CN116041735A

CN116041735A - High-elasticity silk fibroin hydrogel and photo-curing preparation method and application thereof

Info

Publication number: CN116041735A
Application number: CN202211567124.9A
Authority: CN
Inventors: 张强; 黄仁艳; 黄颖; 闫书芹
Original assignee: Wuhan Textile University
Current assignee: Wuhan Textile University
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-05-02

Abstract

The invention relates to a high-elasticity silk fibroin hydrogel, a photocuring preparation method and application thereof. The silk fibroin hydrogel provided by the invention has no organic reagent residue, no biotoxicity, excellent mechanical rebound resilience and good light transmittance, and has good market application prospect in the fields of biomedical materials, 3D printing base materials and the like, and the preparation method provided by the invention greatly shortens the gel forming time, is simple to operate, has short process flow and is easy to realize large-scale production.

Description

High-elasticity silk fibroin hydrogel and photo-curing preparation method and application thereof

Technical Field

The invention belongs to the technical field of polymer gel, and particularly relates to a high-elasticity silk fibroin hydrogel, a photocuring preparation method and application thereof.

Background

Hydrogels refer to materials that can absorb large amounts of water without dissolution, and are formed from hydrophilic natural or synthetic polymers by chemical or physical crosslinking. Because of the high water absorption and water retention properties, the hydrogel has extremely wide application field: such as facial masks, defervescing patches, antistaling agents in the food industry, drug carriers in the medical field, and the like. At present, various preparation methods of hydrogels are reported at home and abroad, such as chemical crosslinking, physical crosslinking, biocatalysis and the like. The hydrogel with different properties can be prepared by different preparation methods, so that the application requirements of different fields are met.

Silk fibroin is a natural protein extracted from silk, has excellent mechanical properties, processability, biocompatibility and biodegradability, has received a great deal of attention in the fields of tissue engineering, drug release and the like, and has been used for developing products in the form of fibers, films, scaffolds, microspheres and the like. For example, CN114409926A provides a preparation method of self-healing anti-freezing conductive silk fibroin hydrogel, which improves three defects of no freezing resistance, no self-healing and uneven distribution of conductive materials of common silk fibroin hydrogel, so that the self-healing anti-freezing conductive silk fibroin hydrogel has wide application prospect in the field of flexible electronic products; CN109824922a provides a silk fibroin hydrogel with infrared light response, and the obtained silk fibroin hydrogel has high-efficiency photo-thermal conversion efficiency; CN109431971a provides a method for preparing an injectable carrier hydrogel, which can be used for local injection, including but not limited to intratumoral injection, paraneoplastic injection and other solid tumor treatments.

The above patent prepares hydrogel based on regenerated silk fibroin, and most of the preparation methods used form a large number of crystallization areas in silk fibroin by chemical methods, and the obtained hydrogel has the problems of poor light transmittance, unsatisfactory mechanical properties and the like although the structure is stable. In order to ensure that the silk fibroin hydrogel has good light transmittance, CN109180964A uses a monomer N-vinyl pyrrolidone to be dissolved in a silk fibroin solution, and polyvinyl pyrrole macromolecules formed by catalyzing and polymerizing the N-vinyl pyrrolidone with horseradish peroxidase are physically entangled with silk fibroin molecular chains to obtain the novel silk fibroin hydrogel with good light transmittance and excellent degradation performance, but the method has a great influence on the biocompatibility of the material due to the fact that more chemical reagents are added into the gel.

In view of the above, the invention prepares the silk fibroin hydrogel based on a silk fibroin photo-curing system, the silk fibroin hydrogel has safe components, no biological toxicity, good mechanical rebound resilience, no organic reagent addition in the preparation process and high gel speed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the high-elasticity silk fibroin hydrogel, the photocuring preparation method and the application thereof, wherein the silk fibroin hydrogel has good light transmittance, excellent biocompatibility and high elasticity, is particularly suitable for the field of biomedical materials, and has short time consumption and high efficiency in the photocuring preparation process.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a high-elasticity silk fibroin hydrogel is provided, wherein the high-elasticity silk fibroin hydrogel is obtained by adding riboflavin and hydrogen peroxide into silk fibroin solution and performing photo-curing under ultraviolet irradiation.

The invention also comprises a preparation method of the high-elasticity silk fibroin hydrogel, which comprises the following specific steps:

s1, preparing a silk fibroin solution: heating and soaking raw silk in sodium carbonate aqueous solution to perform degumming treatment to obtain degummed silk fibroin fibers, immersing the degummed silk fibroin fibers in LiBr aqueous solution to obtain a crude solution by heating and dissolving, placing the crude solution in a dialysis bag, dialyzing in deionized water at low temperature, filtering the dialyzed solution to remove impurities and diluting to obtain a silk fibroin solution;

s2, uniformly mixing the silk fibroin solution obtained in the step S1 with the riboflavin aqueous solution to obtain a mixed solution, and then adding a hydrogen peroxide solution into the mixed solution, and fully mixing to obtain a pre-reaction solution;

and S3, placing the pre-reaction liquid obtained in the step S2 under an ultraviolet lamp for irradiation, and performing a photo-curing reaction to obtain the high-elasticity silk fibroin hydrogel.

According to the scheme, the concentration of the sodium carbonate aqueous solution is 0.1-0.5 wt%, the mass-volume ratio of raw silk to the sodium carbonate aqueous solution is 1 g/10-100 mL, the degumming treatment temperature is 45-100 ℃, and the degumming treatment time is 1-8 h.

According to the scheme, the concentration of the LiBr aqueous solution is 1-10 mol/L, the mass-volume ratio of the degummed silk fibroin fiber to the LiBr aqueous solution is 1 g/1-20 mL, the degummed silk fibroin fiber is immersed into the LiBr aqueous solution, the heating and dissolving temperature is 45-85 ℃, and the heating and dissolving time is 0.5-3 h.

According to the scheme, the low-temperature dialysis temperature of S1 is 0-4 ℃ and the low-temperature dialysis time is 1-5 d.

According to the scheme, the concentration of the silk fibroin solution of S1 is 1.5-7wt%.

According to the scheme, the concentration of the aqueous solution of the riboflavin in the S2 is 0.05-10 mM (mmol/L), and the volume ratio of the silk fibroin solution to the aqueous solution of the riboflavin is 5-20: 1.

according to the scheme, the volume percentage concentration of the hydrogen peroxide solution in the S2 is 5-30%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5 to 30.

According to the scheme, the power of the S3 ultraviolet lamp is 100-900W, and the photo-curing reaction time is 2-30 min.

The invention also comprises application of the high-elasticity silk fibroin hydrogel in the field of biomedical materials. For example, the characteristics of good elasticity, high porosity and short preparation time of the hydrogel are utilized to uniformly disperse cells in the silk fibroin hydrogel for culture, so that the silk fibroin biomedical scaffold is prepared.

The application of the high-elasticity silk fibroin hydrogel in the field of 3D printing base materials.

The invention adopts a photocuring method to prepare the silk fibroin hydrogel, and forms cross-linking through mutual entanglement among silk fibroin macromolecules, and the internal crystal structure is lower, mainly a silk I structure, so that the hydrogel has good light transmittance, extremely high elasticity and excellent biocompatibility.

The invention is based on regenerated silk fibroin, and takes riboflavin and hydrogen peroxide as a photocuring system, and the silk fibroin hydrogel is obtained by curing under ultraviolet irradiation. The riboflavin is capable of being excited to generate a triplet state under ultraviolet irradiation, and reacts with oxygen to form oxygen radicals, and the oxygen radicals can promote mutual entanglement among silk fibroin macromolecules to form gel. However, the gel rate of the photo-crosslinking silk fibroin is very slow by using the riboflavin only, so that the research suggests that the riboflavin and the hydrogen peroxide are taken as a novel photo-curing system, wherein the hydrogen peroxide can be decomposed to generate oxygen under the irradiation of ultraviolet light, and can be taken as an oxygen source to provide more oxygen for the system, so that the reaction is promoted, and the gel forming time is greatly shortened. The hydrogel prepared by the invention has extremely high mechanical rebound performance and extremely good light transmittance. Due to the shortening of the gel time, the mixed solution and cells can be mixed and then subjected to photopolymerization and solidification, and the cells can be uniformly coated in the gel and used as carriers of the cells, so that the application of the silk fibroin-based hydrogel in the biomedical field is enlarged.

In addition, under the photocuring system, the gel forming process is mainly entanglement among silk fibroin macromolecules, and the beta-sheet structure inside the silk fibroin is very few (the silk fibroin hydrogel prepared by the traditional method is provided with a large number of beta-sheet structures inside, and the hydrogel with the beta-sheet structure has higher strength but lower elasticity, so that the hydrogel becomes brittle and is easy to crush after being stressed), so that the hydrogel prepared by the method has good mechanical rebound performance.

The invention has the beneficial effects that: 1. the silk fibroin hydrogel provided by the invention has no organic reagent residue, no biotoxicity, excellent mechanical resilience and good light transmittance, and has good market application prospects in the fields of biomedical materials, 3D printing base materials and the like; 2. the preparation method provided by the invention greatly shortens the gel forming time, is simple to operate, has short process flow and is easy to realize large-scale production.

Drawings

FIG. 1 is a photograph of the pre-reaction solution of example 1 of the present invention before and after photo-curing;

FIG. 2 is an XRD pattern of silk fibroin hydrogels prepared in example 1;

FIG. 3 is a graph showing the rebound resilience of the silk fibroin hydrogel prepared in example 1;

FIG. 4 is a graph showing the real object comparison before and after the silk fibroin hydrogel prepared in example 1 is subjected to compression mechanical test;

FIG. 5 is a graph showing the compressive strength of the silk fibroin hydrogel and pure silk fibroin hydrogel prepared in example 1;

FIG. 6 is a graph showing the biocompatibility test of the silk fibroin hydrogel prepared in example 1;

FIG. 7 is an electron microscope test chart of the silk fibroin hydrogels prepared in examples 1-3 after vacuum lyophilization;

FIG. 8 is a graph showing the comparison of water holdup with silk fibroin hydrogels prepared in examples 1-3;

FIG. 9 is a graph showing the transmittance of 6 silk fibroin hydrogels prepared in example 4.

Detailed Description

The present invention will be described in further detail below with reference to the accompanying drawings, so that those skilled in the art can better understand the technical scheme of the present invention.

Example 1

A preparation method of the high-elasticity silk fibroin hydrogel comprises the following specific steps:

s1, preparing a silk fibroin solution: boiling and soaking 30g of raw silk in 900mL of sodium carbonate aqueous solution with the concentration of 0.1wt% for 1.5h to obtain degummed silk fibroin fibers, immersing the degummed silk fibroin fibers in 300mL of LiBr aqueous solution with the concentration of 10mol/L, heating to 80 ℃ to dissolve for 3h to obtain a crude solution, placing the crude solution in a dialysis bag, dialyzing in deionized water at 0 ℃ for 5 days, filtering the dialyzed solution to remove impurities, and diluting to the concentration of 1.5wt% to obtain a silk fibroin solution for later use;

s2, mixing the silk fibroin solution obtained in the step S1 with 0.5mM riboflavin aqueous solution according to a volume ratio of 20:1, mixing to obtain a mixed solution, and mixing a 30vol% hydrogen peroxide solution with the mixed solution according to the volume ratio of 1:30, adding the mixture into the mixed solution, and fully mixing to obtain a pre-reaction solution;

and S3, placing the pre-reaction liquid obtained in the step S2 under a 400W ultraviolet lamp for irradiation for 30min, and performing a photo-curing reaction to obtain the high-elasticity silk fibroin hydrogel.

Fig. 1 is a photograph of the pre-reaction liquid before and after photo-curing in this example, the photograph before irradiation with ultraviolet lamp on the left, the photograph after irradiation with ultraviolet lamp on the right, and the observation by the tilting method shows that the liquid level is not changed when the bottle body is tilted by the right drawing, indicating that gel has been formed.

Further XRD characterization was performed on the silk fibroin hydrogel prepared in this example, and the result is shown in FIG. 2, it can be seen that the prepared silk fibroin hydrogel has a diffraction peak at 19.7 degrees, which is mainly represented by a silk I structure, which indicates that silk mainly reacts in a non-crystalline region during the hydrogel formation process.

The rebound resilience of the silk fibroin hydrogel prepared in this embodiment is tested, the hydrogel is prepared into a cylinder with a diameter of 2mm and a height of 5mm, and the cylinder is subjected to compression mechanical test on a TMS-PRO type texture analyzer, wherein the test is mainly performed for three times, the compression deformation amount of the first time is 20%, the compression deformation amount of the second time is 40%, the compression deformation amount of the third time is 60%, the stress-strain curve chart in the compression and rebound process of the silk fibroin hydrogel is shown in fig. 3, and it can be seen that the area surrounded by a closed curve formed by a loading curve and an unloading curve is small, namely, the elastic loss in the cyclic compression process is small, so that the photo-cured silk fibroin hydrogel prepared by the invention has high elastic property. Fig. 4 shows the silk fibroin hydrogel prepared in this example before and after the compression mechanical test (physical comparison chart, it can be seen from the chart that the hydrogel has little change before and after compression, indicating that the hydrogel has good compression retraction elasticity.

Further, the compression strength test is performed on the silk fibroin hydrogel prepared in this example, the hydrogel is prepared into a cylinder with a diameter of 2mm and a height of 5mm, the compression mechanical test is performed on a TMS-PRO texture analyzer, the deformation amount of the cylinder is set to 90%, the pure silk fibroin hydrogel is used as a control sample, the pure silk fibroin hydrogel is obtained by ultrasonic treatment with a concentration of 5wt% of silk fibroin solution under 300W power for 1min, the hydrogel breaks when the compression deformation amount of the pure silk fibroin hydrogel reaches 40%, the compression strength of the hydrogel is about 20kPa, and the photocured silk fibroin hydrogel in this example starts to break when the deformation amount of the silk fibroin hydrogel reaches 82%, and the compression strength of the hydrogel reaches about 80 kPa.

Further testing the biocompatibility of the silk fibroin hydrogel prepared in the embodiment, firstly, resuspending endothelial cells with a culture medium, filtering, pouring out waste liquid to obtain endothelial cells, then mixing the endothelial cells with the pre-reaction liquid in the embodiment, performing photo-curing reaction under the condition of the embodiment to form gel, enabling the cells to be wrapped in the gel, then placing the gel in a 37 ℃ incubator for 7 days, and culturing the gel for 7 days, wherein fig. 6 is a tracing and staining photograph of the cultured cells, and the hydrogel is provided with a large number of cells after 7 days of culturing, so that the cells can proliferate in the hydrogel well, thus indicating that the hydrogel has good biocompatibility.

Example 2

s1, preparing a silk fibroin solution: boiling and soaking 30g of raw silk in 900mL of sodium carbonate aqueous solution with the concentration of 0.1wt% for 1.5h to obtain degummed silk fibroin fibers, immersing the degummed silk fibroin fibers in 300mL of LiBr aqueous solution with the concentration of 10mol/L, heating to 80 ℃ to dissolve for 3h to obtain a crude solution, placing the crude solution in a dialysis bag, dialyzing in deionized water at 0 ℃ for 5 days, filtering the dialyzed solution to remove impurities, and diluting to the concentration of 2.5wt% to obtain a silk fibroin solution for later use;

and S3, placing the pre-reaction liquid obtained in the step S2 under a 400W ultraviolet lamp for irradiation for 15min, and performing a photo-curing reaction to obtain the high-elasticity silk fibroin hydrogel.

Example 3

s1, preparing a silk fibroin solution: boiling and soaking 30g of raw silk in 900mL of sodium carbonate aqueous solution with the concentration of 0.1wt% for 1.5h to obtain degummed silk fibroin fibers, immersing the degummed silk fibroin fibers in 300mL of LiBr aqueous solution with the concentration of 10mol/L, heating to 80 ℃ to dissolve for 3h to obtain a crude solution, placing the crude solution in a dialysis bag, dialyzing in deionized water at 0 ℃ for 5 days, filtering the dialyzed solution to remove impurities, and diluting to the concentration of 5wt% to obtain a silk fibroin solution for later use;

and S3, placing the pre-reaction liquid obtained in the step S2 under a 400W ultraviolet lamp for irradiation for 2min, and performing a photo-curing reaction to obtain the high-elasticity silk fibroin hydrogel.

The results of the morphological characterization of the silk fibroin hydrogels prepared in examples 1-3 by electron microscopy after vacuum freeze-drying are shown in fig. 7, a and d in fig. 7 represent the morphological images of the hydrogels prepared in example 1 with a silk fibroin concentration of 1.5%, b and e represent the morphological images of the hydrogels prepared in example 2 with a silk fibroin concentration of 2.5%, c and f represent the morphological images of the hydrogels prepared in example 3 with a silk fibroin concentration of 5%, and it can be seen that the hydrogel prepared in example 3 with a silk fibroin concentration of 1.5% has a pore diameter of 100-300um, the hydrogel prepared in silk fibroin concentration of 2.5% has a pore diameter of 80-200um, and the hydrogel prepared in silk fibroin concentration of 5% has a pore diameter of 50-100um after freeze-drying, and as the silk fibroin concentration increases, the prepared hydrogel has a pore diameter decreasing and the pore diameter becomes smaller and smaller, because the hydrogel has more entangled molecules and has a smaller pore diameter and a smaller cross-linked space with the inside of the gel, as shown in fig. 7. Therefore, the hydrogel with different pore diameters can be prepared by adjusting the concentration of the silk fibroin, and the morphology and structure of the prepared silk fibroin hydrogel can be regulated and controlled to meet different application requirements.

Further, the silk fibroin hydrogels prepared in examples 1 to 3 were tested for water retention, and as shown in FIG. 8, the water retention of the hydrogels was about 1000% when the silk fibroin concentration was 5%, about 2000% when the silk fibroin concentration was 2.5%, and about 3000% when the silk fibroin concentration was reduced to 1.5%. As can be seen from fig. 8, the different concentrations of silk fibroin affect the water holding capacity of the hydrogel, and the higher the concentration of silk fibroin, the lower the water holding capacity of the prepared hydrogel, because the increase of the concentration of silk fibroin increases the cross-linking formed by intermolecular entanglement, so that the pores inside the hydrogel are more compact, the water storage space is reduced, and the water holding capacity is reduced.

Example 4

s1, preparing a silk fibroin solution: boiling and soaking 30g of raw silk in 900mL of sodium carbonate aqueous solution with the concentration of 0.1wt% for 1.5h to obtain degummed silk fibroin fibers, immersing the degummed silk fibroin fibers in 300mL of LiBr aqueous solution with the concentration of 10mol/L, heating to 80 ℃ to dissolve for 3h to obtain a crude solution, placing the crude solution in a dialysis bag, dialyzing in deionized water at 0 ℃ for 5 days, filtering the dialyzed solution to remove impurities, and diluting to the concentration of 1.5wt%, 2.5wt% and 5wt% to obtain a silk fibroin solution for later use;

s2, respectively mixing the three silk fibroin solutions obtained in the step S1 with 0.05mM and 0.5mM riboflavin aqueous solution according to a volume ratio of 20:1 to obtain 6 mixed solutions, mixing 30vol% hydrogen peroxide solution with the 6 mixed solutions according to the volume ratio of 1:30, adding the mixture into the mixed solution, and fully mixing to obtain 6 pre-reaction solutions;

and S3, all the 6 pre-reaction liquids obtained in the step S2 are placed under a 400W ultraviolet lamp to be irradiated for 20min, and the photo-curing reaction is carried out to obtain 6 high-elasticity silk fibroin hydrogels.

The transmittance of the 6 silk fibroin hydrogels prepared in this example was tested and the results are shown in fig. 9. Since riboflavin in this reaction system is a yellow solution, the main factor that affects light transmittance of this system is the concentration of riboflavin. As can be seen from FIG. 9, the higher the riboflavin concentration contained, the more yellow the color of the hydrogel, and the poorer the light transmittance of the hydrogel. When the riboflavin concentration was 0.05mM, the light transmittance of the hydrogel was about 85%, and when the riboflavin concentration was increased to 0.5mM, the light transmittance of the hydrogel was reduced to about 75%. The riboflavin concentration may be adjusted for different applications to achieve a desired balance of photocuring rate and light transmittance.

Comparative example 1

A silk fibroin hydrogel is prepared by adding only riboflavin instead of hydrogen peroxide, and comprises the following specific steps:

s2, mixing the silk fibroin solution obtained in the step S1 with 50mM riboflavin aqueous solution according to a volume ratio of 20:1, fully mixing to obtain a pre-reaction solution;

and S3, placing the pre-reaction liquid obtained in the step S2 under a 400W ultraviolet lamp for irradiation for 3 hours, and performing a photo-curing reaction to obtain the silk fibroin hydrogel.

In the process of preparing the hydrogel in comparative example 1, it is found that if only riboflavin photo-crosslinking silk fibroin is adopted, the concentration of the riboflavin is extremely high, and because the gel rate of the pure riboflavin photo-crosslinking silk fibroin is extremely low, the gel can be formed only by irradiating ultraviolet light for 1-3 hours, the obtained gel is in a viscous state, and the molding is poor.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. The high-elasticity silk fibroin hydrogel is characterized in that the high-elasticity silk fibroin hydrogel is obtained by adding riboflavin and hydrogen peroxide into silk fibroin solution and performing photo-curing under ultraviolet irradiation.

2. A method for preparing the high-elasticity silk fibroin hydrogel according to claim 1, which comprises the following specific steps:

3. The method for preparing the high-elasticity silk fibroin hydrogel according to claim 2, wherein the concentration of the sodium carbonate aqueous solution is 0.1-0.5 wt%, the mass-volume ratio of raw silk to the sodium carbonate aqueous solution is 1 g/10-100 mL, the degumming treatment temperature is 45-100 ℃, and the degumming treatment time is 1-8 h.

4. The preparation method of the high-elasticity silk fibroin hydrogel according to claim 2, wherein the concentration of the LiBr aqueous solution is 1-10 mol/L, the mass volume ratio of degummed silk fibroin fiber to the LiBr aqueous solution is 1 g/1-20 mL, the degummed silk fiber is immersed in the LiBr aqueous solution, the heating dissolution temperature is 45-85 ℃, and the heating dissolution time is 0.5-3 h.

5. The method for preparing a high-elasticity silk fibroin hydrogel according to claim 2, wherein the S1 low-temperature dialysis temperature is 0-4 ℃ and the low-temperature dialysis time is 1-5 d.

6. The method for preparing a high-elasticity silk fibroin hydrogel according to claim 2, wherein the concentration of the silk fibroin solution is 1.5-7wt%.

7. The method for preparing the high-elasticity silk fibroin hydrogel according to claim 2, wherein the concentration of the aqueous solution of the riboflavin in S2 is 0.05-10 mM, and the volume ratio of the silk fibroin solution to the aqueous solution of the riboflavin is 5-20: 1.

8. the method for preparing the high-elasticity silk fibroin hydrogel according to claim 2, wherein the volume percentage concentration of the hydrogen peroxide solution is 5-30%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5 to 30.

9. The method for preparing a high-elasticity silk fibroin hydrogel according to claim 2, wherein the power of the S3 ultraviolet lamp is 100-900W, and the photo-curing reaction time is 2-30 min.

10. Use of the hyperelastic silk fibroin hydrogel according to claim 1 in the field of biomedical materials and in the field of 3D printing substrates.