Chemical modification of Bombyx mori silk fibers with vinyl groups for thiol-ene click chemistry
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Natural Bombyx mori silk fibroin (SF) fibers were modified with 2-methacryloyloxyethyl isocyanate (MOI) first for the introduction of vinyl groups. Then, 1H,1H,2H,2H-perfluorodecanethiol was grafted onto the SF fibers via thiol-ene click chemistry using ultraviolet light. The formations of MOI-modified and PFDT-grafted SF fibers were analyzed using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The morphology of samples was also revealed by a scanning electron microscope. In addition, differential scanning calorimetry results demonstrated that SF fibers did not show significant change in thermal behavior, regardless of the chemical modification. To confirm the cytotoxicity of the prepared SF fibers, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed, and no toxicity was observed with PFDT-grafted SF fibers. The results also showed that PFDT-grafted SF fibers exhibited good antifouling properties when Chlorella vulgaris (C. vulgaris) was selected as a model for algal cells adhesion experiment.
KeywordsBombyx mori silk fibroin Chemical modification Thiol-ene click reaction
X-ray photoelectron spectroscopies
Fourier transform infrared
scanning electron microscopy
differential scanning calorimeter
anhydrous dimethyl sulfoxide
1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride
- C. vulgaris
Silkworm Bombyx mori can secrete silk, which consists of sericin and silk fibroin. The two paralleled fibroin fibers are held together with a layer of sericin on their surfaces . Silk is a natural biopolymer and is highly appreciated for its outstanding characteristics, which majorly result from the properties and structures of silk fibroin. Unlike sericin, silk fibroin in different forms can interact with biological systems without introducing adverse immunological responses, demonstrating good biocompatibility and biodegradability . Among various forms, the fibroin fibers are endowed with a combination of attractive strength, toughness, and thermal stability that surpasses many synthetic and natural fibers . There is a growing interest in introducing more functionalities into silk fibroin fibers while preserving its advantageous intrinsic properties  in order to expand its consumption. One method, chemical modification can imbue silk fibroin fibers with new properties and functions, therefore broadening its applications in the textile and biomedical industries .
In 2000, Tamada et al. reported an approach to introduce a carbon double bond onto silk fibroin with 2-methacryloyloxyethyl isocyanate (MOI) . Because carbon–carbon double bond can react with radicals easily, this approach allows a further modification of silk fibroin based materials, for example, polymerization with azo polymerization initiator . As one example of reactions between ene functional groups and radicals, thiol-ene click reaction involves the addition of a thiyl radical across a carbon–carbon double bond in a regio- and enantio-selective manner . Thiol-ene click reactions are appealing for material modification because they are rapid, largely independent of solvent, in high yield, and no need to require a radical initiator [7, 8]. To the best of our knowledge, the approach for chemical modification of SF fibers based on thiol-ene click chemistry has not been reported previously.
Our manuscript details a technique that can modify silk fibers with thiol-containing molecules based on thiol-ene click chemistry. 1H,1H,2H,2H-perfluorodecanethiol (PFDT) was used as a model molecule as fluorine atom can serve as an elemental tag. MOI, a heterofunctional monomer that combines the versatility of a vinyl double bond and a reactive isocyanate group, was used prior to the PFDT graft. Isocyanate groups can react with hydroxyl, carboxyl, and amino side groups of amino acid residues in silk fibroin, introducing vinyl groups to silk molecules and allowing them to act as reacting points. PFDT molecules were then grafted through these reacting points onto the silk by thiol-ene click reactions. Because PFDT exhibits excellent antibioadhesive properties , an algal cell adhesion experiment was designed to verify the antifouling property of PFDT grafted silk and furtherly prove the successful attachment of PFDT molecules onto silk fiber surface via thiol-ene click chemistry.
Fresh cocoons of Bombyx mori silkworm were obtained from College of Biotechnology, Southwest University. Sodium carbonate anhydrous (Na2CO3), calcium chloride anhydrous (CaCl2), and N,N-dimethylformamide (DMF) were purchased from Kelong Chemical Reagent Factory (Chengdu, China) and at AR grade. Anhydrous ethanol (CH3CH2OH, AR grade) was purchased from Chongqing Chuandong Chemical Group Co., Ltd. (Chongqing, China). 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Solarbio (Beijing, China). Anhydrous dimethyl sulfoxide (DMSO), dibutyltin dilaurate and hydroquinone were purchased from Aladdin Industrial Co., Ltd. (Shanghai, China). 2-Isocyanatoethyl methacrylate (MOI) was purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). 1H,1H,2H,2H-perfluorodecanethiol (PFDT) and 1H,1H,2H,2H-perfluoro-1-decanol (FTOH) were purchased from Sigma-Aldrich (Saint-Louis, USA). Ultrapure water (resistance > 18 MΩ cm−1) was used in all experiments.
Degummed silk fibroin fiber preparation
In preparation for the experiment, Bombyx mori silkworm cocoons were cut into dime-sized pieces. To remove the sericin, those cocoon pieces were added to 500 ml boiled sodium carbonate solution (0.047 M) for 40 min. Then, the degummed silk fibroin was removed from the boiling sodium carbonate solution and cooled by rinsing in ultrapure water three times for 20 min. Each rinsing cycle was carried out in 1 l of ultrapure water in a lab beaker, using a stir bar to circulate the volume within the beaker. After the wash, the excess water was squeezed out of the silk, and the degummed silk was then spread out on a clean piece of print paper, allowing the silk to dry in a fume hood overnight .
Chemical modification by MOI monomer
Once the fibroins were dry, 0.1 g of degummed silk fibers were immersed in 12 ml of anhydrous DMSO under nitrogen protection, and 2.1 g of MOI, 2 μl of hydroquinone and 0.01 g of dibutyltin dilaurate were added to this anhydrous system. The reaction mixture was kept at 35 °C for 24 h. Chemically modified silk fibroin fibers were successively rinsed with DMSO, ethanol and large amounts of ultrapure water thoroughly to remove unreacted reagents and then lyophilized (LGJ-10, Shanghai YuMing Instrument Co., Ltd., China) for 24 h.
Post modification via thiol-ene click reaction
To attach thiol-containing molecules to the MOI-modified SF fibers, each sample was contacted with 0.5 ml of a neat PFDT liquid. A quartz coverslip (0.5 mm thickness, Ted Pella) was placed on the sample to ensure homogeneous coverage of the organic liquid across the sample surface and was illuminated with a 40 W light-emitting diode (LED) UV lamp (UVSP1-A, ShenZhen YuXianDe Science and Technology Ltd., China) with 365 nm wavelength. Each sample was placed 20 mm below the LED UV lamp. Samples were then sonicated in ethanol to remove nonspecifically adsorbed PFDT molecules, rinsed with ultrapure water, and dried under a stream of nitrogen prior to analysis.
Cultivation of algae
Chlorella vulgaris (C. vulgaris) was obtained from Nanjing Health Biological Technology Co. Ltd. (Nanjing, China). The algae cells were cultivated for a period of 6 days, which is when the C. vulgaris population reached a relatively stable phase in the culture medium before the antifouling assay. Cultures were grown at 22 ± 2 °C with 16 h of light exposes and an 8-h of dark photoperiod. Lighting was supplied by a combination of warm and cold fluorescent tubes, giving a luminance range of between 2200 and 2800 Lux. The in vivo absorption of the culture medium containing algal cells in each flask was monitored each day via a UV–Vis spectrophotometer (T6, Beijing Puxi Analytic Instrument Ltd., China) at 660 nm (the growth curve was sigmoid and is presented in Additional file 1: Figure S1).
Silk fibroin fiber characterization
X-ray photoelectron (XP) spectroscopic characterization was carried out in an ultrahigh-vacuum system (Kratos Axis Ultra DLD, UK) with a base pressure of 5 × 10−9 Torr, a monochromatic Al Kα source, and a hemispherical analyzer. Survey (1.0 eV resolution) and highresolution (0.1 eV resolution) spectra were collected at a 0° takeoff angle from surface normal, then analyzed with Kratos Vision 2.0 software. Each high-resolution spectrum was referenced to the C1s peak (284.6 eV) and fit with Voigt functions (70% Gaussian, 30% Lorentzian) after a Shirley background correction.
Scanning electron microscopy (SEM) images were acquired under the desktop SEM Phenom™ Pro (Phenom-World, Netherlands) at an accelerating voltage of 10.0 kV. Infrared spectra of samples were analyzed and recorded using Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet iN10) in KBr pellets.
A differential scanning calorimeter (DSC, HSC-3, Beijing HengJiu Scientific Instrument Factory, China) utilizing N2 gas was used to assess the thermal behavior of the silk fibers under each processing condition. The scanning temperature was raised from room temperature to 390 °C at a heating rate of 10 °C min−1.
Quantification of algal cells adhering to samples
The amount of algal cells adhering to sample was quantified from the chlorophylls released from the adherent algal cells. Briefly, non-adherent algal cells were removed following a gentle wash with PBS, pH = 7.4. The sample was then immersed in 3 ml DMSO, and placed on a rotary shaker with a speed of 150 rpm at 30 °C for 1 h. The extract was filtered through the 0.22 μm filter membrane (Tianjin Jinteng Experimental Equipment Co., Ltd, China), and the UV absorption was recorded using UV spectrophotometry at 428.5 nm.
Results and discussion
Chemical modification by MOI monomer
Silk fibers were chemically modified by MOI monomer in an anhydrous environment. Di-n-butyltin (IV) dilaurate were used as a catalyst effectively promoted the reaction between the isocyanate group and primary alcohol groups in silk fibers. The reaction was proceeded at 35 °C based on a previously published method . We plotted the weight gain in SF fibers modified with MOI monomer against the reaction time at 35 °C. The mass-gain curve was sigmoid (the mass-gain curve is presented in Additional file 1: Figure S2). Weight gain was marked with increasing reaction time, finally plateauing at 20.6 wt% after a reaction time exceeding 24 h. Our reaction was therefore conducted at 35 °C for 24 h .
Graft MOI-modified silk fibers with PFDT
PFDT was grafted at vinyl groups conjugated on the MOI-modified silk fibers using a thiol-ene click reaction. To determine the selectivity of the thiol-ene click reaction, we illuminated MOI-modified silk fibers with PFDT and FTOH molecules. We chose PFDT and FTOH molecules because the fluorine atom can serve as an elemental tag. In addition, as a highly hydrophobic molecule, it is expected that PFDT molecules can provide antifouling behavior against algal cells attachment . Reaction progress was tracked with XPS measurements, and the F/C ratio was used to compare the number of fluorine-containing molecules attached to the surface of each sample.
To determine if PFDT attachment was specific to the MOI modification, we illuminated silk fibers without MOI modification in the presence of PFDT molecules. As a result, a 60 min illumination time resulted in no detectable fluorine electrons (Fig. 2c). The above observation supports the notion that an alkene group is necessary for the surface attachment through thiol-ene click chemistry.
Specifically, we incubated MOI-grafted silk fibers in the presence of PFDT molecules but in the absence of UV illumination before thoroughly rinsing and sonicating. A signal from fluorine electrons was not detectable by XPS (Fig. 2d), indicating that UV illumination of the silk fibers is required for the thiol-ene click reaction to proceed.
Cell viability assay
Algal cells adhesion experiment
To evaluate the antifouling performance of PFDT-grafted silk fibers and confirm the successful binding of PFDT on the surface of silk fibers via thiol-ene click chemistry, we immersed the PFDT-grafted silk fibers in a C. vulgaris culture for 1 week and 2 weeks, respectively. The cell-cultured samples were rinsed with a phosphate-buffered solution (PBS, pH = 7.4) three times to remove all non-adherent cells. The samples were then consecutively immersed in DMSO to extract chlorophyll, as described above. The amount of cells adhered to the samples was evaluated with UV spectrophotometry at a wavelength of 428.5 nm by measuring the chlorophyll content.
In this work, we installed vinyl groups onto the silk fibroin fibers. Then, PFDT, as a model molecule, was grafted onto silk fibers using a thiol-ene click reaction in a heterogeneous system. Through different experimental designs, we specifically determined that (i) a thiol group of the functional molecule is required for attachment; (ii) vinyl groups on the silk fibers are needed for molecule attachment; and (iii) UV illumination of the sample is necessary for the reaction to proceed. C. vulgaris adhesion significantly decreased on PFDT-grafted silk fiber compared to original silk fibers. This result indicates that antifouling property can be lent to silk, and a thiol-ene click reaction initiated by UV illumination provides a method of grafting functional molecules onto silk fibers. This work could provide new perspectives for the application of the chemical modification techniques for silk fibroin and will also expand the spectrum of silk utilization. We believe that further reaction optimization (e.g. design a light source which could illuminate silk fibers with UV light evenly) would realize a controlled graft density of the functional molecules on the silk fibers, therefore facilitating the alternation of chemical and physical properties of silk fibers.
XZ designed and conducted the experiments related to this study and wrote the paper. JL and YL prepared resources for the experiments and collected the data for algal cells adhesion experiment. CD collected the XPS data. ZC performed measurements on DSC. GC reviewed and edited this manuscript. All authors read and approved the final manuscript.
This research was funded by Fundamental Research Funds for the Central Universities (XDJK2018C067), Chongqing Municipal Commission of Commerce (CQ2019JSCC03) and a Start-up Fund of Southwest University Grant (SWU117036). This work was performed in part at the Chapel Hill Analytical and Nanofabrication Laboratory, CHANL, a member of the North Carolina Research Triangle Nanotechnology Network, RTNN, which is supported by the National Science Foundation, Grant ECCS-1542015, as part of the National Nanotechnology Coordinated Infrastructure, NNCI. This work was supported in part by the Chongqing Graduate Student Research Innovation Project (CYS19121).
The authors declare that they have no competing interests.
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