Polypeptide composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a polypeptide composition as well as a preparation method and application thereof. According to the polypeptide composition, sodium carboxymethyl cellulose and xanthan gum are adopted as raw materials, pH-sensitive hydrogel capable of being rapidly cured at a wound is prepared, the hydrogel wraps polypeptide active ingredients and can be rapidly cured to form a layer of hydrogel film after being smeared on the affected part, the hydrogel film firmly covers the skin of the affected part, the environment of the affected part is kept stable and moist, and the wound healing effect is good. Necrotic tissues are softened, scabbing is reduced, active polypeptide components are continuously released, the action time is prolonged, and the repair period is shortened. The polypeptide component in the composition is compounded by arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7 and palmitoyl tripeptide-1, the four polypeptides construct an anti-inflammatory, repair-promoting and anti-scar complete-cycle repair system through multi-target and multi-stage synergy, and the composition is suitable for various skin wound scenes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide composition, a preparation method and an application thereof. Background Art

[0002] As the largest organ in the human body, the skin plays a vital protective role. Frostbite, burns, trauma, diabetic foot, and tumor resection surgery can all lead to skin wounds and defects. The healing of skin wounds is a complex and orderly process of repair and regeneration, involving the diverse cellular behaviors of stem cells, the involvement of multiple cell types, signaling cascade responses, and microenvironmental regulation.

[0003] Peptides are high-molecular-weight compounds between amino acids and proteins, exhibiting strong biological activity. Compared to traditional wound repair drugs, bioactive peptides offer advantages such as high activity, strong specificity, and excellent stability. Some peptides can promote cell proliferation, migration, and differentiation, accelerating granulation tissue growth and epithelial tissue coverage, thereby promoting wound healing.

[0004] Chinese patent application CN 115381930A discloses a skin repair drug formulation and a mixing method thereof. This method involves mixing hydroxybutyl chitosan, the polypeptide SIKVAV, an aqueous extract of lithospermum officinale, and growth factors in water, gelling the mixture, and sterilizing it to produce the skin repair drug formulation. This method, after adding hydroxybutyl chitosan, only stirs at 35-37°C to achieve gelation, which can result in insufficient gel strength and uncontrollable degradation rate, thus affecting the sustained release of the drug. Summary of the Invention

[0005] The present invention aims to provide a polypeptide composition and its preparation method and application. The polypeptide composition uses a pH-sensitive hydrogel to encapsulate the active polypeptide component. The hydrogel is applied to the affected area and quickly solidifies to form a hydrogel film, which maintains a stable and moist environment on the affected area, softens necrotic tissue, reduces scabs, continuously releases active polypeptide components, prolongs the action time, and shortens the repair period. The polypeptide components in the composition are compounded with arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1. The four polypeptides work together through multiple targets and multiple stages to construct a full-cycle repair system that is anti-inflammatory, promotes repair, and resists scarring, and is suitable for a variety of skin trauma scenarios.

[0006] To achieve the above-mentioned object, the present invention provides a polypeptide composition, which comprises a pH-sensitive hydrogel, a composite polypeptide, an antioxidant, an antibacterial adjuvant, and a preservative; the composite 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] Here, n is an integer between 100 and 1000.

[0009] Preferably, the pH-sensitive hydrogel is prepared by reacting modified carboxymethyl cellulose and modified xanthan gum, and the mass ratio of the modified carboxymethyl cellulose, modified xanthan gum, composite 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 auxiliary agent is any one or more of ethylhexylglycerol 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, dispersing sodium carboxymethyl cellulose in deionized water, adding 3-aminopropane-1,2-diol, performing a first reaction, adjusting the pH to 5-6, adding a condensing agent and a catalyst, performing a second reaction, purifying, and freeze-drying to obtain a first intermediate;

[0019] Step S2, dispersing the first intermediate in a first solvent, adjusting the pH to 6-7, adding dimethylaminoethyl methacrylate, an initiator, and a cross-linking agent, reacting to obtain a second intermediate, adding sodium periodate, and oxidizing to obtain modified carboxymethyl cellulose;

[0020] Step S3, dispersing xanthan gum and isophthalic acid hydrazide in a second solvent, adding a condensing agent and a catalyst, adjusting the pH value to 5-6, reacting, purifying, and freeze-drying to obtain modified xanthan gum;

[0021] Step S4, dispersing the arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1 in a third solvent, adding a filler, a stabilizer, and a transdermal adjuvant, and stirring to obtain a composite polypeptide;

[0022] Step S5: dispersing modified carboxymethyl cellulose and modified xanthan gum in deionized water, adding the composite polypeptide, reacting, adding an antioxidant, an antibacterial adjuvant and a preservative to prepare a pH-sensitive hydrogel containing the composite polypeptide.

[0023] Preferably, the purification operation is dialyzed in deionized water for 20 to 24 hours.

[0024] Preferably, the freeze-drying operation is pre-freezing at -20°C to -40°C for 2 to 4 hours, heating to -10°C to 20°C for drying for 12 to 20 hours, and heating to 20°C to 40°C for drying 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 time of the first reaction is 1-2 h.

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

[0029] Preferably, in step S1, the mass ratio of the 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 ethanol aqueous solution 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 cross-linking 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 the cross-linking 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 ethanol aqueous solution, and the mass ratio of water to ethanol is 1:(1-2.5).

[0037] Preferably, in step S3, the mass ratio of the xanthan gum, isophthalic acid 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 a 1,3-propylene glycol aqueous solution, and the mass ratio of 1,3-propylene glycol 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 an application of the polypeptide composition in preparing a medicine for repairing skin wounds.

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

[0043] (1) The polypeptide components in the polypeptide composition prepared in this application are a combination of arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1. The four polypeptides work together through multiple targets and multiple stages to construct a full-cycle repair system of anti-inflammatory, repair-promoting, and anti-scarring, which is suitable for a variety of skin trauma scenarios. In the early stages of inflammation, arginine / lysine peptides are positively charged and adsorbed on the wound surface, reducing bacterial adhesion and 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 scavenges free radicals, reduces oxidative damage, and prevents the expansion of the inflammatory cascade. After the inflammation is relieved, hexapeptide-11 promotes keratinocyte migration and fibronectin synthesis. 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 the homeostasis of the repair environment.

[0044] (2) The polypeptide composition prepared in this application is wrapped in a pH-sensitive hydrogel. The hydrogel is prepared by using sodium carboxymethyl cellulose as the starting material, modified with 3-aminopropane-1,2-diol, and then introducing amino groups and cross-linking with hydrazide-modified xanthan gum. The hydrogel is in a gel-like state in a normal pH environment. When applied to the affected area, the pH-sensitive amino groups in the hydrogel are triggered due to the acidic pH of the affected area. The amino groups accept protons to form positively charged ammonium ions. The charge repulsion causes the polymer chain segments to stretch and swell, cross-linking occurs, and the hydrogel quickly solidifies to form a hydrogel film on the surface of the affected area, triggering the slow release of the drug. The hydrogel can achieve non-invasive coverage and adaptive fit to irregular wounds, overcoming the shortcomings of traditional dressings such as easy displacement, poor air permeability, and adhesion to wounds. The high water content of the hydrogel can maintain a stable and moist environment in the affected area, accelerate epidermal cell migration, and reduce the risk of scar hyperplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The present invention is a flow chart for the preparation of a polypeptide composition.

[0046] Figure 2 Schematic diagram of the synthetic route of modified carboxymethyl cellulose.

[0047] Figure 3 Schematic diagram of the synthetic route of modified xanthan gum.

[0048] Figure 4 Schematic diagram of the synthesis route of pH-sensitive hydrogel containing complex polypeptides. DETAILED DESCRIPTION

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

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

[0051] Example 1

[0052] like Figure 1 As shown, a polypeptide composition, the preparation method of which comprises:

[0053] Step S1, dispersing 10g of sodium carboxymethyl cellulose in 100g of deionized water, adding 3.5g of 3-aminopropane-1,2-diol, reacting at 25°C for 2h, adjusting the pH to 5-6, adding 5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8g of 1-hydroxybenzotriazole, reacting at 25°C for 24h, dialyzing in deionized water for 20h, pre-freezing at -20°C for 4h, heating to -10°C and drying for 20h, heating to 20°C and drying for 10h to obtain a first intermediate.

[0054] Step S2, 10g of the first intermediate was dispersed in 100g of ethanol aqueous solution (80g of water, 20g of ethanol, 0.4g of Tween-80), the pH was adjusted to 6-7, 3.5g of dimethylaminoethyl methacrylate, 0.5g of ammonium persulfate and 0.15g of N,N'-methylenebisacrylamide were added, and the mixture was reacted at 30°C for 3h to obtain a second intermediate, 0.75g of sodium periodate was added, and the mixture was oxidized for 3h to obtain modified carboxymethyl cellulose, such as Figure 2 shown.

[0055] Step S3, dispersing 10 g of xanthan gum and 5 g of isophthalic acid hydrazide in 100 g of ethanol aqueous solution (50 g of water and 50 g of ethanol), adding 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 g of 1-hydroxybenzotriazole, adjusting the pH value to 5-6, reacting for 24 h, dialyzing in deionized water for 20 h, pre-freezing at -20 ° C for 4 h, heating to -10 ° C and drying for 20 h, heating to 20 ° C and drying for 10 h to obtain modified xanthan gum, such as Figure 3 shown.

[0056] Step S4, 10g of arginine / lysine polypeptide, 7.5g of hexapeptide-11, 10g of palmitoyl tetrapeptide-7, and 8g of palmitoyl tripeptide-1 were dispersed in 100g of 1,3-propylene glycol aqueous solution (20g of 1,3-propylene glycol and 80g of water), 0.05g of mannitol, 0.1g of diethylenetriamine pentaacetic acid and 0.5g of transdermal aid (0.25g of caryophyllene and 0.25g of transdermal peptide) were added, and stirred to obtain a composite polypeptide.

[0057] Step S5: 10 g of modified carboxymethyl cellulose and 20 g of modified xanthan gum were dispersed in 100 g of deionized water, 3.5 g of the composite polypeptide was added, and PBS buffer solution was added, and the mixture was reacted at 30° C. for 12 h to obtain a pH-sensitive hydrogel containing the composite polypeptide. Figure 2 As shown, 0.05 g of panthenol, 0.05 g of ethylhexylglycerol and 0.01 g of 1,2-hexanediol were added to obtain a pH-sensitive hydrogel containing a composite polypeptide, as shown in FIG. Figure 4 shown.

[0058] Example 2

[0059] like Figure 1 As shown, a polypeptide composition, the preparation method of which comprises:

[0060] Step S1, dispersing 10g of sodium carboxymethyl cellulose in 120g of deionized water, adding 4g of 3-aminopropane-1,2-diol, reacting at 25°C for 1h, adjusting the pH to 5-6, adding 5.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 8.5g of 1-hydroxybenzotriazole, reacting at 30°C for 22h, dialyzing in deionized water for 22h, pre-freezing at -25°C for 3h, heating to 0°C and drying for 18h, heating to 25°C and drying for 9h, to obtain a first intermediate.

[0061] Step S2, 10g of the first intermediate was dispersed in 120g of ethanol aqueous solution (88g of water, 31g of ethanol, 0.6g of Tween-80), the pH was adjusted to 6-7, 4.5g of dimethylaminoethyl methacrylate, 0.75g of azobisisobutyronitrile and 0.2g of N,N'-methylenebisacrylamide were added, and the reaction was carried out at 40°C for 2.5h to obtain the second intermediate, 1g of sodium periodate was added, and the mixture was oxidized for 2.5h to obtain modified carboxymethyl cellulose, such as Figure 2 shown.

[0062] Step S3, 10g xanthan gum and 6g isophthalic acid hydrazide were dispersed in 120g ethanol aqueous solution (48g water and 72g ethanol), 0.75g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75g 1-hydroxybenzotriazole were added, the pH value was adjusted to 5-6, the reaction was carried out for 22h, the mixture was dialyzed in deionized water for 22h, pre-frozen at -25°C for 3h, heated to 0°C and dried for 18h, heated to 25°C and dried for 9h to obtain modified xanthan gum, such as Figure 3 shown.

[0063] Step S4, 10g of arginine / lysine polypeptide, 9g of hexapeptide-11, 12g of palmitoyl tetrapeptide-7, and 10g of palmitoyl tripeptide-1 were dispersed in 120g of 1,3-propylene glycol aqueous solution (20g of 1,3-propylene glycol and 100g of water), 0.075g of sorbitol, 0.15g of ethylenediaminetetraacetic acid and 0.75g of transdermal aid (0.25g of caryophyllene and 0.5g of transdermal peptide) were added, and stirred to obtain a composite polypeptide.

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

[0065] Example 3

[0066] like Figure 1 As shown, a polypeptide composition, the preparation method of which comprises:

[0067] Step S1, dispersing 10g of sodium carboxymethyl cellulose in 135g of deionized water, adding 5g of 3-aminopropane-1,2-diol, reacting at 25°C for 2h, adjusting the pH to 5-6, adding 6g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 9g of 1-hydroxybenzotriazole, reacting at 25°C for 20h, dialyzing in deionized water for 23h, pre-freezing at -30°C for 3h, heating to 10°C and drying for 16h, heating to 30°C and drying for 9h, to obtain a first intermediate.

[0068] Step S2, 10g of the first intermediate was dispersed in 135g of ethanol aqueous solution (93g of water, 42g of ethanol, 0.84g of Tween-80), the pH was adjusted to 6-7, 6.5gg of dimethylaminoethyl methacrylate, 1g of azobisisobutyronitrile and 0.3g of N,N'-methylenebisacrylamide were added, and the reaction was carried out at 50°C for 2h to obtain the second intermediate, 2g of sodium periodate was added, and the mixture was oxidized for 2h to obtain modified carboxymethyl cellulose, such as Figure 2 shown.

[0069] Step S3, 10g xanthan gum and 5g isophthalic acid hydrazide were dispersed in 135g ethanol aqueous solution (45g water and 90g ethanol), 1g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g 1-hydroxybenzotriazole were added, the pH value was adjusted to 5-6, the reaction was carried out for 20h, the mixture was dialyzed in deionized water for 23h, pre-frozen at -30°C for 3h, heated to 10°C and dried for 16h, heated to 30°C and dried for 9h to obtain modified xanthan gum, such as Figure 3 shown.

[0070] Step S4: 10 g of arginine / lysine polypeptide, 11 g of hexapeptide-11, 13 g of palmitoyl tetrapeptide-7, and 13 g of palmitoyl tripeptide-1 were dispersed in 135 g of 1,3-propylene glycol aqueous solution (16 g of 1,3-propylene glycol and 120 g of water), and 0.09 g of dextran, 0.2 g of diethylenetriamine pentaacetic acid, and 1 g of transdermal aid (0.25 g of caryophyllene and 0.75 g of transdermal peptide) were added, and stirred to obtain a composite polypeptide.

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

[0072] Example 4

[0073] like Figure 1 As shown, a polypeptide composition, the preparation method of which comprises:

[0074] Step S1, dispersing 10g of sodium carboxymethyl cellulose in 150g of deionized water, adding 5.5g of 3-aminopropane-1,2-diol, reacting at 25°C for 10h, adjusting the pH to 5-6, adding 7g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of 1-hydroxybenzotriazole, reacting at 35°C for 18h, dialyzing in deionized water for 24h, pre-freezing at -40°C for 2h, heating to 20°C and drying for 12h, heating to 40°C and drying for 8h to obtain a first intermediate.

[0075] Step S2, 10g of the first intermediate was dispersed in 150g of ethanol aqueous solution (96g of water, 53g of ethanol, 0.96g of Tween-80), the pH was adjusted to 6-7, 7.5g of dimethylaminoethyl methacrylate, 1.1g of ammonium persulfate and 0.33g of N,N'-methylenebisacrylamide were added, and the reaction was carried out at 60°C for 1h to obtain the second intermediate, 2.5g of sodium periodate was added, and the mixture was oxidized for 1h to obtain modified carboxymethyl cellulose, such as Figure 2 shown.

[0076] Step S3, 10g xanthan gum and 7g isophthalic acid hydrazide were dispersed in 150g ethanol aqueous solution (43g water and 107g ethanol), 1.5g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.5g 1-hydroxybenzotriazole were added, the pH value was adjusted to 5-6, the reaction was carried out for 18h, the mixture was dialyzed in deionized water for 24h, pre-frozen at -40°C for 2h, heated to 20°C and dried for 12h, heated to 40°C and dried for 8h to obtain modified xanthan gum, such as Figure 3 shown.

[0077] Step S4, 10g of arginine / lysine polypeptide, 12.5g of hexapeptide-11, 15g of palmitoyl tetrapeptide-7, and 16g of palmitoyl tripeptide-1 were dispersed in 150g of 1,3-propylene glycol aqueous solution (15g of 1,3-propylene glycol and 135g of water), 0.1g of mannitol, 0.25g of ethylenediaminetetraacetic acid and 1.2g of transdermal aid (0.2g of caryophyllene and 1g of transdermal peptide) were added, and stirred to obtain a composite polypeptide.

[0078] Step S5: 10 g of modified carboxymethyl cellulose and 50 g of modified xanthan gum were dispersed in 150 g of deionized water, 7.5 g of the composite polypeptide was added, and PBS buffer solution was added, and the mixture was reacted at 50° C. for 6 h to obtain a pH-sensitive hydrogel containing the composite polypeptide. Figure 2 As shown, 0.15 g of panthenol, 0.075 g of gluconolactone and 0.035 g of 1,2-hexanediol were added to obtain a pH-sensitive hydrogel containing a composite polypeptide. Figure 4 shown.

[0079] Comparative Example 1

[0080] A polypeptide composition, the preparation method of which is different 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 is different 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: Take fibroblasts growing in logarithmic phase and count them according to 8×10 3 Cells were seeded into 96-well plates at a density of 100 μL / well and incubated overnight at 37°C and 5% CO2. The culture medium was removed, and 200 μL of the test substance at a concentration of 0.05% was added to each well. The cells were cultured for another 24 hours, with at least three replicates per group. The supernatant was collected and assayed for FN (fibronectin), COLI (type I collagen), and TIMP-1 (tissue inhibitor of matrix metalloproteinases-1) protein by ELISA. 100 μL of CCK-8 stain was added to the cell plates and incubated in the incubator for 1 hour. Changes in absorbance at 450 nm were measured.

[0085] (2) Skin repair experiment: BALB / c mice weighing 20±2g were used. The model control group consisted of mice with trauma and no drug treatment. The hair on the back of the mice was removed. The mice were treated with skin defect and burn infection models, respectively. The mice were treated with the drug (test sample) or left untreated (model control group). The drug-treated group was given 3-5mg twice a day for 8 consecutive days. The wound surface was quantitatively analyzed to evaluate the healing effect.

[0086] Table 1 Fibroblast detection results

[0087]

[0088] COLⅠ is the most abundant collagen in the dermis. It provides adhesion sites for fibroblasts and endothelial cells, guiding cell migration to the wound area and accelerating granulation tissue formation. High expression indicates that fibroblasts are actively synthesizing the extracellular matrix (ECM), accelerating wound contraction and tissue remodeling, and is a key marker for wound repair entering the proliferation and remodeling phases. FN binds simultaneously to cell surface receptors and the ECM, providing a migration pathway for newly formed endothelial cells, promoting angiogenesis and improving local blood supply to the wound. High FN expression indicates that the wound is in the transition from the inflammatory phase to the proliferation phase. Increased FN synthesis accelerates cell recruitment and matrix remodeling, making it a key signal for initiating wound repair. TIMP-1 inhibits matrix metalloproteinases (MMPs), preventing delayed wound healing caused by excessive ECM degradation, but excessive inhibition may increase the risk of scarring.

[0089] As shown in 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 COL Ⅰ, FN and TIMP-1. After treating cells with the compositions prepared in Comparative Examples 1 to 3, the cell viability 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 Skin defect healing rate test results

[0091]

[0092]

[0093] As shown in 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 rates were all above 15% on the second day, reached above 35% on the fourth day, and exceeded 85% on the eighth day. However, after application of the compositions prepared in Comparative Examples 1 and 2, the healing rates of the wounds on the back of mice were significantly lower than those in Examples 1 to 4, with Comparative Example 2 having a poor effect, with a healing rate of only 6.06% on the second day and a healing rate of only 49.87% on the eighth day.

[0094] In comparative example 1, dimethylaminoethyl methacrylate was not added, and the prepared hydrogel lacked a segment that was sensitive to pH. Therefore, it was difficult for the hydrogel to be quickly cross-linked to form a thin film to cover the wound when applied to the wound. Therefore, the composition was easily rubbed off during the activity of the mouse, and the therapeutic effect was poor. In comparative example 2, the polypeptide was not wrapped with a hydrogel, and a traditional dressing (gauze) was used for administration. The composition degraded too quickly, and the therapeutic effect was significantly lower than that of the group embedded with the hydrogel. At the same time, the gauze was displaced during the activity of the mouse, rubbing the wound, and could not cover the wound well, affecting wound healing. Experiments have shown that the use of pH-sensitive hydrogel to encapsulate polypeptides can achieve long-term sustained-release drugs, form a thin film on the skin, better cover the wound, and continuously administer the drug, while overcoming the limitations of traditional dressings.

[0095] Table 3 Burn infection healing rate test results

[0096]

[0097] As shown in Table 3, the compositions prepared in Examples 1 to 3 were significantly effective in treating burn-infected wounds in mice. The composition prepared in Example 3 showed the best therapeutic effect, achieving 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 were less effective in treating burn-infected wounds in mice. The wound healing rate for Comparative Example 1 on day 8 was only 51.12%, and the wound healing rate for Comparative Example 2 on day 8 was only 45.28%.

[0098] After a period of administration, the composition prepared in Comparative Example 1 will cause the gel to fall off or adhere to other impurities due to the activity of the mice, thereby causing the gel to be unable to continue to be administered. At the same time, it cannot cover the wound well and block external dust and bacteria. Instead, it will cause wound infection due to the adhesion of other impurities. After a period of administration, the active ingredient of the composition prepared in Comparative Example 2 will become ineffective or contaminated, which greatly reduces the therapeutic effect. At the same time, the traditional dressing is easy to move and fall off, and the wound is exposed to the environment, which is prone to secondary infection and aggravates bacterial infection.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide composition, characterized in that: The polypeptide composition includes a pH-sensitive hydrogel, a composite polypeptide, an antioxidant, an antibacterial adjuvant, and a preservative; the composite polypeptide includes 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: Here, n is an integer between 100 and 1000.

2. A polypeptide composition according to claim 1, characterized in that, The pH-sensitive hydrogel is prepared by reacting modified carboxymethyl cellulose and modified xanthan gum. The mass ratio of the modified carboxymethyl cellulose, modified xanthan gum, composite 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); 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).

3. A polypeptide composition according to claim 1, 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; the transdermal adjuvant is caryophyllene and transdermal peptide, and the mass ratio of caryophyllene to transdermal peptide is 1:(1~5).

4. The method for preparing a polypeptide composition according to any one of claims 1 to 3, characterized in that: include: Step S1, dispersing sodium carboxymethyl cellulose in deionized water, adding 3-aminopropane-1,2-diol, performing a first reaction, adjusting the pH to 5-6, adding a condensing agent and a catalyst, performing a second reaction, purifying, and freeze-drying to obtain a first intermediate; Step S2, dispersing the first intermediate in a first solvent, adjusting the pH to 6-7, adding dimethylaminoethyl methacrylate, an initiator, and a cross-linking agent, reacting to obtain a second intermediate, adding sodium periodate, and oxidizing to obtain modified carboxymethyl cellulose; Step S3, dispersing xanthan gum and isophthalic acid hydrazide in a second solvent, adding a condensing agent and a catalyst, adjusting the pH value to 5-6, reacting, purifying, and freeze-drying to obtain modified xanthan gum; Step S4, dispersing the arginine / lysine polypeptide, hexapeptide-11, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-1 in a third solvent, adding a filler, a stabilizer, and a transdermal adjuvant, and stirring to obtain a composite polypeptide; Step S5: dispersing modified carboxymethyl cellulose and modified xanthan gum in deionized water, adding the composite polypeptide, reacting, adding an antioxidant, an antibacterial adjuvant and a preservative to prepare a pH-sensitive hydrogel containing the composite polypeptide.

5. The method for preparing a polypeptide composition according to claim 4, wherein: The purification operation is dialyzed in deionized water for 20 to 24 hours; the freeze-drying operation is pre-freezing at -20°C to -40°C for 2 to 4 hours, heating to -10°C to 20°C for drying for 12 to 20 hours, and heating to 20°C to 40°C for drying for 8 to 10 hours; the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the catalyst is 1-hydroxybenzotriazole.

6. The method for preparing a polypeptide composition according to claim 4, wherein: In step S1, the temperature of the first reaction is 25-30° C., and the time of the first reaction is 1-2 hours; the temperature of the second reaction is 25-35° C., and the time of the second reaction is 18-24 hours; the mass ratio of the 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).

7. The method for preparing a polypeptide composition according to claim 4, wherein: In step S2, the first solvent is an ethanol aqueous 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 initiator is any one or more of ammonium persulfate and azobisisobutyronitrile; the cross-linking agent is N,N'-methylenebisacrylamide; the mass ratio of the first intermediate, the first solvent, dimethylaminoethyl methacrylate, the initiator and the cross-linking 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°C, the reaction time is 1-3 hours, and the oxidation time is 1-3 hours.

8. The method for preparing a polypeptide composition according to claim 4, wherein: In step S3, the second solvent is an ethanol-water solution, and the mass ratio of water to ethanol is 1:(1-2.5); the mass ratio of xanthan gum, isophthalic acid 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); and the reaction time is 18-24 hours.

9. The method for preparing a polypeptide composition according to claim 4, wherein: In step S4, the third solvent is a 1,3-propylene glycol aqueous solution, and the mass ratio of 1,3-propylene glycol to water is 1:(4-9); in step S5, the reaction temperature is 30-50° C., and the reaction time is 6-12 hours.

10. Use of the polypeptide composition according to any one of claims 1 to 3 in the preparation of a drug for repairing skin wounds.

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