Polypeptide combined wound healing preparation with high permeability

Abstract

The invention discloses a polypeptide combination wound healing preparation with high permeability, and particularly relates to a polypeptide combination formed by combining tripeptide-1, hexapeptide-9 and palmitoyl modified peptide, new application of the polypeptide combination to treatment of skin diseases such as wounds, ulcers, burns, scars and gnoids, and the polypeptide combination plays a remarkable synergistic effect on wound healing and has a good application prospect. The effect of effectively preventing and inhibiting scar hyperplasia is achieved; besides, the polypeptide combined wound healing preparation utilizes the wound healing promoting activity of the composite polypeptide and is compounded with a specific penetration enhancer and an antioxidant to prepare the gel with the functions of moisturizing, promoting healing, reducing infection and relieving pain, the skin penetration is greatly increased, the resident dosage of the skin is remarkably increased, and meanwhile the wound healing effect is improved. The ointment has remarkable effects of resisting bacteria, diminishing inflammation, removing slough, promoting granulation, astringing, healing and inhibiting scar hyperplasia, and is particularly suitable for wound healing and reducing scar residues.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide application, and specifically relates to the use of a polypeptide composition as a wound healing agent in treating wounds, ulcers, burns, scars, lumps and other skin diseases. Background Art

[0002] Skin trauma has always been an important topic in surgical research, and the search for drugs that promote skin trauma has been the focus of attention of countless experts and scholars. With the rapid development of molecular biology, basic research on trauma repair has already reached the molecular and genetic levels.

[0003] The healing process of skin and other wounds (specifically, acute wounds such as cuts, abrasions, burns, and chronic wounds such as bedsores) is a complex dynamic process, which 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, which can be divided into three stages: hemostasis and inflammation, proliferation, and remodeling. The three processes are intertwined rather than independent of each other. The hemostasis and inflammation stage is the first process initiated after tissue injury. The reactions that occur include hemodynamic changes, increased vascular permeability, and the infiltration and phagocytosis of neutrophils and monocytes. The cells involved in the hemostasis and inflammation response at the site of injury are mainly platelets, neutrophils, and macrophages. The proliferation stage 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 is promoted by related signals such as nitric oxide, EGF, and KGF. Granulation tissue is a highly proliferative connective tissue composed of cells (mainly fibroblasts and macrophages), extracellular mediators, and capillaries. In the process of wound healing, granulation tissue has the functions of resisting infection and protecting the wound surface, filling wounds and other tissue defects, and organizing blood clots and necrotic tissue. The remodeling period is the process of transformation of granulation tissue into scar tissue. In this process, collagen is constantly renewed, the content of type I collagen increases significantly, the number of various cells in granulation tissue, including myofibroblasts, decreases significantly, and the capillary network gradually disappears. Remodeling will bring about improvements in 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 work together to play a repairing role, including pro-inflammatory factors such as interleukin-β (ILβ), IL-6, tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor alpha (TGF-β), TGF-α, etc. Among them, TGF-β1 is an important influencing factor of fibrotic diseases. It is transduced through the TGF-β / Smads signaling pathway, leading to the early inflammatory response of trauma, wound healing and the formation of pathological scars in the later stage. This pathway is regulated by multiple factors, such as TGF-β type II receptors (TGF-βRII), Smads family proteins, etc.Therefore, studying TGF-β / Smads signal transduction can lay a theoretical foundation for clinical wound healing and the prevention and treatment of pathological scars. During the proliferation period, fibroblasts and capillaries infiltrate the wound site, which promotes the proliferation of fibroblasts and the generation of collagen fibers. As a result, granulation tissue is formed at the wound site during the proliferation period. The granulation tissue formed during the proliferation period will soon retract in the subsequent tissue reconstruction and maturation periods, and will eventually be replaced by healing 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 defects through the proliferation of granulation tissue, and scars are inevitably left after healing. Ordinary scars produced after skin wound healing quickly enter the maturation period after formation, during which cells undergo apoptosis, collagen rearrangement, and vascular density decreases, and the scar color gradually becomes lighter and the texture becomes softer. If the inflammatory response is excessive during the healing process of skin wounds, or the wound surface is subjected to mechanical tension, it may induce the formation of hypertrophic scars. Hypertrophic scars have a distinct proliferative phase, during which cells proliferate actively, collagen is deposited in large quantities, and multiple growth factors (such as transforming growth factor β1, insulin-like growth factor 1, etc.) are abnormally highly expressed, causing the scar to bulge, become red, become hard, and become prone to contracture. This can lead to impaired local appearance and function of the patient and have serious psychological effects, posing 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. Peptides are short-chain molecules formed by amino acids connected by peptide bonds, with advantages such as small molecular weight, good biocompatibility, easy modification and functionalization. Peptides can promote wound healing by mimicking the functions of natural proteins and participating in biological processes such as cell signaling, cell migration and proliferation. In wound healing, peptides can inhibit the release of inflammatory mediators, reduce inflammatory responses at the wound site, and create a favorable environment for wound healing; some peptides have broad-spectrum antibacterial activity, which can effectively inhibit wound infection and prevent complications; peptides can stimulate fibroblasts to synthesize collagen and enhance the mechanical strength and elasticity of wound tissue. Peptides can also reduce scar formation by regulating the activity of fibroblasts and the deposition of collagen. For example, some peptides 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. Peptides have shown a variety of potentials in promoting skin wound repair and prevention, from stimulating cell growth and migration to anti-infection, anti-inflammation and promoting angiogenesis, etc. These properties make peptides a powerful candidate for future skin wound management and treatment.

[0005] At present, a variety of peptides have been developed and applied in the field of wound healing. For example, antimicrobial peptides (AMPs) are widely used to prevent and treat wound infections; self-assembling peptides (SAPs) are used to construct biomimetic scaffolds to promote tissue regeneration. Antimicrobial peptides can effectively inhibit the microbial pathogen contamination of the wound surface in the early stage of trauma. It has been reported that the collagenase degradation of Clostridium histolyticum matrix produces bioactive peptides that can promote the occurrence of microvascular morphology and in vitro wound healing. Among them, Col4-1 and Comb1 peptides can increase the proliferation rate of microvascular endothelial cells by 47% and the in vitro angiogenesis rate by 200% compared with serum. There are also literature reports that Matrikines, which are repair peptides based on the extracellular matrix (ECM), can promote the formation of collagen in fibroblasts. Later, some scholars invented a peptide PA based on the synthesis of simulated 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 diabetic mice induced by streptozotocin (STZ). In addition, GHK tripeptide can form copper complexes, has anti-inflammatory and wound healing properties, improves skin density and firmness, and is widely used in the cosmetics industry.

[0006] With the development of biotechnology, the application prospects of peptides in wound healing are broad. However, the stability, bioavailability and large-scale production cost of peptides are still technical problems that need to be solved. There is an urgent need to develop a drug that can effectively heal skin wounds. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a polypeptide combination wound healing preparation for the treatment of wounds, ulcers, burns, scars, pimples and other skin diseases. The polypeptide combination wound healing preparation has high skin permeability and good stability, and has good antibacterial and anti-inflammatory, tissue repair, astringent healing, and scar hyperplasia inhibition effects. It is particularly suitable for wound healing and reducing scar residues.

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

[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, it is a chitosan-cyclodextrin complex, wherein the mass ratio of chitosan to cyclodextrin is preferably 1:0.5.

[0012] Preferably, the polypeptide combination wound healing preparation is a gel, 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 palmitoyl 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 auxiliary material comprises a gel matrix, a moisturizer, an antioxidant, a pH regulator, and a water 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 lecithin, colloidal silver-gelatin, and collagen; more preferably, colloidal silver-gelatin.

[0017] Preferably, the moisturizing agent is one or more of glycerin, propylene glycol, hyaluronic acid, and butylene glycol, and glycerin is more preferably used; the content of the moisturizing agent is selected according to demand and common 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; further preferably, it is one or a combination of cysteine ​​and vitamin E; particularly preferably, it is a combination of cysteine ​​and vitamin E, wherein the mass ratio of cysteine ​​to vitamin E is preferably 1:1~2.

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

[0020] In a preferred embodiment, the present invention provides a polypeptide composition gel preparation, wherein the components of the gel preparation are, by weight percentage, 1-10% of the polypeptide composition, 5-15% of the penetration enhancer, 1-10% of the gel matrix, 5-15% of the moisturizer, 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, wherein the components of the gel preparation are, by weight percentage, 5% of the polypeptide composition, 10% of a penetration enhancer, 2% of a gel matrix, 10% of a moisturizer, 0.3% of an antioxidant, an appropriate amount of a pH regulator, and an appropriate amount of deionized water.

[0022] In a specific embodiment, the present invention provides a polypeptide composition gel, the components of which are as shown in Table 1: Table 1 Gel formulation of peptide composition

[0023] Among them, the pH value of the gelling agent ranges from 6 to 7.

[0024] In another aspect, the present invention provides a method for preparing a polypeptide combination wound healing preparation, which specifically comprises the following steps: Step 1: Weigh the prescribed amount of gel matrix and evenly sprinkle it on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of the polypeptide composition is dissolved in deionized water, a penetration enhancer is added and stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of moisturizer and antioxidant to the mixed solution in step 3 and mix well. Add an appropriate amount of pH adjuster to adjust the pH value to 6-7. Add purified water to a sufficient amount and stir well to obtain the target product.

[0025] In a preferred embodiment, the preparation steps of the chitosan-cyclodextrin complex include: adding chitosan to a hydrochloric acid solution, adding cyclodextrin to distilled water, pouring the two solutions into a beaker and mixing them, heating to 60°C, slowly adding a 25% glutaraldehyde solution to the beaker, starting constant temperature stirring, filtering the precipitate after the reaction is completed, washing with ethanol and distilled water, and filtering with suction. The solid is dried at 45°C to obtain a brown powder chitosan-cyclodextrin complex.

[0026] The present invention further provides a new use of a polypeptide composition for treating skin diseases related to skin trauma, which is used for preparing products for repairing and regenerating skin trauma (injury), healing of burns and scalds, chronic wounds, and repairing and regenerating diabetic foot ulcers, wherein the composition is administered at least once a day. In some embodiments, the composition is administered once, twice, three times, or four times a day.

[0027] Among them, skin diseases related to skin trauma are skin diseases or conditions caused by acute trauma such as cuts, abrasions, burns, and chronic trauma such as bedsores.

[0028] Gels have multiple advantages in wound healing, such as moisturizing, promoting healing, reducing infection, and relieving pain. They are an efficient and easy-to-use treatment option. Gels can effectively keep wounds moist, accelerate healing, and reduce scab and scar formation. In terms of promoting healing, a moist environment helps cell migration and proliferation, and accelerates tissue repair. Gels can act as a barrier to prevent bacterial invasion and reduce the risk of infection. The cooling and soothing properties of gels can reduce wound pain and discomfort. Gels are easy to apply and are suitable for irregular or difficult-to-handle areas. Gels do not adhere to wounds, reducing pain and tissue damage during dressing changes. Peptides have good biological properties and good affinity with tissues around the wound. They can promote skin and nerve growth by inducing growth factors, facilitate the proliferation and repair of epithelial cells, promote wound healing, and repair skin and tissue defects. The skin also has a good absorption effect on active peptides, which quickly combine into its own collagen to form normal connective tissue, fill and repair damaged skin, and reduce scar formation. Tripeptide-1 can stimulate the production of ECM, accelerate the metabolism of skin cells, promote the repair of damaged tissues, and reduce scars and other signs of skin damage. Hexapeptide-9 can promote the production of type I and IV collagen, integrins, laminin and keratin, and promote the regeneration of key components of the epidermis, epidermal junction layer, and dermis. The research process found that the wound repair effect of tripeptide-1 and hexapeptide-9 alone was not significant. The wound repair effect of tripeptide-1 and hexapeptide-9 was improved, but the scar hyperplasia inhibition effect was not obvious. After adding palmitoyl modified peptide, tripeptide-1, hexapeptide-9, and palmitoyl modified peptide combined, which played a significant synergistic role in wound healing and achieved effective prevention and inhibition of scar hyperplasia.

[0029] Beneficial effects of the present invention: The polypeptide combination wound healing preparation of the present invention utilizes the wound healing 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 relieving pain. It greatly increases skin penetration and significantly improves the resident dose of the skin. At the same time, it has significant antibacterial and anti-inflammatory effects, removes dead tissue and promotes tissue regeneration, astringes and heals, and inhibits scar hyperplasia. It is particularly suitable for wound healing and reduces scar residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : In the cell scratch repair test, the cell morphology of the negative control group, the sample of comparative example 1, and the sample of embodiment 1 was observed under a microscope after 24 hours of culture.

[0031] Figure 2 : Wound healing conditions of the model control group, the drug-treated group in Example 1, and the positive control group after drug administration in the mouse skin wound healing test. DETAILED DESCRIPTION

[0032] The present invention is further described below by way of examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial sources unless otherwise specified.

[0033] In a specific embodiment, 100 g of a gel preparation is prepared according to a prescription, and the preparation method includes: evenly sprinkling a gel matrix on the surface of an appropriate amount of deionized water, allowing it to swell, mixing and stirring the prescribed amount of a polypeptide composition, a penetration enhancer, a moisturizer, and an antioxidant, and then adding the mixture to the gel matrix and mixing, adjusting the pH to obtain the target product. Detailed examples are listed below.

[0034] Example 1 Table 2 Gel formulation of the polypeptide composition of Example 1

[0035] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol, cysteine ​​and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0036] Example 2 Table 3 Gel formulation of the polypeptide composition of Example 2

[0037] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol, cysteine ​​and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0038] Example 3 Table 4 Gel formulation of the polypeptide composition of Example 3

[0039] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0040] Example 4 Table 5 Gel formulation of polypeptide composition of Example 4

[0041] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol and cysteine ​​to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0042] Example 5 Table 6 Gel formulation of the polypeptide composition of Example 5

[0043] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: Add the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex into deionized water to dissolve, then add chitosan, stir and mix to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol and cysteine ​​to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0044] Comparative Example 1 Table 7 Gel formulation of polypeptide composition of Comparative Example 1

[0045] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: Add the prescribed amount of tripeptide-1 into deionized water to dissolve it, then add the chitosan-cyclodextrin complex, stir and mix well to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol, cysteine ​​and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0046] Comparative Example 2, Preparation of Peptide Composition Table 8 Gel prescription of polypeptide composition of comparative example 2

[0047] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: Add the prescribed amount of tripeptide-1 and hexapeptide-9 into deionized water to dissolve, then add chitosan-cyclodextrin complex, stir and mix to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol, cysteine ​​and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0048] Comparative Example 3, Preparation of Peptide Composition Table 9 Gel prescription of polypeptide composition of comparative example 3

[0049] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: Add the prescribed amount of tripeptide-1 and hexapeptide-9 into deionized water to dissolve, then add azone and stir to mix to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount of glycerol, cysteine ​​and vitamin E to the mixed solution in step 3 and mix well. Adjust the pH value to 6-7 with triethanolamine. Add deionized water to a sufficient amount and stir well to obtain the target product.

[0050] Comparative Example 4 Table 10 Gel formulation of polypeptide composition of Comparative Example 4

[0051] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptide complex are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount 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 a sufficient amount and stir well to obtain the target product.

[0052] Comparative Example 5 Table 11 Gel formulation of polypeptide composition of Comparative Example 5

[0053] The specific preparation process is as follows: Step 1: Weigh the prescribed amount of colloidal silver-gelatin and sprinkle it evenly on the surface of an appropriate amount of deionized water, and let it stand to swell; Step 2: After the prescribed amount of tripeptide-1, hexapeptide-9 and carnosine are dissolved in deionized water, chitosan-cyclodextrin complex is added, and the mixture is stirred to prepare a mixed solution; Step 3: Add the mixed solution of step 2 to the gel matrix of step 1 while stirring, and mix well; Step 4: Add the prescribed amount 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 a sufficient amount and stir well to obtain the target product.

[0054] Verification example: Skin permeability test Using ex vivo mouse skin as skin model material, Franz diffusion cell and HPLC were used to determine the transdermal amount of the peptide composition at different time points.

[0055] Preparation of ex vivo mouse skin: The rats were killed by cervical dislocation, the hair of the rats was carefully removed with an appropriate amount of depilatory agent, the skin was rinsed with physiological saline, the fat layer was carefully peeled off, the intact skin was selected, placed in physiological saline, and stored in a refrigerator at 4°C for later use.

[0056] Transdermal test: Franz transdermal tester was used for transdermal test. The rat skin was fixed to the release port of Franz diffusion cell. pH 7.4 phosphate buffer was added to the receiving chamber as the release medium to keep the endothelial layer in close contact with the solution. A quantitative gel (calculated as polypeptide composition, containing about 10 mg of polypeptide composition) was taken and the sample was spread evenly from the center of the membrane to the edge in a radial direction. The water bath temperature of the diffusion cell was 37 ± 0.5 。 C, stirring speed 450 r·min -1 , samples were taken and measured at 2 h, 4 h, 8 h, 12 h and 24 h respectively.

[0057] Peptide content determination method: Chromatographic conditions: Chromatographic column: Agilent Poroshell 120HILIC-Z (100mm×3.0mm, 2.7μm); Chromatographic column temperature: 30℃; Mobile phase flow rate: 0.3mL / min; Fluidity: Phase A 15mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid), Phase B: acetonitrile (containing 0.1% formic acid); Elution program (0min, Phase A 10%, Phase B 90%; 1min, Phase A 10%, Phase B 90%; 8min, Phase A 50%, Phase B 50%; 12min, Phase A 50%, Phase B 50%; 12.1min, Phase A 10%, Phase B 90%; 17min, Phase A 10%, Phase B 90%).

[0058] Calculation of retention volume: The cumulative permeation volume of peptides per unit area (the sum of the content of each peptide) is calculated according to the formula: Q r = C i ×V i / S; where Q r (μg·cm -2 ) represents the cumulative amount of polypeptide permeation per unit area; C i (μg·mL -1 ) represents the peptide concentration in the release medium within time t; V i (mL) represents the volume of the extraction solution (5.0 mL); S (cm 2 ) represents the area of ​​the diffusion cell exposed to the ex vivo skin (1.77 cm 2 ).

[0059] Table 12 Results of peptide permeation test in mice skin

[0060] The above results show that the polypeptide composition gel of the present application has good skin permeability. Adding chitosan and cyclodextrin to the polypeptide composition gel has a permeation-promoting effect, especially the combination of chitosan and cyclodextrin has a better permeation-promoting effect.

[0061] Cell scratch repair assay Plating: Human fibroblasts HFF in the logarithmic growth phase were selected, digested and collected, and added to DMEM medium containing 10% FBS to make a cell suspension. The cell concentration was adjusted to 5x10 5 Cells / mL were inoculated into 24-well plates at 1 mL / well. The cells were cultured at 5% CO2 and 37°C for 24 h to allow the cells to recover and adhere to the wall. The cells were observed under a microscope to see if they were in good condition. If so, the next step of the experiment was carried out.

[0062] Scratch: When the cell plating rate in the 24-well plate reaches about 80%, use a 200μL sterile pipette tip to scratch the sample group and the control group. After the scratch, gently wash twice with PBS, add 2ml of the corresponding sample solution (calculated as peptide composition, buffer solution is prepared at 0.1 mg / ml) according to the group, and place in a 37℃, 5% CO2 incubator for 24 hours. Observe the cell morphology at 0h and 24h under a microscope and take pictures.

[0063] Image J software was used to perform statistical area analysis on the images, and the scratch healing rate was calculated as follows: ; Table 13 Cell scratch healing rate

[0064] According to the above results, the relative healing rate of cell scratches of the synergistic composition of tripeptide-1, hexapeptide-9 and palmitoyl-modified peptide in the present application can be as high as 85.74%, which has a good function of promoting the repair of damaged skin.

[0065] Animal experiments on wound healing effects Animal trauma modeling: Take several mice weighing about 25g, fast for 12 hours, anesthetize each mouse with 300 mg / kg chloral hydrate, remove hair and disinfect the back skin, use a 8 mm diameter puncher to make a round hole on the back of the mouse, cut the full layer of skin, avoid the arteries and veins, deep to the fascia, and perform surgery under sterile conditions. Immediately rinse with sterile saline to stop bleeding, and raise in a single cage after waking up. All mice did not die during the operation, and the postoperative reaction was normal, with no adverse reactions such as diarrhea, and no infection in the wound. 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.

[0066] Grouping and administration: Several mice with successful modeling were randomly divided into a model control group and a model administration group, with 6 mice in each group, half of which were male and half were female; among them, the model control group was not administered or treated; one model 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; the model administration group was smeared with 0.2 g of the polypeptide composition gel 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.

[0067] Determination of skin wound healing rate: Take photos to record the wound healing status. The excision wound healing model is counted as 0 day, and take photos and record on the 4th, 7th, 14th, and 21st days. Use image processing software Image J to analyze and calculate the wound healing rate, observe the scab falling time and wound scar formation. The formula for calculating the wound healing rate is: ; Skin wound histopathological analysis: On the 21st day after surgery, mice were euthanized, and the wound and the full-thickness skin tissue of about 5 mm from the edge were obtained. They were fixed with 10% neutral buffered formalin solution for 24 h and then paraffin sections and HE tissue staining were performed to determine the epidermal thickness index and epidermal thickness ratio. The calculation formula was: ; ; 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, the more uniform, flat and smooth the mouse epidermis has been restored.

[0068] Table 14 Wound healing rate of mice

[0069] In the mouse wound healing test, the granulation tissue regeneration at the wound was good during the healing process, with a bright red color and later covered with a thin layer of epithelium. On the first day after injury, there was a lot of exudate at the wound and an obvious inflammatory response; on the fourth day after injury, the skin trauma in each group showed an overall healing trend, the wound skin had scabs, and granulation tissue had begun to form. Compared with the model control group, the peptide composition gel of the present application had a significant healing effect. During the period of 4 to 21 days after injury, the wound healing rate of mice given the peptide composition gel of the present application was significantly higher than that of the model control group. The appearance of the wound in the mouse group on the 21st day of administration of the peptide composition gel of the present application was significantly improved, and the surface was smooth and flat, but the model control group produced obvious scars.

[0070] Table 15 Mouse epidermal thickness index and epidermal thickness ratio test results

[0071] The test results of mouse epidermal thickness index and epidermal thickness ratio showed that the epidermis of the mice in the peptide composition gel administration group and rhEGF administration group became thinner and flatter 21 days after the injury, while the epidermis of the mice in the model control group was obviously uneven and thickened, with tongue-shaped protrusions at the edges, similar to keloids. The epidermal thickness index showed that the epidermal thickness index of the mice in the Example 1 administration group and the rhEGF gel administration group on the 21st day was 1.25±0.22 and 1.82±0.27, respectively, which was significantly lower than 5.34±0.37 in the model control group. The gel of only a single polypeptide (the epidermal thickness index of Comparative Example 1 was 3.48±0.64) and the tripeptide-1 The gel combined with hexapeptide-9 (epidermal thickness index 2.85±0.52 in comparative example 2) inhibits scar hyperplasia, and the three-combination polypeptide combination gel with palmitoyl-modified peptide plays a significant synergistic role in wound healing, while achieving effective prevention and inhibition of scar hyperplasia. The epidermal thickness ratio of mice in the polypeptide composition gel administration group and rhEGF administration group of the present application is significantly greater than that of mice in the model control group, indicating that the polypeptide composition gel of the present application has a good effect of promoting the restoration of uniform, flat and smooth epidermis.

[0072] It is explained 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 based on the method of the present invention are all within the scope of protection required by the present invention.

Claims

1. A polypeptide combination wound healing preparation, characterized in that: Used for preparing medicines and cosmetics for treating traumatic skin diseases, wherein the polypeptide composition comprises tripeptide-1, hexapeptide-9, and a palmitoyl-modified peptide complex.

2. The polypeptide combination wound healing preparation according to claim 1, characterized in that: The traumatic skin diseases are skin diseases caused by wounds, ulcers, burns, scars, and lumps.

3. The polypeptide combination wound healing preparation according to claim 1, characterized in that: The polypeptide combination wound healing preparation comprises a polypeptide composition, a penetration enhancer, and pharmaceutically acceptable excipients; wherein the polypeptide composition comprises tripeptide-1, hexapeptide-9, and a palmitoyl-modified peptide complex.

4. The polypeptide combination wound healing preparation according to claim 3, characterized in that: 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.

5. The polypeptide combination wound healing preparation according to claim 3, characterized in that: The penetration enhancer is one or a combination of chitosan, cyclodextrin or chitosan-cyclodextrin complex.

6. The polypeptide combination wound healing preparation according to claim 3, characterized in that: The penetration enhancer is a chitosan-cyclodextrin complex, wherein the mass ratio of chitosan to cyclodextrin is 1:0.

5.

7. The polypeptide combination wound healing preparation according to claim 3, characterized in that: The polypeptide combination wound healing preparation is a gel, and the pharmaceutically acceptable excipient is a gel excipient, which comprises a gel matrix, a moisturizer, an antioxidant, a pH regulator, and a water solvent.

8. The polypeptide combination wound healing preparation according to claim 7, characterized in that: The gel preparation comprises, by weight percentage, 1-10% of a polypeptide composition, 5-15% of a penetration enhancer, 1-10% of a gel matrix, 5-15% of a moisturizer, 0.01-0.5% of an antioxidant, an appropriate amount of a pH regulator, and an appropriate amount of deionized water.

9. The polypeptide combination wound healing preparation according to claim 7, 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 lecithin, colloidal silver-gelatin, and collagen.

10. The polypeptide combination wound healing preparation according to claim 7, characterized in that: The antioxidant is one or a combination of cysteine ​​and vitamin E; the mass ratio of cysteine ​​to vitamin E in the cysteine ​​and vitamin E composition is 1:1-2.

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