A polypeptide combination wound healing preparation with high permeability

Abstract

The present invention discloses a polypeptide combination wound healing preparation with high permeability, specifically relating to a polypeptide combination of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide, and a new use for treating skin diseases such as wounds, ulcers, burns, scars, and keloids. While the polypeptide combination plays a significant synergistic effect in wound healing, it can effectively prevent and inhibit scar hyperplasia. In addition, the polypeptide combination wound healing preparation of the present invention utilizes the wound healing promoting activity of the composite polypeptide, and is compounded with specific penetration enhancers and antioxidants to be prepared into a gel with functions of moisturizing, promoting healing, reducing infection, and relieving pain, greatly increasing skin penetration, significantly enhancing the skin retention dose, and having significant antibacterial, anti-inflammatory, eschar-removing and muscle-generating, astringent healing, and scar hyperplasia inhibiting effects, being particularly suitable for wound healing and reducing scar residue.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide applications, and particularly relates to the use of a polypeptide composition as a wound healing agent in the treatment of skin diseases such as wounds, ulcers, burns, scars, and keloids. Background Art

[0002] Skin trauma has always been an important topic in surgical research, and finding drugs to promote skin trauma has been the focus of numerous experts and scholars. With the rapid development of molecular biology, basic research on wound repair has already penetrated to the molecular and genetic levels.

[0003] The healing process of wounds in the skin, etc. (specifically, acute wounds such as incisions, abrasions, burns, etc. and chronic wounds such as pressure ulcers) is a complex dynamic process that involves the participation of various types of cells (such as inflammatory cells, endothelial cells, fibroblasts, etc.), soluble mediators (cytokines and growth factors), and extracellular mediators, and ultimately restores the integrity and dynamic balance of the skin. At the same time, wound healing is a highly ordered process that can be divided into three stages: the hemostatic-inflammatory phase, the proliferative phase, and the remodeling phase. The three processes intersect with each other rather than being independent of each other. The hemostatic-inflammatory phase is the first process initiated after tissue injury, and the reactions that occur include hemodynamic changes, increased vascular permeability, and the exudation and phagocytosis of neutrophils and monocytes. The cells mainly involved in the hemostatic-inflammatory response at the injury site are platelets, neutrophils, and macrophages, etc. The proliferative phase is the most critical stage of wound tissue remodeling, including re-epithelialization and granulation tissue formation. Re-epithelialization is completed through the proliferation, migration, and differentiation of epithelial cells, and the process will be promoted by related signals such as nitric oxide, EGF, KGF, etc. Granulation tissue is a highly proliferative connective tissue composed of cells (mainly fibroblasts and macrophages), extracellular mediators, and capillaries. During the process of wound healing, granulation tissue has the functions of anti-infection and protecting the wound surface, filling the wound and other tissue defects, and organizing blood clots and necrotic tissues. The remodeling phase is the process of granulation tissue transforming into scar tissue. During this process, collagen is continuously updated, the content of type I collagen increases significantly, the number of various types of cells in granulation tissue, including myofibroblasts, decreases significantly, and the capillary network also gradually regresses. Remodeling will bring about the improvement of tissue structure and strength to restore the original structure and function as much as possible. During the whole process of skin wound healing, various factors and ECM cooperate to play a repair function, including pro-inflammatory factors interleukin-β (ILβ), IL-6, tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), TGF-α, etc. Among them, TGF-β1 is an important influencing factor in fibrotic diseases. It is transduced through the TGF-β / Smads signaling pathway and dominates the early inflammatory response of wounds, wound healing, and the formation of pathological scars in the later stage. This pathway is regulated by various factors, such as type II TGF-β receptors (TGF-βRII), Smads family proteins, etc.Therefore, studying TGF-β / Smads signal transduction can lay a theoretical foundation for the prevention and treatment of clinical wound healing and pathological scars. During the proliferation phase, fibroblasts and capillaries infiltrate the wound site, promoting the proliferation of fibroblasts and the production of collagen fibers. As a result, granulation tissue is formed at the wound site during the proliferation phase. The granulation tissue formed during the proliferation phase will soon regress during the subsequent tissue remodeling and maturation phases and will ultimately be replaced by healed tissue. Although the fibrous repair of skin wounds restores the integrity of the skin and the barrier function of the epidermis, it mainly fills the skin defect through granulation tissue hyperplasia, and scars will inevitably be left after healing. The common scars produced after skin wound healing quickly enter the maturation phase. During this period, cells undergo apoptosis, collagen rearrangement, and a decrease in blood vessel density, and the scar color gradually fades and the texture softens. If the inflammatory response is excessive during the skin wound healing process, or the wound surface is mechanically tensioned, it may induce the formation of hypertrophic scars. The proliferation phase of hypertrophic scars is obvious. During this period, cell proliferation is active, a large amount of collagen is deposited, and a variety of growth factors (such as transforming growth factor β1, insulin-like growth factor 1, etc.) are abnormally highly expressed, resulting in scar elevation, red color, hard texture, and easy contracture, which can cause damage to the local appearance and function of patients and have serious psychological effects, which is a major clinical problem.

[0004] Traditional wound treatment methods mainly rely on physical barriers (such as dressings) and drugs (such as antibiotics), but these methods have limitations in promoting cell regeneration and tissue repair. Polypeptides are short-chain molecules formed by amino acids linked by peptide bonds, and have advantages such as small molecular weight, good biocompatibility, easy modification and functionalization. Polypeptides can participate in biological processes such as cell signal transduction, cell migration and proliferation by mimicking the functions of natural proteins, thereby promoting wound healing. In wound healing, polypeptides can create a favorable environment for wound healing by inhibiting the release of inflammatory mediators and reducing the inflammatory response at the wound site; some polypeptides have broad-spectrum antibacterial activity and can effectively inhibit wound infections and prevent the occurrence of complications; polypeptides can stimulate fibroblasts to synthesize collagen and enhance the mechanical strength and elasticity of wound tissue. Polypeptides can also reduce scar formation by regulating the activity of fibroblasts and the deposition of collagen. For example, some polypeptides can reduce scar formation by regulating the activity of fibroblasts and the deposition of collagen, such as inhibiting the excessive proliferation of fibroblasts and the abnormal deposition of collagen, which helps to reduce scar formation and improve the appearance of the skin after healing. Polypeptides show multi-faceted potential in promoting skin wound repair and prevention, from stimulating cell growth and migration to anti-infection, anti-inflammation, and promoting angiogenesis. These properties make polypeptides a promising candidate for future skin wound management and treatment.

[0005] At present, a variety of polypeptides have been developed and applied in the field of wound healing. For example, antimicrobial peptides (AMPs) are widely used for the prevention and treatment of wound infections; self-assembling polypeptides (SAPs) are used to construct bionic scaffolds to promote tissue regeneration. Antimicrobial peptides can effectively inhibit the microbial pathogen contamination of the wound surface in the initial stage of trauma. There are literature reports that bioactive peptides produced by the collagenase degradation matrix of Clostridium histolyticum can promote microvascular morphogenesis and in vitro wound healing. Among them, compared with serum, two peptides, Col4-1 and Comb1, can increase the proliferation rate of microvascular endothelial cells by 47% and the in vitro angiogenesis rate by 200%. There are also literature reports on Matrikines, that is, repair peptides based on the extracellular matrix (ECM), which can promote the formation of collagen in fibroblasts. Later, some scholars invented a polypeptide PA synthesized based on the simulation of heparin. The experimental results showed that PA has the characteristics of heparin promoting angiogenesis, and PA was made into a gel, which was shown to promote re-epithelialization in the treatment of streptozotocin (STZ)-induced diabetic mice. In addition, the GHK tripeptide can form a copper complex, which has anti-inflammatory and wound healing properties, improves skin density and firmness, and has a wide range of applications in the cosmetics industry.

[0006] With the development of biotechnology, the application prospect of polypeptides in wound healing is broad. However, the stability, bioavailability and large-scale production cost of polypeptides are still technical problems to be solved. There is an urgent need in this field to develop a drug that can effectively cure skin trauma. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, and provides a polypeptide combination trauma healing preparation for the use in treating skin diseases such as wounds, ulcers, burns, scars, keloids, etc. The polypeptide combination trauma healing preparation has high skin permeability and good stability, and has good antibacterial, anti-inflammatory, tissue repair, astringent healing and scar hyperplasia inhibition effects, is particularly suitable for wound healing, and reduces scar residue.

[0008] Specifically, the polypeptide combination trauma healing preparation comprises a polypeptide composition, a penetration enhancer, and a pharmaceutically acceptable excipient.

[0009] Preferably, the polypeptide composition comprises tripeptide-1, hexapeptide-9, and a palmitoyl-modified peptide complex.

[0010] Preferably, the palmitoyl-modified peptide complex is selected from one or a combination of palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and palmitoyl tetrapeptide-7.

[0011] Preferably, the penetration enhancer is chitosan, cyclodextrin, or a chitosan-cyclodextrin complex; more preferably a chitosan-cyclodextrin complex, and the mass ratio of chitosan to cyclodextrin is preferably 1:0.5.

[0012] Preferably, the polypeptide combination wound healing preparation is a gel preparation, and the pharmaceutically acceptable excipient is a gel excipient.

[0013] Preferably, the mass ratio of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex in the polypeptide composition is 1:0.1~0.5:0.1~1.

[0014] In a preferred embodiment, the mass ratio of acyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and palmitoyl tetrapeptide-7 in the palmitoyl-modified peptide complex is 1:0.5~1:0.1~0.5:0.1~0.5.

[0015] Preferably, the gel excipient includes a gel matrix, a humectant, an antioxidant, a pH regulator, and an aqueous solvent.

[0016] Preferably, the gel matrix is selected from one or a combination of carbomer, poloxamer, sodium carboxymethyl cellulose, alginate, chitosan, liquid paraffin-hydrogenated soybean phospholipid, colloidal silver-gelatin, and collagen; more preferably colloidal silver-gelatin.

[0017] Preferably, the humectant is one or several of glycerol, propylene glycol, hyaluronic acid, and butylene glycol; more preferably glycerol; its content is selected according to requirements and general knowledge in the art.

[0018] Preferably, the antioxidant is one or a combination of sulfite, cysteine, vitamin A, vitamin C, vitamin E, vitamin K, and potassium sorbate; more preferably one or a combination of cysteine and vitamin E; particularly preferably a composition of cysteine and vitamin E, and the mass ratio of cysteine to vitamin E is preferably 1:1~2.

[0019] Preferably, the pH regulator is one or a combination of triethanolamine and sodium hydroxide.

[0020] In a preferred embodiment, the present invention provides a polypeptide composition gel preparation. By weight percentage, the components of the gel preparation are 1~10% of the polypeptide composition, 5~15% of the penetration enhancer, 1~10% of the gel matrix, 5~15% of the humectant, 0.01~0.5% of the antioxidant, an appropriate amount of pH regulator, and an appropriate amount of deionized water.

[0021] Furthermore, the present invention provides a polypeptide composition gel preparation. By weight percentage, the components of the gel preparation are 5% of the polypeptide composition, 10% of the penetration enhancer, 2% of the gel matrix, 10% of the humectant, 0.3% of the antioxidant, an appropriate amount of pH regulator, and an appropriate amount of deionized water.

[0022] In a specific embodiment, the present invention provides a polypeptide composition gel, and the component contents are as shown in Table 1 below:

[0023] Table 1 Gel Prescription of Polypeptide Composition

[0024]

[0025] Among them, the pH value range of the gelling agent is 6 - 7.

[0026] On the other hand, the present invention provides a preparation method of a polypeptide combination wound healing preparation, which specifically comprises the following steps:

[0027] Step 1: Weigh the prescribed amount of the gelling agent matrix and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling;

[0028] Step 2: After adding the prescribed amount of the polypeptide composition to deionized water and dissolving it clearly, add a penetration enhancer and stir to mix, to prepare a mixed solution;

[0029] Step 3: While stirring, add the mixed solution of Step 2 to the gelling agent matrix of Step 1, and mix evenly;

[0030] Step 4: Add the prescribed amounts of the humectant and antioxidant to the mixed solution of Step 3 and mix well, add an appropriate amount of pH regulator to adjust the pH value to 6 - 7, add purified water to the full amount, stir evenly, to obtain the target product.

[0031] In a preferred embodiment, the preparation step of the chitosan - cyclodextrin complex comprises: adding chitosan to a hydrochloric acid solution, adding cyclodextrin to distilled water, pouring the two solutions into a beaker and mixing well, heating and raising the temperature to 60 °C, then slowly dripping a 25% glutaraldehyde solution into the beaker, starting constant temperature stirring, after the reaction ends, filtering the precipitate, washing with ethanol and distilled water, carrying out suction filtration, and drying the solid at 45 °C to obtain a brown powdery chitosan - cyclodextrin complex.

[0032] The present invention also provides a new use of a polypeptide composition for treating skin diseases related to skin wounds, for use in the preparation of products for the repair and regeneration of skin wounds (injuries), burns and scalds, chronic wound healing, and the repair and regeneration of diabetic foot ulcers, wherein the administration of this composition is at least once a day. In some embodiments, the administration of this composition is once, twice, three times or four times a day.

[0033] Among them, the skin diseases related to skin wounds are skin diseases or disorders such as acute wounds like cuts, abrasions, burns, and chronic wounds like pressure sores.

[0034] Gels have multiple advantages in wound healing, such as moisturizing, promoting healing, reducing infection, and alleviating pain. They are an efficient and easy-to-use treatment option. Gels can effectively keep the wound moist, accelerate healing, reduce scab formation and scarring. In terms of promoting healing, the moist environment helps cell migration and proliferation, accelerating tissue repair. Gels can act as a barrier to prevent bacteria from invading and reduce the risk of infection. The cooling and soothing properties of gels can relieve wound pain and discomfort. Gels are easy to apply and are suitable for irregular or difficult-to-treat areas. Gels do not adhere to the wound, reducing pain and tissue damage during dressing changes. Polypeptides have good biological properties, have good affinity with the tissues around the wound, can promote skin and nerve growth by inducing growth factors, facilitate the proliferation and repair of epithelial cells, promote wound healing, repair skin defects and tissue defects. The skin also has good absorption of active peptides, quickly combines them into its own collagen, thus forming normal connective tissue, filling and repairing the damaged skin, and reducing scar formation. Tripeptide-1 can stimulate the production of ECM, and at the same time can accelerate the metabolism of skin cells, promote the repair of damaged tissues, reduce the signs of scars and other skin injuries. Hexapeptide-9 can promote the production of type I, type IV collagen, integrin, laminin and keratin, and promote the regeneration of key components in the epidermal layer, epidermal-dermal junction layer and dermal layer. During the research process, it was found that the wound repair effect of using tripeptide-1 and hexapeptide-9 alone was not significant. The wound repair effect was improved when tripeptide-1 and hexapeptide-9 were used in combination, but the effect of inhibiting scar hyperplasia was not obvious. After adding palmitoyl-modified peptide, the combination of tripeptide-1, hexapeptide-9 and palmitoyl-modified peptide played a significant synergistic effect in wound healing, and at the same time achieved the effect of effectively preventing and inhibiting scar hyperplasia.

[0035] Advantages of the present invention:

[0036] The polypeptide combination wound healing preparation of the present invention utilizes the wound healing promoting activity of the composite polypeptide, and is compounded with specific penetration enhancers and antioxidants to prepare a gel with the functions of moisturizing, promoting healing, reducing infection and alleviating pain. It greatly increases skin penetration, significantly improves the retention dose in the skin, and at the same time has the effects of significant antibacterial and anti-inflammatory, removing necrotic tissue and promoting granulation, astringing and healing, and inhibiting scar hyperplasia. It is particularly suitable for wound healing and reduces scar residue. Description of the drawings

[0037] Figure 1 : The cell morphology was observed under a microscope after 24 hours of culture in the negative control group, the sample of Comparative Example 1, and the sample of Example 1 in the cell scratch repair test.

[0038] Figure 2 : The wound healing conditions after administration in the model control group, the administration group of Example 1, and the positive control group in the mouse skin wound healing test. Detailed implementation manners

[0039] The present invention will be further illustrated by the following examples. Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0040] In the specific implementation scheme, 100 g of a gel preparation was prepared according to the prescription, and its preparation method included: evenly spreading the gel matrix on the surface of an appropriate amount of deionized water, standing for swelling, mixing the polypeptide composition, penetration enhancer, moisturizer, and antioxidant in the prescribed amounts and stirring evenly, then adding them to the gel matrix and mixing evenly, adjusting the pH to obtain the target product. The specific examples are listed in detail as follows.

[0041] Example 1

[0042] Table 2 Gel Prescription of the Polypeptide Composition in Example 1

[0043]

[0044] The specific preparation process is as follows:

[0045] Step 1: Weigh the prescribed amount of colloidal silver-gelatin and evenly spread it on the surface of an appropriate amount of deionized water, and let it stand for swelling;

[0046] Step 2: Dissolve the prescribed amounts of Tripeptide-1, Hexapeptide-9, and palmitoyl-modified peptide complex in deionized water, then add the chitosan-cyclodextrin complex and stir evenly to prepare a mixed solution;

[0047] Step 3: While stirring, add the mixed solution from Step 2 to the gel matrix in Step 1 and mix evenly;

[0048] Step 4: Add the prescribed amounts of glycerol, cysteine, and vitamin E to the mixed solution in Step 3 and mix evenly. Adjust the pH value to 6-7 with triethanolamine, add deionized water to the full amount, and stir evenly to obtain the target product.

[0049] Example 2

[0050] Table 3 Gel Prescription of the Polypeptide Composition in Example 2

[0051]

[0052] The specific preparation process is as follows:

[0053] Step 1: Weigh the prescribed amount of colloidal silver-gelatin and evenly spread it on the surface of an appropriate amount of deionized water, and let it stand for swelling;

[0054] Step 2: Dissolve the prescribed amounts of Tripeptide-1, Hexapeptide-9, and palmitoyl-modified peptide complex in deionized water, then add the chitosan-cyclodextrin complex and stir evenly to prepare a mixed solution;

[0055] Step 3: While stirring, add the mixture from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0056] Step 4: Add the prescribed amounts of glycerol, cysteine, and vitamin E to the mixture from Step 3, mix well, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0057] Example 3

[0058] Table 4 Gel Prescription of the Polypeptide Composition in Example 3

[0059]

[0060] The specific preparation process is as follows:

[0061] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling.

[0062] Step 2: After dissolving the prescribed amounts of tripeptide - 1, hexapeptide - 9, and palmitoyl - modified peptide complex in deionized water, add the chitosan - cyclodextrin complex and stir well to prepare a mixed solution.

[0063] Step 3: While stirring, add the mixture from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0064] Step 4: Add the prescribed amounts of glycerol and vitamin E to the mixture from Step 3, mix well, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0065] Example 4

[0066] Table 5 Gel Prescription of the Polypeptide Composition in Example 4

[0067]

[0068] The specific preparation process is as follows:

[0069] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling.

[0070] Step 2: After dissolving the prescribed amounts of tripeptide - 1, hexapeptide - 9, and palmitoyl - modified peptide complex in deionized water, add the chitosan - cyclodextrin complex and stir well to prepare a mixed solution.

[0071] Step 3: While stirring, add the mixture from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0072] Step 4: Add the prescribed amount of glycerol and cysteine to the mixture obtained in Step 3, mix well, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0073] Example 5

[0074] Table 6 Gel Prescription of the Polypeptide Composition in Example 5

[0075]

[0076] The specific preparation process is as follows:

[0077] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling.

[0078] Step 2: Dissolve the prescribed amount of Tripeptide - 1, Hexapeptide - 9, and palmitoyl - modified peptide complex in deionized water, then add chitosan and stir well to prepare a mixed solution.

[0079] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0080] Step 4: Add the prescribed amount of glycerol and cysteine to the mixture obtained in Step 3, mix well, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0081] Comparative Example 1

[0082] Table 7 Gel Prescription of the Polypeptide Composition in Comparative Example 1

[0083]

[0084] The specific preparation process is as follows:

[0085] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling.

[0086] Step 2: Dissolve the prescribed amount of Tripeptide - 1 in deionized water, then add chitosan - cyclodextrin complex and stir well to prepare a mixed solution.

[0087] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0088] Step 4: Add the prescribed amount of glycerol, cysteine, and vitamin E to the mixture obtained in Step 3, mix well, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0089] Comparative Example 2, Preparation of the Polypeptide Composition

[0090] Table 8 Gel Prescription of Polypeptide Composition in Comparative Example 2

[0091]

[0092] The specific preparation process is as follows:

[0093] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling;

[0094] Step 2: After dissolving the prescribed amounts of Tripeptide - 1 and Hexapeptide - 9 in deionized water, add chitosan - cyclodextrin complex, and stir to mix evenly to prepare a mixed solution;

[0095] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1, and mix evenly;

[0096] Step 4: Add the prescribed amounts of glycerol, cysteine, and vitamin E to the mixed solution in Step 3, mix evenly, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full amount, and stir evenly to obtain the target product.

[0097] Comparative Example 3, Preparation of Polypeptide Composition

[0098] Table 9 Gel Prescription of Polypeptide Composition in Comparative Example 3

[0099]

[0100] The specific preparation process is as follows:

[0101] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand for swelling;

[0102] Step 2: After dissolving the prescribed amounts of Tripeptide - 1 and Hexapeptide - 9 in deionized water, add azone and stir to mix evenly to prepare a mixed solution;

[0103] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1, and mix evenly;

[0104] Step 4: Add the prescribed amounts of glycerol, cysteine, and vitamin E to the mixed solution in Step 3, mix evenly, adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full amount, and stir evenly to obtain the target product.

[0105] Comparative Example 4

[0106] Table 10 Gel Prescription of Polypeptide Composition in Comparative Example 4

[0107]

[0108] The specific preparation process is as follows:

[0109] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, then let it stand for swelling.

[0110] Step 2: Dissolve the prescribed amounts of Tripeptide - 1, Hexapeptide - 9, and palmitoyl - modified peptide complex in deionized water. After clarification, add the chitosan - cyclodextrin complex and stir well to prepare a mixed solution.

[0111] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0112] Step 4: Add the prescribed amounts of glycerol and potassium sorbate to the mixed solution in Step 3 and mix well. Adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0113] Comparative Example 5

[0114] Table 11 Gel Prescription of the Polypeptide Composition in Comparative Example 5

[0115]

[0116] The specific preparation process is as follows:

[0117] Step 1: Weigh the prescribed amount of colloidal silver - gelatin and evenly sprinkle it on the surface of an appropriate amount of deionized water, then let it stand for swelling.

[0118] Step 2: Dissolve the prescribed amounts of Tripeptide - 1, Hexapeptide - 9, and carnosine in deionized water. After clarification, add the chitosan - cyclodextrin complex and stir well to prepare a mixed solution.

[0119] Step 3: While stirring, add the mixed solution from Step 2 to the gelling agent matrix in Step 1 and mix evenly.

[0120] Step 4: Add the prescribed amounts of glycerol and potassium sorbate to the mixed solution in Step 3 and mix well. Adjust the pH value to 6 - 7 with triethanolamine, add deionized water to the full volume, and stir evenly to obtain the target product.

[0121] Verification Example:

[0122] Skin Permeability Experiment

[0123] Using excised rat skin as the skin model material, the Franz diffusion cell and HPLC method were applied to determine the transdermal amount of the polypeptide composition at different time points.

[0124] Preparation of excised rat skin: Decapitate the rat, carefully remove the hair of the rat with an appropriate amount of depilatory, rinse it thoroughly with physiological saline, cut off the skin, carefully peel off the fat layer, select the intact skin, place it in physiological saline, and store it at 4°C in the refrigerator for later use.

[0125] Transdermal test: The Franz transdermal tester was used for the transdermal test. The rat skin was fixed at the release port of the Franz diffusion cell, and the pH 7.4 phosphate buffer solution was added to the receiving chamber as the release medium to keep the endodermis in close contact with the solution. A quantitative gel (about 10 mg of the polypeptide composition) was taken, and the sample was evenly spread radially from the center of the membrane to the edge. The water bath temperature of the diffusion cell was 37 ± 0.5 。 °C, and the stirring speed was 450 r·min -1 . Samples were taken and measured at 2 h, 4 h, 8 h, 12 h, and 24 h respectively.

[0126] Determination method of polypeptide content: Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 HILIC-Z (100 mm × 3.0 mm, 2.7 μm); Column temperature: 30 °C; Mobile phase flow rate: 0.3 mL / min; Mobile phase: Phase A: 15 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid), Phase B: Acetonitrile (containing 0.1% formic acid); The elution program was (0 min, 10% of Phase A and 90% of Phase B; 1 min, 10% of Phase A and 90% of Phase B; 8 min, 50% of Phase A and 50% of Phase B; 12 min, 50% of Phase A and 50% of Phase B; 12.1 min, 10% of Phase A and 90% of Phase B; 17 min, 10% of Phase A and 90% of Phase B).

[0127] Calculation of residence amount: The cumulative permeation amount of polypeptide per unit area (total content of each peptide) was calculated according to the formula: Q r = C i × V i / S; where Q r (μg·cm -2 ) represents the cumulative permeation amount of polypeptide per unit area; C i (μg·mL -1 ) represents the polypeptide concentration in the release medium at time t; V i (mL) represents the volume of the extraction solution (5.0 mL); S (cm 2 ) represents the exposed area of the diffusion cell to the ex vivo skin (1.77 cm 2 ).

[0128] Table 12 Test results of polypeptide permeation amount in rat skin

[0129]

[0130] The above results show that the polypeptide composition gel of the present application has good skin permeability. The addition of chitosan and cyclodextrin to the polypeptide composition gel has a permeation-promoting effect, especially when chitosan and cyclodextrin are used in combination, the permeation-promoting effect is more obvious.

[0131] Cell scratch repair experiment

[0132] Plating: Select human fibroblasts HFF in the logarithmic growth phase. After digestion and collection of the cells, add them to DMEM medium containing 10% FBS to make a cell suspension. Adjust the cell concentration to 5x10 5 cells / mL, inoculate into 24-well plates at 1 mL / well. Culture the cells in 5% CO2 at 37 °C for 24 h to allow the cells to recover and adhere. Observe the cell status under a microscope. If the status is good, proceed to the next experiment.

[0133] Scratching: When the plating rate of the cells in the 24-well plate reaches about 80%, use a 200 μL sterile pipette tip to perform scratching operations on the sample group and the control group. After scratching, gently wash twice with PBS, add 2 mL of the corresponding sample solution according to the group (calculated as the polypeptide composition, prepared with buffer at 0.1 mg / mL), and culture in an incubator at 37 °C and 5% CO2 for 24 h. Observe and photograph the cell morphology at 0 h and 24 h under a microscope.

[0134] Use Image J software for image statistical area analysis. The formula for calculating the scratch healing rate is:

[0135] ;

[0136] Table 13 Cell scratch healing rate

[0137]

[0138] According to the above results, it can be seen that the relative scratch healing rate of the composition of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide in this application can be as high as 85.74%, and it has a good promoting and repairing function for damaged skin.

[0139] Animal experiment on wound healing effect

[0140] Animal wound modeling: Take several mice with a body weight of about 25 g and fast them for 12 hours. After anesthetizing each mouse with 300 mg / kg chloral hydrate, depilate and disinfect the back skin. Use a punch with a diameter of 8 mm to make a round hole on the back of the mouse, cut the full-thickness skin, avoid large arteries and veins, and cut down to the fascia. Perform the operation under sterile conditions, then immediately rinse with sterile saline, stop bleeding thoroughly, and raise the mice individually in cages after they wake up. None of the mice died during the operation, and their postoperative reactions were normal, without adverse reactions such as diarrhea, and the wounds were not infected. After animal modeling, there was a little tissue fluid exudation and tissue edema in the wound tissue, and no animal infection or accidental death occurred during the healing process.

[0141] Grouping and administration: A number of successfully modeled mice were randomly divided into groups, namely a model control group and a model drug administration group, with 6 mice in each group, half male and half female. Among them, the model control group was not given any drug and was not treated. One group of the model drug administration group was smeared with 0.1 g of recombinant human epidermal growth factor gel (rhEGF) once a day, and the wound healing and scar formation were observed for 21 consecutive days. One group of the model drug administration group was smeared with 0.2 g of the polypeptide composition gels of Examples 1-5 and Comparative Examples 1-5 once a day, and the wound healing and scar formation were observed for 21 consecutive days.

[0142] Determination of skin wound healing rate: The wound healing situation was photographed and recorded. Starting from the excision wound healing model as 0 d, photographs were taken and recorded on the 4th, 7th, 14th, and 21st days thereafter. The Image J image processing software was used to analyze and calculate the wound healing rate, observe the scab falling time, and the formation of wound scars. The formula for calculating the wound healing rate is:

[0143] ;

[0144] Histopathological analysis of skin wound tissue: On the 21st day after the operation, the mice were euthanized, and full-thickness skin tissues about 5 mm around the wound and its edge were taken. After being fixed in 10% neutral buffered formalin solution for 24 h, paraffin sections were made and HE tissue staining was performed to measure the epidermal thickness index and the epidermal thickness ratio. The calculation formulas are:

[0145] ;

[0146] ;

[0147] The closer the epidermal thickness index value is to 1, the closer the epidermal layer is to normal skin. The closer the epidermal thickness ratio value is to 1 numerically, the more uniform, flat, and smooth the mouse epidermis is restored.

[0148] Table 14 Wound healing rate of mice

[0149]

[0150] In the mouse wound healing test, during the healing process, the granulation tissue regeneration at the wound was good, with a bright red color, and then covered by a thin layer of epithelium. On the 1st day after injury, there was more exudate at the wound, and the inflammatory reaction was obvious. On the 4th day after injury, the skin trauma in each group showed a general trend of healing, the wound skin had scabs, and granulation tissue had begun to form. Compared with the model control group, the healing effect of the polypeptide composition gel of the present application was obvious. During the period from 4 to 21 days after injury, the wound healing rate of the mice given the polypeptide composition gel of the present application was significantly higher than that of the model control group. The wound appearance of the mouse group given the polypeptide composition gel of the present application on the 21st day of administration improved significantly, with a smooth and flat surface. However, obvious scars were formed in the model control group.

[0151] Table 15 Test Results of Mouse Epidermal Thickness Index and Epidermal Thickness Ratio

[0152]

[0153] The test results of the mouse epidermal thickness index and epidermal thickness ratio showed that, 21 days after injury, the epidermis of the wound surface in the polypeptide composition gel agent administration group and the rhEGF administration group of this application became thinner and flatter. In the model control group, the epidermis of the mice was significantly uneven and thickened, with a tongue-like protrusion at the edge, similar to keloid. The epidermal thickness index showed that the epidermal thickness indexes of the mice in the administration group of Example 1 and the rhEGF gel administration group on the 21st day were 1.25±0.22 and 1.82±0.27 respectively, which were significantly lower than 5.34±0.37 of the model control group. The gel agent of only a single polypeptide (the epidermal thickness index of Comparative Example 1 was 3.48±0.64) and the gel agent combined with tripeptide-1

[0154] The gel agent combined with hexapeptide-9 (the epidermal thickness index of Comparative Example 2 was 2.85±0.52) had an effect of inhibiting scar hyperplasia. The combined polypeptide combination gel with palmitoyl-modified peptide played a significant synergistic effect in wound healing and achieved an effective effect of preventing and inhibiting scar hyperplasia. The epidermal thickness ratios of the mice in the polypeptide composition gel agent administration group and the rhEGF administration group of this application were significantly greater than those of the mice in the model control group, indicating that the polypeptide composition gel agent of this application had a good effect of promoting the epidermis to recover evenly, flatly and smoothly.

[0155] It should be noted here that the embodiments of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Therefore, simple improvements to the present invention under the premise of the method of the present invention fall within the scope of protection of the present invention.

Claims

1. A polypeptide combination wound healing preparation, characterized in that, Use for preparing drugs and cosmetics for treating traumatic skin diseases. Among them, the polypeptide combination wound healing preparation is a gel preparation. By weight percentage, the components of the gel preparation are 1-10% polypeptide composition, 5-15% penetration enhancer, 1-10% gel matrix, 5-15% humectant, 0.01-0.5% antioxidant, appropriate pH regulator, and appropriate deionized water; Among them, the polypeptide composition contains tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex; the mass ratio of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex in the polypeptide composition is 1:0.1-0.5:0.1-1; The palmitoyl-modified peptide complex consists of palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and palmitoyl tetrapeptide-7, and the mass ratio of palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and palmitoyl tetrapeptide-7 is 1:0.5-1:0.1-0.5:0.1-0.5; The penetration enhancer is a chitosan-cyclodextrin complex, and the mass ratio of chitosan to cyclodextrin is 1:0.5; The antioxidant is a cysteine-vitamin E composition, and the mass ratio of cysteine to vitamin E is 1:1-2.

2. The polypeptide combination wound healing preparation according to claim 1, characterized in that, The gel matrix is selected from one or a combination of carbomer, poloxamer, sodium carboxymethyl cellulose, alginate, chitosan, liquid paraffin-hydrogenated soybean phospholipid, colloidal silver-gelatin, and collagen.

3. The polypeptide combination wound healing preparation according to claim 1, characterized in that The humectant is one or several of glycerol, propylene glycol, hyaluronic acid, and butanediol.

4. The polypeptide combination wound healing preparation according to any one of claims 1-3, characterized in that, Use for preparing drugs or cosmetics for treating traumatic skin diseases related to wounds, ulcers, scars, and bumps.

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