytical grade and used without further purification. De-ionized (DI) water was produced by a Milli-Q Direct-8 purification system (resistivity >18 MΩ cm, Molsheim, France) onsite and used in all experiments.
2.2. Extraction and Purification of Silk Fibroin
Regenerated silk fibroin (RSF) solution was prepared according to the previous protocol developed [
35].
Bombyx mori cocoons were cut to small pieces, and the silkworm chrysalises were removed from the cocoons. Then, 20 g cocoons were boiled in 1 L of 0.02 M Na
2CO
3 solution for 30 min and rinsed with distilled water by a magnetic stirrer (Shanghai Sile Instrument, T09-15, Shanghai, China) for 20 min. The above step was repeated one more time to get degummed silk fibers. In order to obtain RSF solution, degummed silk fibers were immersed in CaCl
2–enthanol–H
2O solution (molar ratio = 1:2:8) at 70 °C until the silk fibers were dissolved completely. Subsequently, the solution was dialyzed against double-distilled water (DI H
2O) for 72 h in order to remove the impurities. The pH of the dialyzed SF solution was adjusted to 6.0 in 0.1M MES solution (containing 0.5 M NaCl) for 24 h. Next, the insoluble impurities were filtered out through the surgical gauze and then centrifuged at 8000 rpm (30 min, 4 °C). Later, the carboxyl groups on SF molecules were activated by EDC/NHS (0.5 mg/mL of EDC with 0.7 mg/mL of NHS in MES buffer) for 15 min at room temperature. Then, GSH was added to a final concentration of 2 g/L in above mixture. The reaction was conducted at room temperature for 15 min in order to couple GSH to the SF molecules covalently. After the completion of GSH coupling, the solution was dialyzed against DI H
2O for another 24 h to remove unbound peptide and chemical remains. The prepared GSH-modified SF (GSH-SF) solution was lyophilized and then stored in a vacuum desiccator over silica gel at room temperature for later usage.
2.3. Hydrogel Preparation
To prepare the hydrogel, the lyophilized SF solid was dissolved in DI H2O to make 10% (w/v) GSH-SF solution, and TCEP·HCl (with a final concentration of 10 mmol/L) was added into GSH-SF solution in order to cleave disulfides. Fifteen min later, 1 mL of the above mixture and 1 mL of PEGDA were mixed in a 5 mL centrifuge cube and stirred using a vortex oscillator (VORTEX-5, Kylin-Bell Lab Instruments Co., Ltd., Haimen, China) for 3 s. Then, 600 μL of above mixture were transferred into a cylindrical mold (12 mm inner diameter) and placed 3 cm below a 30 W light-emitting diode (LED) UV light (405 nm wavelength, Shenzhen YuXianDe Science and Technology Ltd., Shenzhen, China). Each sample was illuminated by UV light for a certain amount of time.
2.4. Compressive Test
The compressive mechanical properties were measured by a universal testing machine (Shanghai Xieqiang Instrument Technology Co., Ltd., Shanghai, China). The compression rate was set at 2 mm/min, and the machine was stopped when the strain attained 50%.
2.5. Swelling Study
The lyophilized PEGDA/SF hydrogels were immersed directly in phosphate-buffered saline (PBS, pH = 7.4) solution for 6 h at 37 °C. The swollen hydrogel was removed from PBS solution at a predetermined time point (t = 30 min, 1 h, 2 h, 3 h, 6 h). The excess liquid was removed by blotting with filter paper. Then, the hydrogels were weighted individually on an analytical balance (Shanghai Jingtian Electronic Instrument Co., Ltd., Shanghai, China), and the swollen ratio (SR) was determined by the following equation [
36]:
where
Wt is the weight of the swollen hydrogel at time
t, and
Wd is the weight of the lyophilized hydrogel.
2.6. Fourier Transform Infrared Spectroscopy (FTIR) Analysis
The lyophilized PEGDA/SF hydrogel was ground into powders and mixed with KBr (ratio = 1:100, w/w). The above mixture was pressed into pellets for FTIR (Nicolet iN10, Thermo Scientific, Waltham, MA, USA) analysis. Each sample was scanned 24 times from 400 cm−1. to 4000 cm−1 with a resolution of 4 cm−1.
2.7. Scanning Electron Microscope (SEM) Observation
The prepared hydrogel sample was cut through to make a cross-section of this sample. The resulting cross-section was lyophilized and sputter-coated (MTI Corporation, Richmond, VA, USA) with gold to improve the conductivity of the surface for SEM (Phenom Pro, Phenom-World) observation.
2.8. Drug Release Test
Rhodamine B (RB) was selected as a model molecule for drug release study. The sustain release performance of RB-loaded hydrogel was tested according to the reference [
37], and the concentration was determined by a UV-vis spectrophotometer (T6, Beijing Puxi Analytic Instrument Ltd., China). Linear regression of the solution concentration (C) and absorbance (A) of RB solution with different concentrations at λ = 555 nm was performed to obtain a standard curvilinear equation: C = 0.08756A-0.0025, R
2 = 0.99914 (
Supplementary Material, Figure S1). RB was mixed with GSH-SF and PEGDA to reach a final concentration of 0.1 mg/mL before gelation. After gelation, the samples were placed in weighing bottles filled with 4 mL of PBS solution (pH = 7.4). The weighing bottles and samples were placed on a shaker (Taicang Experiment Apparatus Ltd., Taicang, China) at 37 °C, with a rotational speed of 40 rpm/min. Then, 4 mL of the release solution was taken out at certain time intervals, and the UV absorbance of that solution at 555 nm was measured. Then, 4 mL of fresh PBS solution was added to the weighing bottles to replace the withdrawn solution.
2.9. Cytotoxicity Assay
The cytotoxicity of the hydrogel against the human embryonic kidney (HEK) 293 cell line was evaluated in vitro using a 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the ISO 10993-5 standard. First, the hydrogels were rinsed by PBS (pH = 7.4) solution three times and then sterilized with high-pressure steam for 2 h. After sterilization, the hydrogels were immersed in Dulbecco’s modified Eagles medium (DMEM) to equilibrate (0.2 g hydrogel/9 mL DMEM) for 72 h in an incubator at 37 °C to obtain the leaching liquor of the prepared hydrogel. Then, the leaching liquor was filtered through the 0.22 μm pore size membrane (Tianjin Jinteng Experimental Equipment Co., Ltd., China). Next, HEK 293 cells were seeded in a 96-well plate with a density of 2 × 104 cells/well in 200 μL complete growth medium (90% DMEM, 10% FBS and 1% penicillin streptomycin) and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Then, the culture medium was removed and replaced with 100 μL of complete growth medium. Later, 10 μL of the leaching liquor, complete growth medium, and phenol solution (64 g/L) was added into each well as experimental groups, blank groups, and positive control groups respectively. After 24 h of incubation, 20 μL of the 5.0 mg/mL MTT solution (dissolved in PBS) was added into each well for a further 4 h of incubation. After the removal of culture medium, the cells in each well were lysed in 200 μL of dimethyl sulfoxide (DMSO) to dissolve the formazan precipitate. After shaking on a Belly Dancer with gentle agitation for 15 min, the optical density at the wavelength of 490 nm was measured using a microplate reader (Biotek Synergy H1, Winooski, VT, USA), and each sample was tested in five replicates.
2.10. Subcutaneous Implantation
To determine the biocompatibility of the PEGDA/SF hydrogel in vivo, the PEGDA/SF hydrogels (1.3 mm in diameter and 0.18 mm in height) were implanted in three healthy female Kunming mice (18–22 g, specific pathogen-free), which were purchased from Tengxin Biotechnology Co., Ltd., (Chongqing, China). All the hydrogels were rinsed with PBS solution (pH = 7.4) three times and then sterilized by high-pressure steam. Then, a 1 cm incision was created on the posterior dorsomedial skin of the mouse, and a small lateral subcutaneous pocket was prepared by scissor dissection subsequently. The hydrogel sample was implanted in the subcutaneous pocket, and the incised wound was sutured. All the mice were individually housed in a sterilized cage with filtered air. The mice were provided autoclaved food and water. After three days, the mice were euthanized, and the hydrogels were retrieved along with the surrounding tissues for further analysis.
2.11. Histological Analysis
After implantation, samples were carefully rinsed with saline for three times and fixed in formaldehyde solution (4%, v/v), embedded in paraffin, and then cut into 5 μm thick sections using a microtome (Leica RM2235, Wetzlar, Germany). Then, the sectioned slides were stained by H&E followed by instruction from the manufacturer and observed under an optical microscope (DM3000, Leica, Wetzlar, Germany). The wounds were evaluated for the inflammation and granulation tissue formation.
The animal experimental design and procedures were executed according to the protocols approved by the Laboratory Animal Ethics Committee of Southwest University (Ethic Permission Code: IACUC-20190117-15; Date of Approval: 17 January 2019). Animal care and use strictly followed the Regulations for the Administration of Affairs Concerning Experimental Animals, National Committee of Science and Technology of China (14 November 1988), and Instructive Notions with Respect to Caring for Laboratory Animals, Ministry of Science and Technology of China (30 September 2006).
5. Conclusions
Here, we developed a new method of PEGDA/SF hydrogel preparation through a thiol–ene click reaction without the use of a photoinitiator. First, 405 nm blue light was used for photocrosslinking, which may improve the implementation of the thiol–ene click reaction in biomedical applications requiring low, cytocompatible doses of light. The thiol–ene click reaction played a key role in the overall gelation process, which installed PEGDA molecules onto SF molecules. Then, PEGDA-installed SF molecules served as focal points or core, participating in the build-up of hydrogel networks. The prepared hydrogel could be obtained in a short and controllable gelation time. Further, the compressive strength of the prepared PEGDA/SF hydrogel was improved by introducing a prior cooling step that can be utilized in the control of hydrogel property.
In this study, FTIR was used to determine the successful grafting of PEGDA to SF and analyze the secondary structure of the prepared PEGDA/SF hydrogel. The compressive test results revealed that hydrogen bonding plays a crucial role in influencing the mechanical properties of the prepared PEGDA/SF hydrogel. The SEM photos showed that the prepared hydrogel is a porous structure, indicating that the PEGDA/SF hydrogel could be used to load drugs or cells. Drug release and swelling ratio experiments confirmed that PEGDA/SF hydrogel possesses the potential to be a drug delivery system. The MTT assay results demonstrated that the prepared PEGDA/SF hydrogel was non-cytotoxic to HEK 293 cells. In addition, the subcutaneous implantation of PEGDA/SF hydrogel did not show any acute inflammatory effects in mice, revealing that the PEGDA/SF hydrogel possessed favorable biocompatibility. We expected that this thiol–ene click reaction-centered hydrogel fabrication method is efficient in SF-based hydrogel preparation for biomedical application.