Injectable sodium hyaluronate-silk fibroin composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable sodium hyaluronate-silk fibroin composite hydrogel and a preparation method and application thereof.The injectable sodium hyaluronate-silk fibroin composite hydrogel is composed of sodium hyaluronate composite gel particles and a sodium hyaluronate solution, and the sodium hyaluronate composite gel particles comprise silk fibroin sponge microspheres and sodium hyaluronate gel particles; the silk fibroin sponge microspheres are loaded on the sodium hyaluronate gel particles, the silk fibroin sponge microspheres are physical crosslinking microspheres, the particle size D50 is 10-30 [mu] m, and the particle size D90 is within 100 [mu] m; the sodium hyaluronate gel particles are chemical cross-linked particles, the particle size D50 is 150-300 microns, and the particle size D90 is within 800 microns. After the sodium hyaluronate gel particles are in contact with tissues, sodium hyaluronate is preferentially degraded, and silk fibroin exposed in the degradation process has better degradation resistance and an effect of stimulating generation of collagen fibers, so that in-situ fixation of the composite gel particles is realized, and an instant and lasting filling effect is achieved; the material is a safe and effective medical beauty filling material.

Description

Technical Field

[0001] The present invention relates to the technical field of medical aesthetic fillers, and particularly relates to an injectable sodium hyaluronate - silk fibroin composite hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] With the continuous development of medical aesthetic plastic surgery technology, injectable filling materials have received extensive attention. Injectable fillers on the market can be divided into biodegradable and non - biodegradable types. Among them, biodegradable fillers have a longer lasting effect (sometimes up to 12 months), can ultimately be metabolized by the body, and have a low risk of adverse prognosis reactions or outcomes. Therefore, they are deeply loved by doctors and patients because of their non - permanence and ease of correction.

[0003] Hydrogels have hydrophilicity, high flexibility, and good biocompatibility, and have extremely high application value in the medical field. Among them, injectable hydrogels have become a research hotspot for medical aesthetic fillers due to their good shaping properties, simple use, minimally invasive nature, etc.

[0004] Currently, the most widely used soft tissue filler on the market is cross - linked sodium hyaluronate gel. Although cross - linked sodium hyaluronate gel has good shaping effects, it degrades relatively quickly in the body, and often needs to be reinjected about 6 months after injection to maintain the filling effect. Sodium hyaluronate has a weak adhesion ability to cells in the body and is difficult to guide tissue regeneration. Therefore, a single sodium hyaluronate filler cannot effectively reduce the treatment cost and adverse feelings of patients.

[0005] Silk fibroin is a natural material obtained by degumming mulberry silk. It was recognized as a biomaterial by the U.S. Food and Drug Administration (FDA) in 1993. Silk fibroin is mainly composed of 18 amino acids such as alanine, glycine, and serine, and belongs to fibrous proteins. It is insoluble in water, but its hydrolysis product is a nutrient for human skin. In recent years, with the gradual deepening of the research and understanding of the structure and properties of silk fibroin, the development and application of silk fibroin in the fields of biomedicine, tissue engineering, etc. have become increasingly extensive. Research data show that silk fibroin has biocompatibility similar to collagen, and at the same time, silk fibroin also has certain advantages such as anti - infectivity, degradation resistance, and no risk of cross - infection. It is an excellent biomaterial. Compared with sodium hyaluronate, silk fibroin has a stronger adhesion to cells and can guide tissue regeneration, but its shaping ability is poor. Therefore, combining the two can make use of their advantages and avoid their disadvantages to prepare an injectable hydrogel filler with strong shaping ability and capable of guiding tissue regeneration.

[0006] Patent CN118370864A discloses a crosslinked sodium hyaluronate gel and its preparation method. Adding a certain proportion of free sodium hyaluronate during the preparation process can play roles in subcutaneous lubrication, nourishment, repairing skin damage, and moisturizing; meanwhile, increasing the proportion of the crosslinking agent and the crosslinking time can appropriately extend the crosslinking degree, making the retention time in the body longer, but the risk of crosslinking agent residue will also increase. From the experimental results of the effectiveness evaluation of the gel prepared from this patent through animal experiments, its degradation rate is slightly lower than that of the mainstream injectable crosslinked sodium hyaluronate gels on the market, but it is still almost completely degraded at about 26 weeks. Therefore, the HA gel still faces the challenge of short retention time.

[0007] Patent CN115671388A discloses an injectable microsphere gel of silk fibroin with adjustable properties and its preparation method. Utilizing the thixotropy of silk fibroin microspheres, they have the rheological behavior of a fluid when subjected to shear force, enabling the injectability of the gel, and can have a certain self-supporting property in the static state, achieving the function of shaping. At the same time, silk fibroin with different molecular weights is screened out in the preparation process to prepare gels with different mechanical properties suitable for different parts. However, the screening method of this gel is relatively complex, and parameters such as the molecular weight of silk fibroin and the gel inducer need to be regulated, which limits the application of the gel. Moreover, the particle size of the silk fibroin particles in this gel is 30 - 800 nm, and it degrades relatively quickly after injection filling, making it difficult to maintain the shaping persistence.

[0008] Patent CN113877001A discloses an injectable silk fibroin composite gel. The hexafluoroisopropanol droplets dissolved with silk fibroin are sprayed into the dispersion liquid by an electrostatic spraying method, sodium hyaluronate is added to the dispersion liquid, and it is left standing at 40 °C for 6 h to form a silk fibroin - hyaluronic acid composite gel material. After dialysis, homogenization, and sieving, it is the injectable silk fibroin composite gel. The composition of this gel is simple, and silk fibroin and sodium hyaluronate only form a gel by standing, with weak bonding force, and it cannot ensure the uniform mixing of the two. Moreover, during the preparation process, it is necessary to heat at 50 °C to volatilize the residual hexafluoroisopropanol solvent in the solution, and the solvent residue is harmful to the human body and water. Summary of the Invention

[0009] The present invention aims at the disadvantages and deficiencies of the prior art, and provides an injectable sodium hyaluronate - silk fibroin composite hydrogel, its preparation method, and application, which solves the problems of short duration of sodium hyaluronate hydrogel, poor mechanical properties, hydrophilicity, and lubrication of silk fibroin hydrogel. Compared with the composite hydrogel prepared by simply blending sodium hyaluronate and silk fibroin, the structure is more stable, and the maintenance time of the injection filling is effectively extended.

[0010] The specific technical solution of the present invention is as follows:

[0011] The present invention discloses an injectable sodium hyaluronate - silk fibroin composite hydrogel, which is composed of sodium hyaluronate composite gel particles and a sodium hyaluronate solution. The sodium hyaluronate composite gel particles include silk fibroin sponge microspheres and sodium hyaluronate gel particles. The silk fibroin sponge microspheres are loaded on the sodium hyaluronate gel particles. The silk fibroin sponge microspheres are physically cross - linked microspheres with a particle size of D50 within 10 - 30 μm and D90 within 100 μm; the sodium hyaluronate gel particles are chemically cross - linked particles with a particle size of D50 within 150 - 300 μm and D90 within 800 μm.

[0012] As a further improvement, the silk fibroin sponge microspheres of the present invention are formed by the self - folding of silk fibroin to form β - crystal regions under solvent stimulation, and the sodium hyaluronate gel particles are formed after the cross - linking reaction of sodium hyaluronate with a chemical cross - linker.

[0013] As a further improvement, the ratio of the mass g of the sodium hyaluronate composite gel particles to the mass g of the sodium hyaluronate solution in the present invention is 4 - 10:1, and the ratio of the mass g of the silk fibroin sponge microspheres to the mass g of the sodium hyaluronate gel particles in the sodium hyaluronate composite gel particles is 0.2 - 1:1.

[0014] As a further improvement, the solvent of the present invention is one or a mixture of two of ethanol, glycerol, and n - butanol, and the chemical cross - linker is one or a combination of two of 1,4 - butanediol diglycidyl ether, divinyl sulfone, and epoxy - terminated multi - arm polyethylene glycol, and the number of arms of the multi - arm polyethylene glycol is one of 2 - arm, 4 - arm, or 8 - arm.

[0015] The present invention also discloses a preparation method of an injectable sodium hyaluronate - silk fibroin composite hydrogel.

[0016] The preparation of the silk fibroin sponge microspheres includes the following steps:

[0017] S1: Preparation of the silk fibroin solution: Add mulberry silk into a sodium carbonate solution for high - temperature alkali washing, water washing, and drying, then dissolve it with a lithium bromide solution, and dialyze the completely dissolved mixture with purified water to obtain a silk fibroin solution;

[0018] S2: Preparation of regenerated silk fibroin: Filter the silk fibroin solution prepared in step S1 to remove impurities, then put it into a refrigerator for freezing. After complete freezing, perform freeze - drying to obtain regenerated silk fibroin;

[0019] S3: Preparation of the silk fibroin sponge microspheres: Re - dissolve the regenerated silk fibroin prepared in step S2 into a regenerated silk fibroin solution, add one or a mixture of two of ethanol, glycerol, and n - butanol for blending, freeze it completely in a refrigerator, and then perform freeze - drying to obtain a silk fibroin sponge. After crushing and sieving, silk fibroin sponge microsphere particles are obtained;

[0020] The preparation of sodium hyaluronate composite gel particles includes the following steps:

[0021] S4: Add the silk fibroin sponge microspheres prepared in step S3 into sodium hydroxide solution, homogenously disperse for 10 - 60 min, then add a crosslinking agent and stir evenly;

[0022] S5: Add sodium hyaluronate to step S4, stir to dissolve and then carry out a heat preservation reaction to obtain a composite gel;

[0023] S6: Dialyze and granulate the composite gel obtained in step S5;

[0024] S7: Compound the gel particles obtained in step S6 with a sodium hyaluronate mobile phase, fill and sterilize to obtain an injectable sodium hyaluronate - silk fibroin composite hydrogel.

[0025] As a further improvement, in step S1 of the present invention, the dialysis temperature is 1 - 4°C; in step S2, the sieve mesh is a 400 - 800 - mesh sieve; in step S3, the concentration of the silk fibroin solution is 5 - 20 wt%, the sieve mesh is 200 - 800 - mesh, the particle size of the silk fibroin sponge microspheres is D50 within 10 - 30 μm, and D90 is within 100 μm.

[0026] As a further improvement, in step S4 of the present invention, the homogenization frequency is 1 - 2.0 Hz, and the crosslinking agent concentration is 0.5 - 10%; in step S5, the mass ratio of silk fibroin (g) to sodium hyaluronate (g) is 0.2 - 1:1; in step S7, the mobile phase is uncrosslinked sodium hyaluronate, the molecular weight of sodium hyaluronate is 1000 - 2000 kDa, the concentration is 1 - 10 wt%, the mixing ratio of the gel phase mass (g) to the mobile phase mass (g) is 4 - 10:1, and the sterilization method is moist heat sterilization.

[0027] The present invention also discloses the application of an injectable sodium hyaluronate - silk fibroin composite hydrogel. The hydrogel is injected into the face through a syringe as a filler to fill facial wrinkles. Among them, after the sodium hyaluronate gel particles loaded with silk fibroin contact the tissue, sodium hyaluronate degrades preferentially. During the degradation process, the exposed silk fibroin has better degradation resistance and the effect of stimulating the generation of collagen fibers, realizing the in - situ fixation of the composite gel particles and achieving an immediate and lasting filling effect.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The scheme of low - temperature dialysis of silk fibroin proposed by the present invention can effectively delay the self - folding speed of silk fibroin, reduce the conformational transition speed of silk fibroin during dialysis, and the obtained freeze - dried silk fibroin has a higher redissolution rate;

[0030] (2) The screening filtration of the silk fibroin solution proposed by the present invention can effectively improve the purity of the silk fibroin raw material. Compared with centrifugal impurity removal, it has the characteristics of simplicity and high efficiency, and is more suitable for industrial applications;

[0031] (3) The sodium hyaluronate-silk fibroin composite hydrogel proposed by the present invention fixes silk fibroin sponge particles with physical cross-linking in the chemical cross-linking network of sodium hyaluronate by chemical cross-linking to form sodium hyaluronate composite gel particles loaded with silk fibroin particles. Compared with cross-linked sodium hyaluronate gel, it has similar extrusion force behavior, better viscoelasticity and the effect of stimulating collagen regeneration. Therefore, it has more excellent filling effect and longer maintenance time compared with cross-linked sodium hyaluronate gel.

[0032] (4) The sodium hyaluronate-silk fibroin composite hydrogel proposed by the present invention is prepared by cross-linking a blend of silk fibroin particles and sodium hyaluronate to form a composite gel. After the sodium hyaluronate gel particles loaded with silk fibroin contact the tissue, sodium hyaluronate degrades preferentially. During the degradation process, the exposed silk fibroin has better degradation resistance and the effect of stimulating the formation of collagen fibers. Therefore, the composite hydrogel will stimulate the generation of collagen and in-situ deposition, realizing the fixation of the composite particles and reducing the free movement of the gel particles at the injection site. Description of the Drawings

[0033] Figure 1 is the gel electrophoresis diagram of the silk fibroin prepared in Example 1;

[0034] Figure 2 is the particle size diagram of the silk fibroin sponge particles prepared in Example 1;

[0035] Figure 3 is the particle size diagram of the sodium hyaluronate-silk fibroin composite hydrogel particles prepared in Example 1;

[0036] Figure 4 is the sample diagram of the injectable sodium hyaluronate-silk fibroin composite hydrogel prepared in Example 1;

[0037] Figure 5 is the extrusion force diagram of the sodium hyaluronate-silk fibroin composite hydrogel in Example 1;

[0038] Figure 6 is the extrusion force diagram of the sodium hyaluronate gel prepared in Comparative Example 1;

[0039] Figure 7 is the cytotoxicity diagram of the gels of Example 1 and Comparative Example 1. Detailed Embodiments

[0040] The present invention discloses an injectable sodium hyaluronate - silk fibroin composite hydrogel, which is composed of sodium hyaluronate composite gel particles and a sodium hyaluronate solution. The sodium hyaluronate composite gel particles include silk fibroin sponge microspheres and sodium hyaluronate gel particles. The silk fibroin sponge microspheres are loaded on the sodium hyaluronate gel particles. The ratio of the mass g of the sodium hyaluronate composite gel particles to the mass g of the sodium hyaluronate solution is 4 - 10:1. Among them, the silk fibroin sponge microspheres are self - folded by silk fibroin under the stimulation of a solvent, and are physical cross - linked microspheres. The particle size of the microspheres is such that D50 is 10 - 30μm and D90 is within 100μm. The sodium hyaluronate composite gel particles are composed of silk fibroin sponge particles, sodium hyaluronate and a chemical cross - linker after a cross - linking reaction, and are chemical cross - linked composite gel particles. The particle size is such that D50 is 150 - 300μm and D90 is within 800μm. The ratio of the mass g of the silk fibroin sponge microspheres to the mass g of the sodium hyaluronate gel particles in the sodium hyaluronate composite gel particles is 0.2 - 1:1.

[0041] The solvent is one or a mixture of two of ethanol, glycerol, and n - butanol. The chemical cross - linker is one or a combination of two of 1,4 - butanediol diglycidyl ether, divinyl sulfone, and epoxy - terminated multi - arm polyethylene glycol. The number of arms of the multi - arm polyethylene glycol is one of 2 - arm, 4 - arm, or 8 - arm.

[0042] The present invention also provides a preparation method for the injectable sodium hyaluronate - silk fibroin composite hydrogel, which includes the following steps:

[0043] S1: Preparation of the silk fibroin solution: Add mulberry silk into a sodium carbonate solution for high - temperature alkali washing several times. After washing with water and drying, dissolve it with a lithium bromide solution. Dialyze the completely dissolved mixture with purified water to obtain a silk fibroin solution. Among them, the concentration of the sodium carbonate solution is 0.2 - 1wt%, the ratio of the volume mL of the sodium carbonate solution to the mass g of the mulberry silk is 10 - 20:1, the alkali washing temperature is 98 - 102°C, the alkali washing time is 30 - 60min, the ratio of the volume mL of the lithium bromide solution to the mass g of the degummed silk is 4 - 5:1, the dissolution temperature is 50 - 60°C, the cut - off molecular weight of the dialysis bag is 8000 - 12000Da, and the dialysis temperature is 1 - 4°C.

[0044] S2: Preparation of regenerated silk fibroin: Filter the silk fibroin solution prepared in step S1 to remove impurities with a sieve, then place it in a refrigerator for freezing. After complete freezing, perform freeze-drying to obtain regenerated silk fibroin; the sieve is a 400 - 800 mesh sieve; in step S3, the molecular weight of silk fibroin ≥ 10 kDa, the concentration of the silk fibroin solution is 5 - 20 wt%, the organic solvent is a mixture of n-butanol and ethanol, and the mixing ratio is about 2:1 in terms of the volume of n-butanol (mL): volume of ethanol (mL). The ratio of the volume of the silk fibroin solution (mL) to the volume of the mixed solvent (mL) is 1 - 2:1, the freezing temperature is -10 to -80 °C, the sieve is 200 - 800 mesh, and the particle size of the silk fibroin sponge microspheres is D50 within 10 - 30 μm and D90 within 100 μm.

[0045] S3: Preparation of silk fibroin sponge microspheres: Re-dissolve the regenerated silk fibroin prepared in step S2 into a regenerated silk fibroin solution with purified water, add an organic solvent for co-blending, freeze completely in a refrigerator, and then perform freeze-drying to obtain a silk fibroin sponge. After pulverization and sieving, silk fibroin sponge microsphere particles are obtained. The molecular weight of silk fibroin ≥ 10 kDa, the concentration of the silk fibroin solution is 5 - 20 wt%, the organic solvent is a mixture of n-butanol and ethanol, and the mixing ratio is 2:1 in terms of the volume of n-butanol (mL): volume of ethanol (mL). The ratio of the volume of the silk fibroin solution (mL) to the volume of the mixed solvent (mL) is 1 - 2:1, the freezing temperature is -10 to -80 °C, the sieve is 200 - 800 mesh, and the particle size of the silk fibroin sponge microspheres is D50 within 10 - 30 μm and D90 within 100 μm.

[0046] S4: Add the silk fibroin sponge microspheres prepared in step S3 into a sodium hydroxide solution, homogenize and disperse for a certain time, then add a cross-linking agent and stir evenly; the concentration of the sodium hydroxide solution is 0.1 - 10 wt%, the homogenization frequency is 1 - 2.0 Hz, the homogenization time is 10 - 60 min, and the cross-linking agent concentration is 0.5 - 10%.

[0047] S5: Add sodium hyaluronate to step S4, stir and dissolve it, and then carry out a heat preservation reaction to obtain a composite gel; the molecular weight of sodium hyaluronate is 200 - 2000 kDa, the ratio of the mass of silk fibroin (g) to the mass of sodium hyaluronate (g) is 0.2 - 1:1, the stirring and dissolving temperature is 5 - 22 °C, the heat preservation temperature is 25 - 60 °C, and the heat preservation time is 1 - 24 h.

[0048] S6: Dialyze and granulate the compliant gel obtained in step S5; the dialysis solution is phosphate buffer, the pH value of the dialysis solution is 4.9 - 7.5, the swelling ratio of the gel after dialysis is 3 - 7, the particle size of the gel after homogenization is D50 within 150 - 300 μm, and D90 is within 800 μm; in step S7, the mobile phase is uncrosslinked sodium hyaluronate, the molecular weight of sodium hyaluronate is 1000 - 2000 kDa, the concentration is 1 - 10 wt%, the mixing ratio of the gel phase mass g to the mobile phase mass g is 4 - 10:1, the sterilization method is moist heat sterilization, and the sterilization condition is F0 = 15.

[0049] S7: Compound the gel particles obtained in step S6 with the sodium hyaluronate mobile phase, fill and sterilize to obtain a sterile sodium hyaluronate - silk fibroin composite hydrogel. The mobile phase is uncrosslinked sodium hyaluronate, the molecular weight of sodium hyaluronate is 1000 - 2000 kDa, the concentration is 1 - 10 wt%, the mixing ratio of the gel phase mass g to the mobile phase mass g is 4 - 10:1, the sterilization method is moist heat sterilization, and the sterilization condition is F0 = 15.

[0050] The present invention also provides an injectable sodium hyaluronate - silk fibroin composite hydrogel, which can be injected into the face through a syringe as a filler to fill facial wrinkles.

[0051] The following will further clearly understand the present invention through specific implementation cases in combination with the accompanying drawings of the specification, but the implementation manners of the present invention are not limited thereto.

[0052] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0053] Example 1

[0054] Preparation of T1 - silk fibroin solution: Dissolve 20 g of sodium carbonate in 2000 mL of purified water. After stirring and dissolving, a 1 wt% sodium carbonate solution is obtained. Take 2 L of the 1 wt% sodium carbonate solution, heat it to boiling, add 20 g of silk, react for 30 min under boiling conditions, then fish out and wring dry. Wash it with 2 L of purified water at 60 °C for 10 min and then fish out. Add the fished - out silk into the boiling 2 L of 1 wt% sodium carbonate solution and continue to react for 30 min. After that, fish out and wring dry, wash it with 2 L of purified water at 60 °C for 10 min, then transfer it to purified water at room temperature and wash it 4 times, 10 min each time. After that, fish out and wring dry, pull the silk into a loose state, dry it at 70 °C, and then take 10 g of degummed silk and put it into 48.4 mL of LiBr solution at 60 °C. Dissolve and react it under stirring at 300 rpm / min for 4 h. Then pour the LiBr solution containing degummed silk into a dialysis bag (cut - off molecular weight: 8000 - 12000 Da), and dialyze it with purified water at 4 °C for 3 days. After dialysis, pour the dialysate into an 800 - mesh sieve for filtration to remove insoluble substances and impurities in the solution;

[0055] Preparation of T2 - regenerated silk fibroin: Freeze the filtrate at - 20 °C overnight, and then put the sample into a freeze - dryer and dry it for 7 days to obtain the solid raw material of regenerated silk fibroin. Among them, the gel electrophoresis pattern of the solid raw material of regenerated silk fibroin is as Figure 1 shown, and the amino acid composition analysis is as shown in the following table (Table 1). The test results show that the regenerated silk fibroin protein contains 16 kinds of amino acids, and the total amino acid content is 790 mg / g, with the characteristic amino acids of silk fibroin, glycine, serine, and alanine.

[0056] Table 1 is the amino acid composition analysis table of the solid raw material of regenerated silk fibroin

[0057] Result table (ESTD - 2977 - 1 - SF_2023_12_19 15_43_23_031 - 570nm)

[0058]

[0059] Preparation of T3 - silk fibroin sponge microspheres: Add 20 g of regenerated silk fibroin into 380 mL of purified water, fully dissolve it to obtain 400 mL of a 5 wt% regenerated silk fibroin solution. Add 200 mL of n - butanol into the regenerated silk fibroin solution, mix it evenly, freeze it at - 20 °C overnight, and then put the sample into a freeze - dryer and dry it for 7 days to obtain silk fibroin sponge. Crush the silk fibroin sponge and pass it through an 800 - mesh sieve to obtain silk fibroin sponge microspheres. The particle size of the silk fibroin sponge microspheres is as Figure 2 shown, and the particle size range is 6.5 - 130 μm, where D50 is 16.5 μm and D90 is 53.2 μm.

[0060] Preparation of T4 - Sodium Hyaluronate - Silk Fibroin Composite Hydrogel: At 22°C, 3.35 g of silk fibroin sponge microspheres prepared in T3 were added to 44 mL of 1 wt% sodium hydroxide solution, dispersed with a homogenizer for 1 h at a homogenization frequency of 1.0 Hz. After mixing evenly, 2805 μL of 1,4 - butanediol diglycidyl ether was added and stirred for 5 min. 7.5 g of sodium hyaluronate with a molecular weight of 200 - 2000 kDa was added to the above solution, stirred at 200 rpm / min for 10 min, and then continued to stir at 150 rpm / min for 1.5 h. After stirring, the mixture was placed in a water bath at 30°C and incubated for 21 h to obtain sodium hyaluronate - silk fibroin composite hydrogel.

[0061] Dialysis and Homogenization of T5 - Sodium Hyaluronate - Silk Fibroin Composite Hydrogel: The gel prepared in T4 was dialyzed in a phosphate buffer solution with a pH value of 6.7. The dialysis solution was changed every 30 min at the beginning of dialysis, and then every 2 h after 3 times until the gel swelling ratio reached 5 to end dialysis. The dialyzed gel was homogenized to obtain composite gel particles.

[0062] Compound and Sterilization of T6 - Sodium Hyaluronate - Silk Fibroin Composite Hydrogel: The composite gel particles obtained in T5 were compounded with a 10 wt% sodium hyaluronate solution at a ratio of 9:1. The compounded mixture was filled and sterilized by moist heat to obtain injectable sodium hyaluronate - silk fibroin composite hydrogel, and its particle size is as Figure 3 shown. The particle size range of the composite gel particles is 51.8 - 454 μm, where D50 is 173.5 μm and D90 is 277.1 μm. Among them, the condition of moist heat sterilization is F0 = 15. The macroscopic diagram of the injectable sodium hyaluronate - silk fibroin composite hydrogel is as Figure 4 shown.

[0063] Example 2

[0064] The difference between this example and Example 1 is that in T3, 10 g of regenerated silk fibroin was added to 190 mL of purified water and fully dissolved to obtain 200 mL of 5 wt% regenerated silk fibroin solution. The volume ratio of the regenerated silk fibroin solution (mL) to the volume of n - butanol (mL) is 1:1, and the rest of the steps remain unchanged.

[0065] Example 3

[0066] The difference between this example and Example 1 is that in T3, the silk fibroin sponge was crushed and passed through a 200 - mesh sieve to obtain silk fibroin sponge microspheres, and the rest of the steps remain unchanged.

[0067] Example 4

[0068] The difference between this example and Example 1 is that in T3, 134 mL of n-butanol and 66 mL of ethanol are blended to obtain a mixed solvent, and 200 mL of the mixed solvent is added to the regenerated silk fibroin solution, with the remaining steps unchanged.

[0069] Example 5

[0070] The difference between this example and Example 1 is that in T4, the heat preservation time is divided into two stages. The first stage: the heat preservation temperature is 50 °C, and the heat preservation reaction time is 1 h. The second stage: the heat preservation temperature is 30 °C, and the heat preservation reaction time is 8 h, with the remaining steps unchanged.

[0071] Example 6

[0072] The difference between this example and Example 1 is that in T5, dialysis ends when the gel swelling ratio is 7, with the remaining steps unchanged.

[0073] Example 7

[0074] The difference between this example and Example 1 is that in T6, the compounding ratio of the composite gel particles to the sodium hyaluronate solution is 6:1, with the remaining steps unchanged.

[0075] Example 8

[0076] The difference between this example and Example 1 is that in T4, the cross-linking agent is divinyl sulfone, and the dosage is 1%, with the remaining steps unchanged. The pushing force of the prepared sodium hyaluronate-silk fibroin composite hydrogel is as Figure 5 shown. When the injection needle is 27G, the average pushing force of the composite hydrogel is 12 N, and the maximum pushing force is 14.7 N. And it can be seen from the figure that the pushing force of the composite hydrogel is relatively uniform

[0077] Comparative Example 1

[0078] This comparative example provides an injectable cross-linked sodium hyaluronate gel and its preparation method. The difference from Example 6 is that it does not contain silk fibroin. The pushing force of the prepared sodium hyaluronate gel is as Figure 6 shown. When the injection needle is 27G, the average pushing force of the composite hydrogel is 12 N, and the maximum pushing force is 12.6 N, indicating that the sodium hyaluronate gel and the sodium hyaluronate-silk fibroin composite hydrogel have similar pushing force behaviors. At the same time, through the cytotoxicity experiment on the prepared injectable gel, it can be known ( Figure 7 ) that the injectable sodium hyaluronate-silk fibroin composite hydrogel has lower cytotoxicity and higher cell activity compared with the injectable cross-linked sodium hyaluronate gel, and its cell activity is equivalent to that of the blank control group, proving that the injectable sodium hyaluronate-silk fibroin composite hydrogel has good biocompatibility.

[0079] Although the present invention has been specifically shown and described in connection with preferred embodiments, there are many ways and means to implement the technical solution. The above description is only a preferred embodiment of the present invention. However, those skilled in the art should understand that various changes can be made to the present invention in form and detail without departing from the spirit and scope of the present invention defined by the appended claims, and all such changes are within the protection scope of the present invention.

Claims

1. An injectable sodium hyaluronate-silk fibroin composite hydrogel, characterized in that: The invention is composed of sodium hyaluronate composite gel particles and sodium hyaluronate solution. The sodium hyaluronate composite gel particles include silk fibroin sponge microspheres and sodium hyaluronate gel particles. The silk fibroin sponge microspheres are loaded on the sodium hyaluronate gel particles. The silk fibroin sponge microspheres are physically cross-linked microspheres with a particle size of D50 within 10-30 μm and D90 within 100 μm. The sodium hyaluronate gel particles are chemically cross-linked particles with a particle size of D50 within 150-300 μm and D90 within 800 μm.

2. The injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 1, characterized in that: The silk fibroin sponge microspheres are formed by self-folding of silk fibroin under the stimulation of a solvent to form a β-crystal region, and the sodium hyaluronate gel particles are formed by a cross-linking reaction between a chemical cross-linking agent and sodium hyaluronate.

3. The injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 2, characterized in that: The mass ratio of the sodium hyaluronate composite gel particles g to the mass ratio of the sodium hyaluronate solution g is 4-10:1, and the mass ratio of the silk fibroin sponge microspheres g in the sodium hyaluronate composite gel particles g to the mass ratio of the sodium hyaluronate gel particles g is 0.2-1:

1.

4. The injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 2, characterized in that: The solvent is one or a mixture of ethanol, glycerol, and n-butanol; the chemical crosslinking agent is one or a combination of two of 1,4-butanediol diglycidyl ether, divinyl sulfone, and epoxy-terminated multi-arm polyethylene glycol; and the number of arms of the multi-arm polyethylene glycol is one of 2 arms, 4 arms, or 8 arms.

5. A method for preparing the injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 1, 2, 3 or 4, characterized in that: The preparation of silk fibroin sponge microspheres comprises the following steps: S1: Preparation of silk fibroin solution: adding mulberry silk to sodium carbonate solution, washing with water, drying at high temperature, dissolving with lithium bromide solution, and dialyzing the completely dissolved mixture with purified water to obtain silk fibroin solution; S2: Preparation of regenerated silk fibroin: The silk fibroin solution prepared in step S1 is filtered through a sieve to remove impurities and then placed in a refrigerator for freezing. After being completely frozen, the solution is freeze-dried to obtain regenerated silk fibroin; S3: Preparation of silk fibroin sponge microspheres: The regenerated silk fibroin prepared in step S2 is re-dissolved in purified water to form a regenerated silk fibroin solution, and one or a mixture of ethanol, glycerol, and n-butanol is added to blend, and the mixture is completely frozen in a refrigerator and freeze-dried to obtain a silk fibroin sponge, and the silk fibroin sponge microsphere particles are obtained after crushing and sieving; The preparation of sodium hyaluronate composite gel particles comprises the following steps: S4: adding the silk fibroin sponge microspheres prepared in step S3 into a sodium hydroxide solution, homogenizing and dispersing for 10-60 minutes, then adding a crosslinking agent and stirring evenly; S5: adding sodium hyaluronate to step S4, stirring to dissolve, and then heat-insulating to react, to obtain a composite gel; S6: dialyzing and granulating the composite gel obtained in step S5; S7: Compounding the gel particles obtained in step S6 with the sodium hyaluronate mobile phase, filling and sterilizing to obtain an injectable sodium hyaluronate-silk fibroin composite hydrogel.

6. The method for preparing the injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 5, characterized in that: In step S1, the dialysis temperature is 1-4°C; in step S2, the screen is 400-800 mesh; in step S3, the concentration of the silk fibroin solution is 5-20wt%, the screen is 200-800 mesh, and the particle size of the silk fibroin sponge microspheres is D50 within 10-30μm and D90 within 100μm.

7. The method for preparing the injectable sodium hyaluronate-silk fibroin composite hydrogel according to claim 6, characterized in that: In the step S4, the homogenization frequency is 1-2.0 Hz, and the cross-linking agent concentration is 0.5-10%; in the step S5, the ratio of silk protein mass g to sodium hyaluronate mass g is 0.2-1:1; in the step S7, the mobile phase is uncross-linked sodium hyaluronate, the molecular weight of sodium hyaluronate is 1000-2000 kDa, the concentration is 1-10wt%, the mixing ratio of gel phase mass g to mobile phase mass g is 4-10:1, and the sterilization method is wet heat sterilization.

8. An application of an injectable sodium hyaluronate-silk fibroin composite hydrogel, characterized in that: The hydrogel is injected into the face via a syringe as a filler to fill facial wrinkles.

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