Preparation method of visible light cured silk fibroin / silk sericin hydrogel

Abstract

The application discloses a preparation method of visible light cured silk fibroin / silk sericin hydrogel, and through adjusting the concentration and time of degumming reagent, silk fibers containing different amounts of silk sericin are obtained, and the silk fibroin / silk sericin composite aqueous solution is prepared by directly dissolving in one step, so that the silk sericin is not needed to be extracted or silk fibroin powder is not needed to be added, the preparation process is greatly shortened, and the cost is reduced; the environmentally-friendly and non-toxic riboflavin (vitamin B2) is used as a photo initiator, and cross-linking is initiated under visible light without using ultraviolet light; the visible light cured silk fibroin / silk sericin hydrogel fully utilizes the performance advantages of silk fibroin and silk sericin in silk fibers, is safe in composition, does not need to add an additional chemical cross-linking agent, is good in light transmittance, is good in biocompatibility, is high in elasticity, is especially suitable for the field of biomedical materials, and is short in preparation process, low in time consumption and high in efficiency.

Description

Technical Field

[0001] This invention belongs to the field of novel materials technology and relates to a method for preparing visible light-cured silk fibroin / sericite hydrogel. Background Technology

[0002] Hydrogel materials, with their excellent water absorption and retention capacity and adjustable physicochemical properties, have shown significant advantages in the fields of medical, environmental protection, and smart materials. Their unique three-dimensional network structure can effectively lock in large amounts of water while maintaining softness and elasticity, making them suitable as carriers for drug sustained release, tissue engineering, water purification, and smart sensing. In recent years, silk fibroin materials, with their excellent biocompatibility, good mechanical properties, and controllable biodegradability, have become advantageous materials in biomedical applications. Silk fibroin hydrogels not only provide ideal carriers for drug controlled release systems but also show application potential in tissue engineering scaffolds and wound repair materials. The preparation methods of silk fibroin-based hydrogels are mainly divided into physical crosslinking and chemical crosslinking. Physical crosslinking mainly forms hydrogels through non-covalent bonding, but suffers from weak mechanical properties, poor stability, and poor long-term preservation. Chemical crosslinking typically involves crosslinking agents or catalysts, inducing silk fibroin molecular chains to connect through chemical bonds or form stable crosslinking points through covalent bonds, ultimately forming a stable three-dimensional network structure. Compared with physical cross-linking, chemical cross-linking of silk fibroin hydrogels can produce hydrogels with stronger mechanical properties and better stability.

[0003] Photocuring, with its advantages of fast curing rate, environmental friendliness and energy saving, high precision, strong controllability, and wide applicability, has shown unique advantages in the preparation of hydrogels using chemical crosslinking methods. Riboflavin, as a bio-photoinitiator, can generate reactive oxygen free radicals through photoexcitation, inducing chemical crosslinking of amino and phenolic groups in the silk fibroin molecular chain to form a stable hydrogel, playing an important role in the photocuring process. For example, patent application CN107118359A describes adding riboflavin to an aqueous solution of silk fibroin and curing it after ultraviolet irradiation to obtain a hydrogel material. However, this method has a slow crosslinking rate, is time-consuming, and the hydrogel material is brittle and has poor mechanical properties. To improve the mechanical properties of hydrogels, patent application CN109134889A utilizes horseradish peroxidase to catalyze a photocrosslinking reaction of riboflavin in an aqueous solution of silk fibroin, resulting in a hydrogel material with high compressive strength and high resilience under ultraviolet light irradiation. Patent application CN116041735A uses riboflavin and hydrogen peroxide as a photocuring system, utilizing the decomposition of hydrogen peroxide under ultraviolet light to provide more oxygen to the system, promoting the curing reaction, shortening the gel formation time, and improving mechanical resilience. While these patent applications improve the mechanical properties of hydrogel materials, their preparation relies heavily on other crosslinking agents or catalysts. These reagents possess potential cytotoxicity, which may affect the biocompatibility of silk fibroin materials. Furthermore, the use of highly radioactive and destructive ultraviolet light poses a threat to the safety of personnel involved in hydrogel preparation.

[0004] It is well known that the simple internal network structure of pure silk fibroin-based hydrogels leads to insufficient mechanical properties and high brittleness. Composite hydrogels composed of two or more materials can effectively construct a dual-network structure, significantly improving the application feasibility of hydrogel materials. Sericin is homologous to silk fibroin and possesses the same excellent biocompatibility, without inducing immune rejection. Furthermore, the molecular conformation of sericin is mainly random coiled, with a loose and disordered molecular spatial structure. This structure endows sericin with good flexibility and plasticity. Moreover, the molecular chains of sericin contain many amino acids with long side chains and hydrophilic groups (such as -OH, -COOH, -NH2, etc.), thus exhibiting excellent moisture-regulating and moisturizing effects. The addition of sericin is expected to improve the brittleness of silk fibroin hydrogels, increasing their toughness and elasticity, thereby adapting to different application requirements. However, the reported preparation processes for silk fibroin / sericin mixed aqueous solutions are quite complex. For example, patent application CN1397354A involves boiling and dissolving silk fibers in deionized water and sodium carbonate solution, purifying them to obtain sericin solution and silk fibroin aqueous solution, and then mixing them to prepare a silk fibroin / sericin aqueous solution. Patent application CN116650727A involves uniformly mixing degummed silk fibroin fibers and sericin powder, then dissolving and dialysis to prepare a mixed solution. However, stepwise extraction involves more steps and condition control, increasing the complexity of the operation and processing costs. Furthermore, other chemical substances may be introduced during the extraction process, affecting the biocompatibility and safety of the proteins.

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

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

[0007] The technical solution of this invention is:

[0008] A method for preparing visible light-curable silk fibroin / seric acid hydrogel includes the following steps:

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

[0010] (2) Dissolve the silk fibers containing different amounts of sericin, dialyze at low temperature, filter to remove impurities, and concentrate to obtain a high-concentration fibroin / sericin complex aqueous solution.

[0011] (3) Under light-protected conditions, riboflavin was added to the high-concentration silk fibroin / sericin composite aqueous solution, mixed evenly, and the air bubbles were removed to obtain a silk fibroin / sericin / riboflavin mixed solution.

[0012] (4) Pour the silk fibroin / sericin / riboflavin mixed solution into a transparent silicone mold, and use a visible light lamp to maintain constant light conditions for photocuring to obtain visible light cured silk fibroin / sericin hydrogel.

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

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

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

[0016] Furthermore, in step (3), the concentration of the high-concentration silk fibroin / sericite composite aqueous solution is 5-20 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 to 2 mg / ml.

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

[0020] Furthermore, in step (4), the illumination 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] This invention provides a method for preparing visible light-curable silk fibroin / seric acid hydrogels. Using silk fibers containing varying amounts of sericin as raw materials, a silk fibroin / seric acid composite aqueous solution is prepared. With the synergistic effect of the photoinitiator riboflavin, the hydrogel material can be obtained in one step. Its advantages are:

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

[0024] (2) This invention utilizes the synergistic effect of sericin and riboflavin on fibroin to regulate the structure and properties of fibroin / sericin hydrogel by adjusting the sericin content and gelation time. By adjusting the intensity and time of the light source, the crosslinking density and mechanical properties of the hydrogel are improved.

[0025] (3) The photocurable hydrogel provided by the present invention does not rely on ultraviolet light with strong radiation. Under safe and low-energy visible light conditions, without the involvement of any chemical crosslinking agent, visible light-curable silk fibroin / sericin hydrogel can be obtained simply, quickly and at low cost. The maximum strain of this hydrogel material is 168%, the water absorption rate can reach 260%, it has good biocompatibility, is biodegradable, and its mechanical properties can be controlled. It has great application potential in the fields of biomedicine, tissue engineering, and 3D printing materials. Attached Figure Description

[0026] Figure 1 The visible light-cured silk fibroin / sericin hydrogel prepared by the method described in this invention is photographed before and after stretching and before and after moisture absorption.

[0027] Figure 2 SEM image of the visible light-cured silk fibroin / sericin hydrogel prepared by the method for preparing visible light-cured silk fibroin / sericin hydrogel according to the present invention after vacuum freeze-drying. Detailed Implementation

[0028] The purpose of this invention is to provide a method for preparing visible light-cured silk fibroin / serin protein hydrogels. Based on the synergistic promoting effect of riboflavin and sericin on the gelation of silk fibroin solutions, an innovative, short-process, green, and eco-friendly method for preparing silk fibroin / serin protein hydrogels is proposed. Using silk fibers containing varying amounts of sericin as raw materials and riboflavin as a photoinitiator, rapid gelation under visible light is achieved. The raw materials used in this invention are natural, environmentally friendly, and abundant, possessing advantages such as controllable mechanical properties, good biocompatibility, and biodegradability.

[0029] The above method includes the following steps:

[0030] (1) By adjusting the concentration of degumming reagent and the degumming time, silk fibers containing different amounts of sericin were obtained;

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

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

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

[0034] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the technical solutions of the present invention are further described below with reference to specific embodiments. However, the present invention is not limited to the listed embodiments, but should also include any other known modifications within the scope of the claims of the present invention.

[0035] The term "an 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 invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Example 1

[0037] The following examples illustrate a method for preparing visible light-cured silk fibroin / seric acid hydrogels, with the specific steps as follows:

[0038] (1) Boil the raw silk in a 0.2 wt% sodium carbonate aqueous solution for 15 min at a bath ratio of 1:100 to obtain silk fibers with a sericin residue of 12%.

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

[0040] (3) The low-concentration silk fibroin / sericin composite aqueous solution was concentrated in 0.1 g / ml polyethylene glycol aqueous solution for 24 h to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 10 wt%.

[0041] (4) Add riboflavin to a high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1.5 mg / ml, stir evenly, and then vacuum to remove air bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution.

[0042] (5) Cast the mixed solution of silk fibroin / sericin / riboflavin into a mold and irradiate it with visible light for 15 minutes at a distance of 10 cm to obtain visible light-cured silk fibroin / sericin hydrogel.

[0043] According to the mechanical tensile test, the fracture strength of the visible light cured silk fibroin hydrogel is 53.38 kPa, the elongation at break is 168%, and the water content is 260%.

[0044] Example 2

[0045] The following examples illustrate a method for preparing visible light-cured silk fibroin / seric acid hydrogels, with the specific steps as follows:

[0046] (1) Boil the raw silk in a 0.3 wt% sodium carbonate aqueous solution for 30 min at a bath ratio of 1:100 to obtain silk fibers with a sericin residue of 7%.

[0047] (2) After dissolving and filtering the silk fibers, pour them into a dialysis bag with a molecular weight cutoff of 14000 Da and immerse them in deionized water. Dialyze at a low temperature of 2℃ for 5 days with a dialysis bath ratio of 1:800 to obtain a low concentration of fibroin / sericin composite aqueous solution.

[0048] (3) The low-concentration silk fibroin / sericin composite aqueous solution was concentrated in 0.1 g / ml polyethylene glycol aqueous solution for 36 h to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 15 wt%.

[0049] (4) Add riboflavin to a high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1 mg / ml, stir evenly, and then vacuum to remove air bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution.

[0050] (5) Cast the mixed solution of silk fibroin / sericin / riboflavin into a mold and irradiate it with visible light for 20 minutes at a distance of 15 cm to obtain visible light-cured silk fibroin / sericin hydrogel.

[0051] Mechanical tensile testing showed that the visible light-cured silk fibroin / seric acid hydrogel had a tensile strength of 54.69 kPa, an elongation at break of 140%, and a water content of 210%.

[0052] Example 3

[0053] The following examples illustrate a method for preparing visible light-cured silk fibroin / seric acid hydrogels, with the specific steps as follows:

[0054] (1) Boil the 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) After dissolving and filtering the silk fibers, pour them into a dialysis bag with a molecular weight cutoff of 14000 Da and immerse them in deionized water. Dialyze at a low temperature of 4℃ for 3 days with a dialysis bath ratio of 1:800 to obtain a low concentration of fibroin / sericin composite aqueous solution.

[0056] (3) The low-concentration silk fibroin / sericin composite aqueous solution was concentrated in 0.15 g / ml polyethylene glycol aqueous solution for 24 h to obtain a high-concentration silk fibroin / sericin composite aqueous solution with a concentration of 10 wt%.

[0057] (4) Add riboflavin to a high-concentration silk fibroin / sericin composite aqueous solution at a concentration of 1.3 mg / ml, stir evenly, and then vacuum to remove air bubbles to obtain a silk fibroin / sericin / riboflavin mixed solution.

[0058] (5) Cast the mixed solution of silk fibroin / sericin / riboflavin into a mold and irradiate it with visible light for 25 minutes at a distance of 20 cm to obtain visible light-cured silk fibroin / sericin hydrogel.

[0059] Mechanical tensile testing revealed that the visible light-cured silk fibroin / seric acid hydrogel exhibited a tensile strength of 20.66 kPa, an elongation at break of 64%, and a water content of 150%.

[0060] Please see Figure 1 and Figure 2 , Figure 1 The visible light-cured silk fibroin / sericin hydrogel prepared by the method described in this invention is photographed before and after stretching and before and after moisture absorption. Figure 2This image shows a SEM image of a visible-light-cured silk fibroin / serice protein hydrogel prepared using the method described in this invention, after vacuum freeze-drying. Figure 1 As shown, the visible light-cured silk fibroin / serice protein hydrogel exhibits good moisture absorption and excellent tensile properties. Figure 2 As shown, the internal structure and mechanical properties of visible light-cured sericin / sericin hydrogels can be controlled according to the sericin content and the light exposure time.

[0061] In summary, the visible light-curable silk fibroin / serice protein hydrogel preparation method of this invention obtains silk fibers containing different amounts of sericin by adjusting the concentration of degumming reagent and degumming time, and directly dissolves them to prepare a silk fibroin / serice protein composite aqueous solution in one step, without the need for additional sericin extraction or the addition of sericin protein powder, which greatly shortens the preparation process and reduces costs. It uses environmentally friendly and non-toxic riboflavin (vitamin B2) as a photoinitiator, eliminating the need for ultraviolet light and initiating cross-linking under visible light. The visible light-curable silk fibroin / serice protein hydrogel fully utilizes the respective performance advantages of silk fibroin and sericin in silk fibers, is safe in composition, requires no additional chemical cross-linking agents, has good light transmittance, good biocompatibility, and high elasticity, making it particularly suitable for the field of biomedical materials. Furthermore, the preparation process is short, time-saving, and highly efficient.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing visible light-cured silk fibroin / seric acid hydrogel, characterized in that, Includes the following steps: (1) Raw silk is boiled in a sodium carbonate aqueous solution for 15-45 minutes. By controlling the concentration of the sodium carbonate aqueous solution and the degumming time, silk fibers containing different amounts of sericin are obtained. The concentration of the sodium carbonate aqueous solution is 0.2-0.5 wt%, the mass-to-volume ratio of the raw silk to the sodium carbonate aqueous solution is 1 g: 100 ml, and the sericin content in the silk fibers is 5-15%. (2) The silk fibers containing different amounts of sericin were dissolved at a temperature of 45-65 °C for 0.2-1 h, dialyzed at a temperature of 4-10 °C for 1-5 days, filtered to remove impurities, and concentrated for 24 h to obtain a high-concentration fibroin / sericin composite aqueous solution. (3) Under light-protected conditions, riboflavin is added to the high-concentration silk fibroin / sericin composite aqueous solution, mixed evenly, and then the air bubbles are removed to obtain a silk fibroin / sericin / riboflavin mixed solution, wherein the concentration of the high-concentration silk fibroin / sericin composite aqueous solution is 5~20 wt%, the concentration of riboflavin is 1~1.5 mg / ml, and the proportion of riboflavin in the silk fibroin / sericin / riboflavin mixed solution is 0.5~2 mg / ml; (4) Pour the silk fibroin / sericin / riboflavin mixed solution into a transparent silicone mold, and use a visible light lamp to maintain constant light conditions for photocuring to obtain visible light cured silk fibroin / sericin hydrogel. The power of the visible light lamp is 100-900 W, the irradiation distance of the visible light lamp is 5-20 cm, and the irradiation time of the visible light lamp is 10-30 min.

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