A gel material for the treatment of keloids with reduced lactic acid production

Abstract

The application discloses a scar treatment gel material capable of reducing lactic acid generation, and belongs to the technical field of scar treatment. The material is prepared by the following steps: first, an intermediate A is obtained by esterification of 9-oxabicyclo[3.3.1]nonane-2,6-diol and S-allyl-L-cysteine, then an intermediate B containing a double bond is prepared by Schiff base reaction of the intermediate A and 5-methoxy-piperonal; then, the intermediate B is copolymerized with methacrylated chitosan and polyethylene glycol diacrylate under light initiation to form a crosslinked network structure; finally, active ingredients such as quercetin-3-O-glucuronide and luteolin-3'-glucuronide are included, and moisturizing agents, preservatives and other auxiliary materials are added to prepare the gel. The gel has a slow-release function, can effectively inhibit glycolysis, reduce lactic acid generation, scavenge free radicals, regulate the inflammatory and fibrotic microenvironment, has good biocompatibility and oxidation response drug release characteristics, and is suitable for the prevention and treatment of scars.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of keloid treatment, and particularly relates to a gel material for reducing lactic acid production in keloid treatment. BACKGROUND

[0002] Keloid is a benign fibroproliferative tumor that occurs after abnormal wound healing of the skin, and its essence is the result of excessive proliferation of fibroblasts and excessive deposition and insufficient degradation of extracellular matrix such as collagen. It often shows persistent growth beyond the original injury boundary, with hard texture and smooth surface, and may be accompanied by symptoms such as pain and itching, which not only affects the appearance, but also may cause dysfunction. Unlike ordinary scars, keloids cannot self-resolve, and the recurrence rate is extremely high after simple surgical resection, so the treatment has always been a clinical problem.

[0003] Traditional views believe that the formation of keloids is closely related to genetic susceptibility, local tension, persistent inflammation, and other factors. Existing research has found that, compared with normal skin scars, the lactic acid content in keloids is abnormally high, the reaction of glycolysis-related enzymes is active, the adenosine triphosphate (ATP) content produced by cells is low, and the level of reactive oxygen species is low. The above evidence shows that the fibroblasts in keloids exist in glycolysis metabolism. Although there is excessive collagen deposition and partially or completely occluded microvessels in keloids, resulting in the formation of a hypoxic microenvironment inside, scholars have found that even under the in vitro culture conditions of normoxia, the fibroblasts in keloids still show higher glucose uptake and lactic acid production, as well as up-regulation of key enzymes of glycolysis. This indicates that the fibroblasts in keloids are actively choosing a glycolysis-based metabolic mode, rather than simply being driven by hypoxia.

[0004] Abnormal accumulation of lactic acid is not only a metabolic end product, but also an active signal molecule that exacerbates the vicious progression of keloids through multiple mechanisms. Studies have found that pro-inflammatory mediators (such as TNF-α) in the inflammatory response can up-regulate glycolysis to produce more lactic acid; and a high-lactic acid environment can in turn exacerbate the inflammatory response by activating inflammasomes and stimulating the production of inflammatory mediators (such as IL-23 / IL-17), forming a vicious cycle of mutual promotion and leading to persistent inflammation in keloids. Lactic acid can interfere with the migration of T cells through specific transporters (Slc5a12 and Slc16a1), causing them to stay at the wound site, reducing their cytolytic ability and promoting the production of pro-inflammatory signals. Regulatory T cells can also promote the synthesis of collagen by fibroblasts in a lactic acid environment. In addition, lactic acid is conducive to the polarization of pro-fibrotic M2 macrophages, which induce the differentiation of myofibroblasts and collagen synthesis by secreting factors such as TGF-β1 and PDGF, and inhibit M1 macrophages with anti-fibrotic effects, leading to an imbalance in the immune microenvironment. Persistent inflammation and activation of M2 macrophages continuously stimulate fibroblast proliferation and excessive collagen secretion, ultimately leading to tissue fibrosis and the formation of keloids.

[0005] In summary, the abnormal accumulation of aerobic glycolysis and its product lactic acid in keloids is a key link connecting persistent inflammation, immune disorders and excessive fibrosis. Therefore, by targeting the glycometabolism of keloid fibroblasts, especially reducing the generation and accumulation of lactic acid, and breaking the vicious cycle triggered thereby, a promising new strategy is provided for the prevention and treatment of keloids. Therefore, it has important application value to develop a gel material that can locally act and effectively reduce lactic acid generation and regulate the keloid microenvironment. SUMMARY

[0006] In view of the above, in order to overcome the defects of the prior art, the present application provides a gel material with sustained-release function, which is suitable for keloid treatment and can effectively reduce lactic acid generation and regulate the keloid microenvironment.

[0007] In order to achieve the above-mentioned purpose, the following technical scheme is adopted: the present application provides a gel material for reducing lactic acid generation in the treatment of keloids, which is prepared by the following steps:

[0008] S1. Add 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine and anhydrous dichloromethane to a reactor, cool to 0-5℃, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after dropwise addition is completed, raise the temperature to 25-30℃ and react for 12-16h, pour the reaction liquid into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol, and vacuum dry to obtain intermediate A;

[0009] S2. To the reactor, intermediate A, 5-methoxy-piperonal and anhydrous ethanol were added, and then a 10% volume fraction of acetic acid solution was added to adjust the pH of the system to 4.0-4.5, followed by heating to 70-75°C, and reacting for 6-8h. The reaction solution was cooled to room temperature and refrigerated at 0-5°C for 8-10h to precipitate the product. After filtration, anhydrous ethanol washing, and vacuum drying, intermediate B was obtained.

[0010] S3. The reactor was treated in the dark, and methyl methacrylated chitosan, polyethylene glycol diacrylate and ultrapure water were added. After stirring, the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added, and stirring was continued for 15-20min. Then intermediate B was added, and stirring was continued at 25-30°C in the dark for 20-30min. The reaction was irradiated with ultraviolet light for 30-45min. After the reaction was completed, the reaction solution was dialyzed in an ultrapure water dialysis bag with a molecular weight cut-off of 3500Da for 3 days. After vacuum drying, the product was crushed and sieved through an 80-100 mesh sieve to obtain the cross-linked modified chitosan-polyethylene glycol diacrylate copolymer.

[0011] S4. Under dark conditions, quercetin-3-O-glucuronide, luteolin-3'-glucuronide and L-carnosine were added to ultrapure water to obtain an active substance dispersion. The cross-linked modified chitosan-polyethylene glycol diacrylate copolymer was added to the dispersion, and then a moisturizing agent, p-hydroxyacetophenone, tocopherol and a pH adjuster were added in sequence. After high-speed homogenization and vacuum degassing, the final product was obtained by standing at 25-30°C for 8-10h.

[0012] Further, in step S1, the feeding ratio of 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and anhydrous dichloromethane was 1-4g: 1.1-2.5g: 1.2-1.8g: 1.5-2.5g: 100-150mL.

[0013] Further, in step S2, the feeding ratio of intermediate A, 5-methoxy-piperonal and anhydrous ethanol was 0.88-1g: 1.02-1.46g: 100-200mL.

[0014] Further, in step S3, the feeding ratio of methyl methacrylated chitosan, polyethylene glycol diacrylate, 2-hydroxy-2-methyl-1-phenyl-1-propanone, intermediate B and ultrapure water was 10-50g: 3-25g: 0.13-1.5g: 8.5-14g: 100-200mL.

[0015] Further, the feeding ratio of quercetin-3-O-glucuronide, luteolin-3'-glucuronide, L-carnosine, cross-linked modified chitosan-polyethylene glycol diacrylate copolymer, humectant, p-hydroxyacetophenone, tocopherol, and ultrapure water in step S4 is 1-2.5 g: 0.8-1.2 g: 0.9-2.2 g: 3.9-4.2 g: 1.3-2.1 g: 0.3-0.8 g: 0.1-0.5 g: 200-400 mL.

[0016] Further, the number average molecular weight of the methacrylated chitosan is 50000-100000.

[0017] Further, the number average molecular weight of the polyethylene glycol diacrylate is 400-600.

[0018] Further, the humectant is at least one of glycerol, propylene glycol, sodium hyaluronate, sodium lactate, and trehalose.

[0019] Further, the pH regulator includes the acidic regulator citric acid, lactic acid, and the basic regulator triethanolamine.

[0020] Further, the pH regulator adjusts the pH of the gel material to 5.5-6.5.

[0021] The beneficial effects of the present application are:

[0022] The present application combines 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine, 5-methoxy-piperonal, methacrylated chitosan, polyethylene glycol diacrylate, and active ingredients such as quercetin-3-O-glucuronide, luteolin-3'-glucuronide, and L-carnosine to construct a gel material with sustained-release function, which is suitable for treating keloids and can effectively reduce lactic acid production and regulate the microenvironment of keloids.

[0023] Specifically, the present application first constructs an intermediate A by esterification of 9-oxabicyclo[3.3.1]nonane-2,6-diol and S-allyl-L-cysteine, introduces a double bond and a thioether bond that can be further reacted, and also retains the rigidity of the oxabicyclo structure and biological activity, intermediate A and 5-methoxy piperonal form intermediate B through Schiff base reaction, while introducing the piperonyl structure, the reactive double bond is retained to provide a site for subsequent photocrosslinking, intermediate B as a bridging molecule, copolymerized with methacrylated chitosan and polyethylene glycol diacrylate by ultraviolet light initiation, to form a cross-linked modified chitosan-polyethylene glycol diacrylate copolymer with a three-dimensional network structure, the network has good biocompatibility and moisturizing property, and can also load quercetin-3-O-glucuronide, luteolin-3'-glucuronide and L-muscle peptide active ingredients through physical inclusion and chemical action, realize slow release function, among the three encapsulated active substances, quercetin-3-O-glucuronide and luteolin-3'-glucuronide are significantly improved in water solubility and transdermal absorption efficiency of quercetin and luteolin through glucuronide modification, so as to better inhibit lactic acid production, scavenge free radicals, reduce oxidative stress, and regulate fibroblast metabolism to inhibit the development of keloid.

[0024] The thioether bond of the cross-linked modified chitosan-polyethylene glycol diacrylate copolymer can be oxidatively responsive cleaved in the high active oxygen environment of the scar tissue, thereby regulating the release of the active substances it encapsulates, and also playing an antibacterial role, in addition, the oxabicyclo[3.3.1]nonane structure can enhance the stability and adhesion of the material, helping to form a persistent protective layer on the surface of the scar, block external stimuli and maintain the stability of the local microenvironment, and the introduction of the piperonyl group further endows the gel material with antibacterial and anti-inflammatory ability, assisting in relieving the inflammatory response during the process of keloid proliferation. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The preparation process equation of intermediate A and intermediate B in the present application and 1 HNMR characterization image;

[0026] Figure 2 The cytotoxicity test results of the gel materials prepared in each example and comparative example;

[0027] Figure 3 The lactic acid production amount test results of the gel materials prepared in each example and comparative example of the present application;

[0028] Figure 4 The in vitro release test results of quercetin-3-O-glucuronide of the gel materials prepared in example 3 and comparative example 2 of the present application;

[0029] Figure 5The results of the in vitro release test of luteolin-3'-glucuronide of the gel material prepared for Example 3 and Comparative Example 2 of the present application;

[0030] Figure 6 The results of the in vitro release test of L-carnosine of the gel material prepared for Example 3 and Comparative Example 2 of the present application;

[0031] Figure 7 The scar treatment effect diagram of the gel material prepared for Example 3, Comparative Example 1 and Comparative Example 2 of the present application.

[0032] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, illustrate the embodiments of the present application and explain the present application, and do not constitute a limitation of the present application. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application are described below clearly and completely. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments of the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0034] Unless otherwise defined, all the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred implementation methods and materials described herein are only used for demonstration, but cannot limit the content of the present application.

[0035] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all purchased from commercial channels. The preparation process equations of intermediate A and intermediate B in the present application and 1 HNMR characterization images are shown in Figure 1 .

[0036] Example 1: A scar treatment gel material for reducing lactic acid production is prepared by the following steps:

[0037] S1. 1 g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 1.1 g of S-allyl-L-cysteine and 100 mL of anhydrous dichloromethane are added to a reactor, cooled to 5°C, and 1.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.5 g of 4-dimethylaminopyridine are added under a nitrogen atmosphere, the temperature is controlled not to exceed 5°C, and after the dropwise addition is completed, the temperature is raised to 25°C and reacted for 12 h. The reaction liquid is poured into ice water to precipitate the product, which is filtered, washed with ice water, recrystallized with ice ethanol, and vacuum dried to obtain intermediate A;

[0038] S2. To the reactor was added 0.88 g of intermediate A, 1.02 g of 5-methoxy-piperonal and 100 mL of anhydrous ethanol, and a 10% by volume acetic acid solution was added dropwise to adjust the pH of the system to 4.5, then the temperature was raised to 75°C, and the reaction was carried out for 8 h. The reaction solution was cooled to room temperature and stored at 5°C for 8 h, and the product was precipitated. After filtration, anhydrous ethanol washing and vacuum drying, intermediate B was obtained;

[0039] S3. The reactor was treated in the dark, 10 g of methacrylated chitosan, 3 g of polyethylene glycol diacrylate and 100 mL of ultrapure water were added, and 0.13 g of a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added by stirring. The stirring was continued for 20 min, then 8.5 g of intermediate B was added, and the stirring was continued at 30°C in the dark for 30 min. The reaction was carried out by ultraviolet light irradiation for 45 min. After the reaction was completed, the reaction solution was dialyzed in an ultrapure water dialysis bag with a molecular weight cut-off of 3500 Da for 3 days, vacuum dried, and then crushed and passed through a 100 mesh sieve to obtain a cross-linked modified chitosan-polyethylene glycol diacrylate copolymer;

[0040] S4. Under dark conditions, 1 g of quercetin-3-O-glucuronide, 0.8 g of luteolin-3'-glucuronide and 0.9 g of L-carnosine were added to 200 mL of ultrapure water to obtain an active substance dispersion. To the dispersion was added 3.9 g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer, and then 1.3 g of a humectant, 0.3 g of p-hydroxyacetophenone, 0.1 g of tocopherol and a pH adjuster were added in sequence. After high-speed homogenization and vacuum degassing, the gel material was obtained by standing at 30°C for 10 h.

[0041] The number average molecular weight of the methacrylated chitosan is 50,000; the number average molecular weight of the polyethylene glycol diacrylate is 400; the humectant is glycerol; the pH adjuster includes an acidic adjuster citric acid and an alkaline adjuster triethanolamine; and the pH adjuster adjusts the pH of the gel material to 5.5.

[0042] Example 2: A scar treatment gel material for reducing lactic acid production, prepared by the following steps:

[0043] S1. To the reactor was added 4 g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 2.5 g of S-allyl-L-cysteine and 150 mL of anhydrous dichloromethane, and the temperature was lowered to 5°C. Under a nitrogen atmosphere, 1.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.5 g of 4-dimethylaminopyridine were added, and the temperature was controlled not to exceed 5°C. After the dropwise addition was completed, the temperature was raised to 30°C and the reaction was carried out for 16 h. The reaction solution was poured into ice water to precipitate the product, which was filtered, washed with ice water, recrystallized with ice ethanol and vacuum dried to obtain intermediate A.

[0044] S2. To the reactor, 1 g of intermediate A, 1.46 g of 5-methoxy-piperonal and 200 mL of anhydrous ethanol were added, and a 10% by volume acetic acid solution was added dropwise to adjust the pH of the system to 4.5, then the temperature was raised to 75℃, and the reaction was carried out for 8 h. The reaction solution was cooled to room temperature and refrigerated at 5℃ for 10 h to precipitate the product. After filtration, anhydrous ethanol washing and vacuum drying, intermediate B was obtained;

[0045] S3. The reactor was treated in the dark, 50 g of methacrylated chitosan, 25 g of polyethylene glycol diacrylate and 200 mL of ultrapure water were added, and 1.5 g of a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added with stirring. The stirring was continued for 20 min, then 14 g of intermediate B was added, and the stirring was continued at 30℃ in the dark for 30 min. The reaction was irradiated with ultraviolet light for 45 min. After the reaction was completed, the reaction solution was dialyzed in an ultrapure water dialysis bag with a molecular weight cut-off of 3500 Da for 3 days. After vacuum drying, the product was crushed and sieved through a 100 mesh sieve to obtain a cross-linked modified chitosan-polyethylene glycol diacrylate copolymer;

[0046] S4. Under dark conditions, 2.5 g of quercetin-3-O-glucuronide, 1.2 g of luteolin-3'-glucuronide and 2.2 g of L-carnosine were added to 400 mL of ultrapure water to obtain an active substance dispersion. To the dispersion, 4.2 g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer was added, and then 2.1 g of a humectant, 0.8 g of p-hydroxyacetophenone, 0.5 g of tocopherol and a pH adjuster were added in sequence. After mixing and homogenizing at high speed, vacuum degassing was performed, and finally the gel material was obtained after standing at 30℃ for 10 h.

[0047] The number average molecular weight of the methacrylated chitosan is 100000; the number average molecular weight of the polyethylene glycol diacrylate is 600; the humectant is obtained by combining glycerol, propylene glycol and sodium hyaluronate in a mass ratio of 2:2:1; the pH adjuster includes an acidic adjuster lactic acid and an alkaline adjuster triethanolamine; and the pH adjuster adjusts the pH of the gel material to 5.5.

[0048] Example 3: A scar treatment gel material for reducing lactic acid production, prepared by the following steps:

[0049] S1. Add 2.5 g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 1.8 g of S-allyl-L-cysteine and 125 mL of anhydrous dichloromethane into a reactor, cool to 2.5℃, and add 1.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0 g of 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after the dropwise addition is completed, raise the temperature to 28℃ and react for 15 h, pour the reaction liquid into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol, and vacuum dry to obtain intermediate A;

[0050] S2. Add 0.94 g of intermediate A, 1.24 g of 5-methoxy-piperonal and 150 mL of anhydrous ethanol into a reactor, then add a 10% by volume acetic acid solution to adjust the pH of the system to 4.25, and then raise the temperature to 72.5℃, react for 7 h, cool the reaction liquid to room temperature and store in a 2.5℃ refrigerator for 9 h to precipitate the product, filter, wash with anhydrous ethanol, and vacuum dry to obtain intermediate B;

[0051] S3. Treat the reactor with light shielding, add 30 g of methacrylated chitosan, 14 g of polyethylene glycol diacrylate and 150 mL of ultrapure water, stir and add 0.82 g of a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, continue stirring for 17.5 min, then add 11.25 g of intermediate B, stir in the dark at 27.5℃ for 25 min, and irradiate with ultraviolet light for 37.5 min, after the reaction is completed, dialyze in ultrapure water for 3 days using a dialysis bag with a molecular weight cut-off of 3500 Da, vacuum dry, crush and pass through a 90-mesh sieve to obtain a cross-linked modified chitosan-polyethylene glycol diacrylate copolymer;

[0052] S4. Add 1.75 g of quercetin-3-O-glucuronide, 1.0 g of luteolin-3'-glucuronide and 1.55 g of L-carnosine to 300 mL of ultrapure water under light shielding conditions to obtain an active substance dispersion liquid, add 4.05 g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer to the dispersion liquid, mix uniformly, then add 1.7 g of a humectant, 0.55 g of p-hydroxyacetophenone, 0.3 g of tocopherol, and a pH adjuster in sequence, high-speed homogenize, vacuum deaerate, and finally stand at 27.5℃ for 9 h to obtain the gel material.

[0053] The number average molecular weight of the methacrylated chitosan is 75,000; the number average molecular weight of the polyethylene glycol diacrylate is 500; the humectant is obtained by combining glycerol, sodium lactate and trehalose in a mass ratio of 2:2:1; the pH adjuster includes the acidic adjuster citric acid and the basic adjuster triethanolamine; and the pH adjuster adjusts the pH of the gel material to 6.0.

[0054] Comparative Example 1: Quercetin-3-O-glucuronide and luteolin-3'-glucuronide were omitted in the preparation process of this comparative example, and the rest were the same as in Example 3.

[0055] Comparative Example 2: In this comparative example, equal amounts of methacrylamide chitosan and polyethylene glycol diacrylate were used to replace the crosslinked modified chitosan-polyethylene glycol diacrylate copolymer. All other aspects were the same as in Example 3.

[0056] Results Analysis

[0057] Intermediate A and Intermediate B of the present invention 1 HNMR image results are as follows Figure 1 As shown, intermediate A 1 In the HNMR image, no proton peak was observed on the carboxyl group (the carboxyl group appears in the range of 10-13 ppm). The proton peak at 8.25 ppm was on the amino group, and the proton peaks at 5.04, 5.29, and 602 ppm were on the double bond. The multiplet at 4.09 ppm was a proton peak on the carbon atom of the oxe ring adjacent to the ether bond in 9-oxabicyclo[3.3.1]nonane-2,6-diol. The multiplet at 4.67 ppm was a proton peak on the carbon atom of the oxe ring adjacent to the orthohydroxy group in 9-oxabicyclo[3.3.1]nonane-2,6-diol. The multiple characteristic peaks in the range of 1.00 ppm-2.00 ppm were proton peaks on the remaining carbon atoms of the oxe ring in 9-oxabicyclo[3.3.1]nonane-2,6-diol. Therefore, it can be seen that the present invention successfully synthesized intermediate A; intermediate A 1 In the HNMR image, the newly appearing peak at 3.83 ppm is the proton peak on the methoxy group in 5-methoxypiperaldehyde, and the newly appearing peak at 7.22 ppm is the proton peak on the benzene ring in 5-methoxypiperaldehyde. The proton peak on the carbon atom connected to the amino group in intermediate A is at 3.72 ppm. Due to the change of the adjacent group from an electron-donating amino group to an electron-withdrawing imine group, a significant shift to a lower field occurs, and the chemical shift value increases, appearing at 4.24 ppm. The broad and diffuse proton peak at the original amino group disappears, and a new sharp single peak appears at 8.75 ppm, indicating the formation of C=N. In summary, this invention successfully synthesized intermediate B.

[0058] The cytotoxicity test of each group of gel materials was performed by BioTek Synergy H1 microplate reader according to the MTT colorimetric method in GB / T 16886.5 “Biological evaluation of medical devices - Part 5: In vitro cytotoxicity tests”. The specific steps are as follows: take well-grown human skin fibroblasts (HSF) and inoculate them in a 96-well plate at a density of 5×10^3 cells / well, add 100 μL of DMEM medium containing 10% FBS to each well, and incubate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely. Discard the original culture medium, and divide the experiment into the following groups: blank control group (replace with fresh culture medium containing 10% FBS), Comparative Example 1 group, Comparative Example 2 group, and Example 1-3 groups of the present application. Replace the medium in the sample groups with medium containing 100% concentration of gel material extract (after 24 hours of extraction in serum-free medium at 37°C at a ratio of 0.1 g / mL, filter through a 0.22 μm filter membrane and dilute to the required concentration), and set two concentration gradients of 50% and 25% to investigate the dose dependence. Six replicate wells are set for each concentration. After 24 hours of continuous culture, add 20 μL of MTT solution (5 mg / mL, prepared with PBS) to each well, and continue to incubate for 4 hours. Carefully aspirate the supernatant in the wells, add 150 μL of DMSO to each well, and shake at low speed for 10 minutes to fully dissolve the formazan crystals. Finally, measure the absorbance value (OD570) of each well at a wavelength of 570 nm on the microplate reader. Calculate the relative growth rate of each group relative to the blank control group, and the test results are shown in Table 1. Figure 2 .

[0059] From Figure 2 It can be seen that the RGR of all example groups and comparative example groups is much higher than 80%, indicating that the gel materials of the present application and each comparative example have no cytotoxicity at the test concentration and have good biocompatibility.

[0060] The effect of each group of gel materials on reducing lactic acid production was tested by BioTek Synergy H1 microplate reader using a lactic acid detection kit according to the guidelines of GB / T 16886.5 “Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test”. The specific steps are as follows: the 3rd-8th generation human keloid fibroblasts in good growth state were inoculated in a 96-well plate at a density of 1×10^4 cells / well, and cultured at 37℃, 5% CO2 incubator for 24 hours to make them completely adhere. The original culture medium was discarded, and the experimental groups were as follows: blank control group (replaced with fresh DMEM high glucose medium containing 10% FBS), comparative example 1 group, comparative example 2 group and inventive examples 1-3 groups (replaced with complete culture medium containing 50% (v / v) corresponding gel material sterile extract, which was prepared by filtering through a 0.22 μm filter membrane after being extracted at 37℃ for 24 hours with complete culture medium as the extraction medium at a ratio of 0.2 g / mL). Each concentration was set with 6 replicate wells. After continuing to culture for 48 hours, strictly according to the kit instructions, 50 μL of each well supernatant was taken and mixed with 100 μL of working solution, 37℃, dark reaction for 30 minutes, then the absorbance value (OD value) of each well was measured at 450 nm wavelength, so as to calculate the lactic acid concentration, and the test results are shown in Figure 3 . In the figure, * indicates that compared with the blank control group, p<0.01, there is a very significant statistical difference.

[0061] From Figure 3 It can be seen that the inventive examples 1-3 groups can significantly reduce the lactic acid production of keloid fibroblasts, and the effect of example 3 is the best. Comparative example 1 lacks key quercetin-3-O-glucuronide and luteolin-3'-glucuronide, and comparative example 2 does not have the cross-linked network of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer, so the effect is significantly weaker than the example group, which proves the synergistic effect of the present application in reducing lactic acid production.

[0062] According to the second method (paddle method) of dissolution and release determination method 0931 of Chinese Pharmacopoeia 2020 edition four general rules, the in vitro release test of each group of gel materials was carried out by TK-12D type intelligent transdermal test instrument. The specific steps are as follows: 1.0 g of gel material prepared by example 3 and comparative example 2 was accurately weighed, and uniformly coated on the semi-permeable membrane (molecular cut-off 3500 Da) between the supply pool and the receiving pool, and the effective diffusion area was 2.8 cm 2The receiving pool is filled with 500 mL of degassed pH = 7.4 phosphate buffer as a release medium, and kept at a constant temperature of 32.0 ± 0.5 °C, and the magnetic stirring speed is 50 ± 5 rpm to simulate the body surface temperature and conditions. At the predetermined time points (1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours), 5 mL of sample is taken (while supplementing with an equal volume of fresh release medium at the same temperature), and the sample is filtered through a 0.45 μm microporous filter. The concentrations of the active substances in the filtrate are detected by high performance liquid chromatography at the maximum absorption wavelengths of the respective active ingredients (quercetin-3-O-glucuronide 370 nm, luteolin-3'-glucuronide 350 nm, and L-carnosine 214 nm) simultaneously. The cumulative release rate is calculated by the external standard method, and the test results are shown in Table 1. Figure 4 、 Figure 5 、 Figure 6 .

[0063] From Figure 4 、 5 , 6, it can be seen that the gel network structure of Comparative Example 2 is not perfect, and the three active ingredients have a sharp burst release within 12 hours, which cannot achieve long-acting sustained release. Example 3 benefits from the formed cross-linked network, effectively encapsulating the active substances, and presents a smooth and slow release behavior without burst release phenomenon within the entire 72 hours, which meets the sustained release requirements.

[0064] Healthy BALB / c mice are selected, and anesthetic is injected intraperitoneally. After the mice are fully anesthetized, a hair remover is used to remove the hair on the back legs, and a depilatory cream is uniformly applied to the exposed skin, and then a sterile cotton swab is used to wipe off to completely expose the clean skin. A sterilized metal ring with a diameter of 10 mm is lightly pressed on the skin surface to create a standardized circular wound, and then 50 μL of a Staphylococcus aureus suspension is added to the center of the wound, and a wound dressing is used to cover the wound to maintain a local moist infection environment to establish a skin wound bacterial infection model. After the skin wound bacterial infection model is established, the mice are divided into three treatment groups, namely, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, and the gel materials prepared in the present application are applied for treatment. The wound area is measured and photographed on days 0, 5, 10, 15, and 20, and the images are shown in Figure 7 .

[0065] Figure 7 As can be clearly seen in the above table, the wound healing effect of the mice in the Example 3 group after 20 days of treatment is obviously better than that of the other groups, and the wound has basically completely healed and no obvious scar exists, while obvious scars can be seen on the wounds of the mice in Comparative Examples 1 and 2, which proves the superiority of the gel materials prepared in the present application in treating keloids.

[0066] While embodiments of the application have been shown and described, it is to be understood that the application is not limited to the details of the embodiments described, since various modifications can be made by those skilled in the art, without departing from the spirit and scope of the application, which is defined by the appended claims and their equivalents.

[0067] The above description of the application and its embodiments is not restrictive, and the embodiments shown are only one of the embodiments of the application, and the actual application is not limited thereto. In general, if a person skilled in the art is inspired by it, without departing from the purpose of the application, without creative design, similar ways and embodiments of the technical solution should belong to the protection scope of the application.

Claims

1. A scar treatment gel material for reducing lactic acid generation, characterized in that it is prepared by the following steps: S1. 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine and anhydrous dichloromethane are added to a reactor, cooled to 0-5°C, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added under a nitrogen atmosphere, controlling the temperature to not exceed 5°C, after dropwise addition is complete, the temperature is raised to 25-30°C and reacted for 12-16 hours, the reaction liquid is poured into ice water to precipitate the product, which is filtered, washed with ice water, recrystallized from ice ethanol, and vacuum dried to obtain intermediate A; The feeding ratio of 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and anhydrous dichloromethane is 1-4 g: 1.1-2.5g:1.2-1.8g:1.5-2.5g:100-150mL; S2. Adding intermediate A, 5-methoxy-piperonal and anhydrous ethanol into the reactor, and then adding 10% acetic acid solution dropwise to adjust the pH of the system to 4.0-4.5, followed by heating to 70-75℃, and reacting for 6-8h, cooling the reaction solution to room temperature and storing at 0-5℃ for 8-10h to precipitate the product, filtering, washing with anhydrous ethanol, and vacuum drying to obtain intermediate B; The feeding ratio of intermediate A, 5-methoxy-piperonal and anhydrous ethanol is 0.88-1g:1.02-1.46g:100-200mL; S3. The reactor is treated in the dark, and methyl methacrylated chitosan, polyethylene glycol diacrylate and ultrapure water are added, and then a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is added with stirring, and stirring is continued for 15-20min, and then intermediate B is added, and stirring is continued in the dark at 25-30℃ for 20-30min, and then the reaction is irradiated with ultraviolet light for 30-45min, and after the reaction is completed, the reaction solution is dialyzed in an ultrapure water dialysis bag with a molecular weight cut-off of 3500Da for 3 days, and then vacuum dried, and then crushed and sieved through an 80-100 mesh sieve to obtain a cross-linked modified chitosan-polyethylene glycol diacrylate copolymer; The feeding ratio of methyl methacrylated chitosan, polyethylene glycol diacrylate, 2-hydroxy-2-methyl-1-phenyl-1-propanone, intermediate B and ultrapure water is 10-50g:3-25g:0.13-1.5g:8.5-14g:100-200mL; the number average molecular weight of the methyl methacrylated chitosan is 50000-100000; and the number average molecular weight of the polyethylene glycol diacrylate is 400-600; S4. Under light shielding conditions, quercetin-3-O-glucuronide, luteolin-3'-glucuronide and L-carnosine are added into ultrapure water to obtain an active substance dispersion, and then the cross-linked modified chitosan-polyethylene glycol diacrylate copolymer is added into the dispersion, and then a humectant, p-hydroxyacetophenone, tocopherol and a pH adjusting agent are sequentially added after uniform mixing, and then high-speed homogenization and vacuum degassing are performed, and finally the gel material is obtained after standing at 25-30℃ for 8-10h.

2. The scar treatment gel material for reducing lactic acid generation according to claim 1, characterized by: The feeding ratio of quercetin-3-O-glucuronide, luteolin-3'-glucuronide, L-carnosine, cross-linked modified chitosan-polyethylene glycol diacrylate copolymer, humectant, p-hydroxyacetophenone, tocopherol and ultrapure water in step S4 is 1-2.5g:0.8-1.2g:0.9-2.2g:3.9-4.2g:1.3-2.1g:0.3-0.8g:0.1-0.5g:200-400mL.

3. The scar treatment gel material for reducing lactic acid generation according to claim 2, characterized by: The humectant is at least one of glycerol, propylene glycol, sodium hyaluronate and sodium lactate.

4. The scar treatment gel material for reducing lactic acid generation according to claim 3, characterized by: The pH adjusting agent includes an acidic adjusting agent citric acid, lactic acid and an alkaline adjusting agent triethanolamine.

5. The scar treatment gel material for reducing lactic acid production according to claim 4, characterized by: The pH adjusting agent adjusts the pH of the gel material to 5.5-6.

5. The pH adjusting agent adjusts the pH of the gel material to 5.5-6.5.

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