Skin wound repair material and preparation method thereof

Abstract

The present invention relates to a skin wound repair material and a preparation method thereof, belonging to the field of biomedicine. The repair material of the present invention contains, among other ingredients, palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin in specific proportions. The cuttlebone extract comprises a product of less than 5 kDa obtained by co-enzymatic hydrolysis of cuttlebone with chitinase and nattokinase, followed by stepwise enzymatic hydrolysis with chymotrypsin and lumbrokinase. Each component is prepared using a special method to obtain a highly effective product, which improves the stability of the ingredients and solves the problem of difficulty in maintaining their activity. Through rational combination, the product has a good skin wound repair effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a skin wound repair material and a preparation method thereof. Background Art

[0002] The skin, as the largest and most vital organ in the human body, is the first line of defense against external aggressions. From a microscopic perspective, the skin is composed of the epidermis, dermis, and subcutaneous tissue, with each layer working closely together. The stratum corneum in the epidermis can effectively resist physical friction, chemical erosion, and microbial invasion; while the dermis is rich in collagen and elastic fibers, which not only give the skin elasticity and toughness, but also contain abundant blood vessels, nerves, and hair follicles, which participate in key physiological processes such as body temperature regulation and sensory perception. In terms of immune defense, the defense cells in the skin can promptly identify and capture invading pathogens, initiate immune responses, and protect the body's health.

[0003] However, the skin is directly exposed to the external environment, constantly facing various risks and extremely vulnerable to various types of trauma. In accidents, there are burns and scalds caused by high temperatures, and cuts caused by sharp objects; in daily life, there are inadvertent abrasions and lacerations during exercise; in bedridden patients, due to continuous pressure on local tissues and obstructed blood circulation, chronic wounds such as pressure sores. These traumas are like ruthless demons, not only causing patients excruciating physical pain, but also seriously interfering with their daily lives and reducing their quality of life. If not handled properly, the wounds are easily infected by pathogens such as bacteria and fungi, and the inflammation continues to spread, leading to serious complications such as sepsis, posing a huge threat to the patient's life safety.

[0004] With the rapid advancement of multidisciplinary fields such as materials science and biomedical engineering, a wide variety of skin wound repair agents are now available on the market. For example, natural polymers like collagen, a key component of the skin, possess a high affinity for human tissue and provide a natural scaffold for cell growth. Chitosan, with its unique antimicrobial properties and excellent biocompatibility, promotes cell adhesion and proliferation. However, these agents also have significant limitations, including susceptibility to enzymatic degradation and poor stability, which significantly compromises their repair effectiveness. Bioactive materials, such as growth factors, can precisely regulate cell proliferation, differentiation, and migration. Extracellular matrix components can mimic the in vivo microenvironment and significantly accelerate wound healing. However, when used alone, growth factor activity is susceptible to factors such as temperature and pH, making it difficult to maintain long-term activity, ultimately compromising repair effectiveness. Furthermore, the varying repair components of complexes and inappropriate compositional combinations can also compromise repair effectiveness. Therefore, there is an urgent need to develop more stable, efficient, and safe skin wound repair agents to provide patients with improved healing products and accelerate their recovery. Summary of the Invention

[0005] In response to the problems of various types of existing skin wound repair drugs, such as susceptibility to degradation, poor stability, difficulty in maintaining activity, and unreasonable ingredient combinations that affect the repair effect, the present invention provides a skin wound repair material and a preparation method thereof. The repair material is designed to contain specific proportions of palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate modified silk fibroin. Each component is prepared using a special method to obtain a high-efficiency product, which improves the stability of the components and solves the problem of difficulty in maintaining activity. After reasonable combination, it has a good skin wound repair effect. The specific technical solution is as follows:

[0006] A skin wound repair material, comprising the following raw materials in parts by weight: 8 to 12 parts of palmitoyl tripeptide-1, 25 to 35 parts of methacryloyl collagen, 8 to 12 parts of cuttlebone extract, 4 to 6 parts of recombinant human epidermal growth factor liposomes, 5 to 10 parts of polydopamine nanoparticles, 5 to 10 parts of hyaluronic acid, and 10 to 15 parts of sodium alginate-modified silk fibroin, with a water content of 65 to 75% by weight.

[0007] The cuttlebone extract comprises the following steps: cutting the cuttlebone by co-enzymatic hydrolysis with chitinase and nattokinase for 1 hour to 1.5 hours, then enzymatic hydrolysis with chymotrypsin for 50 minutes to 70 minutes, and then enzymatic hydrolysis with lumbrokinase for 60 minutes to 80 minutes, to obtain a product with a component below 5 kDa.

[0008] In the above-mentioned repair material, the methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio is methacryloylated type I collagen: methacryloylated type II collagen = (8-10): (3-5).

[0009] In the above-mentioned repair material, the preparation method of the cuttlebone extract includes the following steps: crushing the cuttlebone into powder, adding 8 to 12 times the mass of phosphate buffer, adding chitinase and nattokinase, enzymatically hydrolyzing at 45°C to 55°C for 1 hour to 1.5 hours, inactivating the enzyme, adjusting the pH to 8.0 to 8.5, adding chymotrypsin, enzymatically hydrolyzing at 35°C to 38°C for 50 minutes to 70 minutes, centrifuging, taking the supernatant, adding lumbrokinase, enzymatically hydrolyzing at 50°C to 55°C for 60 minutes to 80 minutes, ultrafiltration using a 5kDa ultrafiltration membrane to obtain components below 5kDa, freeze-drying, and obtaining the cuttlebone extract.

[0010] In the preparation method of the above-mentioned cuttlebone extract, the phosphate buffer is a phosphate buffer with a pH of 6-7 and a concentration of 0.01 mol / L to 0.015 mol / L; the added amounts of the chitinase and nattokinase are both 1% to 2% of the mass of the cuttlebone; the enzyme inactivation is performed at 80°C to 90°C for 10 to 20 minutes; the added amount of the chymotrypsin is 0.5% to 1% of the mass of the cuttlebone; the centrifugation is performed at 10,000 r / min to 12,000 r / min for 20 to 30 minutes; and the added amount of the lumbrokinase is 0.3% to 0.5% of the mass of the cuttlebone.

[0011] In the above-mentioned repair material, the preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio, recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = (1~3): (100~120): (10~12): (5~6): (1~1.5): (0.3~0.6): (3~5), distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, ultrasonicated, and freeze-dried to obtain recombinant human epidermal growth factor liposomes.

[0012] In the above-mentioned method for preparing recombinant human epidermal growth factor liposomes, the power of the ultrasound is 200W to 250W, and the time of the ultrasound is 15 minutes to 20 minutes.

[0013] In the above-mentioned repair material, the preparation method of sodium alginate modified silk fibroin includes the following steps: preparing a sodium alginate aqueous solution with a mass concentration of 2% to 5%; dispersing the silk fibroin in water to prepare an emulsion with a mass concentration of 5% to 8%; preparing a calcium chloride aqueous solution with a concentration of 0.1 mol / L to 0.3 mol / L; adding the emulsion to the sodium alginate aqueous solution at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1: (0.5 to 1.5): (0.03 to 0.05) under stirring, mixing evenly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution under stirring, stirring for cross-linking, and freeze-drying to obtain sodium alginate modified silk fibroin.

[0014] In the above-mentioned preparation method of sodium alginate-modified silk fibroin, the water is all deionized water; the emulsion contains Tween-80, the content of which is 1% to 3% of the mass of the silk fibroin; the stirring speed of the emulsion added to the sodium alginate aqueous solution is 100 r / min to 200 r / min; the stirring speed of the mixed solution added to the calcium chloride aqueous solution is 100 r / min to 200 r / min; the stirring crosslinking is carried out at 30°C to 40°C and a speed of 100 r / min to 200 r / min for 1 hour to 3 hours.

[0015] The above-mentioned method for preparing a skin wound repair material comprises the following steps: adding palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin to deionized water according to parts by mass, mixing, adjusting the mixture to a paste with a water content of 65wt% to 75wt%, and vacuum degassing to obtain a repair material.

[0016] In the above preparation method, the methacryloyl collagen is subjected to swelling and dialysis pretreatment, the methacryloyl collagen is added to deionized water for swelling, and then added to a dialysis bag with a molecular weight cutoff of 5kDa and dialyzed in deionized water at 4°C to 6°C for 50h to 60h; the polydopamine nanoparticles are subjected to dispersion pretreatment, the polydopamine nanoparticles are added to deionized water and ultrasonically dispersed uniformly; the vacuum degassing time is 15min to 20min.

[0017] The present invention provides a skin wound repair material and a preparation method thereof, which have the following beneficial effects:

[0018] 1. Palmitoyl tripeptide-1, as a signal peptide, can stimulate fibroblasts and promote the synthesis of collagen, especially type I and type III collagen, which helps to increase the firmness and elasticity of the skin and reduce the formation of wrinkles. In the repair of skin trauma, it can provide structural support for new tissue and promote the reconstruction of the skin at the wound.

[0019] 2. Methacryloyl-collagen, due to its high affinity with human tissue, can provide a natural growth scaffold for cell adhesion, proliferation and differentiation. Methacryloyl-type I collagen is mainly responsible for maintaining the strength and toughness of the skin, and methacryloyl-type II collagen plays an important role in regulating cell function and tissue repair. The two are matched in an appropriate mass ratio, which is closer to the natural collagen ratio of the skin and can better simulate the physiological environment of the skin.

[0020] 3. Cuttlebone extract is rich in various minerals and bioactive ingredients, has good hemostatic effect, can accelerate platelet aggregation, and is mainly used to promote the coagulation process.

[0021] The present invention designs a cuttlebone extract using a multi-step enzymatic hydrolysis process for cuttlebone, first using chitinase and nattokinase to co-hydrolyze to release small molecule products with biological activity; then it is hydrolyzed step by step by chymotrypsin and lumbrokinase, acting on specific substrates to obtain more effective specific high-activity small molecule peptides and other small molecule active products, fully exerting their biological efficacy. It was found that this specific product can significantly promote cell proliferation and migration in skin repair, accelerate wound healing, and reduce the risk of infection.

[0022] Fourth, in recombinant human epidermal growth factor liposomes, recombinant human epidermal growth factor can specifically bind to cell surface receptors, activate intracellular signaling pathways, regulate cell proliferation, differentiation, and migration, and promote epidermal cell regeneration and repair. As a carrier, liposomes protect recombinant human epidermal growth factor from external environmental factors (enzymatic hydrolysis, pH changes, etc.), prolonging its activity and improving its bioavailability. In particular, the addition of dioleoylphosphatidylethanolamine and phosphatidylserine for protection further effectively enhances the biostability of recombinant human epidermal growth factor liposomes. This structure effectively protects the activity of the growth factor from environmental damage, allowing for stable release in the body and continuously promoting its cell repair effect.

[0023] 5. Polydopamine nanoparticles have excellent adhesion and can adhere tightly to the wound surface, enhancing the fit between the repair material and the wound; at the same time, their good biocompatibility prevents them from causing obvious immune rejection reactions, and can also promote cell adhesion and growth, which is beneficial to wound healing.

[0024] 6. Hyaluronic acid is a natural moisturizer that can absorb and retain a large amount of water, maintaining a moist environment in the local area of the wound. This helps prevent the wound from drying out excessively and forming scabs, promotes cell migration and proliferation, and inhibits inflammatory responses, providing a good microenvironment for wound healing.

[0025] 7. Among the sodium alginate-modified silk fibroin, sodium alginate has strong water absorption and can effectively absorb wound exudate to keep the wound dry; silk fibroin has good biocompatibility and mechanical properties. After the two are combined, sodium alginate-modified silk fibroin with special structure and properties is formed, which has the advantages of both and shows better performance in wound repair. It can not only provide a physical barrier for the wound, but also promote cell adhesion and growth, thereby accelerating wound healing.

[0026] 8. The specific addition ratios of the various components in the present invention are carefully designed. Palmitoyl tripeptide-1 and methacryloylated collagen work synergistically to promote the repair and reconstruction of skin tissue at the cellular and molecular levels; cuttlebone extract and recombinant human epidermal growth factor liposomes play key roles in growth promotion and cell regulation, respectively, accelerating the wound healing process; polydopamine nanoparticles enhance the binding of the repair material to the wound, and hyaluronic acid and sodium alginate-modified silk fibroin jointly maintain the moisture and physical barrier function of the wound. The various components cooperate with each other to form an organic whole, exerting a more powerful repair effect than a single component.

[0027] 9. In the preparation method, methacryloyl-collagen is added to deionized water for swelling so that the collagen can fully absorb water, expand its molecular structure, expose more active sites, and facilitate subsequent interactions with other components; it is then placed in a dialysis bag and dialyzed in low-temperature deionized water. On the one hand, this can remove impurities and small molecules and improve the purity of the collagen; on the other hand, the low-temperature environment helps to improve the stability of the collagen and ensure the integrity of its structure and function.

[0028] 10. In the preparation method, polydopamine nanoparticles are ultrasonically pre-dispersed in deionized water. The cavitation effect of ultrasound breaks down the agglomeration between the polydopamine nanoparticles, allowing them to be evenly dispersed in the water, forming a stable dispersion system. This allows the polydopamine nanoparticles to be evenly distributed throughout the system during the subsequent preparation of the repair material, fully exerting their adhesive properties and promoting cell growth.

[0029] In summary, the repair material designed in the present invention contains palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin in specific proportions. Each component is prepared using a special method to obtain a high-efficiency product, which improves the stability of the components and solves the problem of difficulty in maintaining activity. After reasonable combination, it has good skin wound repair effect, the preparation method is simple, and it has great practical value. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to specific implementation cases, but the present invention is not limited to these embodiments.

[0031] Example 1: A skin wound repair material, comprising the following raw materials in parts by mass: 10 parts of palmitoyl tripeptide-1, 30 parts of methacryloyl collagen, 10 parts of cuttlebone extract, 5 parts of recombinant human epidermal growth factor liposomes, 8 parts of polydopamine nanoparticles, 8 parts of hyaluronic acid, 12 parts of sodium alginate modified silk fibroin, and a water content of 65wt% to 75wt%.

[0032] The methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio of methacryloylated type I collagen:methacryloylated type II collagen is 9:4.

[0033] The preparation method of the cuttlebone extract includes the following steps: crushing the cuttlebone into powder, adding 10 times the mass of pH 6.5, 0.012 mol / L phosphate buffer, adding 1.5% chitinase and 1.5% nattokinase by mass of the cuttlebone, enzymatically hydrolyzing at 50°C for 1 hour, inactivating the enzyme at 85°C for 15 minutes, adjusting the pH to 8.2, adding 0.8% chymotrypsin by mass of the cuttlebone, enzymatically hydrolyzing at 37°C for 60 minutes, centrifuging at 11000 r / min for 25 minutes, taking the supernatant, adding 0.4% lumbrokinase by mass of the cuttlebone, enzymatically hydrolyzing at 52°C for 70 minutes, ultrafiltration using a 5kDa ultrafiltration membrane to obtain components below 5kDa, freeze-drying, and obtaining the cuttlebone extract.

[0034] Among them, the preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio of recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 2:110:11:5.5:1.2:0.5:4, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, and ultrasonication is performed at 220W power for 18 minutes, and freeze-drying is performed to obtain recombinant human epidermal growth factor liposomes.

[0035] The preparation method of sodium alginate-modified silk fibroin comprises the following steps: preparing a 3% sodium alginate aqueous solution; dissolving 2% Tween-80 in water, dispersing the silk fibroin in water to prepare a 6% emulsion; preparing a 0.2 mol / L calcium chloride aqueous solution; adding the emulsion to the sodium alginate aqueous solution at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1:1:0.04, stirring at 150 rpm, and mixing uniformly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution, stirring at 150 rpm, cross-linking at 35°C and 150 rpm for 2 hours, and freeze-drying to obtain the sodium alginate-modified silk fibroin. Deionized water was used.

[0036] The method for preparing the above-mentioned skin wound repair material comprises the following steps:

[0037] Methacryl-collagen was added to deionized water according to mass fraction for swelling, then added to a 5 kDa molecular weight cut-off dialysis bag and dialyzed in deionized water at 4°C for 55 h. The product was then removed for later use. Polydopamine nanoparticles were added to 2.5 times the mass of deionized water and ultrasonically dispersed uniformly for later use.

[0038] Palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin were added to deionized water in parts by mass, mixed, and adjusted to a paste with a water content of 65wt% to 75wt%. The paste was vacuum-degassed for 18 minutes to obtain a repair material.

[0039] For the convenience of detection and comparison, the water content of the restoration samples prepared in this example was controlled at 70 wt %.

[0040] Example 2: A skin wound repair material, comprising the following raw materials in parts by mass: 8 parts of palmitoyl tripeptide-1, 25 parts of methacryloyl collagen, 8 parts of cuttlebone extract, 4 parts of recombinant human epidermal growth factor liposomes, 5 parts of polydopamine nanoparticles, 5 parts of hyaluronic acid, 10 parts of sodium alginate modified silk fibroin, and a water content of 65wt% to 75wt%.

[0041] The methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio of methacryloylated type I collagen:methacryloylated type II collagen is 8:3.

[0042] The preparation method of the cuttlebone extract includes the following steps: crushing the cuttlebone into powder, adding 8 times the mass of pH 6, 0.01 mol / L phosphate buffer, adding 1% chitinase and 1% nattokinase by mass of the cuttlebone, enzymatically hydrolyzing at 45°C for 1 hour, inactivating the enzyme at 80°C for 10 minutes, adjusting the pH to 8.0, adding 0.5% chymotrypsin by mass of the cuttlebone, enzymatically hydrolyzing at 35°C for 50 minutes, centrifuging at 10,000 r / min for 20 minutes, taking the supernatant, adding 0.3% lumbrokinase by mass of the cuttlebone, enzymatically hydrolyzing at 50°C for 60 minutes, ultrafiltration using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, freeze-drying, and obtaining the cuttlebone extract.

[0043] Among them, the preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio of recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 1:100:10:5:1:0.3:3, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, and ultrasonication is performed at a power of 200W for 15 minutes, and freeze-drying is performed to obtain recombinant human epidermal growth factor liposomes.

[0044] The preparation method of sodium alginate-modified silk fibroin comprises the following steps: preparing a 2% sodium alginate aqueous solution; dissolving 1% Tween-80 in water, dispersing the silk fibroin in water to prepare a 5% emulsion; preparing a 0.1 mol / L calcium chloride aqueous solution; adding the emulsion to the sodium alginate aqueous solution at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1:0.5:0.03, stirring at 100 rpm, and mixing uniformly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution, stirring at 100 rpm, cross-linking at 30°C and 100 rpm for 1 hour, and freeze-drying to obtain the sodium alginate-modified silk fibroin. Deionized water was used.

[0045] The method for preparing the above-mentioned skin wound repair material comprises the following steps:

[0046] Methacryl-collagen was added to deionized water according to mass fraction for swelling, then added to a 5 kDa molecular weight cut-off dialysis bag and dialyzed in deionized water at 4°C for 50 h. The product was then removed for later use. Polydopamine nanoparticles were added to 2 times the mass of deionized water and ultrasonically dispersed uniformly for later use.

[0047] Palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin were added to deionized water in parts by mass, mixed, and adjusted to a paste with a water content of 65wt% to 75wt%. The paste was vacuum-degassed for 15 minutes to obtain a repair material.

[0048] For the convenience of detection and comparison, the water content of the restoration samples prepared in this example was controlled at 70 wt %.

[0049] Example 3: A skin wound repair material, comprising the following raw materials in parts by mass: 12 parts of palmitoyl tripeptide-1, 35 parts of methacryloyl collagen, 12 parts of cuttlebone extract, 6 parts of recombinant human epidermal growth factor liposomes, 10 parts of polydopamine nanoparticles, 10 parts of hyaluronic acid, 15 parts of sodium alginate modified silk fibroin, and a water content of 65wt% to 75wt%.

[0050] The methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio of methacryloylated type I collagen:methacryloylated type II collagen is 10:5.

[0051] Among them, the preparation method of the cuttlebone extract includes the following steps: crushing the cuttlebone into powder, adding 12 times the mass of pH 7, 0.015 mol / L phosphate buffer, adding 2% chitinase and 2% nattokinase by mass of the cuttlebone, enzymatically hydrolyzing at 55°C for 1.5 hours, inactivating the enzyme at 90°C for 20 minutes, adjusting the pH to 8.5, adding 1% chymotrypsin by mass of the cuttlebone, enzymatically hydrolyzing at 38°C for 70 minutes, centrifuging at 12000 r / min for 30 minutes, taking the supernatant, adding 0.5% lumbrokinase by mass of the cuttlebone, enzymatically hydrolyzing at 55°C for 80 minutes, ultrafiltration using a 5kDa ultrafiltration membrane to obtain components below 5kDa, freeze-drying, and obtaining the cuttlebone extract.

[0052] Among them, the preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio of recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 3:120:12:6:1.5:0.6:5, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, and ultrasonication is performed at 250W power for 20 minutes, and freeze-drying is performed to obtain recombinant human epidermal growth factor liposomes.

[0053] The preparation method of sodium alginate-modified silk fibroin comprises the following steps: preparing a 5% sodium alginate aqueous solution; dissolving 3% Tween-80 in water, dispersing the silk fibroin in water to prepare an 8% emulsion; preparing a 0.3 mol / L calcium chloride aqueous solution; adding the emulsion to the sodium alginate aqueous solution at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1:1.5:0.05, stirring at 200 rpm, and mixing uniformly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution, stirring at 200 rpm, cross-linking at 40°C and 200 rpm for 3 hours, and freeze-drying to obtain the sodium alginate-modified silk fibroin. Deionized water was used.

[0054] The method for preparing the above-mentioned skin wound repair material comprises the following steps:

[0055] Methacryl-collagen was added to deionized water according to mass fraction for swelling, then added to a 5 kDa molecular weight cut-off dialysis bag and dialyzed in deionized water at 6°C for 60 h. The product was then removed for later use. Polydopamine nanoparticles were added to 3 times the mass of deionized water and ultrasonically dispersed uniformly for later use.

[0056] Palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin were added to deionized water in parts by mass, mixed, and adjusted to a paste with a water content of 65wt% to 75wt%. The paste was vacuum-degassed for 20 minutes to obtain a repair material.

[0057] For the convenience of detection and comparison, the water content of the restoration samples prepared in this example was controlled at 70 wt %.

[0058] Example 4: A skin wound repair material, comprising the following raw materials in parts by mass: 8 parts of palmitoyl tripeptide-1, 35 parts of methacryloyl collagen, 8 parts of cuttlebone extract, 6 parts of recombinant human epidermal growth factor liposomes, 5 parts of polydopamine nanoparticles, 10 parts of hyaluronic acid, 10 parts of sodium alginate modified silk fibroin, and a water content of 65wt% to 75wt%.

[0059] The methacryloylated collagen includes methacryloylated type I collagen and methacryloylated type II collagen, and the mass ratio of methacryloylated type I collagen:methacryloylated type II collagen is 8:5.

[0060] The preparation method of the cuttlebone extract includes the following steps: crushing the cuttlebone into powder, adding 8 times the mass of pH 6.8, 0.01 mol / L phosphate buffer, adding 2% chitinase and 1% nattokinase by mass of the cuttlebone, enzymatically hydrolyzing at 55°C for 1 hour, inactivating the enzyme at 80°C for 15 minutes, adjusting the pH to 8.1, adding 0.6% chymotrypsin by mass of the cuttlebone, enzymatically hydrolyzing at 37°C for 60 minutes, centrifuging at 10,000 r / min for 20 minutes, taking the supernatant, adding 0.5% lumbrokinase by mass of the cuttlebone, enzymatically hydrolyzing at 55°C for 60 minutes, ultrafiltration using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, freeze-drying, and obtaining the cuttlebone extract.

[0061] Among them, the preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio of recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = 3:100:12:5:1.5:0.3:5, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, and ultrasonication is performed at a power of 200W for 15 minutes, and freeze-drying is performed to obtain recombinant human epidermal growth factor liposomes.

[0062] The preparation method of sodium alginate-modified silk fibroin comprises the following steps: preparing a 5% sodium alginate aqueous solution; dissolving 1.5% Tween-80 in water, dispersing the silk fibroin in water to prepare a 7% emulsion; preparing a 0.3 mol / L calcium chloride aqueous solution; adding the emulsion to the sodium alginate aqueous solution at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1:1.2:0.03, stirring at 200 rpm, and mixing uniformly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution, stirring at 200 rpm, cross-linking at 37°C and 200 rpm for 2 hours, and freeze-drying to obtain the sodium alginate-modified silk fibroin. Deionized water was used.

[0063] The method for preparing the above-mentioned skin wound repair material comprises the following steps:

[0064] Methacryl-collagen was added to deionized water according to mass fraction for swelling, then added to a 5 kDa molecular weight cut-off dialysis bag and dialyzed in deionized water at 4°C for 52 h. The product was then removed for later use. Polydopamine nanoparticles were added to 2 times the mass of deionized water and ultrasonically dispersed uniformly for later use.

[0065] Palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin were added to deionized water in parts by mass, mixed, and adjusted to a paste with a water content of 65wt% to 75wt%. The paste was vacuum-degassed for 15 minutes to obtain a repair material.

[0066] For the convenience of detection and comparison, the water content of the restoration samples prepared in this example was controlled at 70 wt %.

[0067] In the above examples: Palmitoyl tripeptide-1 was sourced from Shaanxi Haibo Biotechnology Co., Ltd. Methacryloylated type I collagen and methacryloylated type II collagen were sourced from Shanghai Jiader Chemical Technology Co., Ltd. Cuttlebone was sourced from Bozhou Yuanshengtang Pharmaceutical Co., Ltd., and cuttlefish bone was sourced from Guangzhou Qibao Biotechnology Co., Ltd. Recombinant human epidermal growth factor (EGF) was sourced from Guangzhou Qibao Biotechnology Co., Ltd. Polydopamine nanoparticles were sourced from Beijing Zhongke Keyou Technology Co., Ltd. Hyaluronic acid was sourced from Lanli Biotechnology (Xi'an) Co., Ltd., model LL-TMZS. Sodium alginate was sourced from Qiyuan (Guangdong) Pharmaceutical Chemical Co., Ltd., with a purity exceeding 99%. Silk fibroin was sourced from Xi'an Dongchi Biotechnology Co., Ltd. Silk fibroin is a molecular fibrous protein extracted from silk. Chitinase was sourced from Guangdong Jiudian Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Nattokinase was sourced from Shaanxi Langde Biotechnology Co., Ltd., with an enzyme activity of 40,000 U / g. Chymotrypsin was obtained from Shanghai Yiji Industrial Co., Ltd., α-chymotrypsin, model M0002, with an enzyme activity of 1500 IU / mg. Lumbrokinase was obtained from Tianben Biotechnology (Shanxi) Co., Ltd., with an enzyme activity of 10,000 IU / mg. Phosphatidylcholine was obtained from Shanxi Hengtian Biotechnology Co., Ltd., and egg yolk lecithin. Dioleoylphosphatidylethanolamine (DOPE) was obtained from Shaoguan Chengfeng Chemical Co., Ltd. Cholesterol was obtained from Jiangsu Yingao Biotechnology Co., Ltd., pharmaceutical grade. Phosphatidylserine was obtained from Shaanxi Longzhou Biotechnology Co., Ltd., derived from soybeans. Tween-80 was obtained from Shandong Taixi Chemical Co., Ltd.

[0068] Comparative Example 1

[0069] All methacryloyl-collagen used was methacryloyl-type I collagen; other parameters and methods were the same as those in Example 1.

[0070] Comparative Example 2

[0071] All methacryloyl-collagen used was methacryloyl-type II collagen; other parameters and methods were the same as those in Example 1.

[0072] Comparative Example 3

[0073] The cuttlebone extract was replaced by cuttlebone powder; other parameters and methods were the same as in Example 1.

[0074] Comparative Example 4

[0075] In the preparation method of the cuttlebone extract, chitinase and nattokinase are not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0076] Comparative Example 5

[0077] In the preparation method of the cuttlebone extract, chymotrypsin is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0078] Comparative Example 6

[0079] In the preparation method of the cuttlebone extract, lumbrokinase is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0080] Comparative Example 7

[0081] Recombinant human epidermal growth factor was not prepared into liposomes, but was directly added according to the proportion; other parameters and methods were the same as those in Example 1.

[0082] Comparative Example 8

[0083] In the preparation method of recombinant human epidermal growth factor liposomes, dioleoylphosphatidylethanolamine and phosphatidylserine were not added; other parameters and methods were the same as in Example 1.

[0084] Comparative Example 9

[0085] The silk fibroin was not modified with sodium alginate, and the silk fibroin was directly added in proportion; other parameters and methods were the same as in Example 1.

[0086] Comparative Example 10

[0087] 4 parts of cuttlebone extract were added (too little), and 16 parts of palmitoyl tripeptide-1 were added (too much); other parameters and methods were the same as in Example 1.

[0088] Comparative Example 11

[0089] 12 parts of methacryloyl-collagen were added (too little), and 30 parts of sodium alginate-modified silk fibroin were added (too much); other parameters and methods were the same as in Example 1.

[0090] 1. Cell proliferation rate detection:

[0091] Using the MTT colorimetric assay: Fibroblasts were pre-cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin mixture) at 37°C in a 5% CO2 incubator. For the experiment, fibroblasts in the logarithmic growth phase were dissociated with trypsin and seeded in 96-well plates at a density of 5,000 cells / well in a volume of 200 μL per well. The plates were incubated in the incubator for 24 hours. After the cells attached, the remaining medium was carefully aspirated. The skin wound repair materials from the various examples and comparative examples were diluted 10-fold with the aforementioned DMEM medium to obtain a repair solution. 100 μL of the repair solution was added to each well, with five replicates per group. A control group was also established, receiving only 100 μL of DMEM medium. After 48 hours of culture, 20 μL of MTT solution (5 mg / mL, prepared in PBS buffer) was added to each well and incubated in a dark incubator at 37°C for 4 hours. After incubation, carefully remove the supernatant and add 150 μL of DMSO to each well. Shake the cells at low speed for 10 minutes to fully dissolve the crystals. Finally, measure the absorbance (OD) at 490 nm on a microplate reader. Cell proliferation rate = (OD value of experimental group - OD value of control group) / OD value of control group × 100%. The average results are shown in Table 1 below.

[0092] Table 1 Cell proliferation rate detection results

[0093]

[0094] From the above results, it can be seen that the repair materials of Examples 1 to 4 can promote cell proliferation more efficiently. In Comparative Examples 1 and 2, a single type of methacryloyl-collagen cannot provide a comprehensive and suitable growth microenvironment for cells like the two collagens mixed in a specific ratio in the examples; the structure and composition of collagen affect the adhesion, spreading and proliferation signal transduction of cells, and a single collagen weakens the interaction between cells and materials, thereby inhibiting cell proliferation. In Comparative Example 3, the cuttlebone powder has not been enzymatically hydrolyzed, and the active ingredients inside it, such as polysaccharides and polypeptides, are difficult to release, and cannot effectively stimulate cell proliferation. Comparative Examples 4 to 6 lack enzymatic hydrolysis at different stages during the preparation of the cuttlebone extract, resulting in incomplete extracted components, especially the lack of specific small molecule active peptides, which destroys the synergistic mechanism of promoting cell proliferation in the cuttlebone extract and reduces its ability to promote cell proliferation. In comparative example 7, recombinant human epidermal growth factor was not prepared into liposomes. It was easily affected by factors such as proteases, temperature, and pH in the solution and inactivated. It could not effectively bind to cell surface receptors and start the proliferation signaling pathway in the cell, so the cell proliferation rate was low. In comparative example 8, dioleoylphosphatidylethanolamine and phosphatidylserine were missing when preparing recombinant human epidermal growth factor liposomes. These two components are crucial for maintaining the stability of the double membrane structure of the liposome and promoting the fusion of the liposome with the cell membrane. The lack of them will lead to unstable liposome structure, making recombinant human epidermal growth factor more likely to leak and inactivate, affecting cell proliferation. In comparative example 9, the chemical groups and structure of the surface of unmodified silk fibroin are not conducive to cell adhesion and growth signal transmission. Compared with the modified sodium alginate modified silk fibroin, it has poor compatibility with cells and is not conducive to cell proliferation. Comparative examples 10 and 11 changed the component ratio, breaking the optimal synergistic ratio between the components and affecting the promotion of cell proliferation. Too much or too little of certain components can interfere with cells' uptake of nutrients, signal transduction, and construction of the extracellular matrix, thereby reducing cell proliferation rate.

[0095] 2. Wound healing time detection:

[0096] Healthy adult SD rats weighing 200-250 g were selected and acclimated for one week with free access to food and water. One day before the experiment, the rats' backs were depilated, covering an area approximately 5 cm x 5 cm to avoid skin damage. After depilation, the rats were randomly divided into different groups, with 5 rats in each group. The rats were anesthetized with 3% sodium pentobarbital solution (30 mg / kg) via intraperitoneal injection. After anesthesia, full-thickness skin defects (8 mm circular wounds) were created symmetrically on both sides of the spine on the back of the rats. Two wounds were created for each rat. After the wounds were created, they were rinsed with sterile saline to remove blood and tissue debris. The skin wound repair materials from the various examples and comparative examples were applied to the wounds to a thickness of approximately 1 mm. The wounds were then covered with sterile gauze and secured with medical tape. The gauze was changed daily, and wound healing was observed. Key time points, such as the appearance of scabs, scab shedding, and completion of epithelialization, were recorded. Complete epithelialization and the formation of a complete scar were used as the standard for wound healing, and wound healing time was recorded. The test results are shown in Table 2 below.

[0097] Table 2 Wound healing time test results

[0098]

[0099] The above results demonstrate that the repair materials of Examples 1 to 4 provide sustained, stable, and effective biological activity after wound contact, continuously promoting skin repair, improving repair efficacy, and shortening repair time. In Comparative Examples 1 and 2, the inappropriate ratio of methacryloylated collagen impaired cell adhesion and proliferation. A suitable ratio of methacryloylated type I and type II collagen synergistically promoted fibroblast migration, proliferation, and extracellular matrix synthesis. Single or unbalanced collagen types can lead to abnormal cell-material interactions and delay wound healing. In Comparative Example 3, the active ingredients of the cuttlebone powder were not fully released, lacking specific active small molecules and specific pro-repair small peptides. Consequently, the cuttlebone extract was unable to effectively promote cell proliferation, angiogenesis, and inflammation resolution, hindering wound healing. In Comparative Examples 4 to 6, incomplete enzymatic hydrolysis resulted in an incomplete collection of the active healing-promoting ingredients in the cuttlebone extract, impairing its ability to promote wound healing. Chitinase, nattokinase, chymotrypsin, and lumbrokinase play a key role in the degradation of cuttlebone components and the release of active ingredients at different stages. The lack of specific enzymatic components will reduce their efficacy. In Comparative Example 7, recombinant human epidermal growth factor is unstable and easily inactivated, unable to effectively promote cell proliferation and migration, especially the migration of epithelial cells and coverage of wounds, resulting in prolonged wound healing time. In Comparative Example 8, liposome structural defects affect the activity of recombinant human epidermal growth factor, making it unable to play its role in promoting wound healing; dioleoylphosphatidylethanolamine and phosphatidylserine are essential for maintaining liposome structure and protecting growth factor activity. Their absence will reduce the effectiveness of the growth factor. In Comparative Example 9, unmodified silk fibroin is not conducive to cell migration and proliferation. It cannot provide a good growth microenvironment for cells like sodium alginate-modified silk fibroin, delaying wound healing. In Comparative Examples 10 and 11, the imbalance in the proportion of ingredients affects the overall repair effect. The ratio between the various components has an important impact on cell behavior, inflammatory response and tissue remodeling during wound healing. Changes in the ratio will disrupt the balance of repair and lead to prolonged healing time.

Claims

1. A skin wound repair material, characterized in that: The repair material includes the following raw materials in parts by weight: 8 to 12 parts of palmitoyl tripeptide-1, 25 to 35 parts of methacryloyl collagen, 8 to 12 parts of cuttlebone extract, 4 to 6 parts of recombinant human epidermal growth factor liposomes, 5 to 10 parts of polydopamine nanoparticles, 5 to 10 parts of hyaluronic acid, 10 to 15 parts of sodium alginate modified silk fibroin, and a water content of 65 wt% to 75 wt%; The methacryloyl collagen comprises methacryloyl type I collagen and methacryloyl type II collagen in a mass ratio of (8-10): (3-5); The cuttlebone extract comprises the following steps: cutting the cuttlebone by co-enzymatic hydrolysis with chitinase and nattokinase for 1 hour to 1.5 hours, then enzymatic hydrolysis with chymotrypsin for 50 minutes to 70 minutes, and then enzymatic hydrolysis with lumbrokinase for 60 minutes to 80 minutes, to obtain a product with a component below 5 kDa.

2. A skin wound repair material according to claim 1, characterized in that: The preparation method of the cuttlebone extract comprises the following steps: crushing the cuttlebone into powder, adding 8 to 12 times its mass of phosphate buffer, adding chitinase and nattokinase, performing enzymatic hydrolysis at 45 to 55° C. for 1 to 1.5 hours, inactivating the enzymes, adjusting the pH to 8.0 to 8.5, adding chymotrypsin, performing enzymatic hydrolysis at 35 to 38° C. for 50 to 70 minutes, centrifuging, collecting the supernatant, adding lumbrokinase, performing enzymatic hydrolysis at 50 to 55° C. for 60 to 80 minutes, performing ultrafiltration using a 5 kDa ultrafiltration membrane to obtain components below 5 kDa, and freeze-drying to obtain the cuttlebone extract.

3. The skin wound repair material according to claim 2, characterized in that: The phosphate buffer is a phosphate buffer with a pH of 6 to 7 and a concentration of 0.01 mol / L to 0.015 mol / L. The amounts of chitinase and nattokinase added are both 1% to 2% of the mass of the cuttlebone. The enzyme inactivation step is performed at 80° C. to 90° C. for 10 to 20 minutes. The amount of chymotrypsin added is 0.5% to 1% of the mass of the cuttlebone. The centrifugation step is performed at 10,000 r / min to 12,000 r / min for 20 to 30 minutes. The amount of lumbrokinase added is 0.3% to 0.5% of the mass of the cuttlebone.

4. The skin wound repair material according to claim 1, characterized in that: The preparation method of recombinant human epidermal growth factor liposomes includes the following steps: according to the mass ratio of recombinant human epidermal growth factor: deionized water: phosphatidylcholine: dioleoylphosphatidylethanolamine: cholesterol: phosphatidylserine: glucose = (1-3): (100-120): (10-12): (5-6): (1-1.5): (0.3-0.6): (3-5), distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, cholesterol, phosphatidylserine and glucose are added to deionized water to prepare a mixed solution, and then recombinant human epidermal growth factor is added, ultrasonicated, and freeze-dried to obtain recombinant human epidermal growth factor liposomes.

5. The skin wound repair material according to claim 4, characterized in that: The ultrasonic power is 200W to 250W, and the ultrasonic time is 15min to 20min.

6. The skin wound repair material according to claim 1, characterized in that: The preparation method of sodium alginate modified silk fibroin includes the following steps: preparing a sodium alginate aqueous solution with a mass concentration of 2% to 5%; dispersing silk fibroin in water to prepare an emulsion with a mass concentration of 5% to 8%; preparing a calcium chloride aqueous solution with a concentration of 0.1 mol / L to 0.3 mol / L; adding the emulsion to the sodium alginate aqueous solution under stirring at a mass ratio of silk fibroin: sodium alginate: calcium chloride = 1: (0.5 to 1.5): (0.03 to 0.05), mixing uniformly to obtain a mixed solution; adding the mixed solution to the calcium chloride aqueous solution under stirring, stirring for cross-linking, and freeze-drying to obtain the sodium alginate modified silk fibroin.

7. The skin wound repair material according to claim 6, characterized in that: The water is all deionized water; the emulsion contains Tween-80, the content of which is 1% to 3% of the mass of silk fibroin protein; the stirring speed of the emulsion added to the sodium alginate aqueous solution is 100 r / min to 200 r / min; the stirring speed of the mixed solution added to the calcium chloride aqueous solution is 100 r / min to 200 r / min; the stirring crosslinking is carried out at 30°C to 40°C and a speed of 100 r / min to 200 r / min for 1 hour to 3 hours.

8. The method for preparing a skin wound repair material according to claim 1, characterized in that: The preparation method includes the following steps: adding palmitoyl tripeptide-1, methacryloylated collagen, cuttlebone extract, recombinant human epidermal growth factor liposomes, polydopamine nanoparticles, hyaluronic acid, and sodium alginate-modified silk fibroin into deionized water according to parts by mass, mixing, adjusting the mixture to a paste with a water content of 65wt% to 75wt%, and vacuum degassing to obtain a repair material.

9. The method for preparing a skin wound repair material according to claim 8, characterized in that: The methacryloyl collagen is subjected to swelling and dialysis pretreatment, wherein the methacryloyl collagen is added to deionized water for swelling, and then a dialysis bag with a molecular weight cutoff of 5 kDa is added and dialyzed in deionized water at 4° C. to 6° C. for 50 to 60 hours. The polydopamine nanoparticles are subjected to dispersion pretreatment, wherein the polydopamine nanoparticles are added to deionized water for ultrasonic dispersion. The vacuum degassing time is 15 to 20 minutes.

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