Preparation method of visible light curing fibroin / sericin hydrogel

Abstract

The invention discloses a preparation method of visible-light-cured fibroin / sericin protein hydrogel, silk fibers containing different amounts of sericin are obtained by adjusting the concentration of a degumming reagent and the degumming time, the silk fibers are directly dissolved to prepare a fibroin / sericin protein composite aqueous solution by a one-step method, extra extraction of sericin or addition of sericin protein powder is not needed, and the preparation method is simple and convenient. The cost is reduced while the preparation process is greatly shortened; environment-friendly and nontoxic riboflavin (vitamin B2) is used as a photoinitiator, and crosslinking can be initiated under visible light without using ultraviolet light; the visible light curing fibroin / sericin hydrogel makes full use of respective performance advantages of silk fibroin and sericin in silk fibers, is safe in components, free of additional chemical cross-linking agents, good in light transmission, good in biocompatibility, high in elasticity, especially suitable for the field of biomedical materials, short in preparation process, short in time consumption and high in efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of new materials and relates to a method for preparing a visible light-cured silk fibroin / sericin hydrogel. Background Art

[0002] Hydrogel materials, with their excellent water absorption and retention capacity and adjustable physical and chemical properties, have shown significant advantages in the fields of medical, environmental protection and smart materials. Its unique three-dimensional network structure can effectively lock a large amount of water while maintaining softness and elasticity. It can be used as a carrier for materials such as drug sustained release, tissue engineering, water purification and smart sensing. In recent years, silk fibroin materials have become a dominant material in biomedical applications due to their excellent biocompatibility, good mechanical properties and controllable biodegradability. Silk fibroin hydrogels not only provide an ideal carrier for drug controlled release systems, but also show application potential in the fields of tissue engineering scaffolds and wound repair materials. The preparation methods of silk fibroin-based hydrogels are mainly divided into physical crosslinking and chemical crosslinking. The physical crosslinking method mainly forms hydrogels through non-covalent bonds, which has problems such as weak mechanical properties, poor stability and long-term preservation. The chemical crosslinking method usually involves a crosslinking agent or catalyst to induce the silk fibroin molecular chains to connect to each other through chemical bonds or form stable crosslinking points through covalent bonds, and finally form a stable three-dimensional network structure. Compared with physical cross-linking, chemical cross-linking of silk fibroin hydrogel can obtain hydrogels with stronger mechanical properties and better stability.

[0003] The photocuring method has shown unique advantages in the preparation of hydrogels by chemical crosslinking due to its advantages of fast curing rate, environmental protection and energy saving, high precision, strong controllability and wide application range. Riboflavin, as a biophotoinitiator, can produce reactive oxygen free radicals through photoexcitation, inducing amino groups, phenolic groups and other groups in the silk protein molecular chain to produce chemical crosslinking to form a stable hydrogel, playing an important role in the photocuring process. For example, the patent application with publication number CN107118359A adds riboflavin to the silk protein aqueous solution, and cures it after ultraviolet light irradiation to obtain a hydrogel material, but this method has a slow crosslinking rate, takes a long time, and the hydrogel material is brittle and has poor mechanical properties. In order to improve its mechanical properties, the patent application with publication number CN109134889A uses horseradish peroxidase to catalyze the photocrosslinking reaction of riboflavin in the silk protein aqueous solution, and obtains a hydrogel material with high compressive strength and high resilience under ultraviolet light irradiation; the patent application with publication number CN116041735A uses riboflavin and hydrogen peroxide as a photocuring system, and uses hydrogen peroxide to decompose under ultraviolet light to provide more oxygen for the system, promote the curing reaction, shorten the gel formation time, and improve the mechanical resilience. The above patent applications improve the mechanical properties of hydrogel materials, but they cannot be separated from the assistance of other crosslinking agents or catalysts in the preparation. These reagents have potential cytotoxicity and may affect the biocompatibility of silk protein materials. In addition, ultraviolet light with strong radiation and destructiveness is used, which threatens the life safety of hydrogel preparation personnel.

[0004] As we all know, the single internal network structure of pure silk fibroin-based hydrogels leads to insufficient mechanical properties and high brittleness, while composite hydrogels composed of two or more materials can effectively construct a double network structure, significantly improving the application feasibility of hydrogel materials. Sericin is homologous to silk fibroin and has the same excellent biocompatibility and will not cause immune rejection. In addition, the molecular conformation of sericin is mainly irregular curling, and the molecular spatial structure is loose and disordered. This structure gives sericin good flexibility and plasticity, and there are many amino acids and hydrophilic groups with long side chains (such as -OH, -COOH, -NH2, etc.) on the molecular chain of sericin, so it has excellent humidity regulation and moisturizing effects. The addition of sericin is expected to improve the brittle defects of silk fibroin hydrogels, increase toughness and elasticity, and thus adapt to different application requirements. However, the preparation process of the silk fibroin / sericin mixed aqueous solution reported so far is relatively complicated. For example, in the patent application with publication number CN1397354A, silk is put into deionized water and sodium carbonate solution respectively, boiled and dissolved, purified to obtain sericin solution and silk fibroin aqueous solution, and then mixed to prepare the silk fibroin / sericin aqueous solution; in the patent application with publication number CN116650727A, the degummed silk fibroin fiber and sericin powder are mixed evenly and then dissolved and dialyzed to prepare the mixed solution. However, because the step-by-step extraction involves more steps and condition control, the complexity of the operation and the processing cost are increased. At the same time, other chemical substances may be introduced during the extraction process, affecting the biocompatibility and safety of the protein.

[0005] Therefore, it is necessary to develop a method for preparing visible light-cured silk fibroin / sericin hydrogel to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a visible light-cured silk fibroin / sericin hydrogel, which solves the problems of complex operation, high processing cost, poor biocompatibility and safety of proteins, etc.

[0007] The technical solution of the present invention is:

[0008] A method for preparing a visible light-cured silk fibroin / sericin hydrogel comprises the following steps:

[0009] (1) boiling raw silk in a sodium carbonate aqueous solution, and obtaining silk fibers containing different amounts of sericin by controlling the concentration of the sodium carbonate aqueous solution and the degumming time;

[0010] (2) dissolving the silk fibers containing different amounts of sericin, dialyzing at low temperature, filtering to remove impurities, and concentrating to obtain a high-concentration silk fibroin / sericin composite aqueous solution;

[0011] (3) adding riboflavin to the high-concentration silk fibroin / sericin composite aqueous solution under light-proof conditions, mixing evenly and removing bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution;

[0012] (4) pouring the silk / sericin / riboflavin mixed solution into a light-transmitting silicone mold, and performing light curing using a visible light lamp to maintain constant lighting conditions, thereby obtaining a visible light-cured silk / sericin hydrogel.

[0013] Furthermore, in step (1), the concentration of the sodium carbonate aqueous solution is 0.2-0.5 wt %, the mass volume ratio of the raw silk to the sodium carbonate aqueous solution is 1 g:100 ml, and the content of sericin in the silk fiber is 5-15%.

[0014] Furthermore, in step (1), the boiling time is 15 to 45 minutes.

[0015] Furthermore, in step (2), the temperature of the heating and dissolving is 45-65°C, and the time of the heating and dissolving is 0.2-1h; the temperature of the low-temperature dialysis is 4-10°C, and the time of the low-temperature dialysis is 1-5d; and the time of the concentration is 24h.

[0016] Furthermore, in step (3), the concentration of the high-concentration silk fibroin / sericin composite aqueous solution is 5 to 20 wt %.

[0017] Furthermore, in step (3), the concentration of riboflavin is 1 to 1.5 mg / ml.

[0018] Furthermore, in step (3), the proportion of riboflavin in the silk fibroin / sericin / riboflavin mixed solution is 0.5-2 mg / ml.

[0019] Furthermore, in step (4), the power of the visible light lamp is 100 to 900W.

[0020] Furthermore, in step (4), the irradiation distance of the visible light lamp is 5 to 20 cm.

[0021] Furthermore, in step (4), the irradiation time of the visible light lamp is 10 to 30 minutes.

[0022] The present invention provides a method for preparing a visible light-cured silk fibroin / sericin hydrogel, using silk fibers containing different amounts of sericin as raw materials to prepare a silk fibroin / sericin composite aqueous solution, and under the synergistic effect of a photoinitiator riboflavin, a hydrogel material can be obtained in one step. The advantages are:

[0023] (1) According to the promoting effect of sericin on the gelation of silk protein solution, the present invention uses silk fibers with different sericin contents as raw materials, natural riboflavin (vitamin B2) as a photoinitiator, and visible light for photocuring, thereby realizing a short-process, green and ecological preparation method of silk fibroin / sericin hydrogel. The photocurable hydrogel involved has safe components, good mechanical resilience, fast biodegradation rate, good biocompatibility, simple and easy preparation method, green and environmentally friendly, and low economic cost;

[0024] (2) The present invention utilizes the synergistic effect of sericin and riboflavin on silk fibroin to adjust the content of sericin and the gelation time to achieve the regulation of the structure and performance of silk fibroin / sericin hydrogel, and improves the crosslinking density and mechanical properties of the hydrogel by adjusting the intensity and time of the light source;

[0025] (3) The photocurable hydrogel provided by the present invention does not need to rely on ultraviolet light with strong radiation. Under safe and low-energy visible light conditions, without involving any chemical crosslinking agent, visible light-cured silk / sericin hydrogel can be obtained simply, quickly and at low cost. The maximum strain of the hydrogel material is 168%, the water absorption rate can reach 260%, it has good biocompatibility, is biodegradable, and has adjustable mechanical properties. It has great application potential in the fields of biomedicine, tissue engineering, 3D printing materials, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are photos of the visible light-cured silk / sericin hydrogel prepared by the method for preparing the visible light-cured silk / sericin hydrogel of the present invention before and after stretching and before and after moisture absorption;

[0027] Figure 2 This is a SEM image of the visible light-cured silk / sericin hydrogel after vacuum freeze-drying prepared by the method for preparing the visible light-cured silk / sericin hydrogel described in the present invention. DETAILED DESCRIPTION

[0028] The purpose of the present invention is to provide a method for preparing a visible light-cured silk / sericin hydrogel. According to the synergistic effect of riboflavin and sericin on the gelation of silk protein solution, a short-process, green and ecological method for preparing a silk / sericin hydrogel is innovated. Silk fibers containing different amounts of sericin are used as raw materials, and riboflavin is used as a photoinitiator to achieve rapid gelation under visible light. The raw materials used in the present invention are natural, environmentally friendly and abundant in source, and have the advantages of adjustable mechanical properties, good biocompatibility and biodegradability.

[0029] The above method comprises the following steps:

[0030] (1) obtaining silk fibers containing different amounts of sericin by adjusting the concentration of degumming reagents and degumming time;

[0031] (2) The obtained silk fibers containing different amounts of sericin are dissolved in a LiBr aqueous solution, and then the dialysis bag containing the silk protein solution is immersed in deionized water for low-temperature dialysis to obtain a low-concentration silk fibroin / sericin composite aqueous solution. In order to avoid precipitation and deterioration of silk fibroin and sericin during the preparation process, the dialysis bath ratio needs to be increased in a low-temperature environment according to the different sericin contents. The prepared low-concentration silk fibroin / sericin mixed aqueous solution is placed in a dialysis bag and immersed in a polyethylene glycol aqueous solution to further concentrate to prepare a high-concentration silk fibroin / sericin composite aqueous solution, wherein the concentration of the LiBr aqueous solution is 9.3wt%, and the mass volume ratio of the silk fiber to the LiBr aqueous solution is 1g:10-20ml; the heating and dissolving temperature is 45-65°C, and the heating and dissolving time is 0.2-1h; the temperature of the low-temperature dialysis is 4-10°C, and the low-temperature dialysis time is 1-5d, the molecular weight of the dialysis bag is 8000-14000Da, and the bath ratio of the low-temperature dialysis is 1:800; the concentration of the polyethylene glycol aqueous solution is 0.05-0.15g / ml; and the concentration time is 24h;

[0032] (3) adding riboflavin in a certain proportion to a high concentration of silk fibroin / sericin composite aqueous solution;

[0033] (4) Visible light irradiation molding is used to prepare visible light-cured silk fibroin / sericin hydrogel.

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below in conjunction with specific embodiments. However, the present invention is not limited to the embodiments listed, and should also include any other known changes within the scope of the rights claimed by the present invention.

[0035] The term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0036] Example 1

[0037] The following example shows a method for preparing a visible light-cured silk fibroin / sericin hydrogel, and the specific steps are as follows:

[0038] (1) placing raw silk in a 0.2 wt% sodium carbonate aqueous solution and boiling it for 15 min, with a bath ratio of 1:100, to obtain silk fibers with a sericin residue of 12%;

[0039] (2) dissolving and filtering the silk fibers, pouring them into a dialysis bag with a molecular weight cutoff of 8000 Da, and immersing them in deionized water, dialyzing them at a low temperature of 2° C. for 3 days with a dialysis bath ratio of 1:800 to obtain a low-concentration silk fibroin / sericin composite aqueous solution;

[0040] (3) placing the low-concentration silk fibroin / sericin composite aqueous solution in a 0.1 g / ml polyethylene glycol aqueous solution and concentrating it for 24 h to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 10 wt %;

[0041] (4) adding riboflavin to the high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1.5 mg / ml, stirring evenly, and then evacuating to remove bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution;

[0042] (5) The silk / sericin / riboflavin mixed solution was cast into a mold and irradiated with visible light for 15 min at a visible light irradiation distance of 10 cm to obtain a visible light-cured silk / sericin hydrogel.

[0043] According to the mechanical tensile test, the visible light-cured silk fibroin / sericin hydrogel has a breaking strength of 53.38 kPa, a breaking elongation of 168%, and a water content of 260%.

[0044] Example 2

[0045] The following example shows a method for preparing a visible light-cured silk fibroin / sericin hydrogel, and the specific steps are as follows:

[0046] (1) placing raw silk in a 0.3 wt % sodium carbonate aqueous solution and boiling it for 30 min, with a bath ratio of 1:100, to obtain silk fibers with a sericin residue of 7%;

[0047] (2) dissolving and filtering the silk fibers, pouring them into a dialysis bag with a molecular weight cutoff of 14,000 Da, and immersing them in deionized water. Dialyzing them at a low temperature of 2° C. for 5 days with a dialysis bath ratio of 1:800 to obtain a low-concentration silk fibroin / sericin composite aqueous solution;

[0048] (3) placing the low-concentration silk fibroin / sericin composite aqueous solution in a 0.1 g / ml polyethylene glycol aqueous solution and concentrating it for 36 hours to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 15 wt %;

[0049] (4) adding riboflavin to the high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1 mg / ml, stirring evenly, and then evacuating to remove bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution;

[0050] (5) The silk / sericin / riboflavin mixed solution was cast into a mold and irradiated with visible light for 20 min at a visible light irradiation distance of 15 cm to obtain a visible light-cured silk / sericin hydrogel.

[0051] According to the mechanical tensile test, the breaking strength of the visible light-cured silk fibroin / sericin hydrogel is 54.69 kPa, the breaking elongation is 140%, and the water content is 210%.

[0052] Example 3

[0053] The following example shows a method for preparing a visible light-cured silk fibroin / sericin hydrogel, and the specific steps are as follows:

[0054] (1) boiling raw silk in a 0.5 wt% sodium carbonate aqueous solution for 45 min at a bath ratio of 1:100 to obtain silk fibers with a sericin residue of 4%;

[0055] (2) dissolving and filtering the silk fibers, pouring them into a dialysis bag with a molecular weight cutoff of 14,000 Da, and immersing them in deionized water. The dialysis was performed at a low temperature of 4° C. for 3 days with a dialysis bath ratio of 1:800 to obtain a low-concentration silk fibroin / sericin composite aqueous solution;

[0056] (3) placing the low-concentration silk fibroin / sericin composite aqueous solution in a 0.15 g / ml polyethylene glycol aqueous solution and concentrating it for 24 h to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 10 wt %;

[0057] (4) adding riboflavin to the high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1.3 mg / ml, stirring evenly, and then evacuating to remove bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution;

[0058] (5) The silk / sericin / riboflavin mixed solution was cast into a mold and irradiated with visible light for 25 min at a visible light irradiation distance of 20 cm to obtain a visible light-cured silk / sericin hydrogel.

[0059] According to the mechanical tensile test, the breaking strength of the visible light-cured silk fibroin / sericin hydrogel was 20.66 kPa, the breaking elongation was 64%, and the water content was 150%.

[0060] See also Figure 1 and Figure 2 , Figure 1 These are photos of the visible light-cured silk / sericin hydrogel prepared by the method for preparing the visible light-cured silk / sericin hydrogel of the present invention before and after stretching and before and after moisture absorption; Figure 2The SEM image of the visible light-cured silk / sericin hydrogel prepared by the method for preparing the visible light-cured silk / sericin hydrogel of the present invention after vacuum freeze-drying. Figure 1 As shown in Figure 2, the visible light-cured silk / sericin hydrogel has good hygroscopicity and excellent tensile properties. Figure 2 As shown, the internal structure and mechanical properties of visible light-cured silk fibroin / sericin hydrogel can be regulated according to the sericin content and illumination time.

[0061] In summary, the preparation method of the visible light-cured silk / sericin hydrogel of the present invention obtains silk fibers containing different amounts of sericin by adjusting the concentration of the degumming reagent and the degumming time, and directly dissolves the silk fibers in a one-step method to prepare a silk / sericin composite aqueous solution, without the need for additional extraction of sericin or addition of sericin powder, thereby greatly shortening the preparation process and reducing costs; environmentally friendly and non-toxic riboflavin (vitamin B2) is used as a photoinitiator, and cross-linking can be initiated under visible light without the need for ultraviolet light; the visible light-cured silk / sericin hydrogel fully utilizes the respective performance advantages of silk fibroin and sericin in silk fibers, has safe ingredients, does not require the addition of additional chemical cross-linking agents, has good light transmittance, good biocompatibility, and high elasticity, and is particularly suitable for the field of biomedical materials, and has a short preparation process, less time consumption, and high efficiency.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a visible light-cured silk fibroin / sericin hydrogel, characterized in that: The following steps are involved: (1) boiling raw silk in a sodium carbonate aqueous solution, and obtaining silk fibers containing different amounts of sericin by controlling the concentration of the sodium carbonate aqueous solution and the degumming time; (2) dissolving the silk fibers containing different amounts of sericin, dialyzing at low temperature, filtering to remove impurities, and concentrating to obtain a high-concentration silk fibroin / sericin composite aqueous solution; (3) adding riboflavin to the high-concentration silk fibroin / sericin composite aqueous solution under light-proof conditions, mixing evenly and removing bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution; (4) pouring the silk / sericin / riboflavin mixed solution into a light-transmitting silicone mold, and performing light curing using a visible light lamp to maintain constant lighting conditions, thereby obtaining a visible light-cured silk / sericin hydrogel.

2. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (1), the concentration of the sodium carbonate aqueous solution is 0.2-0.5 wt %, the mass volume ratio of the raw silk to the sodium carbonate aqueous solution is 1 g:100 ml, and the content of sericin in the silk fiber is 5-15%.

3. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (1), the boiling time is 15 to 45 minutes.

4. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (2), the temperature of the heating dissolution is 45-65°C, and the time is 0.2-1h; the temperature of the low-temperature dialysis is 4-10°C, and the time is 1-5d; and the time of the concentration is 24h.

5. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (3), the concentration of the high-concentration silk fibroin / sericin composite aqueous solution is 5-20 wt %.

6. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (3), the concentration of riboflavin is 1 to 1.5 mg / ml.

7. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (3), the proportion of riboflavin in the silk fibroin / sericin / riboflavin mixed solution is 0.5-2 mg / ml.

8. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (4), the power of the visible light lamp is 100 to 900W.

9. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (4), the irradiation distance of the visible light lamp is 5 to 20 cm.

10. The method for preparing a visible light-cured silk fibroin / sericin hydrogel according to claim 1, characterized in that: In step (4), the irradiation time of the visible light lamp is 10 to 30 minutes.

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