Gel material for treating keloid capable of reducing generation of lactic acid

Abstract

The invention discloses a keloid treatment gel material capable of reducing lactic acid generation, and belongs to the technical field of keloid treatment. The material is prepared by the following steps: firstly, carrying out esterification on 9-oxabicyclo [3.3. 1] nonane-2, 6-diol and S-allyl-L-cysteine to obtain an intermediate A, and then carrying out Schiff base reaction on the intermediate A and 5-methoxy heliotropin to obtain an intermediate B containing double bonds; then, copolymerizing with methylacryloylated chitosan and polyethylene glycol diacrylate under photo-initiation to form a cross-linked network structure; finally, active ingredients such as quercetin-3-O-glucuronide and luteolin-3 '-glucuronide are included, and auxiliary materials such as a humectant and a preservative are added, so that the composition is prepared. The gel has a slow release function, can effectively inhibit glycolysis, reduce lactic acid generation, scavenge free radicals and regulate inflammation and fibrosis microenvironment, has good biocompatibility and oxidation response drug release characteristics, and is suitable for prevention and treatment of keloids.

Description

Technical Field

[0001] This invention belongs to the field of keloid treatment technology, and particularly relates to a gel material for keloid treatment that reduces lactic acid production. Background Technology

[0002] Keloids are benign fibroproliferative tumors that occur after abnormal wound healing. They are essentially the result of excessive proliferation of fibroblasts and insufficient deposition and degradation of extracellular matrix such as collagen. They often manifest as persistent growth extending beyond the original lesion boundary, with a hard texture, smooth surface, and may be accompanied by symptoms such as pain and itching. They not only affect appearance but can also lead to functional impairment. Unlike ordinary scars, keloids do not resolve spontaneously, and the recurrence rate after simple surgical excision is extremely high, making treatment a persistent clinical challenge.

[0003] Traditional views hold that keloid formation is closely related to multiple factors, including genetic susceptibility, local tension, and persistent inflammation. Current research has found that compared to normal skin scars, keloids exhibit abnormally high lactic acid levels, active glycolysis-related enzymes, low ATP production, and low levels of reactive oxygen species. This evidence suggests that keloid fibroblasts engage in glycolytic metabolism. Although excessive collagen deposition and partially or completely occluded microvessels in keloids create a hypoxic microenvironment, researchers have found that even under normoxic in vitro culture conditions, keloid fibroblasts still exhibit higher glucose uptake and lactic acid production, as well as upregulation of key glycolytic enzymes. This indicates that keloid fibroblasts actively choose a glycolytic-based metabolic mode, rather than being simply driven by hypoxia.

[0004] Abnormally accumulated lactic acid is not merely a metabolic end product; it is also an active signaling molecule that exacerbates the malignant progression of keloids through multiple mechanisms. Studies have found that pro-inflammatory mediators in the inflammatory response (such as TNF-α) can upregulate glycolysis, producing more lactic acid. Conversely, a high lactic acid environment can further exacerbate the inflammatory response by activating inflammasomes and stimulating the production of inflammatory mediators (such as IL-23 / IL-17), forming a mutually reinforcing vicious cycle that leads to persistent inflammation in keloids. Lactic acid can interfere with T cell migration through specific transport proteins (Slc5a12 and Slc16a1), causing them to remain at the wound site, reducing their cytolytic capacity, and promoting the generation of pro-inflammatory signals. Regulatory T cells can also promote collagen synthesis in fibroblasts under lactic acid conditions. Furthermore, lactic acid favors the polarization of pro-fibrotic M2 macrophages, which induce myofibroblast differentiation and collagen synthesis by secreting factors such as TGF-β1 and PDGF, inhibiting 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 keloid formation.

[0005] In summary, the aerobic glycolysis and its product lactic acid accumulation in keloids are core components linking persistent inflammation, immune dysregulation, and excessive fibrosis. Therefore, targeting and regulating the glucose metabolism of keloid fibroblasts, particularly reducing lactic acid production and accumulation, to break the vicious cycle, offers a promising new strategy for the prevention and treatment of keloids. Thus, developing a gel material that can act locally, effectively reduce lactic acid production, and regulate the scar microenvironment has significant application value. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention constructs a gel material with sustained-release function, which is suitable for the treatment of keloids, and can effectively reduce lactic acid production and regulate the scar microenvironment.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides a gel material for treating keloids that reduces lactic acid production, prepared through the following steps: S1. Add 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine ​​and anhydrous dichloromethane to the reactor, cool to 0-5℃, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after the addition is complete, raise the temperature to 25-30℃ and react for 12-16h, pour the reaction solution into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol and vacuum dry to obtain intermediate A; S2. Add intermediate A, 5-methoxypiperaldehyde and anhydrous ethanol to the reactor, then add 10% acetic acid solution to adjust the pH of the system to 4.0-4.5. Then heat to 70-75℃ and react for 6-8 hours. Cool the reaction solution to room temperature and refrigerate at 0-5℃ for 8-10 hours to allow the product to precipitate. After filtration, washing with anhydrous ethanol and vacuum drying, intermediate B is obtained. S3. Protect the reactor from light, add methacrylamide chitosan, polyethylene glycol diacrylate and ultrapure water, stir and add photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, continue stirring for 15-20 min, then add intermediate B, stir at 25-30℃ in the dark for 20-30 min, react with ultraviolet light for 30-45 min, after the reaction is completed, dialyze in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3500 Da, vacuum dry, then pulverize and pass through an 80-100 mesh sieve to obtain cross-linked modified chitosan-polyethylene glycol diacrylate copolymer; S4. Quercetin-3-O-glucuronide, luteolin-3'-glucuronide, and L-carnosine are added to ultrapure water under light-protected conditions to obtain an active substance dispersion. Crosslinked modified chitosan-polyethylene glycol diacrylate copolymer is added to the dispersion and mixed evenly. Then, a humectant, p-hydroxyacetophenone, tocopherol, and pH adjuster are added sequentially. After high-speed homogenization and vacuum degassing, the mixture is finally allowed to stand at 25-30°C for 8-10 hours to obtain the gel material.

[0008] 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 is 1-4g: 1.1-2.5g: 1.2-1.8g: 1.5-2.5g: 100-150mL.

[0009] Further, in step S2, the feeding ratio of intermediate A, 5-methoxypiperaldehyde and anhydrous ethanol is 0.88-1g: 1.02-1.46g: 100-200mL.

[0010] Further, in step S3, the feeding ratio of 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.

[0011] Further, in step S4, 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 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.

[0012] Furthermore, the number average molecular weight of the methacrylamide chitosan is 50,000-100,000.

[0013] Furthermore, the number average molecular weight of the polyethylene glycol diacrylate is 400-600.

[0014] Furthermore, the moisturizer is at least one of glycerin, propylene glycol, sodium hyaluronate, sodium lactate, and trehalose.

[0015] Furthermore, the pH adjuster includes acidic adjusters citric acid and lactic acid, and alkaline adjuster triethanolamine.

[0016] Furthermore, the pH adjuster adjusts the pH of the gel material to 5.5-6.5.

[0017] The beneficial effects of this invention are: This invention constructs a sustained-release gel material by combining active ingredients such as 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine, 5-methoxypiperaldehyde, methacryloxychitosan, polyethylene glycol diacrylate, quercetin-3-O-glucuronide, luteolin-3'-glucuronide, and L-carnosine. This material is suitable for the treatment of keloids and is particularly effective in reducing lactic acid production and regulating the scar microenvironment.

[0018] Specifically, this invention first constructs intermediate A through the esterification reaction of 9-oxabicyclo[3.3.1]nonane-2,6-diol with S-allyl-L-cysteine, introducing further reactive double bonds and thioether bonds while retaining the rigidity and bioactivity of the oxabicyclo structure. Intermediate A reacts with 5-methoxypiperaldehyde via a Schiff base reaction to form intermediate B, introducing a piperine structure while retaining reactive double bonds, providing sites for subsequent photocrosslinking. Intermediate B, as a bridging molecule, copolymerizes with methacrylamide chitosan and polyethylene glycol diacrylate via UV initiation to form a crosslinked modified chitosan with a three-dimensional network structure. The polyethylene glycol diacrylate copolymer network exhibits excellent biocompatibility and moisturizing properties. It can also load quercetin-3-O-glucuronide, luteolin-3'-glucuronide, and L-carnosine active ingredients through physical inclusion and chemical action, achieving sustained-release function. Among the three encapsulated active substances, quercetin-3-O-glucuronide and luteolin-3'-glucuronide, through glucuronylylation modification, significantly improve the water solubility and transdermal absorption efficiency of quercetin and luteolin, thereby better inhibiting lactic acid production, scavenging free radicals, reducing oxidative stress, regulating fibroblast metabolism, and inhibiting keloid development.

[0019] The thioether bonds of the cross-linked modified chitosan-polyethylene glycol diacrylate copolymer can undergo oxidative responsive cleavage in the highly reactive oxygen environment of scar tissue, thereby regulating the release of its encapsulated active substances and exerting antibacterial effects. In addition, the oxabicyclo[3.3.1]nonane structure can enhance the stability and adhesion of the material, helping to form a durable protective layer on the scar surface, blocking external stimuli and maintaining the stability of the local microenvironment. The introduction of piperine further endows the gel material with antibacterial and anti-inflammatory capabilities, helping to alleviate the inflammatory response during scar hyperplasia. Attached Figure Description

[0020] Figure 1 The preparation process equations for intermediates A and B of this invention are as follows: 1 HNMR characterization images; Figure 2 The results of cytotoxicity tests are shown for the gel materials prepared in each embodiment and comparative example; Figure 3 The results of lactic acid production tests on the gel materials prepared in the various embodiments and comparative examples of the present invention; Figure 4 The results of the in vitro release test of quercetin-3-O-glucuronide from the gel materials prepared in Example 3 and Comparative Example 2 of this invention; Figure 5 The results of the in vitro release test of luteolin-3'-glucuronide from the gel materials prepared in Example 3 and Comparative Example 2 of this invention; Figure 6 The results of the in vitro release test of L-carnosine from the gel materials prepared in Example 3 and Comparative Example 2 of this invention; Figure 7 The images show the scar treatment effects of the gel materials prepared in Example 3, Comparative Example 1, and Comparative Example 2 of this invention.

[0021] 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

[0022] 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.

[0023] 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.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are commercially available. The preparation process equations for intermediates A and B in this invention are as follows: 1 HNMR characterization images are shown below Figure 1 .

[0025] Example 1: A gel material for treating keloids that reduces lactic acid production, prepared through the following steps: S1. Add 1g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 1.1g of S-allyl-L-cysteine ​​and 100mL of anhydrous dichloromethane to the reactor, cool to 5℃, add 1.2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.5g of 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after the addition is complete, raise the temperature to 25℃ and react for 12h, pour the reaction solution into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol, and vacuum dry to obtain intermediate A; S2. Add 0.88g of intermediate A, 1.02g of 5-methoxypiperaldehyde and 100mL of anhydrous ethanol to the reactor, then add 10% acetic acid solution to adjust the pH of the system to 4.5. Then heat to 75℃ and react for 8h. Cool the reaction solution to room temperature and refrigerate at 5℃ for 8h to allow the product to precipitate. After filtration, washing with anhydrous ethanol and vacuum drying, intermediate B is obtained. S3. Protect the reactor from light, add 10g of methacrylamide chitosan, 3g of polyethylene glycol diacrylate and 100mL of ultrapure water, stir and add 0.13g of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, continue stirring for 20min, then add 8.5g of intermediate B, stir at 30℃ in the dark for 30min, react with ultraviolet light for 45min, after the reaction is completed, dialyze in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da, vacuum dry, then pulverize and pass through a 100-mesh sieve to obtain cross-linked modified chitosan-polyethylene glycol diacrylate copolymer; S4. Under light-protected conditions, add 1g of quercetin-3-O-glucuronide, 0.8g of luteolin-3'-glucuronide, and 0.9g of L-carnosine to 200mL of ultrapure water to obtain an active substance dispersion. Add 3.9g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer to the dispersion, mix well, and then add 1.3g of humectant, 0.3g of p-hydroxyacetophenone, 0.1g of tocopherol, and pH adjuster in sequence. After high-speed homogenization and vacuum degassing, finally let it stand at 30℃ for 10h to obtain the gel material.

[0026] The number average molecular weight of the methacrylamide chitosan is 50,000; the number average molecular weight of the polyethylene glycol diacrylate is 400; the humectant is glycerin; the pH adjuster includes the acidic adjuster citric acid and the alkaline adjuster triethanolamine; the pH adjuster adjusts the pH of the gel material to 5.5.

[0027] Example 2: A gel material for treating keloids that reduces lactic acid production, prepared through the following steps: S1. Add 4g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 2.5g of S-allyl-L-cysteine ​​and 150mL of anhydrous dichloromethane to the reactor, cool to 5℃, add 1.8g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.5g of 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after the addition is complete, raise the temperature to 30℃ and react for 16h, pour the reaction solution into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol and vacuum dry to obtain intermediate A; S2. Add 1g of intermediate A, 1.46g of 5-methoxypiperaldehyde and 200mL of anhydrous ethanol to the reactor, then add 10% acetic acid solution to adjust the pH of the system to 4.5. Then heat to 75℃ and react for 8h. Cool the reaction solution to room temperature and refrigerate at 5℃ for 10h to allow the product to precipitate. After filtration, washing with anhydrous ethanol and vacuum drying, intermediate B is obtained. S3. Protect the reactor from light, add 50g of methacrylamide chitosan, 25g of polyethylene glycol diacrylate and 200mL of ultrapure water, stir and add 1.5g of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, continue stirring for 20min, then add 14g of intermediate B, stir at 30℃ in the dark for 30min, react with ultraviolet light for 45min, after the reaction is completed, dialyze in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da, vacuum dry, then pulverize and pass through a 100-mesh sieve to obtain cross-linked modified chitosan-polyethylene glycol diacrylate copolymer; S4. Under light-protected conditions, add 2.5g of quercetin-3-O-glucuronide, 1.2g of luteolin-3'-glucuronide, and 2.2g of L-carnosine to 400mL of ultrapure water to obtain an active substance dispersion. Add 4.2g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer to the dispersion, mix well, and then add 2.1g of humectant, 0.8g of p-hydroxyacetophenone, 0.5g of tocopherol, and pH adjuster in sequence. After high-speed homogenization and vacuum degassing, finally let it stand at 30℃ for 10h to obtain the gel material.

[0028] The number average molecular weight of the methacryloylchitosan is 100,000; the number average molecular weight of the polyethylene glycol diacrylate is 600; the humectant is obtained by combining glycerin, propylene glycol, and sodium hyaluronate in a mass ratio of 2:2:1; the pH adjuster includes the acidic adjuster lactic acid and the alkaline adjuster triethanolamine; the pH adjuster adjusts the pH of the gel material to 5.5.

[0029] Example 3: A gel material for treating keloids that reduces lactic acid production, prepared through the following steps: S1. Add 2.5g of 9-oxabicyclo[3.3.1]nonane-2,6-diol, 1.8g of S-allyl-L-cysteine ​​and 125mL of anhydrous dichloromethane to the reactor, cool to 2.5℃, add 1.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0g of 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, and after the addition is complete, raise the temperature to 28℃ and react for 15h. Pour the reaction solution into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol, and vacuum dry to obtain intermediate A; S2. Add 0.94g of intermediate A, 1.24g of 5-methoxypiperaldehyde and 150mL of anhydrous ethanol to the reactor, then add 10% acetic acid solution to adjust the pH of the system to 4.25. Then heat to 72.5℃ and react for 7h. Cool the reaction solution to room temperature and refrigerate at 2.5℃ for 9h to allow the product to precipitate. After filtration, washing with anhydrous ethanol and vacuum drying, intermediate B is obtained. S3. The reactor was protected from light. 30g of methacrylamide chitosan, 14g of polyethylene glycol diacrylate and 150mL of ultrapure water were added. 0.82g of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added and stirred for 17.5min. Then 11.25g of intermediate B was added and stirred at 27.5℃ in the dark for 25min. The reaction was carried out under ultraviolet light for 37.5min. After the reaction was completed, the mixture was dialyzed in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da. After vacuum drying, the mixture was pulverized and passed through a 90-mesh sieve to obtain the cross-linked modified chitosan-polyethylene glycol diacrylate copolymer. S4. Under light-protected conditions, 1.75g ​​of quercetin-3-O-glucuronide, 1.0g of luteolin-3'-glucuronide, and 1.55g of L-carnosine were added to 300mL of ultrapure water to obtain an active substance dispersion. 4.05g of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer was added to the dispersion, and after mixing evenly, 1.7g of humectant, 0.55g of p-hydroxyacetophenone, 0.3g of tocopherol, and pH adjuster were added sequentially. After high-speed homogenization and vacuum degassing, the mixture was finally allowed to stand at 27.5℃ for 9h to obtain the gel material.

[0030] The number average molecular weight of the methacryloylchitosan is 75,000; the number average molecular weight of the polyethylene glycol diacrylate is 500; the humectant is obtained by combining glycerin, sodium lactate, and trehalose in a mass ratio of 2:2:1; the pH adjuster includes citric acid as an acidic adjuster and triethanolamine as an alkaline adjuster; the pH adjuster adjusts the pH of the gel material to 6.0.

[0031] 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.

[0032] 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.

[0033] Results Analysis Intermediate A and Intermediate B of the present invention 1 HNMR image results are as follows Figure 1 As shown, intermediate A1 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.

[0034] The cytotoxicity of each group of gel materials was tested using the MTT assay in GB / T 16886.5 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests" via a BioTek Synergy H1 microplate reader. The specific steps were as follows: Well-grown human skin fibroblasts (HSF) were seeded at a density of 5 × 10^3 cells / well in 96-well plates. 100 μL of DMEM medium containing 10% FBS was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 hours to allow complete cell adhesion. The original culture medium was discarded, and the experimental groups were as follows: blank control group (replaced with fresh medium containing 10% FBS), comparative example group 1, comparative example group 2, and groups 1-3 of the present invention. All test samples were replaced with a culture medium containing 100% concentration of gel material extract (extracted at 0.1 g / mL in serum-free medium at 37°C for 24 hours, filtered through a 0.22 μm filter and diluted to the required concentration), with two concentration gradients of 50% and 25% to investigate dose dependence. Each concentration was used in six replicates. After culturing for another 24 hours, 20 μL MTT solution (5 mg / mL, prepared with PBS) was added to each well, and incubation continued for another 4 hours. The supernatant was carefully aspirated from the wells, and 150 μL LDMSO was added to each well, followed by low-speed shaking for 10 minutes to fully dissolve the formazan crystals. Finally, the absorbance (OD570) of each well was measured at 570 nm using a microplate reader. The relative proliferation rate of each cell group compared to the blank control group was calculated, and the results are shown below. Figure 2 .

[0035] from Figure 2 It can be seen that the RGR of all example groups and comparative examples is much higher than 80%, indicating that the gel material of the present invention and each comparative example have no cytotoxicity and good biocompatibility at the test concentration.

[0036] Following the guidelines of GB / T 16886.5 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests", the effects of each gel material on reducing lactate production were tested using a BioTek Synergy H1 microplate reader and a lactate detection kit. The specific steps are as follows: Human keloid fibroblasts in good growth condition (passages 3-8) were seeded in 96-well plates at a density of 1×10^4 cells / well and cultured at 37°C in a 5% CO2 incubator for 24 hours to ensure complete adhesion. 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, comparative example 2, and examples 1-3 of this invention (replaced with complete culture medium containing 50% (v / v) of the corresponding sterile extract of the gel material; the sterile extract was prepared by extraction at 0.2 g / mL at 37°C for 24 hours, followed by filtration through a 0.22 μm filter membrane). Six replicates were set up for each concentration. After culturing for another 48 hours, following the kit instructions strictly, 50 μL of cell supernatant from each well was mixed with 100 μL of working solution and incubated at 37°C in the dark for 30 minutes. The absorbance (OD) value of each well was then measured at 450 nm to calculate the lactate concentration. The test results are shown below. Figure 3 * in the figure indicates a highly statistically significant difference compared to the blank control group (p < 0.01).

[0037] Depend on Figure 3 It can be seen that all three groups of Examples 1-3 of the present invention can significantly reduce lactic acid production in keloid fibroblasts. Among them, Example 3 has the best effect. Comparative Example 1 lacks the key quercetin-3-O-glucuronide and luteolin-3'-glucuronide, while Comparative Example 2 does not have the cross-linking network of cross-linked modified chitosan-polyethylene glycol diacrylate copolymer. Therefore, the effect of Comparative Example 1 is significantly weaker than that of the Example groups, which proves the synergistic effect of the present invention in reducing lactic acid production.

[0038] According to Method 2 (Paddle Method) of Dissolution and Release Determination in General Chapter 0931 of Part IV of the 2020 Edition of the Pharmacopoeia of the People's Republic of China, in vitro release tests of each group of gel materials were conducted using a TK-12D intelligent transdermal testing instrument. The specific steps are as follows: 1.0 g of each of the gel materials prepared in Example 3 and Comparative Example 2 were accurately weighed and evenly coated onto the semi-permeable membrane (molecular cutoff 3500 Da) between the supply and receiving cells, with an effective diffusion area of ​​2.8 cm². 2500 mL of degassed pH 7.4 phosphate buffer solution was added to the receiving tank as the release medium, and the temperature was maintained at a constant 32.0 ± 0.5 °C with a magnetic stirrer speed of 50 ± 5 rpm to simulate body surface temperature and conditions. At predetermined time points (1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours), 5 mL samples were taken (with simultaneous replenishment of isothermal and equal volume of fresh release medium). After filtration through a 0.45 μm microporous membrane, high-performance liquid chromatography (HPLC) was used to simultaneously detect the concentration of each active substance in the filtrate at its maximum absorption wavelength (quercetin-3-O-glucuronide 370 nm, luteolin-3'-glucuronide 350 nm, L-carnosine 214 nm). The cumulative release rate was calculated using the external standard method. The test results are shown in [Figure 1]. Figure 4 , Figure 5 , Figure 6 .

[0039] from Figure 4 , 5 As can be seen from Figures 6 and 7, in Comparative Example 2, due to the imperfect gel network structure, the three active ingredients underwent a violent burst release within 12 hours, failing to achieve long-term sustained release. In contrast, Example 3, thanks to the formed cross-linked network, effectively encapsulated the active substances, exhibiting a stable and slow release behavior throughout 72 hours, without burst release, thus meeting the requirements for sustained release.

[0040] Healthy BALB / c mice were selected and injected intraperitoneally with an anesthetic. After the mice were fully anesthetized, the hair on their hind legs was removed using a shaver. Hair removal cream was then evenly applied to the exposed skin and wiped away with a sterile cotton swab to completely expose the clean skin. A sterilized 10mm diameter metal ring was gently pressed onto the skin surface to create a standardized circular wound. 50 μL of Staphylococcus aureus suspension was then dripped into the center of the wound. The wound was covered with a wound dressing to maintain a moist environment and establish a bacterial infection model of the skin wound. After establishing the bacterial infection model, the mice were divided into three treatment groups: Example 3 group, Comparative Example 1 group, and Comparative Example 2 group. The gel material prepared in this application was applied for treatment. The wound area was measured and photographed on days 0, 5, 10, 15, and 20. Images are shown below. Figure 7 .

[0041] Figure 7 As can be clearly seen, after 20 days of treatment, the wounds of mice in Example 3 showed significantly better healing than those in other groups, with almost complete healing and no obvious scarring. In contrast, obvious scars were visible on the wounds of mice in Comparative Examples 1 and 2, demonstrating the superiority of the gel material prepared in this invention in the treatment of keloids.

[0042] 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.

[0043] 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 gel material for treating keloids that reduces lactic acid production, characterized in that: Prepared by the following steps: S1. Add 9-oxabicyclo[3.3.1]nonane-2,6-diol, S-allyl-L-cysteine ​​and anhydrous dichloromethane to the reactor, cool to 0-5℃, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine under a nitrogen atmosphere, control the temperature not to exceed 5℃, after the addition is complete, raise the temperature to 25-30℃ and react for 12-16h, pour the reaction solution into ice water to precipitate the product, filter, wash with ice water, recrystallize with ice ethanol and vacuum dry to obtain intermediate A; S2. Add intermediate A, 5-methoxypiperaldehyde and anhydrous ethanol to the reactor, then add 10% acetic acid solution to adjust the pH of the system to 4.0-4.

5. Then heat to 70-75℃ and react for 6-8 hours. Cool the reaction solution to room temperature and refrigerate at 0-5℃ for 8-10 hours to allow the product to precipitate. After filtration, washing with anhydrous ethanol and vacuum drying, intermediate B is obtained. S3. Protect the reactor from light, add methacrylamide chitosan, polyethylene glycol diacrylate and ultrapure water, stir and add photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, continue stirring for 15-20 min, then add intermediate B, stir at 25-30℃ in the dark for 20-30 min, react with ultraviolet light for 30-45 min, after the reaction is completed, dialyze in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3500 Da, vacuum dry, then pulverize and pass through an 80-100 mesh sieve to obtain cross-linked modified chitosan-polyethylene glycol diacrylate copolymer; S4. Quercetin-3-O-glucuronide, luteolin-3'-glucuronide, and L-carnosine are added to ultrapure water under light-protected conditions to obtain an active substance dispersion. Crosslinked modified chitosan-polyethylene glycol diacrylate copolymer is added to the dispersion and mixed evenly. Then, a humectant, p-hydroxyacetophenone, tocopherol, and pH adjuster are added sequentially. After high-speed homogenization and vacuum degassing, the mixture is finally allowed to stand at 25-30°C for 8-10 hours to obtain the gel material.

2. The gel material for treating keloids that reduces lactic acid production according to claim 1, characterized in that: In step S1, the feed 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 1-4g. 1.1-2.5g: 1.2-1.8g: 1.5-2.5g: 100-150mL.

3. The gel material for treating keloids that reduces lactic acid production according to claim 2, characterized in that: In step S2, the feeding ratio of intermediate A, 5-methoxypiperaldehyde and anhydrous ethanol is 0.88-1g: 1.02-1.46g: 100-200mL.

4. The gel material for treating keloids that reduces lactic acid production according to claim 3, characterized in that: In step S3, the feeding ratio of 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.

5. The gel material for treating keloids that reduces lactic acid production according to claim 4, characterized in that: In step S4, 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 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.

6. The gel material for treating keloids that reduces lactic acid production according to claim 5, characterized in that: The number average molecular weight of the methacrylated chitosan is 50,000-100,000.

7. The gel material for treating keloids that reduces lactic acid production according to claim 6, characterized in that: The number average molecular weight of the polyethylene glycol diacrylate is 400-600.

8. The gel material for treating keloids that reduces lactic acid production according to claim 7, characterized in that: The moisturizer is at least one of glycerin, propylene glycol, sodium hyaluronate, sodium lactate, and trehalose.

9. The gel material for treating keloids that reduces lactic acid production according to claim 8, characterized in that: The pH adjusters include acidic adjusters citric acid and lactic acid, and alkaline adjusters triethanolamine.

10. The gel material for treating keloids that reduces lactic acid production according to claim 9, characterized in that: The pH adjuster adjusts the pH of the gel material to 5.5-6.5.

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