CN114539564B - Preparation method of rapid-gel silk fibroin-based strong hydrogel - Google Patents

Abstract

The invention provides a preparation method of a quick-gelling silk fibroin-based strong hydrogel, which comprises the steps of cleaning degummed silk with deionized water, drying, dissolving and dialyzing to obtain a silk fibroin solution; and then adding a cross-linking agent into the silk fibroin solution for chemical cross-linking, uniformly mixing with a gelatin solution, and standing for a certain time under the low-temperature condition to obtain the silk fibroin/gelatin hydrogel. Through the combined use of chemical crosslinking and physical crosslinking, the high-efficiency combination between the silk fibroin and the gelatin is realized, and the integration of high performance and controllability is realized; by utilizing the salt solution to induce the molecular structure of the silk fibroin and the gelatin, the mechanical property of the hydrogel is improved. The hydrogel prepared by the preparation method has the advantages of uniform three-dimensional porous structure, high elasticity, strong mechanical property, good biocompatibility and biodegradability.

Description

Preparation method of fast gelling silk fibroin-based strong hydrogel

Technical field

The present invention relates to the technical field of biomaterials, and in particular to a method for preparing a fast-gelling silk fibroin-based strong hydrogel.

Background technique

Gel is a form between solid and liquid. According to the properties of dispersed particles and the characteristics of the connection between particles when forming a gel structure, gel can be divided into elastic gel and inelastic gel. Hydrogel is an elastic gel formed from a solution of polymer compounds. It uses water as the dispersion medium, can absorb and retain a large amount of water, and has a cross-linked network structure. It is composed of hydrophilic polymer compounds through ionic bonds, It is cross-linked by covalent bonds, hydrogen bonds, etc., and has polymer electrolyte characteristics and three-dimensional structure. Hydrogel has a higher water content and more internal void structures. Its interconnected porous structure provides channels for the transportation of water, nutrients and drugs, and provides a good environment for cell migration and proliferation. It is known as It is considered to be a good material for the encapsulation and delivery of cells and bioactive molecules, and can be used in tissue engineering and cell therapy.

Silk fibroin is a natural polymer protein extracted from silk. Its internal structure is divided into crystalline and amorphous regions. The antiparallel β-sheet structure constitutes the crystalline region, and the irregularly coiled structure belongs to the amorphous region. The crystalline region is generally relatively evenly dispersed in the amorphous region. The molecular chains in the amorphous region and microcrystalline region follow the filaments. The direction of the long axis of the fiber is arranged in an orderly manner. Based on the staggered existence of crystalline regions and amorphous regions and the directional arrangement of macromolecules, silk fibers have good mechanical properties and soft luster. The physical and chemical properties of silk fibroin are determined by the arrangement of amino acids that affects the molecular conformation. Silk fibroin has good physical and mechanical properties, adjustable biodegradability and excellent biocompatibility. Currently, silk fibroin is widely used in tissue engineering, regenerative medicine and other fields. In the existing technology, silk fibroin hydrogels prepared through physical cross-linking are slow to gel, have poor structural stability, and have poor mechanical properties; silk fibroin hydrogels prepared through chemical cross-linking have poor elasticity. , the above shortcomings limit the application of silk fibroin hydrogels in regenerative medicine, tissue engineering and other fields. Therefore, it is of great significance to find a method to prepare silk fibroin hydrogels with both excellent mechanical properties and biocompatibility.

Chinese patent application number 200910025440.1 was published on July 25, 2012. The patent titled "A preparation method of silk fibroin hydrogel" discloses a preparation method of silk fibroin hydrogel. In the above method, the silk fibroin solution is obtained by degumming, dissolving, and dialyzing the silk fibroin with a mass concentration of 1 to 30%; then, ultrasonic oscillation is performed at 37°C to obtain a silk fibroin hydrogel; and finally , pour the treated silk fibroin solution into the mold, and leave it at room temperature for 5 minutes to obtain the silk fibroin solution. In the above technical solution, the method of ultrasonic oscillation is used to prepare silk fibroin hydrogel. The silk fibroin molecules are physically cross-linked under the action of ultrasound, and the silk fibroin forms a β-sheet crystal structure, but the formed silk fibroin hydrogel The gel is very hard and brittle, and its strength and flexibility cannot meet the subsequent application requirements of high-strength and high-tenacity silk fibroin materials.

Chinese patent application number 201810662604.0, published on June 2, 2020, discloses double network cross-linked fibers in the patent titled "Double network cross-linked cellulose/silk fibroin high-strength hydrogel and its preparation and application" High-strength fibroin/silk fibroin hydrogel and its preparation and application. In the above technical solution, the cellulose solution, silk fibroin solution, and epichlorohydrin are mixed evenly and cross-linked under stirring conditions to obtain a cross-linked solution; the cross-linked solution is centrifuged to remove bubbles and then poured into the mold for reaction and solidification. Finally, a low-density chemically cross-linked cellulose/silk fibroin hydrogel is obtained; the obtained cellulose/silk fibroin hydrogel is placed in a carbon dioxide incubator for atmospheric physical cross-linking; rinsed with running water, a double network can be obtained Cross-linked cellulose/silk fibroin high-strength hydrogel. Although the above technical solution can produce high-strength hydrogel, it requires the use of alkaline solutions and toxic organic solutions during the preparation process. The preparation process is complicated and the gel requires a long time.

In view of this, it is necessary to design an improved preparation method for fast gelling silk fibroin-based strong hydrogels to solve the above problems.

Contents of the invention

The object of the present invention is to provide a method for preparing a silk fibroin-based strong hydrogel that is simple and convenient, has fast gelation speed, strong mechanical properties, and high elasticity and can quickly gel.

In order to achieve the above-mentioned object of the invention, the present invention provides a method for preparing a fast-gelling silk fibroin-based strong hydrogel, which includes the following steps:

S1. Preparation of silk fibroin solution: clean the degummed silk with deionized water and dry it to obtain dry silk; dissolve the dry silk and undergo dialysis treatment to obtain silk fibroin solution I;

S2. Preparation of silk fibroin-based hydrogel: Add a cross-linking agent to the silk fibroin solution I obtained in step S1 for chemical cross-linking to obtain a chemically cross-linked silk fibroin solution II, and then The silk fibroin solution II and the gelatin solution are mixed uniformly to obtain a mixed solution III; the mixed solution III is allowed to stand under low temperature conditions to obtain a silk fibroin/gelatin hydrogel.

Preferably, the preparation method of the fast gelling silk fibroin-based strong hydrogel further includes the following steps:

S3. Soak the silk fibroin/gelatin hydrogel obtained in step S2 in (NH ₄ ) ₂ SO ₄ solution and deionized water in sequence to obtain silk fibroin-based hydrogel.

Preferably, the mass fraction of the (NH ₄ ) ₂ SO ₄ solution is 10 to 20%.

Preferably, the silk fibroin/gelatin hydrogel is soaked in the (NH ₄ ) ₂ SO ₄ solution for 10 to 12 hours.

Preferably, the mass fraction of the silk fibroin solution I is 5-10%, and the mass fraction of the gelatin solution is 5-20%.

Preferably, the mixing volume of the silk fibroin solution II and the gelatin solution is (1-2): (0.5-2).

Preferably, the temperature of the dialysis treatment is 0-4°C and the time is 24-72 hours.

Preferably, in step S2, the low temperature condition is 0 to 4°C, and the time of standing under the low temperature condition is 3 to 10 minutes.

Preferably, the cross-linking agent is one or more of MES, NHS and EDC.

Preferably, in step S3, the soaking time of the silk fibroin/gelatin hydrogel in deionized water is 24h to 96h.

The beneficial effects of the present invention are:

1. The method for preparing a fast-gelling silk fibroin-based strong hydrogel provided by the present invention uses gelatin and silk fibroin as raw materials to prepare a double-crosslinked high-strength hydrogel through processes such as blending, chemical cross-linking, and low-temperature treatment. Compared with the process of preparing hydrogels in the prior art, the present invention greatly shortens the time required for gelation. It only takes 3 minutes to achieve gelation, and The prepared hydrogel has strong mechanical properties, high elasticity and good biocompatibility.

2. The preparation method of the fast-gelling silk fibroin-based strong hydrogel provided by the present invention realizes the control of the mechanical strength of the hydrogel by adjusting the experimental conditions to achieve controllable preparation of the gel, which satisfies the actual needs. The demand for hydrogels with different mechanical strengths; making full use of salt solution to regulate the structure of silk fibroin and gelatin, improving the mechanical properties of the produced hydrogel, making the hydrogel better meet application needs; by first Silk fibroin is chemically cross-linked and then physically cross-linked with gelatin to achieve the combination between silk fibroin and gelatin. At the same time, the combination of chemical cross-linking and non-covalent physical cross-linking can effectively achieve high performance. Integrated preparation with controllability.

3. The preparation method of the fast-gelling silk fibroin-based strong hydrogel provided by the present invention realizes the regulation of the mechanical strength of the hydrogel by adjusting the cross-linking method and the proportional content of the two components used in the experimental process. It realizes the controllable preparation of gels and meets the actual demand for hydrogels with different mechanical strengths. It has the advantages of fast, mild preparation conditions and simple preparation process. In the controllable preparation of high-strength functionalized hydrogels of great significance.

Description of drawings

Figure 1 is an SEM image of the hydrogel prepared in Example 1 of the preparation method of the fast-gelling silk fibroin-based strong hydrogel of the present invention;

Figure 2 is a SEM image of the hydrogel prepared in Example 2;

Figure 3 is an SEM image of the hydrogel prepared in Comparative Example 2;

Figure 4 is an SEM image of the hydrogel prepared in Comparative Example 3;

Figure 5 is a stress-strain curve diagram of hydrogels prepared in Examples 1 to 2 and Comparative Examples 1 to 3 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below with reference to the drawings and specific embodiments.

Here, it should also be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the solution of the present invention are shown in the drawings, and the details related to the present invention are omitted. Invent other details that are less relevant.

Additionally, it should be noted that the terms "comprises," "comprises," or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also It also includes other elements not expressly listed or that are inherent to the process, method, article or equipment.

The preparation method of the fast-gelling silk fibroin-based strong hydrogel provided by the invention includes the following steps:

S1. Preparation of silk fibroin solution: clean the degummed silk with deionized water and dry it at 60°C to obtain dry silk; dissolve the above dry silk and undergo dialysis to obtain silk fibroin. Solution I;

S2. Preparation of silk fibroin-based hydrogel: Add a cross-linking agent to the silk fibroin solution I prepared in step S1 to perform cross-linking treatment to obtain a chemically cross-linked silk fibroin solution II, and then add the silk fibroin solution I The protein solution II and the gelatin solution are evenly mixed to obtain a mixed solution III; after the mixed solution III is allowed to stand under low temperature conditions, a silk fibroin/gelatin hydrogel is obtained.

S3. Soak the silk fibroin/gelatin hydrogel obtained in step S2 in (NH ₄ ) ₂ SO ₄ solution and deionized water in sequence to prepare silk fibroin-based hydrogel.

Among them, in step S1, the degumming process includes the following steps:

S11. Immerse 100g of mulberry silk in 5L of sodium carbonate solution with a mass fraction of 0.1%, and boil it for 30 minutes at 98-100°C. After each boiling, take out the cocoons and wash them with deionized water;

S12. Add the washed silkworm cocoons in step S1 to the newly prepared 5L sodium carbonate solution with a mass fraction of 0.1%, boil for 30 minutes in an environment of 98-100°C, and repeat the above steps three times.

Specifically, in step S1, the temperature of the dialysis treatment is 0-4°C and the time is 24h-72h; the mass fraction of the silk fibroin solution I is 5-10%, and the drying time is 72h.

Specifically, in step S2, the chemical cross-linking time is 5 to 10 minutes; the silk fibroin solution II and the gelatin solution are mixed evenly according to the volume ratio (1 to 2): (0.5 to 2).

Among them, step S2 also requires injecting mixed solution III into the mold, and then placing the mold at a low temperature of 0 to 4°C for 3 to 10 minutes to prepare silk fibroin/gelatin hydrogel.

Wherein, in step S2, the added cross-linking agent is one or more of fatty acid methyl ester sulfonate (MES), N-hydroxysuccinimide (NHS) and dichloroethane (EDC); gelatin The mass fraction of the solution is 5 to 20%.

Specifically, in step S3, the mass fraction of the (NH ₄ ) ₂ SO ₄ solution is 10 to 20%, and the time for the silk fibroin/gelatin hydrogel to be soaked in the (NH ₄ ) ₂ SO ₄ solution is 10 to 12 hours. , the volume of (NH ₄ ) ₂ SO ₄ solution used was 100 mL, and the silk fibroin/gelatin hydrogel was soaked in deionized water for 24 to 96 hours.

Example 1

Immerse 100g of silk in 5L of sodium carbonate solution with a mass fraction of 0.1%, and boil it for 30 minutes at 98-100°C. After each boiling, take out the cocoons and wash them with deionized water; then, wash the washed cocoons. Add 5L of 0.1% sodium carbonate solution to the cocoons, repeat the above steps three times until the cocoons are degummed, wash thoroughly with deionized water, and dry in an oven for 72 hours at 60°C to obtain pure silk fibroin fiber.

Next, 5 g of the pure silk fibroin fiber prepared above was dissolved in 25 mL of lithium bromide solution to obtain a silk fibroin solution with a mass fraction of 5%. The silk fibroin solution was then dialyzed at 4°C for 72 hours; after the dialysis, the silk fibroin solution was added to the silk fibroin solution. Add 20% of the mass of the silk fibroin solution to MES, 20% of the mass of the silk fibroin solution to NHS, and 10% of the mass of the silk fibroin solution to EDC as cross-linking agents for chemical cross-linking. The cross-linking time is For 5 minutes, a cross-linked silk fibroin solution was obtained; then, a 10% gelatin solution with a mass fraction of 10% was added to the cross-linked silk fibroin solution. The volume ratio of the silk fibroin solution to the gelatin solution was 1: 1. After mixing evenly, pour the mixture into the mold, put it in the refrigerator, and let it stand for 3 minutes at 4°C to prepare the silk fibroin/gelatin hydrogel; finally, put the prepared silk fibroin/gelatin hydrogel into the mold. The gelatin hydrogel was soaked in 100 mL (NH ₄ ) ₂ SO ₄ with a mass fraction of 15% for 12 hours to enhance the mechanical properties of the hydrogel. After the soaking treatment, the resulting hydrogel was soaked in deionized water for 72 hours to remove the hydrogel. (NH ₄ ) ₂ SO ₄ adsorbed internally and on the surface is used to prepare silk fibroin-based strong hydrogel. The SEM image of the prepared hydrogel is shown in Figure 1. From the figure, it can be seen that the hydrogel is three-dimensional. Porous structure, pore size is uniform and dense.

Example 2

The only difference between Example 2 and Example 1 is that the prepared silk fibroin/gelatin hydrogel is not soaked in (NH ₄ ) ₂ SO ₄ solution. The remaining steps are basically the same as those in Example 1 and will not be repeated here. Repeat. The SEM image of the prepared hydrogel is shown in Figure 2. It can be seen from the figure that the hydrogel has a three-dimensional pore structure with uniform pore size. Compared with the hydrogel prepared in Example 1, the pore size is increased.

Comparative example 1

The only difference between Comparative Example 1 and Example 1 is that the silk fibroin solution is chemically cross-linked by MES/NHS/EDC and then directly frozen in a 4°C refrigerator for 3 minutes to obtain a silk fibroin hydrogel. The remaining steps are the same as those in the Examples. 1 are basically the same and will not be repeated here.

Comparative example 2

The only difference between Comparative Example 2 and Example 1 is that the prepared silk fibroin solution is directly mixed with the gelatin solution at a volume ratio of 1:1 to obtain a mixed solution of silk fibroin solution and gelatin solution, and then the above mixed solution is Add into the injection mold, and freeze in a 4°C refrigerator for 10 minutes. The remaining steps are basically the same as in Example 1 and will not be described again. The SEM image of the prepared hydrogel is shown in Figure 3. From Figure 3, it can be seen that the interior of the hydrogel has a three-dimensional pore structure with uniform pore size and dense pore structure.

Comparative example 3

The only difference between Comparative Example 3 and Example 1 is that the prepared silk fibroin solution is directly injected into the mold, and dried in an oven for 72 hours at 60°C. The remaining steps are basically the same as in Example 1. I won’t go into details here. The SEM image of the prepared hydrogel is shown in Figure 4. From Figure 4, it can be seen that the interior of the hydrogel has a three-dimensional pore structure, the pore size distribution is uneven, and cracks can be observed inside the material within the lens field of view; The stress-strain curves of the hydrogels prepared in Examples 1 to 2 and Comparative Examples 1 to 3 are shown in Figure 5. It can be seen from the figure that the hydrogel prepared by the method in Example 1 has the highest strength. , the strength of the hydrogels in Comparative Example 2 and Comparative Example 3 is almost 0.

Examples 3 to 7

The only difference between Examples 3 to 7 and Example 1 is that the volume ratio of the chemically cross-linked silk fibroin solution and the gelatin solution is set differently. The remaining steps are basically the same as in Example 1 and will not be repeated here. Different volume ratios The strength of the silk fibroin/gelatin hydrogel prepared under The resulting hydrogel has the highest strength.

Table 1 Volume ratio settings of Examples 3-7 and the strength of the silk fibroin/gelatin hydrogel prepared under these conditions

Examples 8 to 12

The only difference between Examples 8 to 12 and Example 1 is that the mass fraction of the silk fibroin solution and the mass fraction of the gelatin solution are set differently. The remaining steps are basically the same as in Example 1 and will not be repeated here. Under different mass fraction ratios The strength of the prepared silk fibroin/gelatin hydrogel is shown in Table 2: From the data in the table, it can be seen that when the mass fraction of the silk fibroin solution and the gelatin solution is 5%:10%, the The hydrogel has the highest strength.

Table 2 The mass fraction settings of Examples 8-12 and the strength of the silk fibroin/gelatin hydrogel prepared under these conditions

The formation mechanism of silk fibroin-based hydrogel is explained below:

In the presence of a cross-linking agent, the residues of silk fibroin react with the cross-linking agent to promote the chemical bond between fibrin and the cross-linking agent; then, gelatin is added to the chemically cross-linked silk fibroin solution. The solution is left to stand under low temperature conditions. Under low temperature conditions, silk fibroin changes from a random structure to a β-sheet structure. At the same time, the silk fibroin solution and the gelatin solution intersperse with each other to form a macromolecular structure. In the above two Under the action of (NH 4 ) 2 SO 4 solution, gelation can be quickly achieved to prepare silk fibroin/gelatin hydrogel; when the silk fibroin/gelatin hydrogel is soaked in (NH ₄ ) ₂ SO ₄ solution, due to (NH ₄ ) ₂ SO ₄ can induce chain entanglements, hydrophobic interactions, and microphase separation regions in the hydrogel network within the hydrogel, thereby improving the mechanical properties of the hydrogel.

Those skilled in the art should understand that the mass fraction of (NH ₄ ) ₂ SO ₄ can also be 10% or 20%, which is not limited here and only needs to be set according to actual needs.

In summary, the present invention provides a method for preparing a fast-gelling silk fibroin-based strong hydrogel, which uses gelatin and silk fibroin as raw materials to prepare a double-crosslinked hydrogel through processes such as blending, chemical cross-linking, and low-temperature treatment. A high-strength hydrogel with a three-dimensional cross-linked network structure. The produced hydrogel has strong mechanical properties and high elasticity. By adjusting the experimental conditions, the mechanical strength of the hydrogel can be controlled to achieve the desired properties of the gel. The controllable preparation meets the actual needs for hydrogels with different mechanical strengths; making full use of the influence of the salt solution on the structure of silk fibroin and gelatin, further improving the mechanical properties of the prepared hydrogel, making the water Gels are more suitable for applications; by combining chemical cross-linking with non-covalent physical cross-linking, high performance and controllability are effectively achieved. Compared with the scheme of preparing hydrogel in the prior art, the present invention can achieve rapid gelation, and the formation of hydrogel only takes 3 minutes. At the same time, the method provided by the present invention also has the advantages of wide sources of raw materials, mild and green preparation conditions The advantages of , fast and simple preparation process are of great significance in the controllable preparation of high-strength functional hydrogels.

The above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently substituted. without departing from the spirit and scope of the technical solution of the present invention.

Claims (4)

1. The preparation method of the rapid-gel silk fibroin-based strong hydrogel is characterized by comprising the following steps:

s1, preparing a silk fibroin solution: washing degummed silk with deionized water, and drying to obtain dry silk; dissolving the dry silk and performing dialysis treatment to obtain a silk fibroin solution I;

s2, preparing silk fibroin-based hydrogel: adding a cross-linking agent into the silk fibroin solution I obtained in the step S1 to carry out chemical cross-linking to obtain a silk fibroin solution II subjected to chemical cross-linking, and uniformly mixing the silk fibroin solution II with a gelatin solution to obtain a mixed solution III; standing the mixed solution III at a low temperature to obtain silk fibroin/gelatin hydrogel;

the mass fraction of the silk fibroin solution I is 5-10%, and the mass fraction of the gelatin solution is 5-20%; the volume of the silk fibroin solution II and the gelatin solution is (1-2): (0.5-2); the low temperature condition is 0-4 ℃, and the standing time is 3-10 min under the low temperature condition; the cross-linking agent is one or more of MES, NHS and EDC;

s3, sequentially soaking the silk fibroin/gelatin hydrogel obtained in the step S2 in (NH) ₄ ) ₂ SO ₄ Obtaining silk fibroin-based hydrogel in the solution and deionized water; said (NH) ₄ ) ₂ SO ₄ The mass fraction of the solution is 10-20%.

2. The method of preparing a rapid gelling silk fibroin-based strong hydrogel according to claim 1, wherein the silk fibroin/gelatin hydrogel is immersed in (NH ₄ ) ₂ SO ₄ The time in the solution is 10-12 hours.

3. The method for preparing the rapid gel silk fibroin-based strong hydrogel according to claim 1, wherein the dialysis treatment is performed at a temperature of 0-4 ℃ for 24-72 hours.

4. The method for preparing a rapid gel silk fibroin-based strong hydrogel according to claim 1, wherein in step S3, the soaking time of the silk fibroin/gelatin hydrogel in deionized water is 24h to 96h.