A polypeptide composition, its preparation method and use

Abstract

The present application belongs to the technical field of biological medicine, and relates to a polypeptide composition and a preparation method and application thereof. The polypeptide composition uses sodium carboxymethyl cellulose and xanthan gum as raw materials to prepare a pH-sensitive hydrogel capable of rapidly solidifying at a wound site. The hydrogel wraps polypeptide active ingredients, is applied to an affected part, rapidly solidifies to form a hydrogel film, firmly covers the skin of the affected part, maintains the stable and moist environment of the affected part, softens necrotic tissue, reduces scabbing, continuously releases active polypeptide ingredients, prolongs the action time, and shortens the repair period. The polypeptide ingredients in the composition are compounded by arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7 and palmitoyl tripeptide-1. The four polypeptides synergize through multiple targets and multiple stages to construct a whole-cycle repair system with anti-inflammatory, repair-promoting and anti-scarring functions, and are suitable for various skin wound scenes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide composition, its preparation method, and its application. Background Technology

[0002] As the largest organ in the human body, the skin plays a vital protective role. Frostbite, burns, external injuries, diabetic foot, and tumor resection surgery can all lead to skin wounds and defects. Skin wound healing is a complex and orderly repair and regeneration process involving various cellular behaviors of stem cells, the participation of multiple cell types, signal cascade responses, and microenvironment regulation.

[0003] Polypeptides are high-molecular-weight compounds that lie between amino acids and proteins, possessing strong biological activity. Compared with traditional wound healing drugs, bioactive peptides have advantages such as high activity, strong specificity, and good stability. Some peptides can promote cell proliferation, migration, and differentiation, accelerate granulation tissue growth and epithelial tissue coverage, thereby promoting wound healing.

[0004] Chinese patent application CN 115381930A discloses a skin repair drug formulation and its mixing method. This technical solution involves mixing hydroxybutyl chitosan, the polypeptide SIKVAV, water extract of Lithospermum erythrorhizon, and growth factors in water, followed by gelation and sterilization to obtain the skin repair drug formulation. In this technical solution, gelation is achieved only by stirring at 35–37°C after the addition of hydroxybutyl chitosan, which may result in insufficient gel strength and an uncontrollable degradation rate, thus affecting the sustained-release effect of the drug. Summary of the Invention

[0005] This invention aims to provide a polypeptide composition, its preparation method, and its application. The polypeptide composition utilizes a pH-sensitive hydrogel to encapsulate active polypeptide components. When applied to the affected area, the hydrogel rapidly solidifies to form a hydrogel film, maintaining a stable and moist environment, softening necrotic tissue, reducing crusting, continuously releasing active polypeptide components, prolonging the duration of action, and shortening the repair period. The polypeptide components in the composition are a combination of arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1. These four polypeptides work synergistically across multiple targets and stages to construct a full-cycle repair system that is anti-inflammatory, promotes repair, and prevents scarring, making it suitable for various skin trauma scenarios.

[0006] To achieve the above objectives, the present invention provides a polypeptide composition comprising a pH-sensitive hydrogel, a complex polypeptide, an antioxidant, an antibacterial adjuvant, and a preservative; the complex polypeptide comprises an arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, a filler, a stabilizer, and a transdermal adjuvant; the structure of the pH-sensitive hydrogel is shown below:

[0007]

[0008] Where n takes the value of an integer between 100 and 1000.

[0009] Preferably, the pH-sensitive hydrogel is prepared by reacting modified carboxymethyl cellulose and modified xanthan gum, wherein the mass ratio of the modified carboxymethyl cellulose, modified xanthan gum, complex polypeptide, antioxidant, antibacterial adjuvant and preservative is 1:(2-5):(0.35-0.75):(0.005-0.015):(0.005-0.0075):(0.001-0.0035).

[0010] Preferably, the mass ratio of the arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, filler, stabilizer and transdermal adjuvant is 1:(0.75-1.25):(1-1.5):(0.8-1.6):(0.005-0.01):(0.01-0.025):(0.05-0.12).

[0011] Preferably, the antioxidant is any one or more of panthenol, bisabolol, and dipotassium glycyrrhizate.

[0012] Preferably, the preservative is any one or more of 1,2-hexanediol, phenoxyethanol, and methylparaben.

[0013] Preferably, the antibacterial adjuvant is any one or more of ethylhexylglycerin and gluconolactone.

[0014] Preferably, the filler is any one or more of mannitol, sorbitol, and dextran.

[0015] Preferably, the stabilizer is any one or more of diethylenetriaminepentaacetic acid and ethylenediaminetetraacetic acid.

[0016] Preferably, the transdermal adjuvant is caryophyllene and transdermal peptide, and the mass ratio of caryophyllene to transdermal peptide is 1:(1-5).

[0017] The present invention also provides a method for preparing a polypeptide composition, comprising:

[0018] Step S1: Disperse sodium carboxymethyl cellulose in deionized water, add 3-aminopropane-1,2-diol, and after the first reaction, adjust the pH to 5-6, add condensing agent and catalyst, and after the second reaction, purify and freeze dry to obtain the first intermediate;

[0019] Step S2: Disperse the first intermediate in the first solvent, adjust the pH to 6-7, add dimethylaminoethyl methacrylate, initiator and crosslinking agent, react to obtain the second intermediate, add sodium periodate, and oxidize to obtain modified carboxymethyl cellulose;

[0020] Step S3: Disperse xanthan gum and isophthalic hydrazide in a second solvent, add condensing agent and catalyst, adjust pH to 5-6, react, purify, freeze dry to obtain modified xanthan gum;

[0021] Step S4: Disperse arginine / lysine peptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1 in a third solvent, add filler, stabilizer, and transdermal adjuvant, and stir to obtain composite peptide;

[0022] Step S5: Disperse modified carboxymethyl cellulose and modified xanthan gum in deionized water, add the complex peptide, react, add antioxidants, antibacterial auxiliaries and preservatives, and prepare a pH-sensitive hydrogel containing the complex peptide.

[0023] Preferably, the purification process involves dialyzing in deionized water for 20–24 hours.

[0024] Preferably, the freeze-drying operation involves pre-freezing at -20℃ to -40℃ for 2 to 4 hours, heating to -10℃ to 20℃ for 12 to 20 hours, and heating to 20℃ to 40℃ for 8 to 10 hours.

[0025] Preferably, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0026] Preferably, the catalyst is 1-hydroxybenzotriazole.

[0027] Preferably, in step S1, the temperature of the first reaction is 25-30°C, and the reaction time is 1-2 hours.

[0028] Preferably, in step S1, the temperature of the second reaction is 25-35°C, and the reaction time is 18-24 hours.

[0029] Preferably, in step S1, the mass ratio of sodium carboxymethyl cellulose, deionized water, 3-aminopropane-1,2-diol, condensing agent and catalyst is 1:(10-15):(0.35-0.55):(0.5-0.7):(0.8-1).

[0030] Preferably, in step S2, the first solvent is an aqueous solution of ethanol containing Tween-80, and the mass ratio of water, ethanol and Tween-80 is 1:(0.25~0.55):(0.005~0.01).

[0031] Preferably, in step S2, the initiator is any one or more of ammonium persulfate and azobisisobutyronitrile.

[0032] Preferably, in step S2, the crosslinking agent is N,N'-methylenebisacrylamide.

[0033] Preferably, in step S2, the mass ratio of the first intermediate, the first solvent, dimethylaminoethyl methacrylate, the initiator and crosslinking agent and sodium periodate is 1:(10-15):(0.35-0.75):(0.05-0.11):(0.015-0.033):(0.075-0.25).

[0034] Preferably, in step S2, the reaction temperature is 30–60°C and the reaction time is 1–3 hours.

[0035] Preferably, in step S2, the oxidation time is 1 to 3 hours.

[0036] Preferably, in step S3, the second solvent is an aqueous ethanol solution, and the mass ratio of water to ethanol is 1:(1-2.5).

[0037] Preferably, in step S3, the mass ratio of xanthan gum, isophthalic hydrazide, the second solvent, the condensing agent, and the catalyst is 1:(0.5-0.7):(10-15):(0.05-0.15):(0.05-0.15).

[0038] Preferably, in step S3, the reaction time is 18 to 24 hours.

[0039] Preferably, in step S4, the third solvent is an aqueous solution of 1,3-propanediol, and the mass ratio of 1,3-propanediol to water is 1:(4-9).

[0040] Preferably, in step S5, the reaction temperature is 30–50°C and the reaction time is 6–12 h.

[0041] The present invention also provides the use of a polypeptide composition in the preparation of a medicament for skin wound repair.

[0042] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0043] (1) The polypeptide composition prepared in this application uses arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7 and palmitoyl tripeptide-1 as a compound. The four polypeptides work together through multiple targets and multiple stages to construct a full-cycle repair system of anti-inflammatory-promoting repair-anti-scarring, which is suitable for various skin trauma scenarios. In the early stages of inflammation, arginine / lysine peptides adsorb onto the wound surface with a positive charge, reducing bacterial adhesion and lowering the risk of infection. Palmitoyl tetrapeptide-7 inhibits pro-inflammatory factors such as IL-6 and IL-8, reducing redness and swelling. Palmitoyl tripeptide-1 scavenge free radicals, reducing oxidative damage and preventing the spread of the inflammatory cascade. After inflammation subsides, hexapeptide-11 promotes keratinocyte migration and fibronectin synthesis, while arginine / lysine peptides activate fibroblasts to synthesize collagen, synergistically accelerating epidermal regeneration and dermal reconstruction. During the wound healing stage, palmitoyl tetrapeptide-7 inhibits excessive collagen degradation by MMPs, and hexapeptide-11 regulates the orderly arrangement of collagen fibers, jointly reducing scar hyperplasia and promoting scar softening. Arginine / lysine peptides continuously improve local microcirculation, accelerate the excretion of metabolic waste, and maintain homeostasis in the repair environment.

[0044] (2) The polypeptide composition prepared in this application is encapsulated in a pH-sensitive hydrogel. This hydrogel is prepared by using sodium carboxymethyl cellulose as the starting material, modifying it with 3-aminopropane-1,2-diol, introducing amino groups, and crosslinking it with xanthan gum modified with hydrazide. The hydrogel exhibits a gel-like state in a normal pH environment. When applied to the affected area, due to the slightly acidic pH of the affected area, the pH-sensitive amino groups in the hydrogel are triggered. The amino groups accept protons, forming positively charged ammonium ions. The repulsive force of the charges causes the polymer chains to extend and expand, resulting in crosslinking and rapid solidification on the surface of the affected area to form a hydrogel film, triggering the slow release of the drug. This hydrogel can achieve non-invasive coverage and adaptive fit to irregular wounds, overcoming the shortcomings of traditional dressings such as easy displacement, poor breathability, and adhesion to the wound. The high water content of the hydrogel can maintain a stable and moist environment at the affected area, accelerate epidermal cell migration, and reduce the risk of scar hyperplasia. Attached Figure Description

[0045] Figure 1 This is a flowchart illustrating the preparation process of a polypeptide composition.

[0046] Figure 2 This is a schematic diagram of the synthetic route for modified carboxymethyl cellulose.

[0047] Figure 3 This is a schematic diagram of the synthetic route for modified xanthan gum.

[0048] Figure 4 This is a schematic diagram of the synthesis route for pH-sensitive hydrogels containing complex peptides. Detailed Implementation

[0049] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0050] Unless otherwise specified, all reagents and equipment used in the following examples were purchased from commercial channels.

[0051] Example 1

[0052] like Figure 1 As shown, a polypeptide composition is prepared by means of:

[0053] Step S1: Disperse 10g of sodium carboxymethyl cellulose in 100g of deionized water, add 3.5g of 3-aminopropane-1,2-diol, react at 25℃ for 2h, adjust the pH to 5-6, add 5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8g of 1-hydroxybenzotriazole, react at 25℃ for 24h, dialyze in deionized water for 20h, pre-freeze at -20℃ for 4h, heat to -10℃ and dry for 20h, heat to 20℃ and dry for 10h to obtain the first intermediate.

[0054] Step S2: Disperse 10g of the first intermediate in 100g of an ethanol-water solution (80g water, 20g ethanol, 0.4g Tween-80), adjust the pH to 6-7, add 3.5g of dimethylaminoethyl methacrylate, 0.5g of ammonium persulfate, and 0.15g of N,N'-methylenebisacrylamide, react at 30°C for 3h to obtain the second intermediate, add 0.75g of sodium periodate, oxidize for 3h to obtain modified carboxymethyl cellulose, as shown below. Figure 2 As shown.

[0055] Step S3: Disperse 10g xanthan gum and 5g isophthalohydrazide in 100g of ethanol aqueous solution (50g water and 50g ethanol), add 0.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5g of 1-hydroxybenzotriazole, adjust the pH to 5-6, react for 24h, dialyze in deionized water for 20h, pre-freeze at -20℃ for 4h, heat to -10℃ for drying for 20h, heat to 20℃ for drying for 10h, to obtain modified xanthan gum, as shown. Figure 3 As shown.

[0056] Step S4: Disperse 10g of arginine / lysine polypeptide, 7.5g of hexapeptide-11, 10g of palmitoyl tetrapeptide-7, and 8g of palmitoyl tripeptide-1 in 100g of 1,3-propanediol aqueous solution (20g of 1,3-propanediol and 80g of water), add 0.05g of mannitol, 0.1g of diethylenetriaminepentaacetic acid, and 0.5g of transdermal adjuvant (0.25g of caryophyllene and 0.25g of transdermal peptide), and stir to obtain the composite polypeptide.

[0057] Step S5: Disperse 10g of modified carboxymethyl cellulose and 20g of modified xanthan gum in 100g of deionized water, add 3.5g of the complex peptide, add PBS buffer solution, and react at 30℃ for 12h to obtain a pH-sensitive hydrogel containing the complex peptide, as shown below. Figure 2 As shown, adding 0.05 g panthenol, 0.05 g ethylhexylglycerin, and 0.01 g 1,2-hexanediol yields a pH-sensitive hydrogel containing a complex polypeptide, as shown. Figure 4 As shown.

[0058] Example 2

[0059] like Figure 1 As shown, a polypeptide composition is prepared by means of:

[0060] Step S1: Disperse 10g of sodium carboxymethyl cellulose in 120g of deionized water, add 4g of 3-aminopropane-1,2-diol, react at 25℃ for 1h, adjust the pH to 5-6, add 5.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8.5g of 1-hydroxybenzotriazole, react at 30℃ for 22h, dialyze in deionized water for 22h, pre-freeze at -25℃ for 3h, heat to 0℃ and dry for 18h, heat to 25℃ and dry for 9h to obtain the first intermediate.

[0061] Step S2: Disperse 10g of the first intermediate in 120g of an aqueous ethanol solution (88g water, 31g ethanol, 0.6g Tween-80), adjust the pH to 6-7, add 4.5g of dimethylaminoethyl methacrylate, 0.75g of azobisisobutyronitrile, and 0.2g of N,N'-methylenebisacrylamide, react at 40℃ for 2.5h to obtain the second intermediate, add 1g of sodium periodate, and oxidize for 2.5h to obtain modified carboxymethyl cellulose, as shown below. Figure 2 As shown.

[0062] Step S3: Disperse 10g xanthan gum and 6g isophthalohydrazide in 120g of ethanol aqueous solution (48g water and 72g ethanol), add 0.75g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75g of 1-hydroxybenzotriazole, adjust the pH to 5-6, react for 22h, dialyze in deionized water for 22h, pre-freeze at -25℃ for 3h, heat to 0℃ and dry for 18h, heat to 25℃ and dry for 9h to obtain modified xanthan gum, as shown. Figure 3 As shown.

[0063] Step S4: Disperse 10g of arginine / lysine polypeptide, 9g of hexapeptide-11, 12g of palmitoyl tetrapeptide-7, and 10g of palmitoyl tripeptide-1 in 120g of 1,3-propanediol aqueous solution (20g of 1,3-propanediol and 100g of water), add 0.075g of sorbitol, 0.15g of ethylenediaminetetraacetic acid, and 0.75g of transdermal adjuvant (0.25g of caryophyllene and 0.5g of transdermal peptide), and stir to obtain the composite polypeptide.

[0064] Step S5: Disperse 10g of modified carboxymethyl cellulose and 30g of modified xanthan gum in 120g of deionized water, add 5g of the complex peptide, add PBS buffer solution, and react at 35℃ for 10h to obtain a pH-sensitive hydrogel containing the complex peptide, as shown below. Figure 2 As shown, 0.075 g bisabolol, 0.06 g gluconolactone, and 0.02 g phenoxyethanol were added. A pH-sensitive hydrogel containing a complex polypeptide was obtained, as shown. Figure 4 As shown.

[0065] Example 3

[0066] like Figure 1 As shown, a polypeptide composition is prepared by means of:

[0067] Step S1: Disperse 10g of sodium carboxymethyl cellulose in 135g of deionized water, add 5g of 3-aminopropane-1,2-diol, react at 25℃ for 2h, adjust the pH to 5-6, add 6g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 9g of 1-hydroxybenzotriazole, react at 25℃ for 20h, dialyze in deionized water for 23h, pre-freeze at -30℃ for 3h, heat to 10℃ and dry for 16h, heat to 30℃ and dry for 9h to obtain the first intermediate.

[0068] Step S2: Disperse 10g of the first intermediate in 135g of an ethanol-water solution (93g water, 42g ethanol, 0.84g Tween-80), adjust the pH to 6-7, add 6.5g of dimethylaminoethyl methacrylate, 1g of azobisisobutyronitrile, and 0.3g of N,N'-methylenebisacrylamide, react at 50°C for 2h to obtain the second intermediate, add 2g of sodium periodate, oxidize for 2h to obtain modified carboxymethyl cellulose, as shown below. Figure 2 As shown.

[0069] Step S3: Disperse 10g xanthan gum and 5g isophthalohydrazide in 135g of ethanol aqueous solution (45g water and 90g ethanol), add 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of 1-hydroxybenzotriazole, adjust the pH to 5-6, react for 20h, dialyze in deionized water for 23h, pre-freeze at -30℃ for 3h, heat to 10℃ and dry for 16h, heat to 30℃ and dry for 9h to obtain modified xanthan gum, as shown. Figure 3 As shown.

[0070] Step S4: Disperse 10g of arginine / lysine polypeptide, 11g of hexapeptide-11, 13g of palmitoyl tetrapeptide-7, and 13g of palmitoyl tripeptide-1 in 135g of 1,3-propanediol aqueous solution (16g of 1,3-propanediol and 120g of water), add 0.09g of dextran, 0.2g of diethylenetriaminepentaacetic acid, and 1g of transdermal adjuvant (0.25g of caryophyllene and 0.75g of transdermal peptide), and stir to obtain the composite polypeptide.

[0071] Step S5: Disperse 10g of modified carboxymethyl cellulose and 40g of modified xanthan gum in 135g of deionized water, add 6.5g of the complex peptide, add PBS buffer solution, and react at 40℃ for 8h to obtain a pH-sensitive hydrogel containing the complex peptide. Figure 2 As shown, 0.1 g dipotassium glycyrrhizate, 0.07 g ethylhexylglycerin, and 0.03 g methylparaben were added. A pH-sensitive hydrogel containing a complex polypeptide was obtained, as shown. Figure 4 As shown.

[0072] Example 4

[0073] like Figure 1 As shown, a polypeptide composition is prepared by means of:

[0074] Step S1: Disperse 10g of sodium carboxymethyl cellulose in 150g of deionized water, add 5.5g of 3-aminopropane-1,2-diol, react at 25℃ for 10h, adjust the pH to 5-6, add 7g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of 1-hydroxybenzotriazole, react at 35℃ for 18h, dialyze in deionized water for 24h, pre-freeze at -40℃ for 2h, heat to 20℃ and dry for 12h, heat to 40℃ and dry for 8h to obtain the first intermediate.

[0075] Step S2: Disperse 10g of the first intermediate in 150g of an ethanol-water solution (96g water, 53g ethanol, 0.96g Tween-80), adjust the pH to 6-7, add 7.5g of dimethylaminoethyl methacrylate, 1.1g of ammonium persulfate, and 0.33g of N,N'-methylenebisacrylamide, react at 60℃ for 1h to obtain the second intermediate, add 2.5g of sodium periodate, oxidize for 1h to obtain modified carboxymethyl cellulose, as shown below. Figure 2 As shown.

[0076] Step S3: Disperse 10g xanthan gum and 7g isophthalohydrazide in 150g of ethanol aqueous solution (43g water and 107g ethanol), add 1.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.5g of 1-hydroxybenzotriazole, adjust the pH to 5-6, react for 18h, dialyze in deionized water for 24h, pre-freeze at -40℃ for 2h, heat to 20℃ and dry for 12h, heat to 40℃ and dry for 8h to obtain modified xanthan gum, as shown. Figure 3 As shown.

[0077] Step S4: Disperse 10g of arginine / lysine polypeptide, 12.5g of hexapeptide-11, 15g of palmitoyl tetrapeptide-7, and 16g of palmitoyl tripeptide-1 in 150g of 1,3-propanediol aqueous solution (15g of 1,3-propanediol and 135g of water), add 0.1g of mannitol, 0.25g of ethylenediaminetetraacetic acid, and 1.2g of transdermal adjuvant (0.2g of caryophyllene and 1g of transdermal peptide), and stir to obtain the composite polypeptide.

[0078] Step S5: Disperse 10g of modified carboxymethyl cellulose and 50g of modified xanthan gum in 150g of deionized water, add 7.5g of the complex peptide, add PBS buffer solution, and react at 50℃ for 6h to obtain a pH-sensitive hydrogel containing the complex peptide. Figure 2 As shown, 0.15 g panthenol, 0.075 g gluconolactone, and 0.035 g 1,2-hexanediol were added. A pH-sensitive hydrogel containing a complex polypeptide was obtained, as shown. Figure 4 As shown.

[0079] Comparative Example 1

[0080] A polypeptide composition, the preparation method of which differs from that of Example 3 in that dimethylaminoethyl methacrylate is not added in step S2.

[0081] Comparative Example 2

[0082] A polypeptide composition, the preparation method of which differs from that of Example 3 in that modified carboxymethyl cellulose and modified xanthan gum are not added in step S5.

[0083] Efficacy test:

[0084] (1) Fibroblast detection: Fibroblasts in the logarithmic growth phase were collected and analyzed at a concentration of 8 × 10⁻⁶ cells / mL. 3 The cells were seeded at a density of 100 μL / well in 96-well plates and incubated overnight at 37°C with 5% CO2. The culture medium was removed, and 200 μL of the test substance (0.05%) was added to each well. The plates were incubated for another 24 hours, with at least three replicates per group. The supernatant was collected for ELISA detection of FN (fibronectin), COLI (type I collagen), and TIMP-1 (tissue inhibitor of metalloproteinases-1). The cells were also incubated with 100 μL of CCK-8 staining solution for 1 hour, and the absorbance at 450 nm was measured.

[0085] (2) Skin repair experiment: BALB / c mice weighing 20±2g were used, and the model control group consisted of mice with trauma and no drug treatment. The hair on the back of the mice was removed to establish mouse models of skin defects and burn infection, respectively. The mice were treated with drugs (test samples) or without treatment (model control group). The drug treatment group was treated with drugs twice a day, 3-5mg each time, for 8 consecutive days. The wounds were quantitatively analyzed to evaluate the healing effect.

[0086] Table 1. Results of fibroblast detection

[0087]

[0088] Collagen I (COLⅠ) is the most abundant collagen in the dermis of the skin. It provides adhesion sites for fibroblasts and endothelial cells, guides cell migration to the wound area, and accelerates granulation tissue formation. High COLⅠ expression indicates that fibroblasts are actively synthesizing extracellular matrix (ECM), accelerating wound contraction and tissue remodeling, and is an important marker of the proliferative and remodeling phases of wound repair. FN (fibrinolytic nuclease) binds to both cell surface receptors and ECM, providing migration pathways for newly formed vascular endothelial cells, promoting angiogenesis, and improving local blood supply to the wound. High FN expression indicates that the wound is in a transitional phase from inflammation to proliferation. Increased FN synthesis can accelerate cell recruitment and matrix remodeling, and is a key signal for initiating wound repair. TIMP-1 inhibits matrix metalloproteinases (MMPs), preventing excessive ECM degradation that could delay wound healing; however, excessive inhibition may increase the risk of scar hyperplasia.

[0089] According to the data in Table 1, the compositions prepared in Examples 1 to 4 can improve the viability of fibroblasts and significantly increase the expression levels of COLⅠ, FN and TIMP-1. The cell viability of the compositions prepared in Examples 1 to 3 after treatment was significantly lower than that in Examples 1 to 3, and the expression levels of the three proteins were also significantly lower than those in Examples 1 to 3.

[0090] Table 2 Results of skin defect healing rate test

[0091]

[0092]

[0093] According to Table 2, when the compositions prepared in Examples 1 to 4 were applied to the skin defects on the back of mice, the wound healing rate was above 15% on the second day, above 35% on the fourth day, and above 85% on the eighth day. However, the healing rate of the wounds on the back of mice after applying the compositions prepared in Comparative Examples 1 and 2 was significantly lower than that in Examples 1 to 4. Among them, Comparative Example 2 was less effective, with a healing rate of only 6.06% on the second day and only 49.87% on the eighth day.

[0094] In Comparative Example 1, without the addition of dimethylaminoethyl methacrylate, the prepared hydrogel lacked pH-sensitive segments. Therefore, when applied to the wound, the hydrogel struggled to quickly cross-link and form a thin film to cover the wound. Consequently, the composition was easily rubbed off during mouse activity, resulting in poor therapeutic efficacy. In Comparative Example 2, the peptide was not encapsulated in a hydrogel and was administered using traditional dressings (gauze). The composition degraded too quickly, and the therapeutic effect was significantly lower than the group encapsulated in the hydrogel. Furthermore, the gauze shifted during mouse activity, rubbing against the wound and failing to adequately cover it, thus hindering wound healing. The experiments demonstrate that encapsulating the peptide in a pH-sensitive hydrogel enables long-acting, sustained drug release, forming a thin film on the skin for better wound coverage and continuous drug delivery, while overcoming the limitations of traditional dressings.

[0095] Table 3 Results of Burn Infection Healing Rate Test

[0096]

[0097] As shown in Table 3, the compositions prepared in Examples 1 to 3 have significant effects on treating infected burn wounds in mice. The composition prepared in Example 3 showed the best therapeutic effect, with a wound healing rate of 40.58% on day 4 and 83.25% on day 8. The compositions prepared in Comparative Examples 1 and 2 showed poorer therapeutic effects on infected burn wounds in mice; the wound healing rate in Comparative Example 1 was only 51.12% on day 8, and in Comparative Example 2 it was only 45.28%.

[0098] The composition prepared in Comparative Example 1, after a period of administration, caused the gel to detach or adhere to other impurities due to the activity of the mice, which prevented the continuous administration of the gel. At the same time, it could not effectively cover the wound and block external dust and bacteria. Instead, the adhesion of other impurities led to wound infection. The composition prepared in Comparative Example 2, after a period of administration, showed that the active ingredients were ineffective or contaminated, which greatly reduced the therapeutic effect. In addition, traditional dressings are prone to displacement and detachment, exposing the wound to the environment, which can easily lead to secondary infection and aggravate bacterial infection.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide composition, characterized in that, The polypeptide composition comprises a pH-sensitive hydrogel, a complex polypeptide, an antioxidant, an antibacterial adjuvant, and a preservative; the complex polypeptide comprises an arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, a filler, a stabilizer, and a transdermal adjuvant; the structure of the pH-sensitive hydrogel is shown below: ， Where n takes the value of an integer between 100 and 1000.

2. The polypeptide composition according to claim 1, characterized in that, The pH-sensitive hydrogel was prepared by reacting modified carboxymethyl cellulose and modified xanthan gum; the mass ratio of the modified carboxymethyl cellulose, modified xanthan gum, composite peptide, antioxidant, antibacterial adjuvant and preservative was 1:(2~5):(0.35~0.75):(0.005~0.015):(0.005~0.0075):(0.001~0.0035); the mass ratio of the arginine / lysine peptide, hexapeptide-11, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, filler, stabilizer and transdermal adjuvant was 1:(0.75~1.25):(1~1.5):(0.8~1.6):(0.005~0.01):(0.01~0.025):(0.05~0.12). The method for preparing the modified carboxymethyl cellulose includes: step S1, dispersing sodium carboxymethyl cellulose in deionized water, adding 3-aminopropane-1, 2-Diol, after a first reaction, the pH is adjusted to 5-6, a condensing agent and a catalyst are added, and after a second reaction, purification and freeze-drying are performed to obtain a first intermediate; Step S2, the first intermediate is dispersed in a first solvent, the pH is adjusted to 6-7, dimethylaminoethyl methacrylate, an initiator and a crosslinking agent are added, and the reaction is carried out to obtain a second intermediate, sodium periodate is added, and oxidation is performed to obtain modified carboxymethyl cellulose; the first solvent is an aqueous ethanol solution containing Tween-80, and the mass ratio of water, ethanol and Tween-80 is 1:(0.25-0.55):(0.005-0.01); The preparation method of the modified xanthan gum includes: Step S3, xanthan gum and isophthalohydrazide are dispersed in a second solvent, a condensing agent and a catalyst are added, the pH is adjusted to 5-6, the reaction is carried out, purification is performed, and freeze-drying is performed to obtain modified xanthan gum; the second solvent is an aqueous ethanol solution, and the mass ratio of water and ethanol is 1:(1-2.5).

3. A polypeptide composition according to claim 1 or 2, characterized in that, The antioxidant is any one or more of panthenol, bisabolol, and dipotassium glycyrrhizate; the preservative is any one or more of 1,2-hexanediol, phenoxyethanol, and methylparaben; the antibacterial adjuvant is any one or more of ethylhexylglycerin and gluconolactone; the filler is any one or more of mannitol, sorbitol, and dextran; the stabilizer is any one or more of diethylenetriaminepentaacetic acid and ethylenediaminetetraacetic acid; and the transdermal adjuvant is caryophyllene and transdermal peptide, with a mass ratio of caryophyllene to transdermal peptide of 1:(1~5).

4. The polypeptide composition according to claim 2, characterized in that, In step S1, the purification operation involves dialyzing in deionized water for 20-24 h; the freeze-drying operation involves pre-freezing at -20℃ to -40℃ for 2-4 h, heating to -10℃ to 20℃ for 12-20 h, and heating to 20℃ to 40℃ for 8-10 h; the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the catalyst is 1-hydroxybenzotriazole; the temperature of the first reaction is 25-30℃, and the time of the first reaction is 1-2 h; the temperature of the second reaction is 25-35℃, and the time of the second reaction is 18-24 h; the mass ratio of sodium carboxymethyl cellulose, deionized water, 3-aminopropane-1,2-diol, condensing agent, and catalyst is 1:(10-15):(0.35-0.55):(0.5-0.7):(0.8-1).

5. The polypeptide composition according to claim 2, characterized in that, In step S2, the initiator is any one or more of ammonium persulfate and azobisisobutyronitrile; the crosslinking agent is N,N'-methylenebisacrylamide; the mass ratio of the first intermediate, the first solvent, dimethylaminoethyl methacrylate, the initiator, the crosslinking agent, and sodium periodate is 1:(10~15):(0.35~0.75):(0.05~0.11):(0.015~0.033):(0.075~0.25); the reaction temperature is 30~60℃, the reaction time is 1~3 h; and the oxidation time is 1~3 h.

6. The polypeptide composition according to claim 2, characterized in that, In step S3, the mass ratio of xanthan gum, isophthalohydrazide, the second solvent, the condensing agent, and the catalyst is 1:(0.5~0.7):(10~15):(0.05~0.15):(0.05~0.15); the reaction time is 18~24 h.

7. A method for preparing a polypeptide composition according to any one of claims 1 to 6, characterized in that, The preparation method includes steps S1, S2, and S3 as described in claim 2, and further includes: Step S4: Disperse arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1 in a third solvent, add filler, stabilizer, and transdermal adjuvant, and stir to obtain a composite polypeptide; the third solvent is an aqueous solution of 1,3-propanediol. Step S5: Disperse modified carboxymethyl cellulose and modified xanthan gum in deionized water, add the complex peptide, react, add antioxidants, antibacterial auxiliaries and preservatives, and prepare a pH-sensitive hydrogel containing the complex peptide.

8. The method for preparing a polypeptide composition according to claim 7, characterized in that, In step S4, the mass ratio of 1,3-propanediol to water in the 1,3-propanediol aqueous solution is 1:(4~9).

9. A method for preparing a polypeptide composition according to claim 7, characterized in that, In step S5, the reaction temperature is 30~50℃ and the reaction time is 6~12 h.

10. The use of a polypeptide composition according to any one of claims 1 to 6 in the preparation of a medicament for skin wound repair.

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