Sodium hyaluronate gel complex for wound repair and preparation process thereof

Abstract

This invention provides a sodium hyaluronate gel complex for wound repair and its preparation process, belonging to the field of gel complex preparation technology. The preparation process includes the following steps: preparation of a Poria cocos polysaccharide-antimicrobial peptide complex; preparation of catechol-grafted modified gelatin; preparation of oxidized sodium alginate; preparation of a sodium alginate complex; and preparation of a sodium hyaluronate gel complex. This invention prepares a sodium alginate complex with double dynamic bond crosslinking by side-linking catechol groups to gelatin, through the coordination between ferric ions and catechol groups, and the Schiff base reaction between the amino groups on gelatin and the aldehyde groups on oxidized sodium alginate. Furthermore, the sodium alginate complex is doped with the Poria cocos polysaccharide-antimicrobial peptide complex. The resulting sodium hyaluronate gel complex, prepared by compounding with thiolated sodium hyaluronate, exhibits good biocompatibility, high antibacterial activity, and significantly promotes wound healing.

Description

Technical Field

[0001] This invention relates to the field of gel complex preparation technology, specifically to a sodium hyaluronate gel complex for wound repair and its preparation process. Background Technology

[0002] In the field of trauma treatment, wound repair is a complex and crucial process that affects patients' recovery progress, quality of life, and medical costs. Ideal wound repair materials need to possess multiple properties, including good biocompatibility, moisture retention, antibacterial properties, promotion of cell proliferation and migration, and suitable mechanical properties, to meet the treatment needs of different types of wounds. Currently, although various wound repair materials exist on the market, many challenges remain. Developing novel and highly efficient wound repair materials has significant clinical importance and broad market prospects.

[0003] Gauze, as the most common traditional wound dressing, has the advantages of low cost and ease of use. However, it also has several disadvantages. On the one hand, gauze has poor breathability and moisture retention. During the wound healing process, excessive water loss can lead to wound dryness, hindering the migration and proliferation of epithelial cells, prolonging healing time, and potentially causing pain and secondary injury. On the other hand, gauze tends to stick to the wound, tearing newly formed tissue when changing dressings, exacerbating patient pain, and increasing the risk of infection.

[0004] Some natural polymer materials, such as collagen and chitosan, have found some application in wound repair due to their good biocompatibility. However, they also have their own problems. Collagen has limited sources, complex extraction and purification processes, high costs, and is prone to triggering immune responses. Although chitosan has antibacterial properties, its degradation rate in vivo is difficult to control precisely, degradation products may have adverse effects on tissues, and its mechanical properties are relatively weak, making it difficult to meet the repair needs of complex wounds when used alone.

[0005] Sodium hyaluronate is a widely used biomaterial for wound repair, possessing excellent moisturizing properties that maintain a moist wound environment and promote cell migration and proliferation. However, pure sodium hyaluronate gel suffers from poor mechanical properties and limited antibacterial activity. To improve these properties, researchers have attempted to modify sodium hyaluronate or combine it with other materials, but current compounding methods and modification techniques still have certain limitations. For example, some simple physical mixing methods fail to achieve good synergistic effects between sodium hyaluronate and other materials, resulting in limited improvement in the overall performance of the composite; some chemical modification methods may damage the original structure and bioactivity of sodium hyaluronate, affecting its efficacy in wound repair.

[0006] With social development and changes in lifestyle, the incidence of trauma is on the rise. Various types of wounds, including burns, lacerations, and diabetic foot ulcers, cause immense suffering to patients. Clinically, there is an urgent need for wound repair materials that can rapidly promote wound healing, reduce infection, minimize scar formation, and are easy to use. This is especially true for special populations such as diabetic patients and the elderly, whose healing abilities are weaker, making the demand for high-quality wound repair materials even more pronounced. Furthermore, with advancements in medical technology, patients have increasingly higher expectations for the aesthetics and functional recovery after wound repair, prompting researchers to continuously explore and develop new wound repair materials and preparation processes.

[0007] Therefore, we propose a sodium hyaluronate gel complex with good biocompatibility, high antibacterial properties, high mechanical strength, and the ability to significantly promote wound healing, as well as its preparation process. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a sodium hyaluronate gel complex for wound repair and its preparation process.

[0009] A process for preparing a sodium hyaluronate gel complex for wound repair includes the following steps:

[0010] S1: Preparation of Poria cocos polysaccharide-antimicrobial peptide complex

[0011] Microwave-ultrasound assisted extraction of Poria cocos polysaccharide was used. Then, Poria cocos polysaccharide and antimicrobial peptide LL-37 were covalently linked by 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester crosslinking agent to obtain Poria cocos polysaccharide-antimicrobial peptide complex.

[0012] S2: Preparation of catechol-grafted modified gelatin

[0013] NHS and EDC were added to a solution of 3-3-(3,4-dihydroxyphenyl)propionic acid, mixed, and then added to a gelatin solution. The mixture was reacted under an argon atmosphere in the dark to prepare catechol-grafted modified gelatin.

[0014] S3: Preparation of sodium alginate oxidized

[0015] Sodium oxidized alginate was prepared by reacting sodium metaperiodate with sodium alginate.

[0016] S4: Preparation of sodium alginate complex

[0017] A solution of catechol-grafted modified gelatin was mixed with FeCl3 solution to obtain a mixture. Then, the Poria cocos polysaccharide-antimicrobial peptide complex was added to the sodium alginate oxidized solution and mixed with the mixture to prepare the sodium alginate complex.

[0018] S5: Preparation of sodium hyaluronate gel complex

[0019] Thiolized sodium hyaluronate was prepared by mixing N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, cysteine ​​hydrochloride and lyophilized sodium hyaluronate powder. Then, the thiolated sodium hyaluronate was compounded with sodium alginate to prepare sodium hyaluronate gel complex.

[0020] Further, the preparation of the Poria cocos polysaccharide-antimicrobial peptide complex in step S1 specifically includes the following steps:

[0021] S1.1: Select Poria cocos pieces with uniform color and no mold spots, dry them in an oven at 50-60℃, pulverize them with a high-speed pulverizer, and pass them through a 40-50 mesh sieve to obtain Poria cocos powder.

[0022] S1.2: Add 10-20 parts by weight of Poria cocos powder to pure water at a material-liquid ratio of 1:25-30, then place it in a microwave-ultrasonic extractor. After microwave-ultrasonic extraction, centrifuge at 3000-4000 r / min for 20-30 min, filter the supernatant, freeze dry, and obtain Poria cocos polysaccharide.

[0023] S1.3: Add Poria cocos polysaccharide to PBS buffer at pH 7.4 to obtain a Poria cocos polysaccharide solution with a concentration of 2-3% w / v. Add antimicrobial peptide LL-37 to PBS buffer at pH 7.4 to obtain an antimicrobial peptide LL-37 solution with a concentration of 1-2% w / v.

[0024] S1.4: Add 0.01-0.02 parts by weight of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester to 20-30 parts by weight of Poria cocos polysaccharide solution, then add 4-5 parts by weight of antimicrobial peptide LL-37 solution, shake and react at 37-38℃ for 4-5 hours. After the reaction is completed, dialyze and freeze dry to obtain Poria cocos polysaccharide-antimicrobial peptide complex.

[0025] Furthermore, the preparation of catechol-grafted modified gelatin in step S2 specifically includes the following steps:

[0026] S2.1: Dissolve 5-8 parts by weight of gelatin in 50-60 parts by weight of pH 5.5 phosphate buffer at 37-38℃ to obtain a gelatin solution; dissolve 0.23-0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50-60 parts by weight of pH 5.5 phosphate buffer at 37-38℃ to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution.

[0027] S2.2: Add 0.2-0.3 parts by weight of NHS and 0.4-0.5 parts by weight of EDC to a 3-3-(3,4-dihydroxyphenyl)propionic acid solution and stir and mix for 20-30 min under an argon atmosphere. Then add the mixture to a gelatin solution and react under an argon atmosphere in the dark for 20-24 h. After dialysis and freeze-drying, catechol-grafted modified gelatin is obtained.

[0028] Furthermore, step S3, the preparation of oxidized sodium alginate, specifically includes the following steps:

[0029] S3.1: Disperse 6-8 parts by weight of sodium alginate in 30-40 parts by weight of ethanol. Under vigorous stirring, slowly add 30-40 parts by weight of sodium metaperiodate solution with a concentration of 17-20%. Then react in the dark for 4-6 hours. After that, add 2-3 parts by weight of ethylene glycol and stir for 20-30 minutes to obtain the reaction solution.

[0030] S3.2: Dialyze the reaction solution with distilled water through a dialysis bag with a molecular weight cutoff of 3500 Da for 3-4 days, and then freeze-dry the purified solution to obtain sodium oxidized alginate.

[0031] Furthermore, the preparation of the sodium alginate complex in step S4 specifically includes the following steps:

[0032] S4.1: Prepare a 250 mg / mL catechol-grafted modified gelatin solution and a 150 mg / mL sodium alginate oxidase solution using distilled water. Mix the catechol-grafted modified gelatin solution with a 5 mg / mL FeCl3 solution to obtain a mixed solution.

[0033] S4.2: Add 2-3 parts by weight of the Poria cocos polysaccharide-antimicrobial peptide complex to 20-30 parts by weight of the sodium alginate oxidized solution, then add 2-3 mol / L sodium hydroxide solution to adjust the pH to 8-10, and then add 10-20 parts by weight of the mixture and premix using a vortex shaker for 2-3 hours to obtain the sodium alginate complex.

[0034] Furthermore, the preparation of the sodium hyaluronate gel complex in step S5 specifically includes the following steps:

[0035] S5.1: Add 4-5 parts by weight of sodium hyaluronate lyophilized powder to 80-100 parts by weight of deionized water, then add 0.23-0.25 parts by weight of N-hydroxysuccinimide and 0.57-0.59 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. After stirring and mixing, adjust the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then react at room temperature for 2-3 hours to obtain a mixed reaction solution.

[0036] S5.2: Add 0.34-0.36 parts by weight of cysteine ​​hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57 with 1 mol / L sodium hydroxide solution, react at room temperature in the dark for 24-25 h, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3-4 days, and finally freeze dry to obtain thiolated sodium hyaluronate;

[0037] S5.3: Dissolve thiolated sodium hyaluronate in PBS solution at pH 7.4 to prepare a 2% (w / v) precursor solution. Then add sodium alginate complex to the precursor solution and stir for 2-3 hours to obtain sodium hyaluronate gel complex.

[0038] Furthermore, the parameters of the microwave-ultrasonic extraction instrument in step S1.2 are as follows: ultrasonic power 100-120w, ultrasonic interval time 2-3s, microwave power 100-120w, temperature 45-50℃, and time 30-40min.

[0039] Furthermore, in step S4.1, Fe 3+ The molar ratio of the catechol group to the hydroxyl group is 1:3-4.

[0040] Furthermore, in step S5.3, the amount of sodium alginate complex added is 2-3% of the precursor solution.

[0041] A sodium hyaluronate gel complex for wound repair, prepared by any one of the preparation processes of a sodium hyaluronate gel complex for wound repair as described in any one of the claims.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] 1. In this invention, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester is a bifunctional crosslinking agent with two different active groups. One end can react with active groups such as amino groups on Poria cocos polysaccharide, and the other end can react with active groups such as thiol groups on antimicrobial peptide LL-37, thereby covalently linking Poria cocos polysaccharide and antimicrobial peptide LL-37 together to form a Poria cocos polysaccharide-antimicrobial peptide complex. Antimicrobial peptide LL-37 itself has good antimicrobial activity. After being compounded with Poria cocos polysaccharide, it is used in the preparation of sodium hyaluronate gel. When combined, the antimicrobial peptides can exert a sustained antimicrobial effect, effectively inhibiting the growth and reproduction of bacteria and other microorganisms in the gel system, reducing the risk of infection, and improving the safety and stability of the gel. Poria cocos polysaccharides have a variety of biological activities, such as immunomodulation and antitumor activity. Introducing the Poria cocos polysaccharide-antimicrobial peptide complex into sodium hyaluronate gel allows the gel complex to not only have antibacterial properties and good biocompatibility, but also to obtain other biological activities brought by Poria cocos polysaccharides, thereby regulating the local immune microenvironment, promoting cell proliferation, differentiation and tissue repair, and thus significantly promoting wound healing.

[0044] 2. This invention prepares a sodium alginate complex with double dynamic bonds by side-linking catechol groups to gelatin and then coordinating the ferric ions with the catechol groups and the Schiff base reaction between the amino groups on the gelatin and the aldehyde groups on the oxidized sodium alginate. The sodium alginate complex is further doped with a poria cocos polysaccharide-antimicrobial peptide complex. This complex is then compounded with thiolated sodium hyaluronate to prepare a sodium hyaluronate gel complex. The coordination between the ferric ions and the catechol groups, and the Schiff base reaction between the amino groups on the gelatin and the aldehyde groups on the oxidized sodium alginate, form a double dynamic bond cross-linked porous structure. This cross-linking method gives the sodium alginate complex a unique network structure, effectively improving the mechanical strength of the gel, making it more resilient and less prone to breakage. The porous structure is beneficial for cell proliferation and migration. The presence of these substances endows the gel with self-repairing properties. When the gel is damaged by external forces, the coordination bonds between ferric ions and catechol groups, as well as the Schiff base bonds, can automatically restore the damaged structure and properties to a certain extent through dynamic dissociation and recombination processes, thereby extending the gel's lifespan. The gel complex can promote the migration and proliferation of wound cells such as fibroblasts and epithelial cells, accelerating wound healing. Sodium hyaluronate can absorb a large amount of water and retain it in the gel, providing a moist environment for the skin or tissue, which helps wound healing and tissue repair. When thiolated sodium hyaluronate is combined with sodium alginate complex and Poria cocos polysaccharide-antimicrobial peptide complex, it can produce a synergistic effect, further enhancing the gel's bioactivity and function, and can jointly regulate cell behavior, promoting tissue regeneration and repair. Attached Figure Description

[0045] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate embodiments of the invention and, together with the specification, further serve to explain the principles of the invention and enable those skilled in the art to practice and use the invention.

[0046] Figure 1 This is a flowchart illustrating the preparation process of a sodium hyaluronate gel complex for wound repair used in an embodiment of the present invention. Detailed Implementation

[0047] The preparation process of a sodium hyaluronate gel complex for wound repair provided by the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0048] Example 1

[0049] A preparation process for a sodium hyaluronate gel complex for wound repair, such as... Figure 1 As shown, it includes the following steps:

[0050] S1: Preparation of Poria cocos polysaccharide-antimicrobial peptide complex

[0051] S1.1: Select Poria cocos pieces with uniform color and no mold spots, dry them in an oven at 50℃, pulverize them with a high-speed pulverizer, and pass them through a 40-mesh sieve to obtain Poria cocos powder.

[0052] S1.2: Add 10 parts by weight of Poria cocos powder to pure water at a material-liquid ratio of 1:25, and then place it in a microwave-ultrasonic extractor. Set the parameters as follows: ultrasonic power 100w, ultrasonic interval time 2s, microwave power 100w, temperature 45℃, time 30min, and then centrifuge at 3000r / min for 20min. Filter the supernatant and freeze-dry it to obtain Poria cocos polysaccharide.

[0053] S1.3: Add Poria cocos polysaccharide to PBS buffer at pH 7.4 to obtain a Poria cocos polysaccharide solution with a concentration of 2% w / v. Add antimicrobial peptide LL-37 to PBS buffer at pH 7.4 to obtain an antimicrobial peptide LL-37 solution with a concentration of 1% w / v.

[0054] S1.4: Add 0.01 parts by weight of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester to 20 parts by weight of Poria cocos polysaccharide solution, then add 4 parts by weight of antimicrobial peptide LL-37 solution, shake at 37°C for 4 hours, dialyze after the reaction is completed, freeze dry to obtain Poria cocos polysaccharide-antimicrobial peptide complex;

[0055] S2: Preparation of catechol-grafted modified gelatin

[0056] S2.1: Dissolve 5 parts by weight of gelatin in 50 parts by weight of pH 5.5 phosphate buffer at 37°C to obtain a gelatin solution; dissolve 0.23 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50 parts by weight of pH 5.5 phosphate buffer at 37°C to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution.

[0057] S2.2: 0.2 parts by weight of NHS and 0.4 parts by weight of EDC were added to a 3-3-(3,4-dihydroxyphenyl)propionic acid solution and stirred for 20 min under an argon atmosphere. Then, the mixture was added to a gelatin solution and reacted for 20 h under an argon atmosphere in the dark. After dialysis and freeze-drying, catechol-grafted modified gelatin was obtained.

[0058] S3: Preparation of sodium alginate oxidized

[0059] S3.1: Disperse 6 parts by weight of sodium alginate in 30 parts by weight of ethanol. Under vigorous stirring, slowly add 30 parts by weight of sodium metaperiodate solution with a concentration of 17%. Then react in the dark for 4 hours. After that, add 2 parts by weight of ethylene glycol and stir for 20 minutes to obtain the reaction solution.

[0060] S3.2: The reaction solution was dialyzed with distilled water for 3 days through a dialysis bag with a molecular weight cutoff of 3500 Da. The purified solution was then freeze-dried to obtain sodium oxidized alginate.

[0061] S4: Preparation of sodium alginate complex

[0062] S4.1: Prepare a 250 mg / mL solution of catechol-grafted modified gelatin and a 150 mg / mL solution of oxidized sodium alginate using distilled water. Mix the catechol-grafted modified gelatin solution with a 5 mg / mL FeCl3 solution. 3+ A mixture was obtained by setting the molar ratio of catechol groups to catechol groups at 1:3.

[0063] S4.2: Add 2 parts by weight of Poria cocos polysaccharide-antimicrobial peptide complex to 20 parts by weight of sodium alginate solution, then add 2 mol / L sodium hydroxide solution, adjust the pH to 8, then add 10 parts by weight of the mixture and premix using a vortex shaker for 2 hours to obtain sodium alginate complex.

[0064] S5: Preparation of sodium hyaluronate gel complex

[0065] S5.1: Add 4 parts by weight of sodium hyaluronate lyophilized powder to 80 parts by weight of deionized water, then add 0.23 parts by weight of N-hydroxysuccinimide and 0.57 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir and mix, adjust the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then react at room temperature for 2 h to obtain a mixed reaction solution;

[0066] S5.2: Add 0.34 parts by weight of cysteine ​​hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 24 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3 days, and finally freeze dry to obtain thiolated sodium hyaluronate.

[0067] S5.3: Dissolve thiolated sodium hyaluronate in PBS solution at pH 7.4 to prepare a 2% (w / v) precursor solution. Then add sodium alginate complex to the precursor solution at a concentration of 2% of the precursor solution. Stir and mix for 2 hours to obtain sodium hyaluronate gel complex.

[0068] Example 2

[0069] A preparation process for a sodium hyaluronate gel complex for wound repair, such as... Figure 1 As shown, it includes the following steps:

[0070] S1: Preparation of Poria cocos polysaccharide-antimicrobial peptide complex

[0071] S1.1: Select Poria cocos pieces with uniform color and no mold spots, dry them in an oven at 50℃, pulverize them with a high-speed pulverizer, and pass them through a 40-mesh sieve to obtain Poria cocos powder.

[0072] S1.2: Add 20 parts by weight of Poria cocos powder to pure water at a material-liquid ratio of 1:30, and then place it in a microwave-ultrasonic extractor. Set the parameters as follows: ultrasonic power 100w, ultrasonic interval time 2s, microwave power -120w, temperature 45℃, time 30min, and then centrifuge at 3000r / min for 20min. Filter the supernatant and freeze dry to obtain Poria cocos polysaccharide.

[0073] S1.3: Add Poria cocos polysaccharide to PBS buffer at pH 7.4 to obtain a 3% w / v Poria cocos polysaccharide solution. Add antimicrobial peptide LL-37 to PBS buffer at pH 7.4 to obtain a 2% w / v antimicrobial peptide LL-37 solution.

[0074] S1.4: Add 0.02 parts by weight of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester to 30 parts by weight of Poria cocos polysaccharide solution, then add 5 parts by weight of antimicrobial peptide LL-37 solution, shake at 37℃ for 4 hours, dialyze after the reaction is completed, freeze dry to obtain Poria cocos polysaccharide-antimicrobial peptide complex;

[0075] S2: Preparation of catechol-grafted modified gelatin

[0076] S2.1: Dissolve 8 parts by weight of gelatin in 60 parts by weight of pH 5.5 phosphate buffer at 37°C to obtain a gelatin solution; dissolve 0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 60 parts by weight of pH 5.5 phosphate buffer at 37°C to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution.

[0077] S2.2: 0.3 parts by weight of NHS and 0.5 parts by weight of EDC were added to a 3-3-(3,4-dihydroxyphenyl)propionic acid solution and stirred for 20 min under an argon atmosphere. Then, the mixture was added to a gelatin solution and reacted for 20 h under an argon atmosphere in the dark. After dialysis and freeze-drying, catechol-grafted modified gelatin was obtained.

[0078] S3: Preparation of sodium alginate oxidized

[0079] S3.1: Disperse 8 parts by weight of sodium alginate in 40 parts by weight of ethanol. Under vigorous stirring, slowly add 40 parts by weight of sodium metaperiodate solution with a concentration of 20%. Then react in the dark for 4 hours. After that, add 3 parts by weight of ethylene glycol and stir for 20 minutes to obtain the reaction solution.

[0080] S3.2: The reaction solution was dialyzed with distilled water for 3 days through a dialysis bag with a molecular weight cutoff of 3500 Da. The purified solution was then freeze-dried to obtain sodium oxidized alginate.

[0081] S4: Preparation of sodium alginate complex

[0082] S4.1: Prepare a 250 mg / mL solution of catechol-grafted modified gelatin and a 150 mg / mL solution of oxidized sodium alginate using distilled water. Mix the catechol-grafted modified gelatin solution with a 5 mg / mL FeCl3 solution. 3+ A mixture was obtained by setting the molar ratio of catechol groups to catechol groups at 1:4.

[0083] S4.2: Add 3 parts by weight of Poria cocos polysaccharide-antimicrobial peptide complex to 30 parts by weight of sodium alginate solution, then add 3 mol / L sodium hydroxide solution to adjust the pH to 10, then add 20 parts by weight of the mixture and premix using a vortex shaker for 2 hours to obtain sodium alginate complex.

[0084] S5: Preparation of sodium hyaluronate gel complex

[0085] S5.1: Add 5 parts by weight of sodium hyaluronate lyophilized powder to 100 parts by weight of deionized water, then add 0.25 parts by weight of N-hydroxysuccinimide and 0.59 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir and mix, adjust the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then react at room temperature for 2 h to obtain a mixed reaction solution;

[0086] S5.2: Add 0.36 parts by weight of cysteine ​​hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 24 hours, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3 days, and finally freeze dry to obtain thiolated sodium hyaluronate.

[0087] S5.3: Dissolve thiolated sodium hyaluronate in PBS solution at pH 7.4 to prepare a 2% (w / v) precursor solution. Then add sodium alginate complex to the precursor solution at an amount of 2-3% of the precursor solution. Stir and mix for 2 hours to obtain sodium hyaluronate gel complex.

[0088] Example 3

[0089] A preparation process for a sodium hyaluronate gel complex for wound repair, such as... Figure 1 As shown, it includes the following steps:

[0090] S1: Preparation of Poria cocos polysaccharide-antimicrobial peptide complex

[0091] S1.1: Select Poria cocos pieces with uniform color and no mold spots, dry them in an oven at 60℃, pulverize them with a high-speed pulverizer, and pass them through a 50-mesh sieve to obtain Poria cocos powder.

[0092] S1.2: Add 10 parts by weight of Poria cocos powder to pure water at a material-liquid ratio of 1:25, and then place it in a microwave-ultrasonic extractor. Set the parameters as follows: ultrasonic power 120w, ultrasonic interval time 3s, microwave power 120w, temperature 50℃, time 40min. Then centrifuge at 4000r / min for 30min. Filter the supernatant and freeze dry to obtain Poria cocos polysaccharide.

[0093] S1.3: Add Poria cocos polysaccharide to PBS buffer at pH 7.4 to obtain a Poria cocos polysaccharide solution with a concentration of 2% w / v. Add antimicrobial peptide LL-37 to PBS buffer at pH 7.4 to obtain an antimicrobial peptide LL-37 solution with a concentration of 1% w / v.

[0094] S1.4: Add 0.01 parts by weight of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester to 20 parts by weight of Poria cocos polysaccharide solution, then add 4 parts by weight of antimicrobial peptide LL-37 solution, shake at 38°C for 5 hours, dialyze after the reaction is complete, freeze dry to obtain Poria cocos polysaccharide-antimicrobial peptide complex;

[0095] S2: Preparation of catechol-grafted modified gelatin

[0096] S2.1: Dissolve 5 parts by weight of gelatin in 50 parts by weight of pH 5.5 phosphate buffer at 38°C to obtain a gelatin solution; dissolve 0.23 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50 parts by weight of pH 5.5 phosphate buffer at 38°C to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution.

[0097] S2.2: 0.2 parts by weight of NHS and 0.4 parts by weight of EDC were added to a 3-3-(3,4-dihydroxyphenyl)propionic acid solution and stirred for 30 min under an argon atmosphere. Then, the mixture was added to a gelatin solution and reacted for 24 h under an argon atmosphere in the dark. After dialysis and freeze-drying, catechol-grafted modified gelatin was obtained.

[0098] S3: Preparation of sodium alginate oxidized

[0099] S3.1: Disperse 6 parts by weight of sodium alginate in 30 parts by weight of ethanol. Under vigorous stirring, slowly add 30 parts by weight of sodium metaperiodate solution with a concentration of 17%. Then react in the dark for 6 hours. After that, add 2 parts by weight of ethylene glycol and stir for 30 minutes to obtain the reaction solution.

[0100] S3.2: The reaction solution was dialyzed with distilled water for 4 days through a dialysis bag with a molecular weight cutoff of 3500 Da. The purified solution was then freeze-dried to obtain sodium oxidized alginate.

[0101] S4: Preparation of sodium alginate complex

[0102] S4.1: Prepare a 250 mg / mL solution of catechol-grafted modified gelatin and a 150 mg / mL solution of oxidized sodium alginate using distilled water. Mix the catechol-grafted modified gelatin solution with a 5 mg / mL FeCl3 solution. 3+ A mixture was obtained by setting the molar ratio of catechol groups to catechol groups at 1:3.

[0103] S4.2: Add 2 parts by weight of Poria cocos polysaccharide-antimicrobial peptide complex to 20 parts by weight of sodium alginate oxidized solution, then add 2 mol / L sodium hydroxide solution, adjust the pH to 8, then add 10 parts by weight of the mixture and premix using a vortex shaker for 3 hours to obtain sodium alginate complex.

[0104] S5: Preparation of sodium hyaluronate gel complex

[0105] S5.1: Add 4 parts by weight of sodium hyaluronate lyophilized powder to 80 parts by weight of deionized water, then add 0.23 parts by weight of N-hydroxysuccinimide and 0.57 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir and mix, adjust the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then react at room temperature for 3 h to obtain a mixed reaction solution;

[0106] S5.2: Add 0.34 parts by weight of cysteine ​​hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57, react at room temperature in the dark for 25 h, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 4 days, and finally freeze dry to obtain thiolated sodium hyaluronate.

[0107] S5.3: Dissolve thiolated sodium hyaluronate in PBS solution at pH 7.4 to prepare a 2% (w / v) precursor solution. Then add sodium alginate complex to the precursor solution at a concentration of 2% of the precursor solution. Stir and mix for 3 hours to obtain sodium hyaluronate gel complex.

[0108] Comparative Example 1

[0109] Compared with Example 1, Comparative Example 1 differs in that the Poria cocos polysaccharide-antimicrobial peptide complex in steps S1 and S4.2 is removed, while the remaining steps remain unchanged to prepare the sodium hyaluronate gel complex, which is referred to as Comparative Example 1.

[0110] Comparative Example 2

[0111] Compared with Example 1, the difference of Comparative Example 2 is that steps S2-S4 are removed in Comparative Example 2, and the sodium alginate complex in step S5.3 is prepared by equal weight of sodium alginate and Poria cocos polysaccharide-antimicrobial peptide complex, while the other steps remain unchanged to prepare sodium hyaluronate gel complex, which is referred to as Comparative Example 2.

[0112] Comparative Example 3

[0113] Compared with Example 1, Comparative Example 3 differs in that steps S5.1-S5.2 are removed, and the thiolated sodium hyaluronate in step S5.3 is replaced with sodium hyaluronate, while the remaining steps remain unchanged to prepare the sodium hyaluronate gel complex, which is referred to as Comparative Example 3.

[0114] Antibacterial test results:

[0115] Table 1. Results of antibacterial performance testing in Examples and Comparative Example 1

[0116]

[0117] As can be seen from the data in Table 1, the introduction of the Poria cocos polysaccharide-antimicrobial peptide complex into sodium hyaluronate gel in this invention can effectively inhibit the growth and reproduction of bacteria and other microorganisms in the gel system, thereby reducing the risk of infection.

[0118] Antibacterial test results:

[0119] Table 2. Results of antibacterial performance testing in Examples and Comparative Example 1

[0120]

[0121] As can be seen from the data in Table 2, the sodium alginate complex with double dynamic bond crosslinking introduced into the sodium hyaluronate gel complex prepared by the present invention can effectively improve the mechanical strength of the gel, making it tougher, less prone to breakage, and improving mechanical strength.

[0122] Determination of healing rate in a rat burn model:

[0123] Healthy adult rats, typically weighing between 200-250g, were selected. They were acclimatized for one week prior to the experiment to ensure good health. The rats were anesthetized with an anesthetic, and their backs were shaved to expose the skin. Standardized deep second-degree burns were caused by applying 90°C hot water to the skin for a specified period. The rats were randomly divided into seven groups of 10 each. The first six groups were treated with the sodium hyaluronate gel complexes prepared in Examples 1-3 and Comparative Examples 1-3. The seventh group served as a blank control group, receiving only physiological saline treatment. The healing rate was measured.

[0124] Healing rate (%) = [(initial wound area - current wound area) / initial wound area] × 100%. See Table 3 for the measurement results.

[0125] Table 3. Results of healing rate determination in Examples 1-3 and Comparative Examples 1-3

[0126]

[0127] As can be seen from the data in Table 3, the data in Comparative Example 1 show that the introduction of the Poria cocos polysaccharide-antimicrobial peptide complex into sodium hyaluronate gel in this invention can promote cell proliferation, differentiation and tissue repair, thereby significantly promoting wound healing.

[0128] The data from Comparative Example 2 show that the prepared sodium alginate complex with dual dynamic bond crosslinking can improve wound healing.

[0129] The data from Comparative Example 3 show that the combination of thiolated sodium hyaluronate, sodium alginate, and Poria cocos polysaccharide-antimicrobial peptide complex can produce a synergistic effect, promoting tissue regeneration and repair.

[0130] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A preparation process for a sodium hyaluronate gel complex for wound repair, characterized in that, Includes the following steps: S1: Preparation of Poria cocos polysaccharide-antimicrobial peptide complex Microwave-ultrasound assisted extraction of Poria cocos polysaccharide was used. Then, Poria cocos polysaccharide and antimicrobial peptide LL-37 were covalently linked by 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester crosslinking agent to obtain Poria cocos polysaccharide-antimicrobial peptide complex. S2: Preparation of catechol-grafted modified gelatin NHS and EDC were added to a solution of 3-3-(3,4-dihydroxyphenyl)propionic acid, mixed, and then added to a gelatin solution. The mixture was reacted under an argon atmosphere in the dark to prepare catechol-grafted modified gelatin. S3: Preparation of sodium alginate oxidized Sodium oxidized alginate was prepared by reacting sodium metaperiodate with sodium alginate. S4: Preparation of sodium alginate complex A solution of catechol-grafted modified gelatin was mixed with FeCl3 solution to obtain a mixture. Then, the Poria cocos polysaccharide-antimicrobial peptide complex was added to the sodium alginate oxidized solution and mixed with the mixture to prepare the sodium alginate complex. S5: Preparation of sodium hyaluronate gel complex Thiolized sodium hyaluronate was prepared by mixing N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, cysteine ​​hydrochloride and lyophilized sodium hyaluronate powder. Then, the thiolated sodium hyaluronate was compounded with sodium alginate to prepare sodium hyaluronate gel complex.

2. The preparation process of the sodium hyaluronate gel complex for wound repair according to claim 1, characterized in that, Step S1, the preparation of the Poria cocos polysaccharide-antimicrobial peptide complex, specifically includes the following steps: S1.1: Select Poria cocos pieces with uniform color and no mold spots, dry them in an oven at 50-60℃, pulverize them with a high-speed pulverizer, and pass them through a 40-50 mesh sieve to obtain Poria cocos powder. S1.2: Add 10-20 parts by weight of Poria cocos powder to pure water at a material-liquid ratio of 1:25-30, then place it in a microwave-ultrasonic extractor. After microwave-ultrasonic extraction, centrifuge at 3000-4000 r / min for 20-30 min, filter the supernatant, freeze dry, and obtain Poria cocos polysaccharide. S1.3: Add Poria cocos polysaccharide to PBS buffer at pH 7.4 to obtain a Poria cocos polysaccharide solution with a concentration of 2-3% w / v. Add antimicrobial peptide LL-37 to PBS buffer at pH 7.4 to obtain an antimicrobial peptide LL-37 solution with a concentration of 1-2% w / v. S1.4: Add 0.01-0.02 parts by weight of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester to 20-30 parts by weight of Poria cocos polysaccharide solution, then add 4-5 parts by weight of antimicrobial peptide LL-37 solution, shake and react at 37-38℃ for 4-5 hours, after the reaction is completed, dialyze and freeze dry to obtain Poria cocos polysaccharide-antimicrobial peptide complex.

3. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 2, characterized in that, Step S2, the preparation of catechol-grafted modified gelatin, specifically includes the following steps: S2.1: Dissolve 5-8 parts by weight of gelatin in 50-60 parts by weight of pH 5.5 phosphate buffer at 37-38℃ to obtain a gelatin solution; dissolve 0.23-0.25 parts by weight of 3-(3,4-dihydroxyphenyl)propionic acid in 50-60 parts by weight of pH 5.5 phosphate buffer at 37-38℃ to obtain a 3-(3,4-dihydroxyphenyl)propionic acid solution. S2.2: Add 0.2-0.3 parts by weight of NHS and 0.4-0.5 parts by weight of EDC to a 3-3-(3,4-dihydroxyphenyl)propionic acid solution and stir and mix for 20-30 min under an argon atmosphere. Then add the mixture to a gelatin solution and react under an argon atmosphere in the dark for 20-24 h. After dialysis and freeze-drying, catechol-grafted modified gelatin is obtained.

4. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 3, characterized in that, Step S3, the preparation of oxidized sodium alginate, specifically includes the following steps: S3.1: Disperse 6-8 parts by weight of sodium alginate in 30-40 parts by weight of ethanol. Under vigorous stirring, slowly add 30-40 parts by weight of sodium metaperiodate solution with a concentration of 17-20%. Then react in the dark for 4-6 hours. After that, add 2-3 parts by weight of ethylene glycol and stir for 20-30 minutes to obtain the reaction solution. S3.2: Dialyze the reaction solution with distilled water through a dialysis bag with a molecular weight cutoff of 3500 Da for 3-4 days, and then freeze-dry the purified solution to obtain sodium oxidized alginate.

5. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 4, characterized in that, Step S4, the preparation of the sodium alginate complex, specifically includes the following steps: S4.1: Prepare a 250 mg / mL catechol-grafted modified gelatin solution and a 150 mg / mL sodium alginate oxidase solution using distilled water. Mix the catechol-grafted modified gelatin solution with a 5 mg / mL FeCl3 solution to obtain a mixed solution. S4.2: Add 2-3 parts by weight of the Poria cocos polysaccharide-antimicrobial peptide complex to 20-30 parts by weight of the sodium alginate oxidized solution, then add 2-3 mol / L sodium hydroxide solution to adjust the pH to 8-10, and then add 10-20 parts by weight of the mixture and premix using a vortex shaker for 2-3 hours to obtain the sodium alginate complex.

6. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 5, characterized in that, Step S5, the preparation of the sodium hyaluronate gel complex, specifically includes the following steps: S5.1: Add 4-5 parts by weight of sodium hyaluronate lyophilized powder to 80-100 parts by weight of deionized water, then add 0.23-0.25 parts by weight of N-hydroxysuccinimide and 0.57-0.59 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. After stirring and mixing, adjust the pH to 4.57 with 1 mol / L hydrogen chloride solution, and then react at room temperature for 2-3 hours to obtain a mixed reaction solution. S5.2: Add 0.34-0.36 parts by weight of cysteine ​​hydrochloride to the mixed reaction solution, stir and mix, adjust the pH to 4.57 with 1 mol / L sodium hydroxide solution, react at room temperature in the dark for 24-25 h, then transfer to a dialysis bag, dialyze with deionized water at pH 3.5 at room temperature in the dark for 3-4 days, and finally freeze dry to obtain thiolated sodium hyaluronate; S5.3: Dissolve thiolated sodium hyaluronate in PBS solution at pH 7.4 to prepare a 2% (w / v) precursor solution. Then add sodium alginate complex to the precursor solution and stir for 2-3 hours to obtain sodium hyaluronate gel complex.

7. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 2, characterized in that, The parameters of the microwave-ultrasonic extraction instrument in step S1.2 are: ultrasonic power 100-120w, ultrasonic interval time 2-3s, microwave power 100-120w, temperature 45-50℃, and time 30-40min.

8. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 5, characterized in that, Fe in step S4.1 3+ The molar ratio of the catechol group to the hydroxyl group is 1:3-4.

9. The preparation process of a sodium hyaluronate gel complex for wound repair according to claim 6, characterized in that, In step S5.3, the amount of sodium alginate complex added is 2-3% of the precursor solution.

10. A sodium hyaluronate gel complex for wound repair, characterized in that, It is prepared by the preparation process of a sodium hyaluronate gel complex for wound repair as described in any one of claims 1-9.

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