A regenerated silk fibroin composite sodium hyaluronate gel and a preparation method and application thereof

Abstract

The application discloses a regenerated silk fibroin composite sodium hyaluronate gel and a preparation method and application thereof, and the preparation method comprises the following steps: step 1, stirring a regenerated silk fibroin solution, filtering, adding a gel promoter into the filtrate, uniformly mixing, sealing, and carrying out heat preservation treatment to obtain a regenerated silk fibroin gel; and step 2, adding a sodium hyaluronate solution into the regenerated silk fibroin gel obtained in step 1, uniformly mixing the system, and sterilizing to obtain the regenerated silk fibroin composite sodium hyaluronate gel. The preparation method is characterized in that the regenerated silk fibroin composite sodium hyaluronate gel is prepared without adding a chemical cross-linking agent by utilizing the shear resistance of the regenerated silk fibroin, the prepared regenerated silk fibroin composite sodium hyaluronate gel has excellent biocompatibility, and has the effects of soothing, repairing, anti-inflammation and skin tightening; and the preparation process is controllable, and the gel suitable for medical repair products, functional skin care products and injectable soft tissue fillers can be conveniently and quickly prepared according to the requirements.

Description

Technical Field

[0001] This invention relates to the field of biopolymer materials, specifically to a regenerated silk fibroin composite sodium hyaluronate gel, its preparation method, and its application. Background Technology

[0002] Silk fibroin (SF), as a natural polymer material, has shown broad application prospects in tissue engineering scaffolds, drug delivery carriers and wound repair due to its excellent biocompatibility, biodegradability and tunable mechanical properties.

[0003] Regenerated silk fibroin gel can form a protective film on the wound surface, thereby preventing wound infection, accelerating skin healing, and reducing scar formation. It is especially suitable for skin repair after minimally invasive cosmetic procedures such as laser therapy and microneedling. Furthermore, by adjusting the preparation process, injectable regenerated silk fibroin gels with certain mechanical strength can be obtained as soft tissue fillers. The publication of YY / T 1950-2024 "Tissue Engineering Medical Devices: Silk Fiber Protein" also provides standardized guidance for the application of silk fibroin.

[0004] However, the preparation of regenerated silk fibroin gels in existing technologies still faces some key bottlenecks. There is a trade-off between the safety and efficiency of cross-linking technologies: while chemical cross-linking agents (such as epichlorohydrin and glutaraldehyde) can shorten the gelation time (the time required for the silk fibroin solution to transform into a gel) to several hours, residual cross-linking agents may impair bioactivity. Physical cross-linking technologies (such as ultrasound-induced β-sheet structure formation) avoid chemical toxicity, but existing equipment parameters (such as ultrasound power > 200 W / cm², processing time > 30 min) easily lead to protein denaturation and precipitation, and gel uniformity is difficult to guarantee in large-scale production (the problem of uneven mixing is mentioned in patent CN116284869 A).

[0005] Currently, there is a lack of cross-linking agent-free, easily manufactured silk fibroin composite gels suitable for wound repair and soft tissue filling. Sodium hyaluronate, due to its excellent biocompatibility and immunogenicity, has been widely used in wound dressings and soft tissue filling. Therefore, there is a need for an injectable silk fibroin gel that can be easily and quickly prepared without the addition of additional cross-linking agents and can be used for wound repair and soft tissue filling. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a method for preparing a regenerated silk fibroin composite sodium hyaluronate gel. This method utilizes the protein properties of silk fibroin itself to prepare a regenerated silk fibroin composite sodium hyaluronate gel that does not require the addition of an additional cross-linking agent, exhibits good biocompatibility, promotes cell proliferation, has controllable mechanical strength, can be used as a soft tissue filler and wound repair agent, and has no toxic side effects.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A method for preparing a regenerated silk fibroin composite sodium hyaluronate gel includes the following steps:

[0009] Step 1: The regenerated silk fibroin solution is stirred and sheared, filtered, and a gelation promoter is added to the filtrate. The mixture is then mixed, sealed, and kept warm to obtain the regenerated silk fibroin gel. This step utilizes the shear-intolerant property of silk fibroin by stirring and shearing the regenerated silk fibroin solution. The shear force can promote the conformational change of the regenerated silk fibroin to a certain extent and promote the gelation of the regenerated silk fibroin solution. The solution after stirring and shearing is then naturally filtered through a filter screen to remove a small amount of precipitated protein.

[0010] Step 2: Add sodium hyaluronate solution to the regenerated silk fibroin gel obtained in Step 1, mix well, and sterilize to obtain the final product.

[0011] Preferably, the preparation method of the regenerated silk fibroin is as follows: 5A grade silk is mixed with a 1-20 g / L sodium carbonate aqueous solution at a mass-to-volume ratio of 1 g: 50-200 mL. The mixture is boiled to degummify for 20-50 minutes, drained, and the degumming process is repeated. After two degumming cycles, the degummed silk is drained, washed with water, and dried in a constant temperature drying oven at 60-80°C to obtain degummed silk. The degummed silk is then mixed with an 8-9.5 M lithium bromide aqueous solution at a mass-to-volume ratio of 1 g: 8-20 mL and stirred at 60-90°C for 0.5-5 hours to obtain a silk fibroin-lithium bromide solution. This solution is then purified by dialysis using a 10 kDa-50 kDa dialysis bag, with pure water as the dialysis solution. After dialysis purification, the solution is freeze-dried to obtain soluble regenerated silk fibroin.

[0012] In step 1, the solvent of the regenerated silk fibroin solution is a phosphate buffer with a pH of 5.0 to 8.0 and a concentration of 0.1 to 0.2 M; the phosphate buffer contains sodium chloride, so that the osmotic pressure of the phosphate buffer is equal to that of animals or humans; the phosphate buffer is preferably a sodium phosphate buffer; the water used to prepare the phosphate buffer is water for injection.

[0013] In step 1, the concentration of the regenerated silk fibroin solution is 5-50 g / L. If the concentration is too low, it is difficult to form a gel, and if the concentration is too high, the mechanical strength of the gel is too high. The endotoxin content in the regenerated silk fibroin is less than 0.05 EU / mg, and the regenerated silk fibroin is sterile.

[0014] In step 1, the stirring speed is 100-4000 rpm, and the time is 1 min-3 h; the stirring is preferably magnetic stirring or mechanical stirring. Shear force can promote the conformational change of regenerated silk fibroin to a certain extent, but excessive stirring speed and / or excessive stirring time will lead to a large amount of regenerated silk fibroin agglomeration and precipitation, reducing the yield and affecting the properties of the gel.

[0015] In step 1, a filter screen is used for filtration, and the filter screen has a mesh size of 100 to 300 (pore size of 48 to 150 μm).

[0016] In step 1, the gelation accelerator is glycerol or polyethylene glycol; preferably, the glycerol is injection-grade glycerol; the amount of gelation accelerator added is 10~300 mL / L. Glycerol induces gelation through hydrogen bond competition and water displacement, and glycerol has good biocompatibility.

[0017] In step 1, the temperature for the heat preservation treatment is 60-90℃, and the time is 3-24 h; the heat preservation treatment is preferably a water bath treatment or a treatment in an electric heating drying oven. High temperature can promote the conformational transition rate of regenerated silk fibroin, thereby promoting the transition of silk fibroin solution to gelation, but excessively high temperature can easily lead to protein denaturation and precipitation.

[0018] In step 2, the solvent of the sodium hyaluronate solution is the same as the solvent of the regenerated silk fibroin solution; the sodium hyaluronate is injection-grade sodium hyaluronate.

[0019] In step 2, the amount of sodium hyaluronate solution added is controlled so that the concentration of sodium hyaluronate in the mixture of regenerated silk fibroin gel and sodium hyaluronate solution is 1 ~ 20 g / L.

[0020] In step 2, the sterilization is preferably radiation sterilization or moist heat sterilization; preferably, the temperature of the moist heat sterilization is 121~123℃ and the time is 15~20 min.

[0021] In step 1 and / or step 2 of the method of the present invention, regenerated silk fibroin composite sodium hyaluronate gel can be prepared without the addition of an additional chemical crosslinking agent. The chemical crosslinking agent is, for example, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polypropylene glycol diglycidyl ether, diglycidyl ether, or diethylene sulfone.

[0022] The present invention also provides a regenerated silk fibroin composite sodium hyaluronate gel prepared by the above preparation method.

[0023] This invention also provides the application of the regenerated silk fibroin composite sodium hyaluronate gel prepared by the above method in the preparation of medical repair products, functional skin care products or injectable soft tissue fillers.

[0024] Preferably, the medical repair products include wound repair agents, post-operative (laser, microneedling, hyaluronic acid injection, etc.) skin repair gels, medical cooling gels, etc.; the functional skin care products include repair essences, anti-inflammatory acne-removing gels, firming and anti-wrinkle eye gels, skin soothing gels, and gel-like sheet masks, etc.

[0025] Beneficial effects:

[0026] (1) The regenerated silk fibroin composite sodium hyaluronate gel prepared by the method of the present invention contains no chemical cross-linking agents, has no cytotoxicity, good biocompatibility, and will not cause irritation or skin sensitization when injected into the skin. Its components are all injection-grade raw materials, which are safe and reliable and can be used to prepare injectable soft tissue fillers.

[0027] (2) The regenerated silk fibroin complex sodium hyaluronate gel prepared by the method of the present invention has a good moisturizing effect. Skin efficacy test shows that it has the effects of soothing, repairing, anti-inflammatory and firming the skin. It can be used to prepare medical repair products and functional skin care products.

[0028] (3) The method of the present invention can adjust the mechanical strength of the composite gel by adjusting the concentration of regenerated silk fibroin. The preparation process is controllable, and the regenerated silk fibroin gel has the characteristics of shear thinning, high water content and good film-forming properties. It can be conveniently and quickly prepared as needed to obtain gels suitable for medical repair products, functional skin care products and injectable soft tissue fillers. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1 This is a fluorescence inverted microscope image of the MTT cytotoxicity assay of sample 1 in Example 4.

[0031] Figure 2 This is a fluorescence inverted microscope image of sample 2 in Example 4, obtained from the MTT cytotoxicity assay.

[0032] Figure 3 These are photos of regenerated silk fibroin composite sodium hyaluronate gels with different regenerated silk fibroin contents before and after moist heat sterilization in Example 5.

[0033] Figure 4 This is a photograph of the regenerated silk fibroin composite sodium hyaluronate gel after high-temperature drying treatment in Example 5.

[0034] Figure 5 These are photos of the regenerated silk fibroin composite sodium hyaluronate gel before and after moist heat sterilization in Example 5.

[0035] Figure 6 This is a photograph of the regenerated silk fibroin composite sodium hyaluronate gel encapsulated in a syringe and sterilized by moist heat in Example 5.

[0036] Figure 7 This is a typical image of the test method for testing the soothing effect of regenerated silk fibroin composite sodium hyaluronate gel in Example 6 (the yellow dotted line area is the quantitative region).

[0037] Figure 8 This is a graph showing the TEWL (Tissue Surface Energy) trend of the skin after slight damage to the skin barrier in Example 7, with and without the application of regenerating silk fibroin complex sodium hyaluronate gel.

[0038] Figure 9 This is a graph showing the change rate of TEWL in the skin after slight damage to the skin barrier in Example 7, with and without the application of regenerating silk fibroin complex sodium hyaluronate gel.

[0039] Figure 10 This is a graph showing the trend of skin stratum corneum moisture content changes after applying regenerating silk fibroin complex sodium hyaluronate gel following slight skin barrier damage in Example 7.

[0040] Figure 11 This is a graph showing the change in skin stratum corneum moisture content after applying regenerating silk fibroin composite sodium hyaluronate gel following slight skin barrier damage in Example 7.

[0041] Figure 12 The image shows the mean optical density of skin erythema before and after continuous use of regenerated silk fibroin composite sodium hyaluronate gel for 28 days in Example 8.

[0042] Figure 13 The image shows the skin firmness (F4) values ​​before and after continuous use of the regenerated silk fibroin complex sodium hyaluronate gel for 28 days in Example 8.

[0043] Figure 14 These are facial images of two patients before and after continuous use of regenerated silk fibroin complex sodium hyaluronate gel for 28 days in Example 8, as measured by VISIA CR. Detailed Implementation

[0044] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0045] The preparation method of regenerated silk fibroin used in the following examples is as follows: 5A grade silk and 5 g / L sodium carbonate aqueous solution are mixed at a mass-to-volume ratio of 1 g: 100 mL. The mixture is boiled and degummed for 30 minutes, then drained. This degumming step is repeated twice. After degumming, the degummed silk is drained, washed with water, and dried in a constant temperature drying oven at 60°C to obtain degummed silk. The degummed silk is mixed with 9 M lithium bromide aqueous solution at a mass-to-volume ratio of 1 g: 10 mL, and stirred at 60°C for 2 hours to obtain a silk fibroin-lithium bromide solution. This solution is purified by dialysis in pure water using a 30 kDa dialysis bag. After dialysis purification, the purified solution is freeze-dried to obtain soluble regenerated silk fibroin.

[0046] Example 1

[0047] Step 1: Dissolve the soluble regenerated silk fibroin in a 0.1 M sodium phosphate buffer (containing 9 g / L sodium chloride) with a pH of 6.8 to prepare a regenerated silk fibroin solution with a volume of 800 mL and a concentration of 15 g / L.

[0048] Step 2: The regenerated silk fibroin solution was mechanically stirred and sheared with a paddle at a speed of 800 rpm for 10 minutes. The stirred and sheared regenerated silk fibroin solution was then naturally filtered through a 100-mesh filter to obtain approximately 600 mL of a slightly whitish regenerated silk fibroin solution.

[0049] Step 3: Add 20 mL of injection-grade glycerol to the filtered regenerated silk fibroin solution to make the initial concentration of glycerol in the mixture approximately 32 mL / L. After mixing evenly, seal the mixture and place it in a 70℃ water bath for 8 hours to obtain regenerated silk fibroin gel.

[0050] Step 4: Prepare 200 mL of 12 g / L sodium hyaluronate solution using the sodium phosphate buffer from Step 1, add it to the regenerated silk fibroin gel, stir well, and sterilize by moist heat at 121℃ for 15 min to obtain a regenerated silk fibroin composite sodium hyaluronate gel, in which the regenerated silk fibroin content is approximately 10 mg / mL.

[0051] Example 2

[0052] Step 1: Dissolve the soluble regenerated silk fibroin in a 0.2 M sodium phosphate buffer (containing 9 g / L sodium chloride) with a pH of 7.2 to prepare a 1200 mL regenerated silk fibroin solution with a concentration of 20 g / L.

[0053] Step 2: The regenerated silk fibroin solution was mechanically stirred and sheared with a paddle at a speed of 2000 rpm for 40 minutes. The stirred and sheared regenerated silk fibroin solution was then naturally filtered through a 200-mesh filter to obtain approximately 900 mL of a slightly whitish regenerated silk fibroin solution.

[0054] Step 3: Add 50 mL of injection-grade glycerol to the filtered regenerated silk fibroin solution to make the initial concentration of glycerol in the mixture approximately 50 mL / L. After mixing evenly, seal the mixture and place it in an 80℃ electric heating drying oven for 15 hours to obtain regenerated silk fibroin gel.

[0055] Step 4: Prepare 300 mL of 10 g / L sodium hyaluronate solution using the sodium phosphate buffer from Step 1, add it to the regenerated silk fibroin gel, stir well, and sterilize by moist heat at 121℃ for 15 min to obtain a regenerated silk fibroin composite sodium hyaluronate gel, in which the regenerated silk fibroin content is approximately 15 mg / mL.

[0056] Example 3

[0057] Step 1: Dissolve the soluble regenerated silk fibroin in a 0.2 M sodium phosphate buffer (containing 9 g / L sodium chloride) with a pH of 6.5 to prepare a 1500 mL regenerated silk fibroin solution with a concentration of 40 g / L.

[0058] Step 2: The regenerated silk fibroin solution was mechanically stirred and sheared with a paddle at a speed of 1500 rpm for 20 minutes. The stirred and sheared regenerated silk fibroin solution was then naturally filtered through a 200-mesh filter to obtain approximately 1200 mL of a slightly whitish regenerated silk fibroin solution.

[0059] Step 3: Add 20 mL of injection-grade glycerol to the filtered regenerated silk fibroin solution to make the initial concentration of glycerol in the mixture approximately 15 mL / L. After mixing evenly, seal the mixture and place it in an 85℃ electric heating drying oven for 5 hours to obtain regenerated silk fibroin gel.

[0060] Step 4: Prepare 400 mL of 9 g / L sodium hyaluronate solution using the sodium phosphate buffer from Step 1, add it to the regenerated silk fibroin gel, stir well, and sterilize by moist heat at 121℃ for 15 min to obtain a regenerated silk fibroin composite sodium hyaluronate gel, in which the regenerated silk fibroin content is approximately 30 mg / mL.

[0061] Example 4

[0062] The regenerated silk fibroin gel samples 1 and 2 prepared in Examples 2 and 3 were subjected to three basic biological tests: cytotoxicity, intradermal reaction, and skin sensitization. Specific testing methods and results are shown in Table 1. The experimental results indicate that the regenerated silk fibroin gel prepared by the method of this invention has no cytotoxicity (e.g., ...). Figure 1 and Figure 2 As shown in the figure, the regenerated silk fibroin gel prepared by this invention does not produce irritation or skin sensitization when injected into the skin, and has excellent biocompatibility.

[0063] Table 1. Cytotoxicity, intradermal reaction, and skin sensitization tests for Sample 1 and Sample 2.

[0064]

[0065] Example 5

[0066] The mechanical strength of the composite gel can be adjusted by regulating the concentration of silk fibroin in the composite gel. The regenerated silk fibroin gel has shear-thinning properties, high water content and good film-forming properties. The resulting composite gel can be used as an excellent soft tissue filler and wound repair agent.

[0067] The mechanical strength of the composite gel can be adjusted by regulating the content of regenerated silk fibroin in the composite gel to meet the needs of different indications, such as... Figure 3 The images shown are of the composite gels prepared in Examples 1, 2, and 3 before and after sterilization. From left to right, they are composite gels with regenerated silk fibroin contents of 10 mg / mL, 15 mg / mL, and 30 mg / mL, respectively, prepared in Examples 1, 2, and 3. The composite gel with a low regenerated silk fibroin content (e.g., 10 mg / mL) has a soft texture and a certain degree of fluidity, and can be used as a wound dressing. The composite gel with a high regenerated silk fibroin content (e.g., 30 mg / mL) has stronger mechanical properties and can be used as a soft tissue filler.

[0068] The composite gel prepared in Example 2 above was spread evenly in a glass petri dish and dried at 60°C for 2 hours to obtain the following result: Figure 4 The protein film shown has a certain mechanical strength, indicating that the regenerated silk fibroin composite gel of the present invention can absorb and lock in moisture well. When applied to the wound surface as a wound repair agent, it can form a protective film on the wound surface, which helps to keep the wound moist and helps to reduce the formation of scars.

[0069] The composite gel prepared in Example 2 above, either alone or encapsulated in a syringe, was sterilized by moist heat at 121°C for 15 minutes. The results are as follows: Figure 5 and 6As shown, the properties of the composite gel did not change significantly before and after sterilization, and its mechanical properties remained good. This indicates that the composite gel prepared by the present invention has good stability and certain mechanical strength, and can be used as an injectable soft tissue filler.

[0070] Example 6

[0071] The composite gel prepared in Example 2 was tested using a zebrafish cosmetic soothing efficacy assay to verify its soothing effects. The experimental principle is that sodium dodecyl sulfate (SLS) may trigger a stimulating response in zebrafish. The stimulant enters the zebrafish's body, inducing an inflammatory response, causing neutrophils to initiate an immune response, migrate to the skin epidermis, and aggregate. The composite gel of this invention was used to treat transgenic zebrafish (MPX) with green fluorescent neutrophils that had undergone a SLS-induced stimulating response. The change in the number of neutrophils in the MPX skin before and after treatment with the composite gel of this invention was used to verify whether the composite gel of this invention has a soothing effect.

[0072] Zebrafish MPX were randomly selected and placed in 6-well plates, 15 fish per well. A zebrafish skin inflammation model was established by pre-administering SLS in water. The composite gel prepared in Example 2 was administered in water to a concentration of 20 mg / mL to obtain the sample group. A normal control group (containing juvenile zebrafish and standard dilution water) and a model control group (containing juvenile zebrafish and sodium dodecyl sulfate solution) were also set up. Each well had a volume of 3 mL, and the samples were incubated at 28 ℃ in the dark for 18 h. Ten zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, analyzing the number of neutrophils (N) in the zebrafish skin. The soothing efficacy of the samples was calculated according to a formula to determine whether they possessed a soothing effect.

[0073]

[0074] In this embodiment, the calculated soothing efficacy of the sample (the composite gel prepared in Example 2) was 44%. Figure 7 As shown, the number of neutrophils in the sample regenerated silk fibroin combined with sodium hyaluronate gel was significantly reduced compared with the model control group, revealing that the sample has a soothing effect.

[0075] Example 7

[0076] Transepidermal water loss (TEWL) is one of the indicators for evaluating skin repair efficacy. The higher the value of this indicator, the faster the water loss rate from the skin surface and the worse the skin barrier function. In a model where the skin barrier is slightly damaged after being peeled off with tape, if the TEWL in the area where the product was used is significantly lower than that in the area where the product was not used, it indicates that the product has a repairing effect.

[0077] On day 0, two 3cm×3cm areas were marked on the flexed side of the human arm as test areas (model control area and sample area). The area was equilibrated in a constant temperature and humidity environment for 20 minutes. The skin stratum corneum moisture content and TEWL of the test areas were measured. Then, the skin was peeled off with tape in both test areas, and each test area was peeled off 6 times. Then, 1 g of the composite gel prepared in Example 3 was applied to the sample area, and no product was used in the model control area.

[0078] On days 1-2, the two test areas were peeled off with tape 6 times each day; then 1 g of composite gel was applied to the sample area once a day, and no product was used on the model control area.

[0079] From day 3 to day 6, apply 1 g of the composite gel to the sample area once a day. Do not use the product on the model control area.

[0080] On day 7, after equilibration in a constant temperature and humidity environment for 20 minutes, the skin stratum corneum moisture content and TEWL of the two test areas were measured.

[0081] like Figure 8 As shown in the TEWL trend graph, the TEWL value of the sample area on day 7 was significantly lower than that of the model control area.

[0082] like Figure 9 As shown in the TEWL change rate plot, the TEWL change rate in the sample area on day 7 was significantly lower than that in the model control area.

[0083] like Figure 10 As shown in the trend graph of skin stratum corneum moisture content, the moisture content of the skin stratum corneum in the sample area was significantly higher than that in the model control area on day 7.

[0084] like Figure 11 As shown in the graph of the rate of change in skin stratum corneum moisture content, the rate of change in skin stratum corneum moisture content in the sample area on day 7 was significantly higher than that in the model control area.

[0085] For three consecutive days, the skin on the flexor surface of a human arm was peeled off with tape, and the composite gel prepared according to this invention was applied once daily. By day 7, the average TEWL in the control area increased by approximately 52.2% compared to before skin barrier damage on day 0. The average TEWL in the area using the composite gel prepared according to this invention (test sample) increased by approximately 19.7% compared to before skin barrier damage on day 0. The proportion of skin TEWL increase caused by using the test sample decreased by approximately 62.3%. The TEWL measurement value in the test sample area was significantly lower than that in the control area, and the increase in TEWL in the test sample area was significantly less than that in the control area. Therefore, the regenerated silk fibroin composite sodium hyaluronate gel prepared according to this invention has excellent skin moisturizing and repairing effects.

[0086] The regenerated silk fibroin composite sodium hyaluronate gel prepared by this invention has good repair effects in skin efficacy tests due to its excellent moisturizing and film-forming properties.

[0087] Example 8

[0088] The regenerated silk fibroin composite sodium hyaluronate gel prepared in Example 2 was continuously applied to the affected areas of patients with erythema inflammatoryum for 28 days, serving as the sample group; a separate control group was established in which patients with erythema inflammatoryum did not use any product on their affected areas. After 28 days, the mean optical density of the erythema area and skin firmness were measured in both groups on day 0 (D0) and day 28 (D28) to evaluate the anti-inflammatory and skin-firming effects of the regenerated silk fibroin composite sodium hyaluronate gel prepared in this invention.

[0089] Images of the patient's face were taken using VISIA CR, and the optical density of the skin erythema was analyzed using IPP (Image-Pro Plus) software. The optical density of the skin erythema reflects the condition of facial capillaries and indicates the degree of skin inflammation. The lower the value, the lower the degree of skin inflammation, and the better the sample's effect on improving skin erythema.

[0090] The patient's facial skin firmness (F4) was measured using a Cutometer MPA580 skin elasticity tester. The lower the test value, the better the skin firmness.

[0091] The mean optical density of facial skin erythema and skin firmness (F4) were measured on day 0 and after 28 days of continuous use, respectively. Figure 12 Mean optical density map of skin erythema and Figure 14 As shown in the VISIA CR facial image, after 28 days of continuous use, the skin erythema was significantly improved. This demonstrates that the regenerated silk fibroin composite sodium hyaluronate gel prepared in this invention has excellent anti-inflammatory effects on the skin.

[0092] like Figure 13 As shown in the statistical chart of skin firmness (F4) values, the skin firmness of the sample was significantly improved after 28 days of continuous use, proving that the regenerated silk fibroin composite sodium hyaluronate gel prepared in this invention has a good skin firming effect.

[0093] This invention provides a regenerated silk fibroin composite sodium hyaluronate gel and its preparation method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a regenerated silk fibroin composite sodium hyaluronate gel, characterized in that, Includes the following steps: Step 1: Stir the regenerated silk fibroin solution, filter it, add a gelation promoter to the filtrate, mix well, seal and heat-preserve to obtain regenerated silk fibroin gel. Step 2: Add sodium hyaluronate solution to the regenerated silk fibroin gel obtained in Step 1, mix the system well, and sterilize to obtain the final product. In step 1, the gelation accelerator is glycerol or polyethylene glycol; the number average molecular weight of the polyethylene glycol is 200-1000 Da; the concentration of the regenerated silk fibroin solution is 5-50 g / L; the stirring speed is 100-4000 rpm, and the time is 1 min-3 h; the amount of gelation accelerator added is 10-300 mL / L.

2. The method according to claim 1, characterized in that, In step 1, the solvent for the regenerated silk fibroin solution is a phosphate buffer with a pH of 5.0 to 8.

0.

3. The method according to claim 1, characterized in that, In step 1, a filter screen is used for filtration, and the mesh size of the filter screen is 100 to 300.

4. The method according to claim 1, characterized in that, In step 1, the temperature of the heat preservation treatment is 60 ~ 90℃, and the time is 3 ~ 24 h.

5. The method according to claim 2, characterized in that, In step 2, the solvent of the sodium hyaluronate solution is the same as the solvent of the regenerated silk fibroin solution.

6. The method according to claim 1, characterized in that, In step 2, the amount of sodium hyaluronate solution added is controlled so that the concentration of sodium hyaluronate in the mixing system is 1 ~ 20 g / L.

7. The regenerated silk fibroin composite sodium hyaluronate gel prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the regenerated silk fibroin composite sodium hyaluronate gel according to claim 7 in the preparation of medical repair products, functional skin care products or injectable soft tissue fillers.

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