阅读说明：本技术 一种丝胶蛋白水凝胶及其制备方法和应用 (Sericin hydrogel and preparation method and application thereof ) 是由 张业顺 沈聪昳 张国政 吴堂凤 钱平 朱大林 方瑷 曹喜涛 冉素晴 于 2020-12-08 设计创作，主要内容包括：本发明公开了一种丝胶蛋白水凝胶。该丝胶蛋白水凝胶由丝胶蛋白水溶液和交联剂水溶液反应制得,制备方法是：将不含丝素的蚕茧洗净、干燥、粉碎得到蚕茧粉,加水置于100～180℃反应0.5～10小时,离心并去除不溶物质,收集澄清溶液,获得浓度为2～18％的丝胶蛋白水溶液；配制交联剂水溶液,滤膜过滤除菌,加入丝胶蛋白水溶液中混合均匀,摇床震荡20～120分钟,即可。该丝胶蛋白水凝胶具有优良的力学性能、多孔微观结构、形变记忆性且无菌,可作为药物或生长因子的载体,冻干得到的丝胶蛋白生物支架,可应用于组织损伤的修复和疾病的治疗。本发明的制备方法简单,成胶过程可控且成胶时间可通过调节丝胶蛋白的浓度而调控。(The invention discloses a sericin hydrogel. The sericin hydrogel is prepared by the reaction of a sericin aqueous solution and a cross-linking agent aqueous solution, and the preparation method comprises the following steps: cleaning, drying and crushing silk cocoons without fibroin to obtain silk cocoon powder, adding water, reacting at 100-180 ℃ for 0.5-10 hours, centrifuging, removing insoluble substances, and collecting a clear solution to obtain a sericin aqueous solution with the concentration of 2-18%; and (3) preparing a cross-linking agent aqueous solution, filtering and sterilizing by using a filter membrane, adding the cross-linking agent aqueous solution into a sericin aqueous solution, uniformly mixing, and shaking for 20-120 minutes by using a shaking table. The sericin hydrogel has excellent mechanical property, porous microstructure, deformation memory and asepsis, can be used as a carrier of a medicament or a growth factor, is a sericin biological scaffold obtained by freeze-drying, and can be applied to the repair of tissue injury and the treatment of diseases. The preparation method is simple, the gelling process is controllable, and the gelling time can be regulated and controlled by regulating the concentration of the sericin.)

1. A sericin hydrogel, which is prepared by the reaction of a sericin aqueous solution and a cross-linking agent aqueous solution, wherein the cross-linking agent aqueous solution is genipin, glutaraldehyde, malondialdehyde, succinaldehyde, dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, glycol diglycidyl ether, transglutaminase or H₂O₂Any of the aqueous solutions or H therein₂O₂Mixing the aqueous solution with another; the concentration of the sericin aqueous solution is 2-18 wt%; the concentration of the glutaraldehyde or malonaldehyde or succinaldehyde aqueous solution is 0.1-50 wt%, and the dosage of the glutaraldehyde or malonaldehyde aqueous solution is 1-500 mu L per ml of sericin aqueous solution; the genipin or dimethylol urea, oxidized glutathione, bis (3,5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, glycol diglycidyl ether and transglutaminase aqueous solution, wherein the concentrations of the 5-dibromo-salicylic acid) fumarate, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the glycol diglycidyl ether and the transglutaminase aqueous solution are 0.1 to 4 wt%, and the amount of the 5 to 5000 mu L of the aqueous solution of sericin per milliliter is used; said H₂O₂The concentration of the aqueous solution is 0.03-30 wt%, and the dosage of the aqueous solution is 1-1000 mu L per ml of sericin aqueous solution.

2. The method for preparing the sericin hydrogel according to claim 1, which comprises the steps of:

(1) preparation of silk fibroin-free Bombyx mori cocoons

Selecting a silkworm variety without silk fibroin, feeding the silkworm variety with fresh mulberry leaves at the whole age after the silkworm eggs are hatched, and strictly controlling environmental parameters during the silk spinning period of the silkworms when the silkworms bunch up: the temperature is controlled to be 20-30 ℃, and the humidity is controlled to be 30-70%; after spinning is finished and before the sericin aqueous solution is prepared, the temperature of the silkworm cocoons is kept at 20-30 ℃, and the humidity is kept at 30-70%;

(2) preparation of aqueous sericin solution

Cleaning, drying and crushing silk cocoons without fibroin to obtain silk cocoon powder, adding water to the silk cocoon powder, reacting at 100-180 ℃ for 0.5-10 hours, centrifuging, removing insoluble substances, and collecting a clear solution to obtain a sericin protein aqueous solution with the concentration of 2-18 wt%;

(3) preparation of sericin hydrogel

Preparing a cross-linking agent aqueous solution, filtering the cross-linking agent aqueous solution with a filter membrane for sterilization, then adding the cross-linking agent aqueous solution into a sericin aqueous solution, uniformly mixing, and shaking for 20-120 minutes in a shaking table to obtain the sericin hydrogel;

the cross-linking agent aqueous solution is genipin, glutaraldehyde, malondialdehyde, succinaldehyde, dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride, glycol diglycidyl ether, transglutaminase or H₂O₂Any of the aqueous solutions or H therein₂O₂A mixture of an aqueous solution with another; the glutaraldehyde or the propaneThe concentration of dialdehyde or succinaldehyde aqueous solution is 0.1-50 wt%, and the dosage is 1-500 mu L per ml of sericin aqueous solution; the concentration of the genipin or dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride, glycol diglycidyl ether and transglutaminase aqueous solution is 0.1-4 wt%, and the dosage of the genipin or dimethylol urea is 5-5000 mu L added in each milliliter of sericin aqueous solution; said H₂O₂The concentration of the aqueous solution is 0.03-30 wt%, and the dosage of the aqueous solution is 1-1000 mu L per ml of sericin aqueous solution.

3. The method for preparing the sericin hydrogel according to claim 2, wherein in the step (1), after the silkworm cocoons are crushed, 5 to 45mL of water is added to each gram of silkworm cocoon powder.

4. The method for preparing the sericin hydrogel according to claim 2, wherein the particle size of the silkworm cocoon powder in the step (1) is not less than 50 meshes.

5. The method for preparing the sericin hydrogel according to claim 2, wherein the rotation speed of the centrifugation in the step (1) is 3000-10000 rpm, and the centrifugation time is 5-30 min.

6. The method for preparing the sericin hydrogel according to claim 2, wherein the pore size of the filter membrane in the step (2) is not more than 0.22 μm.

7. The sericin hydrogel according to claim 1 is used for preparing a biomedical material.

8. The sericin hydrogel according to claim 1 is used for preparing a drug and a growth factor carrier.

9. The sericin hydrogel according to claim 1 is used for preparing a cell carrier.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a sericin hydrogel and a preparation method and application thereof.

Background

Sericin is a natural adhesive protein secreted by silkworm, consists of polypeptides with molecular weight of 24-400 kDa and contains 18 amino acids. In recent years, the research shows that sericin has no immunogenicity, and has a plurality of excellent characteristics, such as degradability, antibiosis, anticancer, antioxidation, uvioresistant, anticoagulation, tyrosinase activity inhibition, hydrophily, moisture retention, cell adhesion and proliferation promotion and the like; in addition, sericin has a large number of active groups such as amino, hydroxyl, carboxyl and the like which are convenient for people to modify or crosslink. At present, sericin becomes a novel biomedical material which is favored by people.

Currently, sericin has been used for preparing various forms of tissue engineering materials including sericin microspheres, sericin membranes, sericin scaffolds, sericin hydrogels, etc., with sericin hydrogels being particularly spotlighted. At present, people adopt various preparation processes and prepare sericin hydrogel with different characteristics, and meanwhile, the sericin hydrogel is used for repairing and treating research on various tissue injuries such as skin injury, nerve injury, muscle injury, bone injury and the like, and shows that the sericin hydrogel has great potential value in the field of tissue engineering regenerative medicine.

In the early stage, aiming at the problems that the sericin hydrogel has low mechanical strength, poor transparency, weaker fluorescence characteristic, cross-linking agent toxicity, serious damage to the structure and performance of the hydrogel caused by the degerming and degerming process, and the like, the sericin hydrogel is prepared by adopting ultrasonic induction, self-assembly and sericin obtained from silkworm bodies, and the problems in the aspects of transparency, mechanical strength and the like are solved to a certain extent. However, in the case of a sterile sericin hydrogel, although it can be directly prepared by self-assembly, it is poor in mechanical properties, lacks elasticity and is fragile. On the other hand, it has been considered that sericin obtained by high temperature and high pressure has high water solubility for a long time, and it is difficult to prepare a pure sericin hydrogel by direct crosslinking. On the other hand, the pure sericin hydrogel prepared based on sericin extracted from silkworm cocoons at present has low mechanical strength, while the hydrogel prepared from sericin extracted from silkworm bodies has strong mechanical strength, the sericin obtained from silkworm bodies wastes time and labor, is difficult to operate and is difficult to carry out large-scale production; meanwhile, there is a great difference in structure and performance between sericin in silkworm bodies and sericin separated from silkworm cocoons, and therefore, even if hydrogels having the same mechanical strength are prepared based on sericin obtained from silkworm bodies and sericin separated from silkworm cocoons, hydrogels prepared from both of them will still have great difference in other physicochemical properties and biological activities. In addition, the study on the performance and extraction efficiency of sericin in silkworm cocoons by silkworm feeding conditions and silking environment is rarely reported. At present, the preparation of pure sericin hydrogel with high mechanical strength based on the separation of sericin from silkworm cocoons still faces technical difficulties.

Disclosure of Invention

One of the objects of the present invention is to provide a sericin hydrogel. The specific technical scheme is as follows:

a sericin hydrogel is prepared by the reaction of a sericin aqueous solution and a cross-linking agent aqueous solution, wherein the cross-linking agent aqueous solution is genipin, glutaraldehyde, malondialdehyde, succinaldehyde, dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl aminopropyl) carbodiimide hydrochloride, glycol diglycidyl ether, transglutaminase or H₂O₂Any of the aqueous solutions or H therein₂O₂Mixing the aqueous solution with another; the concentration of the sericin aqueous solution is 2-18 wt%; the concentration of the glutaraldehyde or malonaldehyde or succinaldehyde aqueous solution is 0.1-50 wt%, and the dosage of the glutaraldehyde or malonaldehyde aqueous solution is 1-500 mu L per ml of sericin aqueous solution; the concentration of the genipin or dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride, glycol diglycidyl ether and transglutaminase aqueous solution is 0.1-4 wt%, and the dosage of the genipin or dimethylol urea is 5-5000 mu L added in each milliliter of sericin aqueous solution; said H₂O₂The concentration of the aqueous solution is 0.03-30 wt%, and the dosage of the aqueous solution is 1-1000 mu L per ml of sericin aqueous solution. The silkworm cocoon used for preparing the sericin protein aqueous solution is a silkworm cocoon of a silkworm variety without fibroin, and is fed by mulberry leaves, the temperature of a spinning environment is controlled to be 20-30 ℃, and the humidity is controlled to be 30-70%. The silkworm cocoons for preparing the sericin hydrogel are stored under the environment that the temperature is 20-30 ℃ and the humidity is 30-70%.

The other purpose of the invention is to provide a preparation method of the sericin hydrogel. The specific technical scheme is as follows:

the preparation method of the sericin hydrogel comprises the following steps:

(1) preparation of silk fibroin-free Bombyx mori cocoons

Selecting a silkworm variety without silk fibroin, feeding the silkworm variety with fresh mulberry leaves at the whole age after the silkworm eggs are hatched, and strictly controlling environmental parameters during the silk spinning period of the silkworm when the silkworm is clustered: the temperature is controlled to be 20-30 ℃, and the humidity is controlled to be 30-70%; after spinning is finished and before the sericin aqueous solution is prepared, the temperature of the silkworm cocoons is kept at 20-30 ℃, and the humidity is kept at 30-70%;

(2) preparation of aqueous sericin solution

Cleaning, drying and crushing silk cocoons without fibroin to obtain silk cocoon powder, adding water to the silk cocoon powder, reacting at 100-180 ℃ for 0.5-10 hours, centrifuging, removing insoluble substances, and collecting a clear solution to obtain a sericin aqueous solution with the concentration of 2-18 wt%;

(3) preparation of sericin hydrogel

Preparing a cross-linking agent aqueous solution, filtering the cross-linking agent aqueous solution with a filter membrane for sterilization, then adding the cross-linking agent aqueous solution into a sericin aqueous solution, uniformly mixing, and shaking for 20-120 minutes in a shaking table to obtain the sericin hydrogel;

the cross-linking agent aqueous solution is genipin, glutaraldehyde, malondialdehyde, succinaldehyde, dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride, glycol diglycidyl ether, transglutaminase or H₂O₂Any of the aqueous solutions or H therein₂O₂A mixture of an aqueous solution with another; the concentration of the glutaraldehyde or malonaldehyde or succinaldehyde aqueous solution is 0.1-50 wt%, and the dosage of the glutaraldehyde or malonaldehyde aqueous solution is 1-500 mu L per ml of sericin aqueous solution; the concentration of the genipin or dimethylol urea, oxidized glutathione, bis (3, 5-dibromo-salicylic acid) fumarate, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride, glycol diglycidyl ether and transglutaminase aqueous solution is 0.1-4 wt%, and the dosage is that each milliliter of sericin aqueous solution is added with the sericin aqueous solution5-5000 mu L of the mixture is added; said H₂O₂The concentration of the aqueous solution is 0.03-30 wt%, and the dosage of the aqueous solution is 1-1000 mu L per ml of sericin aqueous solution.

Preferably, in the step (1), after the silkworm cocoons are crushed, 5-45 mL of water is added to each gram of silkworm cocoon powder.

Preferably, the particle size of the silkworm cocoon powder in the step (1) is more than or equal to 50 meshes.

Preferably, the rotation speed of the centrifugation in the step (1) is 3000-10000 rpm, and the centrifugation time is 5-30 min.

Preferably, the pore size of the filter membrane in the step (2) is less than or equal to 0.22 mu m.

The invention also aims to provide application of the sericin hydrogel. The specific technical scheme is as follows:

the sericin hydrogel is used for preparing biomedical materials.

Preferably, the sericin hydrogel is used for preparing a drug and a growth factor carrier.

Preferably, the sericin hydrogel is used for preparing a cell carrier.

The invention has the beneficial effects that:

(1) the method selects the silkworm variety without fibroin, feeds the silkworm variety with fresh mulberry leaves at the whole age, and strictly controls the environmental parameters (the temperature is controlled to be 20-30 ℃ and the humidity is controlled to be 30-70%) and the silkworm cocoon storage environment (the temperature is 20-30 ℃ and the humidity is kept to be 30-70%) during the silking period of the silkworm. Lays a foundation for obtaining sericin with high extraction efficiency and high dissolution rate;

(1) according to the invention, the sericin is obtained at high temperature and high pressure for the first time to act with a cross-linking agent (through covalent cross-linking), the sericin hydrogel with excellent mechanical property, porous microstructure and deformation memory is prepared, and the problem that sericin extracted by a high-temperature and high-pressure method is difficult to directly cross-link to form hydrogel with good mechanical property for a long time is solved;

(2) the sericin hydrogel prepared by the method has simple preparation method and can directly realize sterility; the gelling time can be regulated and controlled by regulating the concentration of sericin;

(3) the invention adopts high-temperature and high-pressure technology to obtain sericin with the extraction efficiency of 96% after the silk fibroin-free silkworm cocoons are ground, and simultaneously directly obtains a sericin solution with the concentration of 18% (w/v); the elastic modulus of the pure silk glue gel prepared by the invention can reach 2560Kpa, which is far higher than the hardness of the pure silk glue gel reported in the early stage.

(4) The sericin hydrogel can be used as a carrier of a medicament or a growth factor and has good medicament controlled release function; the sericin hydrogel and the three-dimensional porous sericin biological scaffold obtained by freeze-drying the sericin hydrogel can be used as an extracellular matrix to support cell growth and promote nutrient exchange, and can be applied to repair of various tissue injuries and treatment of diseases.

Drawings

FIG. 1 is a flow chart of the preparation of a sericin hydrogel according to the present invention;

FIG. 2 is a microscopic structural view of the sericin hydrogel prepared in example 1; wherein FIG. 2A is a photograph of a sample lyophilized after treatment of hydrogel at-20 deg.C, and FIG. 2B is a photograph of a sample lyophilized after treatment of hydrogel at-80 deg.C; FIG. 2C is a photograph of a sample lyophilized after treatment of hydrogel at-196 deg.C;

FIG. 3 is an SDS-Page graph of sericin extracted in example 1;

FIG. 4 is a graph of gelling time for different concentrations of sericin in example 1 with the aid of a crosslinking agent;

FIG. 5 is a graph showing the water swelling rate (37 ℃) of the sericin hydrogel in example 1 under different pH conditions;

FIG. 6 is a graph of the degradation profile of the sericin hydrogel in example 1 under different pH conditions;

FIG. 7A, FIG. 7B and FIG. 7C are the infrared spectra before and after gelling of sericin in example 1; FIG. D is an X-ray diffraction pattern of sericin in example 1 before and after gelling;

FIG. 8 is a graph of the controlled release of the sericin hydrogel loading mode drug in example 1;

FIG. 9 is a graph showing the mechanical properties of the sericin hydrogel in example 1, wherein FIG. 9A is a graph showing the mechanical properties before stretching, FIG. 9B is a graph showing the mechanical properties after stretching, FIG. 9C is a graph showing the mechanical properties before and after compression, and FIG. 9D is a graph showing the mechanical properties before and after compression

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

A preparation method of sericin hydrogel comprises the following steps:

first, silkworm cocoon acquisition

Selecting a silkworm variety (preserved by a silkworm research institute of the Chinese academy of agricultural sciences and provided for commercialization) without silk fibroin, feeding the silkworm with fresh mulberry leaves at all ages after hatching, and strictly controlling the silk-spinning environmental parameters of the silkworms before the silkworms cluster: the temperature is controlled to be 20-30 ℃, and the humidity is controlled to be 30-70%. And after spinning is finished and before the sericin aqueous solution is prepared, keeping the temperature of the silkworm cocoons at 20-30 ℃ and the humidity at 30-70%. The main chemical components of the silkworm cocoon are as follows: silkworm silk glue protein.

Secondly, extraction of sericin and preparation of hydrogel

(1) Cleaning silk cocoons without fibroin with ultrapure water, then placing in a clean tray, drying at room temperature, crushing, and sieving with a 100-mesh sieve to obtain silk cocoon powder;

(2) weighing the silkworm cocoon powder obtained in the step (1), transferring the silkworm cocoon powder into a blue-covered bottle, and adding a proper amount of ultrapure water to ensure that the mass ratio of the silkworm cocoon powder to water is 15: 100. filling powder, stirring, mixing, placing the blue-cap bottle in a high-pressure cooker at 120 deg.C, and dissolving sericin at high pressure for 0.5 hr;

(3) transferring the solution obtained in the step (2) into a centrifugal tube, centrifuging at 6000rpm for 10 minutes, and removing insoluble substances to obtain a sericin solution;

(4) preparing a genipin aqueous solution with the mass percentage concentration of 2%, and filtering and sterilizing through a filter membrane with the diameter of 0.22 mu m.

(5) Adjusting the mass percentage concentration of the sericin aqueous solution obtained in the step (3) to 14% by using ultrapure water, and then fully and uniformly mixing a proper amount of sericin liquid with the concentration and the genipin aqueous solution obtained in the step (4) at room temperature;

(6) and (5) placing the uniformly mixed sample in the step (5) on a shaking table to shake, and gelling at room temperature for tens of minutes to obtain the sericin protein hydrogel.

Third, detection

The protein concentration detection adopts a sericin aqueous solution drying method; transferring 1mL of sericin aqueous solution into a clean vessel for drying. The dried sericin sample is then taken off and weighed with forceps. The concentration of the sericin solution can be known according to the mass-to-volume ratio.

The molecular weight of the protein is detected by SDS-PAGE. 20 mu L of the mixture is used for molecular weight detection, and the rest is stored in a refrigerator at 4 ℃ for later use.

Fourth, result analysis

1. Color of sericin hydrogel

The sericin hydrogel is obtained by crosslinking sericin and genipin, and the color of the hydrogel is dark blue as shown in figure 1.

2. Molecular weight distribution of sericin

The molecular weight distribution of the extracted sericin was examined by SDS-Page gel electrophoresis, and the results are shown in FIG. 2. The obtained sericin has a wide molecular weight distribution range, and has a distribution from 15kD to more than 180kD, wherein the molecular weight is mainly distributed to more than 40 kD.

3. The sericin hydrogel is in a porous structure

The sericin hydrogel is frozen at-20 deg.C, -80 deg.C and-196 deg.C, then lyophilized at low temperature under vacuum, and then observed under scanning electron microscope. As shown in fig. 2: the lyophilized sericin hydrogel is a three-dimensional porous scaffold. Hydrogel is processed at different temperatures, the obtained scaffold has obvious difference in pore size, and the hydrogel scaffold obtained at lower processing temperature has smaller pore size.

4. Porosity detection of sericin hydrogel

Lyophilizing the hydrogel at-80 deg.C, and placing the hydrogel sample at a volume V₁In ultrapure water, the total volume of sample and water at this time is denoted as V₂Then the hydrogel sample was removed and the volume of remaining water was recorded as V₃. The porosity of the sericin sample is (V)₂-V₁)/(V₃-V₁) X 100%. The hydrogel prepared from sericin at a concentration of 10% (w/v) had a porosity of 60%.

5. Effect of sericin concentration on hydrogel gelling time

By regulating the concentration of the sericin solution, the gelling time of sericin with different concentrations is detected, and the result is shown in fig. 4, wherein the gelling time is faster as the concentration of sericin is higher. The gelling time was 66 min when the sericin concentration was 2% (w/v), and was shortened to 23min when the sericin concentration was 10% (w/v), indicating that the gelling time can be controlled by controlling the sericin concentration.

6. Effect of pH on swelling Rate of sericin hydrogel

The sericin hydrogel was lyophilized, weighed, and soaked in three kinds of PBS solutions of different pH values (pH 3.0, pH7.4, pH 11.0), and at different time points, taken out and the water swelling rate thereof was measured according to the following formula. (wherein Ws is the weight in the swollen state and Wd is the dry weight)

As shown in fig. 5: the sericin hydrogel rapidly rose in swelling rate within the first 18 hours and was substantially stable after 220 hours. The maximum swelling rates were 4, 7 and 8 times in the pH 3.0, pH7.4 and pH 11.0 environments, respectively. From the figure, it can be seen that the swelling characteristics of the sericin hydrogel are pH-responsive.

7. Degradation rates of sericin hydrogel in different pH environments

For testing the effect of pH environment on degradation, the sericin hydrogel was soaked in PBS solutions of different pH values (pH 3.0, pH7.4, pH 11.0), the PBS solution was changed once a day, taken out at a preset time point, dried, and weighed, and the results are shown in fig. 6.

As shown in fig. 6: the hydrogel degradation has pH responsiveness, wherein the degradation rate is fastest under the alkaline condition of pH 11.0, the 3-week cumulative degradation rate is 45%, the 3-week cumulative degradation rate is 34% after the time of neutral environment (pH7.4), the degradation rate is slowest under the acidic condition (pH 3.0), and the 3-week degradation rate is 24%.

8. Infrared spectroscopy and X-ray diffraction analysis of sericin hydrogel and sericin

Measurement of sericin hydrogel at 4000-^-1Characteristic peak of (2).

As shown in fig. 7A, 7B, 7C: the secondary structure of sericin has no obvious change before and after crosslinking; as shown in fig. 7D, the crystallinity of sericin did not change much before and after crosslinking.

9. Sericin hydrogel used as drug or growth factor carrier

Horseradish peroxidase (HRP) was chosen as the model drug. 2mg/ml horseradish peroxidase solution and genipin (1%, w/v) were first prepared and filter sterilized, and then sericin solution (12%, w/v), genipin (1.5%, w/v) and horseradish peroxidase solution (2mg/ml) were mixed at 125: 20: 5, the mixture was thoroughly mixed, the mixed solution was transferred to an EP tube in an amount of 300. mu.L per tube before gelling, 1mL of fresh PBS (pH7.4) was added after gelling, and then sample 37 was added^℃And (4) under the environment. The amount of HRP released from the hydrogel was measured at a preset time point. As shown in FIG. 8, HRP released more rapidly in the initial stage and the drug release rate was slower and slower with time. With drug release rates of 23.38%, 36.74% and 47.45% during the first 6 hours, 12 hours and 24 hours, respectively, after which the release rate slowed significantly and a cumulative release of 58.65% of the drug over 192 hours. The result shows that the sericin hydrogel can maintain the function of drug release and can be used as a carrier for drug controlled release.

10. Detection of mechanical properties of sericin hydrogel

Preparing a strip with the thickness of 3mm and the width of 10mm in a die for a tensile test; cylinders of 12mm diameter were prepared for compression testing and the testing was performed on a universal material testing machine. As shown in FIGS. 9A to 9D, the hydrogel prepared by sericin prepared at high temperature and high pressure according to the present invention under the action of a crosslinking agent has excellent elasticity, and when the sericin concentration is 10%, the sericin hydrogel has a tensile rate of 17%, and the elastic modulus is up to 121KPa without breaking; when the depth of the sericin is 18%, the elastic modulus of the sericin hydrogel reaches 2560Kpa which is far higher than the related reported parameters of pure sericin hydrogel, which indicates that the sericin hydrogel with high hardness can be prepared by the method. Furthermore, it can be seen from the compression results that the hydrogel remained intact without breakage when the compression rate exceeded 50%. The results show that the sericin hydrogel has excellent elasticity and good mechanical properties. Breaks through the view that sericin obtained by high temperature and high pressure is difficult to directly crosslink to form pure sericin hydrogel for a long time. Furthermore, it is even more surprising that the sericin hydrogel prepared has excellent mechanical properties.