A bioactive composite gel dressing for skin scar repair that inhibits lactic acid production

Abstract

This invention discloses a bioactive composite gel dressing for skin scar repair that inhibits lactic acid production, belonging to the technical field of scar repair gel dressings. The gel dressing is obtained by mixing methacrylamide-modified hyaluronic acid, methacrylamide-modified carboxymethyl chitosan, and mesoporous polydopamine, followed by a cross-linking reaction initiated by a cross-linking monomer and a photoinitiator. The mesoporous polydopamine is loaded with berberine and resveratrol. The cross-linking monomer can gradually degrade in the weakly acidic, highly reactive oxygen species microenvironment of scars, achieving controlled release of active substances. The mesoporous polydopamine combines drug carrier and antioxidant functions; its three-dimensional porous network structure contains numerous carboxyl groups, providing a moist environment for the wound. The long-chain alkane structure of the cross-linking monomer enhances the compatibility and transdermal properties of hydrophobic drugs. The active substances berberine and resveratrol inhibit lactic acid production and abnormal fibroblast proliferation. This dressing has high biocompatibility, and its decomposition products can participate in skin protein synthesis and barrier repair.

Description

Technical Field

[0001] This invention belongs to the field of scar repair gel dressing technology, and particularly relates to a bioactive composite gel dressing for skin scar repair that inhibits lactic acid production. Background Technology

[0002] Skin scars are a common pathological outcome of the natural healing process of wounds or lesions following trauma. They are essentially caused by an imbalance between excessive proliferation of fibroblasts and abnormal deposition and remodeling of the extracellular matrix (especially type I and type III collagen). Pathological scars (such as hypertrophic scars and keloids) not only affect appearance but may also be accompanied by itching, pain, and functional impairment, placing a heavy physiological and psychological burden on patients. Currently, treatment methods for scars include surgical excision, laser therapy, radiation therapy, pressure therapy, and drug injections. However, these methods often have limitations. For example, surgical treatment carries the risk of recurrence; laser and radiation therapies are expensive and may cause side effects such as pigmentation; and hormonal drug injections may cause adverse reactions such as skin atrophy and telangiectasia. Therefore, developing a novel, efficient, safe, and easy-to-use scar management program is of significant clinical importance.

[0003] Recent studies have revealed that metabolic reprogramming within the scar microenvironment is a key driver of scar formation. Compared to normal skin fibroblasts, fibroblasts, even under oxygen-rich conditions, tend to engage in highly efficient aerobic glycolysis, producing large amounts of lactic acid. Lactic acid accumulation not only directly stimulates collagen synthesis but also continuously promotes fibroblast proliferation and differentiation and inhibits collagenase activity through multiple mechanisms, including acidifying the microenvironment, stabilizing hypoxia-inducible factor-1α (HIF-1α), and activating the transforming growth factor-β (TGF-β) signaling pathway. This creates a vicious cycle, leading to scar hyperplasia and sclerosis. Therefore, targeting and inhibiting glycolysis and reducing lactic acid production is considered a promising new strategy for addressing scar formation at its metabolic root.

[0004] Berberine and resveratrol are two bioactive substances derived from natural plants. Numerous studies have demonstrated their pharmacological activities in regulating cellular metabolism, inhibiting glycolysis and lactic acid production, and they exhibit high biocompatibility. Berberine can reduce glucose uptake and metabolic flux by inhibiting the activity of mitochondrial complex I and key glycolytic enzymes (such as hexokinase and pyruvate kinase M2). Resveratrol, on the other hand, can activate the AMP-activated protein kinase (AMPK) signaling pathway, promoting mitochondrial biosynthesis and oxidative phosphorylation while inhibiting the expression of glycolysis-related genes. The two have the potential for synergistic and complementary effects in metabolic regulation. However, the main technical challenge for their successful application in scar treatment lies in how to efficiently and targetedly deliver these two hydrophobic bioactive substances deep into scar tissue and achieve controlled release within the abnormal scar microenvironment. Summary of the Invention

[0005] In view of the above situation and to overcome the defects of the prior art, the present invention has bioactivity, wherein the mesoporous polydopamine is loaded with the active substances berberine and resveratrol, contains a three-dimensional spatial network with a porous structure, contains a large number of carboxyl groups, has a good water absorption rate, and can slowly release berberine and resveratrol to inhibit lactic acid production, inhibit glycolysis, thereby inhibiting lactic acid production and promoting scar repair.

[0006] To achieve the above objectives, the following technical solution is adopted: This invention provides a bioactive composite gel dressing for skin scar repair that inhibits lactic acid production. It is obtained by mixing methacrylamide hyaluronic acid, methacrylamide carboxymethyl chitosan, and mesoporous polydopamine and then initiating a crosslinking reaction with a crosslinking monomer and a photoinitiator. The mesoporous polydopamine is loaded with the active substances berberine and resveratrol.

[0007] Berberine and resveratrol, as active substances, inhibit lactate production through multi-target action. Berberine reduces glucose metabolic flux by inhibiting the activity of mitochondrial complex I and key glycolytic enzymes (such as hexokinase); resveratrol activates the AMPK signaling pathway, regulates cellular energy metabolism, promotes oxidative phosphorylation, and inhibits glycolysis. The two work synergistically to reduce lactate levels in the scar microenvironment at its source, thereby correcting the abnormal metabolic state of fibroblasts, inhibiting their excessive proliferation and collagen deposition, and promoting scar softening and repair.

[0008] Mesoporous polydopamine not only serves as a drug carrier but also possesses excellent antioxidant activity, capable of clearing excess ROS from scar sites and reducing oxidative stress damage. Simultaneously, the adhesive properties of polydopamine help dressings adhere closely to the skin, forming a physical barrier, reducing moisture loss, and providing a moist healing environment for the wound.

[0009] Furthermore, the crosslinking monomer is prepared by the following steps:

[0010] S1. Dissolve carboxymethylcysteine ​​and 3,4-dihydroxy-2'-chloroacetophenone in N,N-dimethylformamide solvent, add potassium carbonate, and stir the reaction at 60-80℃ for 6-12h under nitrogen protection. After the reaction is completed, cool to room temperature, adjust the pH to 3-4 with dilute hydrochloric acid, precipitate, filter, wash with deionized water, and vacuum dry to obtain intermediate A;

[0011] S2. Intermediate A and 4-boron-DL-phenylalanine were added to a mixed solvent of toluene and ethanol, and 2 mol / L sodium carbonate solution was added. The mixture was refluxed at 80-90℃ for 8-16 h under nitrogen protection. After the reaction was completed, the mixture was cooled, and dilute hydrochloric acid was added to adjust the pH to neutral. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, concentrated, and vacuum dried to obtain intermediate B.

[0012] S3. Add intermediate B and 10-undecenal to dichloromethane, add glacial acetic acid, and stir the reaction at room temperature for 12-24 h. After the reaction is complete, wash with saturated sodium bicarbonate solution, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain the crosslinking monomer.

[0013] Furthermore, the composite gel dressing is prepared through the following steps:

[0014] (1) Berberine and resveratrol were added to deionized water and ultrasonically dispersed for 30 min to obtain an active substance dispersion. Mesoporous polydopamine was added to deionized water and ultrasonically dispersed for 30 min to obtain a polydopamine dispersion. The two dispersions were mixed and stirred at 25-37℃ for 12-24 h. Then, the mixture was centrifuged at 8000-10000 rpm for 10-15 min. The solid was collected, washed with deionized water, and vacuum dried to obtain mesoporous polydopamine loaded with active substances.

[0015] (2) Take methacrylamide hyaluronic acid and add it to PBS buffer at pH 7.4. Stir and dissolve at 4°C for 4-6 hours to obtain a hyaluronic acid solution with a concentration of 2.5-7.5% w / v. Take methacrylamide carboxymethyl chitosan and mesoporous polydopamine loaded with active substances and add them to deionized water. Disperse by ultrasonication for 1 hour and then stir for 2-4 hours to obtain a uniform chitosan-polydopamine dispersion.

[0016] (3) Mix hyaluronic acid solution with chitosan-polydopamine dispersion to obtain a mixture, add crosslinking monomer and photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, stir evenly, inject into mold, and irradiate under ultraviolet light with wavelength of 365nm and intensity of 10-20mW / cm2 for 1-5min to perform photocrosslinking, and obtain the composite gel dressing after drying.

[0017] Further, in step S1, the feeding ratio of carboxymethylcysteine, 3,4-dihydroxy-2'-chloroacetophenone, N,N-dimethylformamide, and potassium carbonate is 10-15g: 12-18g: 100-200mL: 20-30g.

[0018] Further, in step S2, the feeding ratio of intermediate A, 4-boron-DL-phenylalanine, toluene, ethanol, and sodium carbonate aqueous solution is: 15-25g: 18-30g: 150-200mL: 150-250mL: 80-150mL.

[0019] Furthermore, in step S3, the feeding ratio of intermediate B, 10-undecenal, dichloromethane, and glacial acetic acid is 20-30g: 15-25mL: 200-300mL: 25-40g.

[0020] Furthermore, the feeding ratio of berberine, resveratrol, and deionized water in the active substance dispersion is 0.2-1.5g:0.2-1.5g:50-100mL; the feeding ratio of mesoporous polydopamine and deionized water in the polydopamine dispersion is 0.5-3.0g:20-50mL; and the feeding volume ratio of the active substance dispersion to the polydopamine dispersion is 1:1-3:1.

[0021] Furthermore, the ratio of methacrylamide hyaluronic acid to PBS buffer in the hyaluronic acid solution is 5-15g:100-200mL, and the ratio of methacrylamide carboxymethyl chitosan, mesoporous polydopamine loaded with active substances, to deionized water in the chitosan-polydopamine dispersion is 5-15g:1-5g:50-100mL.

[0022] Furthermore, in step (3), the feeding ratio of crosslinking monomer and 2-hydroxy-2-methyl-1-phenyl-1-propanone is 1-5g: 0.1-0.5mL.

[0023] Furthermore, the molecular weight of the methacrylamide hyaluronic acid is 100kDa-300kDa, and the molecular weight of the methacrylamide carboxymethyl chitosan is 100kDa-200kDa.

[0024] Furthermore, the specific surface area of ​​the mesoporous polydopamine is 200-600 m² / g. 2 / g, with an average pore size of 2-10nm.

[0025] The beneficial effects of this invention are:

[0026] The composite gel dressing of the present invention is obtained by mixing methacrylamide hyaluronic acid, methacrylamide carboxymethyl chitosan, and mesoporous polydopamine and then initiating a cross-linking reaction with a cross-linking monomer and a photoinitiator. It has bioactivity, wherein the mesoporous polydopamine is loaded with the active substances berberine and resveratrol, contains a porous three-dimensional network, contains more carboxyl groups, has a good water absorption rate, and can slowly release berberine and resveratrol to inhibit lactic acid production, inhibit glycolysis, thereby inhibiting lactic acid production and promoting scar repair.

[0027] The design of thioether and borate ester bonds in the crosslinking monomers has environmentally responsive characteristics. Scar tissue is usually weakly acidic and has a high level of reactive oxygen species. Thioether bonds are easily broken under acidic conditions, while borate ester bonds are sensitive to ROS. This dual-response mechanism enables the gel network to degrade gradually in the scar microenvironment, achieving the controlled release of active substances berberine and resveratrol, improving drug utilization and targeting, and avoiding the side effects caused by burst release.

[0028] The 10-undecenal long-chain alkane structure introduced into the crosslinking monomer can enhance the compatibility and loading capacity of the gel for hydrophobic drugs. At the same time, the long-chain alkane can interfere with the arrangement of the lipid bilayer of the stratum corneum, improve the transdermal performance of the dressing, and promote the penetration of active substances into the dermis to act on deep fibroblasts in scars.

[0029] The cross-linked monomer contains unsaturated double bonds and carbonyl groups. The unsaturated double bonds can undergo photo-initiated polymerization with methacrylamide hyaluronic acid and methacrylamide carboxymethyl chitosan, while the carbonyl groups can undergo Schiff base reactions with the amino groups on methacrylamide carboxymethyl chitosan to carry out secondary cross-linking, thereby forming a three-dimensional spatial network structure with good water absorption, which can provide a suitable moist healing environment for wounds.

[0030] In addition, the gel dressing and its decomposition products are highly biocompatible. The amino acids such as cysteine ​​and phenylalanine and their derivatives produced by the decomposition of cross-linked monomers are small molecule amino acids required for skin repair, which can be directly utilized by cells and participate in protein synthesis and barrier repair. Attached Figure Description

[0031] Figure 1 This is a diagram illustrating the preparation equation for the crosslinking monomer of this invention;

[0032] Figure 2 Intermediate A, intermediate B, and crosslinking monomer prepared according to the present invention 1 HNMR characterization images;

[0033] Figure 3 This is a comparative graph showing the in vitro drug release behavior test results of the gel dressing of the present invention;

[0034] Figure 4 This is a comparative graph showing the in vitro cytotoxicity and antiproliferative activity evaluation results of the gel dressing of the present invention;

[0035] Figure 5 This is a comparison chart showing the in vitro efficacy of the gel dressing of the present invention in inhibiting lactic acid production.

[0036] Figure 6 This is a comparison chart of the test results of the mechanical properties and swelling behavior of the gel dressing of the present invention;

[0037] Figure 7 This is a comparative diagram showing the experimental results of the gel dressing for rabbit ear scar repair according to the present invention.

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels. Specifically, human skin fibroblasts and DMEM culture medium were purchased from Wuhan Shangen Biotechnology Co., Ltd., CCK-8 reagent was purchased from Shanghai Beibo Biotechnology Co., Ltd., TGF-β1 was purchased from Zhengzhou Sensu Biotechnology Co., Ltd., and New Zealand white rabbits were purchased from Qingdao Kangda Biotechnology Co., Ltd.

[0042] The preparation equations of the crosslinking monomers in various embodiments of the present invention are as follows: Figure 1 As shown.

[0043] Example 1:

[0044] A bioactive composite gel dressing for skin scar repair that inhibits lactic acid production is obtained by mixing methacrylamide hyaluronic acid, methacrylamide carboxymethyl chitosan, and mesoporous polydopamine and then initiating a crosslinking reaction with a crosslinking monomer and a photoinitiator. The mesoporous polydopamine is loaded with the active substances berberine and resveratrol.

[0045] The crosslinking monomer is prepared by the following steps:

[0046] S1. Dissolve 10g of carboxymethylcysteine ​​and 12g of 3,4-dihydroxy-2'-chloroacetophenone in 100mL of N,N-dimethylformamide solvent, add 20g of potassium carbonate, and stir the mixture at 60℃ for 6h under nitrogen protection. After the reaction is completed, cool to room temperature, adjust the pH to 3 with dilute hydrochloric acid, precipitate out, filter, wash with deionized water, and dry under vacuum to obtain intermediate A;

[0047] S2. Add 15g of intermediate A and 18g of 4-boron-DL-phenylalanine to a mixed solvent of 150mL toluene and 150mL ethanol, add 80mL of 2mol / L sodium carbonate solution, and reflux at 80℃ for 8h under nitrogen protection. After the reaction is complete, cool, add dilute hydrochloric acid to adjust the pH to neutral, separate the organic layer, extract the aqueous layer with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain intermediate B;

[0048] S3. Add 20g of intermediate B and 15mL of 10-undecenal to 200mL of dichloromethane, add 25g of glacial acetic acid, stir and react at room temperature for 12h. After the reaction is complete, wash with saturated sodium bicarbonate solution, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain the crosslinking monomer.

[0049] The composite gel dressing is prepared through the following steps:

[0050] (1) Take 0.2g of berberine and 0.2g of resveratrol and add them to 50mL of deionized water. Disperse them by sonication for 30min to obtain an active substance dispersion. Take 0.5g of mesoporous polydopamine and add it to 20mL of deionized water. Disperse it by sonication for 30min to obtain a polydopamine dispersion. Mix 50mL of active substance dispersion with 50mL of polydopamine dispersion and stir at 25℃ for 12h. Then centrifuge at 8000rpm for 10min to separate the solid. Wash it with deionized water and vacuum dry it to obtain mesoporous polydopamine loaded with active substances.

[0051] (2) Take 5g of methacrylamide hyaluronic acid and add it to 100mL of pH7.4 PBS buffer. Stir and dissolve at 4℃ for 4h to obtain hyaluronic acid solution. Take 5g of methacrylamide carboxymethyl chitosan and 1g of mesoporous polydopamine loaded with active substances and add them to 50mL of deionized water. Disperse by ultrasonication for 1h and then stir for 2h to obtain a uniform chitosan-polydopamine dispersion.

[0052] (3) Mix hyaluronic acid solution with chitosan-polydopamine dispersion to obtain a mixture, add 1g crosslinking monomer and 0.1mL photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, stir evenly, inject into mold, and irradiate for 1min under ultraviolet light with wavelength of 365nm and intensity of 10mW / cm2 for photocrosslinking. After drying, the composite gel dressing is obtained.

[0053] The methacrylamide-modified hyaluronic acid has a molecular weight of 100 kDa, and the methacrylamide-modified carboxymethyl chitosan has a molecular weight of 100 kDa; the mesoporous polydopamine has a specific surface area of ​​200 m². 2 / g, with an average pore size of 2nm.

[0054] Implementation: 2:

[0055] A bioactive composite gel dressing for skin scar repair that inhibits lactic acid production is obtained by mixing methacrylamide hyaluronic acid, methacrylamide carboxymethyl chitosan, and mesoporous polydopamine and then initiating a crosslinking reaction with a crosslinking monomer and a photoinitiator. The mesoporous polydopamine is loaded with the active substances berberine and resveratrol.

[0056] The crosslinking monomer is prepared by the following steps:

[0057] S1. Dissolve 15g of carboxymethylcysteine ​​and 18g of 3,4-dihydroxy-2'-chloroacetophenone in 200mL of N,N-dimethylformamide solvent, add 30g of potassium carbonate, and stir the mixture at 80℃ for 12h under nitrogen protection. After the reaction is completed, cool to room temperature, adjust the pH to 4 with dilute hydrochloric acid, precipitate out, filter, wash with deionized water, and dry under vacuum to obtain intermediate A;

[0058] S2. Add 25g of intermediate A and 30g of 4-boron-DL-phenylalanine to a mixed solvent of 200mL toluene and 250mL ethanol, add 150mL of 2mol / L sodium carbonate solution, and reflux at 90℃ for 16h under nitrogen protection. After the reaction is complete, cool, add dilute hydrochloric acid to adjust the pH to neutral, separate the organic layer, extract the aqueous layer with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain intermediate B;

[0059] S3. Add 30g of intermediate B and 25mL of 10-undecenal to 300mL of dichloromethane, add 40g of glacial acetic acid, stir and react at room temperature for 24h. After the reaction is complete, wash with saturated sodium bicarbonate solution, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain the crosslinking monomer.

[0060] The composite gel dressing is prepared through the following steps:

[0061] (1) Take 1.5g of berberine and 1.5g of resveratrol and add them to 100mL of deionized water. Disperse them by sonication for 30min to obtain an active substance dispersion. Take 3.0g of mesoporous polydopamine and add it to 50mL of deionized water. Disperse it by sonication for 30min to obtain a polydopamine dispersion. Mix 100mL of active substance dispersion with 100mL of polydopamine dispersion and stir at 37℃ for 24h. Then centrifuge at 10000rpm for 15min to separate the solid. Wash it with deionized water and vacuum dry it to obtain mesoporous polydopamine loaded with active substances.

[0062] (2) Take 15g of methacrylamide hyaluronic acid and add it to 200mL of pH7.4 PBS buffer. Stir and dissolve at 4℃ for 6h to obtain hyaluronic acid solution. Take 15g of methacrylamide carboxymethyl chitosan and 5g of mesoporous polydopamine loaded with active substances and add them to 100mL of deionized water. Disperse by ultrasonication for 1h and then stir for 4h to obtain a uniform chitosan-polydopamine dispersion.

[0063] (3) Mix hyaluronic acid solution with chitosan-polydopamine dispersion to obtain a mixture, add 5g crosslinking monomer and 0.5mL photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, stir evenly, inject into mold, and irradiate under ultraviolet light with wavelength of 365nm and intensity of 20mW / cm2 for 5min to perform photocrosslinking. After drying, the composite gel dressing is obtained.

[0064] The methacrylamide-modified hyaluronic acid has a molecular weight of 300 kDa, and the methacrylamide-modified carboxymethyl chitosan has a molecular weight of 200 kDa; the mesoporous polydopamine has a specific surface area of ​​600 m². 2 / g, with an average pore size of 10nm.

[0065] Example 3:

[0066] A bioactive composite gel dressing for skin scar repair that inhibits lactic acid production is obtained by mixing methacrylamide hyaluronic acid, methacrylamide carboxymethyl chitosan, and mesoporous polydopamine and then initiating a crosslinking reaction with a crosslinking monomer and a photoinitiator. The mesoporous polydopamine is loaded with the active substances berberine and resveratrol.

[0067] The crosslinking monomer is prepared by the following steps:

[0068] S1. Dissolve 12.5g of carboxymethylcysteine ​​and 15g of 3,4-dihydroxy-2'-chloroacetophenone in 150mL of N,N-dimethylformamide solvent, add 25g of potassium carbonate, and stir the mixture at 70℃ for 9h under nitrogen protection. After the reaction is complete, cool to room temperature, adjust the pH to 3.5 with dilute hydrochloric acid, precipitate out, filter, wash with deionized water, and dry under vacuum to obtain intermediate A;

[0069] S2. Add 20g of intermediate A and 24g of 4-boron-DL-phenylalanine to a mixed solvent of 175mL toluene and 200mL ethanol, add 115mL of 2mol / L sodium carbonate solution, and reflux at 85℃ for 12h under nitrogen protection. After the reaction is complete, cool, add dilute hydrochloric acid to adjust the pH to neutral, separate the organic layer, extract the aqueous layer with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain intermediate B;

[0070] S3. Add 25g of intermediate B and 20mL of 10-undecenal to 250mL of dichloromethane, add 32g of glacial acetic acid, stir and react at room temperature for 18h. After the reaction is complete, wash with saturated sodium bicarbonate solution, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain the crosslinking monomer.

[0071] The composite gel dressing is prepared through the following steps:

[0072] (1) Take 0.8g of berberine and 0.8g of resveratrol and add them to 75mL of deionized water. Disperse them by sonication for 30min to obtain an active substance dispersion. Take 1.75g ​​of mesoporous polydopamine and add it to 35mL of deionized water. Disperse it by sonication for 30min to obtain a polydopamine dispersion. Mix 75mL of active substance dispersion with 75mL of polydopamine dispersion and stir at 31℃ for 18h. Then centrifuge at 9000rpm for 12.5min to separate the solid. Collect the solid, wash it with deionized water, and vacuum dry it to obtain mesoporous polydopamine loaded with active substances.

[0073] (2) Take 10g of methacrylamide hyaluronic acid and add it to 150mL of pH7.4 PBS buffer. Stir and dissolve at 4℃ for 5h to obtain hyaluronic acid solution. Take 10g of methacrylamide carboxymethyl chitosan and 3g of mesoporous polydopamine loaded with active substances and add them to 75mL of deionized water. Disperse by ultrasonication for 1h and then stir for 3h to obtain a uniform chitosan-polydopamine dispersion.

[0074] (3) Mix hyaluronic acid solution with chitosan-polydopamine dispersion to obtain a mixture, add 3g crosslinking monomer and 0.3mL photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, stir evenly, inject into mold, and irradiate under ultraviolet light with wavelength of 365nm and intensity of 15mW / cm2 for 3min to perform photocrosslinking. After drying, the composite gel dressing is obtained.

[0075] The methacrylamide-modified hyaluronic acid has a molecular weight of 200 kDa, and the methacrylamide-modified carboxymethyl chitosan has a molecular weight of 150 kDa; the mesoporous polydopamine has a specific surface area of ​​400 m². 2 / g, with an average pore size of 6nm.

[0076] Comparative Example 1:

[0077] A composite gel dressing differs from Example 3 in that: the crosslinking monomer is not added, and in step (3), the hyaluronic acid solution is directly mixed with the chitosan-polydopamine dispersion, while the rest is the same as in Example 3.

[0078] Comparative Example 2:

[0079] A composite gel dressing differs from Example 3 in that it does not use mesoporous polydopamine to load the active substance. In step (1), the preparation step of the mesoporous polydopamine dispersion is omitted, and berberine and resveratrol are directly added to the chitosan-polydopamine dispersion in step (2). The rest is the same as in Example 3.

[0080] Comparative Example 3:

[0081] A composite gel dressing differs from Example 3 in that the mesoporous polydopamine is not loaded with any substance, while the rest is the same as Example 3.

[0082] Results Analysis

[0083] Intermediate A, intermediate B, and crosslinking monomer prepared in Example 3 of this invention were subjected to... 1 HNMR characterization, images as follows Figure 2 As shown, (a), (b), and (c) correspond to intermediate A, intermediate B, and crosslinking monomer, respectively. Figure 2 In (a), the proton peak at 3.32 ppm is the proton peak of the secondary amine, the proton peaks at 12.34 ppm and 12.39 ppm are the proton peaks of the two carboxyl groups introduced by carboxymethyl cysteine, and the proton peak at 9.48 ppm is the proton peak of the phenolic hydroxyl group introduced by 3,4-dihydroxy-2'-chloroacetophenone. Figure 2(b) The proton peak of the phenolic hydroxyl group at 9.48 ppm disappears, and the amino proton peak at 8.71 ppm is introduced by 4-boron-DL-phenylalanine. A new proton peak on the carboxyl group is generated at 12.89 ppm, which is introduced by 4-boron-DL-phenylalanine. This indicates that the catechol structure of intermediate A reacts with the boric acid in 4-boron-DL-phenylalanine. Figure 2 In (c), the proton peak at 8.71 ppm disappears, and a proton peak consistent with that on the alkane in 10-undecenal appears in the range of 1.5-2 ppm. This indicates that intermediate B and 10-undecenal underwent a Schiff base reaction. In summary, this invention demonstrates the successful synthesis of a substance with attached... Figure 1 Intermediate A, intermediate B, and crosslinking monomers in the intermediate structure.

[0084] Test Example 1:

[0085] In vitro drug release behavior test of gel dressing

[0086] According to the General Chapter 0931, Method II of the 2020 edition of the Pharmacopoeia of the People's Republic of China, drug release tests simulating the scar microenvironment were conducted using an RC806 dissolution tester. The specific steps are as follows: The composite gel dressings prepared in Examples 1-3 and Comparative Examples 1-2 were cut into 10mm diameter discs and placed in dissolution cups containing 100mL of release medium. One set of release medium consisted of pH 7.4 PBS buffer (simulating normal tissue), and the other set consisted of pH 5.5 PBS buffer containing 1mmol / L hydrogen peroxide (simulating the scar microenvironment). The experiment was conducted at 37℃ and 50rpm. Samples were taken after 24h and 48h, and an equal amount of fresh medium was added. The concentrations of berberine and resveratrol were determined by high-performance liquid chromatography. The results are shown in [Figure 1]. Figure 3 .

[0087] from Figure 3 As can be seen, Examples 1-3 release faster in the simulated scar microenvironment, indicating environmentally responsive release. Comparative Example 1 shows a burst release phenomenon because the absence of cross-linked monomers prevents the formation of a three-dimensional spatial network structure. Comparative Example 2 releases faster but has poor controllability.

[0088] Test Example 2:

[0089] Evaluation of in vitro cytotoxicity and antiproliferative activity of gel dressing

[0090] Human skin fibroblast (HSF) viability was tested using a microplate reader via the CCK-8 assay according to GB / T16886.5-2017 standard. The specific steps were as follows: Extracts were extracted from each group of gel dressings. After centrifugation, the supernatant was used as the 100% extract. The 100% extract was diluted with DMEM medium containing 10% fetal bovine serum to create three different concentration gradients: 50%, 25%, and 12.5%. These were co-cultured with HSF cells for 24 h and 48 h, followed by incubation with the CCK-8 reagent. The absorbance at 450 nm was measured, and the relative cell proliferation rate was calculated to obtain the cell viability. Results are shown below. Figure 4 .

[0091] from Figure 4 The results showed that the relative cell proliferation rate was above 90% at all tested concentrations, and no obvious dose-dependent toxicity was observed. This indicates that the controllable release of the drug was achieved through mesoporous polydopamine loading and intelligent cross-linking network, avoiding the cytotoxicity caused by the burst release of high-concentration drugs, and demonstrating excellent biocompatibility.

[0092] Test Example 3:

[0093] Verification of the in vitro effect of gel dressing on inhibiting lactic acid production

[0094] Lactate content in cell supernatant was determined using a spectrophotometer and a lactate detection kit. The specific steps were as follows: HSF cells were seeded in culture plates. When cell confluence reached 60-70%, the medium was replaced with medium containing 10 ng / mL TGF-β1, and cultured for 48 hours to successfully induce a pathological model. The TGF-β1-induced HSF cells were co-cultured with the gel extracts from each group in Test Example 2 for 24 hours. Cell supernatant was collected, and absorbance was measured at 530 nm according to the kit instructions. Lactate concentration was calculated based on the standard curve to obtain the lactate production inhibition rate. Results are shown in [Figure number missing]. Figure 5 .

[0095] exist Figure 5 In the examples, the extracts of Examples 1-3 significantly inhibited lactic acid production, indicating that the gel dressing of the present invention can effectively inhibit abnormal glycolysis of fibroblasts, thereby promoting scar repair.

[0096] Test Example 4:

[0097] Determination of Mechanical Properties and Swelling Behavior of Gel Dressings

[0098] Compression modulus and swelling ratio were tested using a texture analyzer. The specific steps are as follows: The gel was made into a standard cylinder, and its stress-strain curve was measured when it was compressed to 70% of its original height. The compression modulus was calculated. The dried gel was weighed (Wd) and immersed in PBS at pH 7.4. It was removed at 37℃ at different time points, the surface moisture was absorbed, and the gel was weighed again (Ws). The swelling ratio (SR% = (Ws - Wd) / Wd × 100%) was calculated to obtain the equilibrium swelling ratio. The results are shown in [Figure number missing]. Figure 6 .

[0099] exist Figure 6 In the examples, Examples 1-3 exhibit moderate compression modulus and swelling ratio, indicating good mechanical properties and water absorption capacity. Comparative Example 1, lacking crosslinking monomers, has a low modulus and high swelling ratio, but a weaker structure. Comparative Examples 2 and 3, however, have poor mechanical properties, affecting the stability of the dressing.

[0100] Test Example 5:

[0101] Experiment on rabbit ear scar repair with gel dressing

[0102] Twelve healthy male New Zealand white rabbits (6-8 weeks old, weighing 2.0-2.5 kg) were selected. After anesthesia via intravenous injection of sodium pentobarbital (30 mg / kg) into the ear margin, the surgical area on the ventral side of both ears was prepared and disinfected. Under aseptic conditions, a circular full-thickness skin defect was prepared on the non-vascular area of ​​the ventral side of each rabbit's ear using an 8 mm diameter skin biopsy punch. Hemostasis was achieved by applying pressure with sterile cotton balls, followed by disinfection with povidone-iodine. The experimental animals were then randomly divided into four groups, each receiving a gel dressing prepared according to Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of this invention, with dressing changes performed daily. The morphology of the rabbit ear scars was observed and photographed on postoperative days 14, 28, and 42 to assess scar hyperplasia and repair. The results are shown in the table below. Figure 7 .

[0103] from Figure 7 As can be seen, the rabbit ear scars in Example 3 were the smallest in size and the color closest to normal skin. Comparative Example 1 showed a certain inhibitory effect in the early stage of the experiment, but the scar recovery rate was not as stable and significant as that in Example 3 at the end of the experiment. Slight improvements were observed in Comparative Examples 2 and 3, but the scar proliferation inhibition effect was significantly inferior to that in Example 3, and the images showed that the scars were more obvious.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0105] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A bioactive composite gel dressing for skin scar repair that inhibits lactic acid production, characterized in that: It is obtained by mixing methacrylamide-modified hyaluronic acid, methacrylamide-modified carboxymethyl chitosan, and mesoporous polydopamine and then initiating a crosslinking reaction with a crosslinking monomer and a photoinitiator. The mesoporous polydopamine is loaded with the active substances berberine and resveratrol. The crosslinking monomer is prepared by the following steps: S1. Dissolve carboxymethylcysteine ​​and 3,4-dihydroxy-2'-chloroacetophenone in N,N-dimethylformamide solvent, add potassium carbonate, and stir the reaction at 60-80℃ for 6-12h under nitrogen protection. After the reaction is completed, cool to room temperature, adjust the pH to 3-4 with dilute hydrochloric acid, precipitate, filter, wash with deionized water, and vacuum dry to obtain intermediate A; S2. Intermediate A and 4-boron-DL-phenylalanine were added to a mixed solvent of toluene and ethanol, and 2 mol / L sodium carbonate solution was added. The mixture was refluxed at 80-90℃ for 8-16 h under nitrogen protection. After the reaction was completed, the mixture was cooled, and dilute hydrochloric acid was added to adjust the pH to neutral. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, concentrated, and vacuum dried to obtain intermediate B. S3. Add intermediate B and 10-undecenal to dichloromethane, add glacial acetic acid, and stir the reaction at room temperature for 12-24 h. After the reaction is complete, wash with saturated sodium bicarbonate solution, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain the crosslinking monomer.

2. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 1, characterized in that: Prepared by the following steps: (1) Berberine and resveratrol were added to deionized water and ultrasonically dispersed for 30 min to obtain an active substance dispersion. Mesoporous polydopamine was added to deionized water and ultrasonically dispersed for 30 min to obtain a polydopamine dispersion. The two dispersions were mixed and stirred at 25-37℃ for 12-24 h. Then, the mixture was centrifuged at 8000-10000 rpm for 10-15 min. The solid was collected, washed with deionized water, and vacuum dried to obtain mesoporous polydopamine loaded with active substances. (2) Take methacrylamide hyaluronic acid and add it to PBS buffer at pH 7.

4. Stir and dissolve at 4°C for 4-6 hours to obtain a hyaluronic acid solution with a concentration of 2.5-7.5% w / v. Take methacrylamide carboxymethyl chitosan and mesoporous polydopamine loaded with active substances and add them to deionized water. Disperse by ultrasonication for 1 hour and then stir for 2-4 hours to obtain a uniform chitosan-polydopamine dispersion. (3) A mixture of hyaluronic acid solution and chitosan-polydopamine dispersion was prepared. Crosslinking monomers and photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone were added, and the mixture was stirred evenly and poured into a mold. The mixture was then subjected to a photoinitiator at a wavelength of 365 nm and an intensity of 10-20 mW / cm. 2 The composite gel dressing is obtained by photocrosslinking under ultraviolet light for 1-5 minutes and then drying.

3. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 2, characterized in that: In step S1, the feeding ratio of carboxymethylcysteine, 3,4-dihydroxy-2'-chloroacetophenone, N,N-dimethylformamide, and potassium carbonate is 10-15g: 12-18g: 100-200mL: 20-30g.

4. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 3, characterized in that: In step S2, the feeding ratio of intermediate A, 4-boron-DL-phenylalanine, toluene, ethanol, and sodium carbonate aqueous solution is: 15-25g: 18-30g: 150-200mL: 150-250mL: 80-150mL.

5. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 4, characterized in that: In step S3, the feeding ratio of intermediate B, 10-undecenal, dichloromethane, and glacial acetic acid is 20-30g: 15-25mL: 200-300mL: 25-40g.

6. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 5, characterized in that: The feeding ratio of berberine, resveratrol, and deionized water in the active substance dispersion is 0.2-1.5g:0.2-1.5g:50-100mL; the feeding ratio of mesoporous polydopamine and deionized water in the polydopamine dispersion is 0.5-3.0g:20-50mL; and the volume ratio of the active substance dispersion to the polydopamine dispersion is 1:1-3:

1.

7. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 6, characterized in that: The ratio of methacrylamide-modified hyaluronic acid to PBS buffer in the hyaluronic acid solution is 5-15g:100-200mL. The ratio of methacrylamide-modified carboxymethyl chitosan, mesoporous polydopamine loaded with active substances, and deionized water in the chitosan-polydopamine dispersion is 5-15g:1-5g:50-100mL.

8. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 7, characterized in that: In step (3), the feeding ratio of crosslinking monomer and 2-hydroxy-2-methyl-1-phenyl-1-propanone is 1-5g: 0.1-0.5mL.

9. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 8, characterized in that: The molecular weight of the methacrylamide hyaluronic acid is 100kDa-300kDa, and the molecular weight of the methacrylamide carboxymethyl chitosan is 100kDa-200kDa.

10. The bioactive composite gel dressing for skin scar repair that inhibits lactic acid production according to claim 9, characterized in that: The specific surface area of ​​the mesoporous polydopamine is 200-600 m². 2 / g, with an average pore size of 2-10nm.

Images