Preparation of modified hyaluronic acid and application of modified hyaluronic acid in gynecological gel

Abstract

The invention belongs to the technical field of biological materials, and relates to hyaluronic acid, in particular to preparation of modified hyaluronic acid and application of the modified hyaluronic acid to gynecological gel. The prepared modified hyaluronic acid improves the hydrophobicity and stability of hyaluronic acid, and the modified hyaluronic acid is beneficial to formation of gel and has good oxidation resistance and antibacterial property. The preparation process is simple to operate, and the prepared product is low in price. The prepared modified hyaluronic acid is good in crosslinking degree, excellent in lubricity and antibacterial property, free of anaphylaxis and high in biocompatibility, can provide lasting lubricating and moisturizing effects when being used for preparing gynecological gel, improves the comfort level of vagina, and relieves symptoms such as vaginal dryness, sexual intercourse pain and climacteric.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, relates to a hyaluronic acid, and particularly relates to the preparation of a modified hyaluronic acid and its application in a gynecological gel. Background Art

[0002] Hyaluronic Acid (HA) is a high-molecular-weight polysaccharide that widely exists in animal tissues, especially in the skin, joints, and eyes. Hyaluronic acid has good moisture retention and biocompatibility and is widely used in fields such as cosmetics, medicine, and tissue engineering. Hyaluronic acid is a water-soluble linear polysaccharide polymer that widely exists in the connective tissues of organisms and is an important component of the extracellular matrix. The HA molecule formed by the repeated arrangement of D-glucuronic acid and N-acetylglucosamine units can entangle with each other to form a continuous three-dimensional hydrogen bond network structure, which acts by binding to specific receptors on the cell surface and has a strong adsorption capacity for water. In addition to functions such as water retention and lubrication, hyaluronic acid also has advantages such as non-allergic and high biocompatibility. This also means that hyaluronic acid can form intermolecular or intramolecular hydrogen bonds with water or substances containing hydrophilic groups, and can also undergo chemical reactions with various types of polymers to construct new systems. In terms of biological functions, hyaluronic acid has advantages such as regulating proteins, acting on the blood vessel wall, lubricating joints, and promoting wound healing. For wound repair, hyaluronic acid can act with cell surface receptors, mediate cell signals, and promote cell proliferation and migration, thereby accelerating tissue regeneration. In short, hyaluronic acid is a multifunctional biomaterial with excellent moisture retention, biocompatibility, and biodegradability. Hyaluronic acid can be prepared by microbial fermentation, chemical synthesis, and animal tissue extraction methods. Hyaluronic acid has a wide range of applications in fields such as cosmetics, medicine, and tissue engineering, and the market demand continues to grow.

[0003] Today, hyaluronic acid has been proven to inhibit platelet adhesion and aggregation and avoid thrombus formation. According to the degree of crosslinking, it is divided into two types: single-phase and double-phase. The single-phase filler has good stability and no particles. It is prepared by changing the quantity ratio of high-molecular-weight hyaluronic acid and low-molecular-weight hyaluronic acid. It has low strength, is easily changed by external forces, and has no granular texture touch. The double-phase filler is the most widely used product on the market. It consists of stable hyaluronic acid gel particles and uncrosslinked hyaluronic acid. The crosslinked hyaluronic acid particles are suspended in the uncrosslinked hyaluronic acid to play a lubricating role. Natural hyaluronic acid has physical and chemical properties such as being easily soluble in water, being absorbed quickly, and having a short residence time in tissues. However, its overly strong hydrophilicity and poor mechanical properties also bring some limitations to the application of hyaluronic acid. Modified hyaluronic acid is modified by chemical or physical methods to improve its performance or endow it with new functions. The present invention prepares high-purity and high-molecular-weight modified hyaluronic acid by optimizing the preparation conditions and purification methods, meeting the application requirements in different fields, which is crucial for the development of the industry. Summary of the Invention

[0004] In view of the above problems, the present invention provides a hyaluronic acid with antibacterial and collagen regeneration-promoting effects and its application in gynecological gels. The specific preparation method is as follows:

[0005] S1. Dissolve 28 - 36 g of D-glucuronic acid in 280 - 320 mL of deionized water, dissolve 32 - 43 g of N-acetylglucosamine in 296 - 338 mL of deionized water, mix the two solutions, then add 3 - 5 g of glucuronyltransferase, 5 - 8 g of N,N'-dicyclohexylcarbodiimide (DCC), 2.1 - 3.3 g of terephthalic acid, and 4 - 6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Ultrasonically treat for 25 - 30 min at a temperature of 33 - 45 °C, then continue to add 1 - 3 M hydrochloric acid to adjust the pH value of the system to 4 - 6. Continue to stir for 23 - 32 min, control the reaction temperature at 68 - 88 °C, and react for 26 - 45 h. Dialyze the reaction solution with a dialysis bag for 35 - 40 h to remove unreacted monomers and by-products. Further purify the hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities. Then, make the purified solution into a powder by freeze-drying to obtain hyaluronic acid powder. In this step, hydrochloric acid is used to promote the polymerization reaction of monomers. Terephthalic acid not only participates in the reaction but also can play a role in adjusting the pH value. Glucuronyltransferase is used to catalyze the biosynthesis reaction. N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used to promote the esterification or amidation reaction in the reaction.

[0006] S2. Preparation of antibacterial nanoparticles: Take 2.5 - 2.9 g of silver acetylacetonate, 1.1 - 1.8 g of zinc sulfate, 1.7 - 2.3 g of copper acetylacetonate, 1.8 - 2.4 g of sodium dodecylbenzenesulfonate (LAS) and 5 - 7 g of reduced graphene oxide, dissolve them in 18 - 35 mL of ethylene glycol and 55 - 62 mL of distilled water. After stirring for 25 - 45 min, transfer the mixture to a microwave reactor, set the microwave power to 300 - 600 W, the temperature to 185 - 215 °C, and the reaction time to 35 - 40 min. After the reaction, obtain the antibacterial nanoparticles by centrifugation, washing and drying. In this step, taking advantage of the characteristic of rapid heat release of microwave, silver, zinc and copper are quickly and evenly attached to the lamellar reduced graphene oxide, increasing the antibacterial property of the material. In addition, the antibacterial effects of silver, zinc and copper combined with the advantages of reduced graphene oxide also have better effects in stimulating collagen regeneration.

[0007] S3. Dissolve 23 - 35 g of the hyaluronic acid prepared in step S1 in 138 - 185 mL of deionized water, add 15 - 28 g of stearic acid, react at 48 - 56 °C for 3 - 5 h, then add 5 - 9 g of 1,4 - butanediol diglycidyl ether (BDDGE), 6 - 8 g of polyethylene glycol (PEG) and 1 - 3 g of ammonium persulfate (APS). After stirring evenly, add 0.9 - 1.6 g of the antibacterial nanoparticles prepared in step S2. Expose this solution to a γ - ray radiation source (Co - 60), continue to stir while ultrasonicating for 40 - 50 min, and control the irradiation dose to 10 - 50 kGy. After the reaction, remove the unreacted by - products by dialysis, and make the purified modified hyaluronic acid solution into powder by spray - drying method, which is the modified hyaluronic acid powder. The purpose of adding stearic acid in this step is to improve the hydrophobicity and stability of hyaluronic acid. The purpose of adding 1,4 - butanediol diglycidyl ether is to improve the mechanical strength and stability of hyaluronic acid, forming a gel or hydrogel. The purpose of adding polyethylene glycol is to improve the new antioxidant property of hyaluronic acid. The purpose of irradiation in this step is to improve the cross - linking degree and stability of hyaluronic acid. The in - situ addition of antibacterial nanoparticles can make them better fuse in the modified hyaluronic acid, enhancing the antibacterial performance of the material. Combining with the moisturizing property of hyaluronic acid can promote the regeneration of skin cells. In addition, the modified hyaluronic acid prepared in this step has good moisturizing property, and can be used to prepare gynecological gels to provide long - lasting lubrication and moisturizing effects, improving vaginal comfort and relieving symptoms such as vaginal dryness, dyspareunia, menopause, etc.

[0008] S4. Take 25 - 42 g of carbomer (C 3 H 4 O 2 ) nDissolve it in 303 - 348 mL of water to form a gel matrix solution; add 18 - 31 g of the modified hyaluronic acid powder prepared in step S3 into the above gel matrix solution, stir evenly to ensure complete dissolution of hyaluronic acid; then add 3 - 5 g of metronidazole, 1 - 3 g of clotrimazole, 0.8 - 1.2 g of sodium benzoate or potassium sorbate, and 3 - 6 g of vitamin C into the gel matrix solution, stir evenly by ultrasonic wave and then add 60 - 80 mL of glycerol, adjust the pH value of the gel to 4.5 - 5.5 with citric acid; finally, fill the prepared materials into appropriate containers, seal and package to ensure sterility, then the gynecological gel can be obtained. The control of the pH value in this step makes it close to the natural pH value of the vagina. The function of adding vitamin C is to promote the synthesis of collagen, accelerate tissue repair, have antioxidant effect, and enhance immunity; the purpose of adding sodium benzoate or potassium sorbate is to ensure the stability of the gel. Combined with the modified hyaluronic acid prepared in step S3, the gynecological gel prepared by the present invention has the effects of antibacterial and promoting collagen regeneration, and has broad application prospects in vaginal lubrication, antibacterial treatment and tissue repair.

[0009] Preferably: In step S1, dissolve 28 g of D - glucuronic acid in 280 mL of deionized water, dissolve 32 g of N - acetylglucosamine in 296 mL of deionized water, mix the two solutions, then add 3 g of glucuronic acid transferase, 5 g of N,N'-dicyclohexylcarbodiimide (DCC), 2.1 g of terephthalic acid, and 4 g of 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide (EDC), perform ultrasonic treatment at a temperature of 33 °C for 25 min, then continue to add 1 M hydrochloric acid to adjust the pH value of the system to 4, continue to stir for 23 min, control the reaction temperature at 68 °C, react for 26 h, dialyze the reaction solution with a dialysis bag for 35 h to remove unreacted monomers and by - products, further purify hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities, and then make the purified solution into powder by freeze - drying to obtain hyaluronic acid powder.

[0010] Preferably: In step S1, 36 g of D-glucuronic acid is dissolved in 320 mL of deionized water, and 43 g of N-acetylglucosamine is dissolved in 338 mL of deionized water. The two solutions are mixed, and then 5 g of glucuronyl transferase, 8 g of N,N'-dicyclohexylcarbodiimide (DCC), 3.3 g of terephthalic acid, and 6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are added. The mixture is ultrasonically treated at a temperature of 45°C for 30 min, and then 3 M hydrochloric acid is continuously added to adjust the pH value of the system to 6. Stirring is continued for 32 min, the reaction temperature is controlled at 88°C, and the reaction is carried out for 45 h. The reaction solution is dialyzed through a dialysis bag for 40 h to remove unreacted monomers and by-products. The hyaluronic acid is further purified through an ultrafiltration membrane to remove macromolecular impurities. Then, the purified solution is made into a powder by freeze-drying to obtain hyaluronic acid powder.

[0011] Preferably: In step S2, 2.5 g of silver acetylacetonate, 1.1 g of zinc sulfate, 1.7 g of copper acetylacetonate, 1.8 g of sodium dodecylbenzenesulfonate (LAS), and 5 g of reduced graphene oxide are dissolved in 18 mL of ethylene glycol and 55 mL of distilled water. After stirring for 25 min, the mixture is transferred to a microwave reaction kettle. The microwave power is set to 300 W, the temperature is 185°C, and the reaction time is 35 min. After the reaction is completed, the antibacterial nanoparticles can be obtained after centrifugation, washing, and drying.

[0012] Preferably: In step S2, 2.9 g of silver acetylacetonate, 1.8 g of zinc sulfate, 2.3 g of copper acetylacetonate, 2.4 g of sodium dodecylbenzenesulfonate (LAS), and 7 g of reduced graphene oxide are dissolved in 35 mL of ethylene glycol and 62 mL of distilled water. After stirring for 45 min, the mixture is transferred to a microwave reaction kettle. The microwave power is set to 600 W, the temperature is 215°C, and the reaction time is 40 min. After the reaction is completed, the antibacterial nanoparticles can be obtained after centrifugation, washing, and drying.

[0013] Preferably: In step S3, 23 g of the hyaluronic acid prepared in step S1 is dissolved in 138 mL of deionized water, 15 g of stearic acid is added, and the reaction is carried out at 48°C for 3 h. Then, 5 g of 1,4-butanediol diglycidyl ether (BDDGE), 6 g of polyethylene glycol (PEG), and 1 g of ammonium persulfate (APS) are added. After stirring evenly, 0.9 g of the antibacterial nanoparticles prepared in step S2 is added. The solution is exposed to a γ-ray radiation source (Co-60), and stirring is continued while ultrasonically treating for 40 min, and the irradiation dose is controlled at 10 kGy. After the reaction is completed, unreacted by-products are removed by dialysis, and the purified modified hyaluronic acid solution is made into a powder by spray drying to obtain modified hyaluronic acid powder.

[0014] Preferably: In step S3, 35 g of the hyaluronic acid prepared in step S1 is dissolved in 185 mL of deionized water, 28 g of stearic acid is added, and the reaction is carried out at 56 °C for 5 h. Then, 9 g of 1,4-butanediol diglycidyl ether (BDDGE), 8 g of polyethylene glycol (PEG), and 3 g of ammonium persulfate (APS) are added. After stirring evenly, 1.6 g of the antibacterial nanoparticles prepared in step S2 is added. The solution is exposed to a γ-ray radiation source (Co-60), and stirring is continued while ultrasonically treating for 50 min, with the irradiation dose controlled at 50 kGy. After the reaction, unreacted by-products are removed by dialysis, and the purified modified hyaluronic acid solution is made into a powder by spray drying to obtain the modified hyaluronic acid powder.

[0015] Preferably: In step S4, 25 g of carbomer (C 3 H 4 O 2 ) n is dissolved in 303 mL of water to form a gel matrix solution; 18 g of the modified hyaluronic acid powder prepared in step S3 is added to the above gel matrix solution, and stirred evenly to ensure that the hyaluronic acid is completely dissolved. Then, 3 g of metronidazole, 1 g of clotrimazole, 0.8 g of potassium sorbate, and 3 g of vitamin C are added to the gel matrix solution. After ultrasonically stirring evenly, 60 mL of glycerol is added, and the pH value of the gel is adjusted to 4.5 using citric acid. Finally, the preparation material is filled into an appropriate container, sealed and packaged to ensure sterility, and the gynecological gel can be obtained.

[0016] Preferably: In step S4, 42 g of carbomer (C 3 H 4 O 2 ) n is dissolved in 348 mL of water to form a gel matrix solution; 31 g of the modified hyaluronic acid powder prepared in step S3 is added to the above gel matrix solution, and stirred evenly to ensure that the hyaluronic acid is completely dissolved. Then, 5 g of metronidazole, 3 g of clotrimazole, 1.2 g of sodium benzoate, and 6 g of vitamin C are added to the gel matrix solution. After ultrasonically stirring evenly, 80 mL of glycerol is added, and the pH value of the gel is adjusted to 5.5 using citric acid. Finally, the preparation material is filled into an appropriate container, sealed and packaged to ensure sterility, and the gynecological gel can be obtained.

[0017] Advantages of the present invention:

[0018] 1. The modified hyaluronic acid prepared in the present invention improves the hydrophobicity and stability of hyaluronic acid.

[0019] 2. The modified hyaluronic acid prepared in the present invention improves the mechanical strength of hyaluronic acid, which is beneficial to the formation of gels or hydrogels.

[0020] 3. The modified hyaluronic acid prepared by the present invention has good antioxidant and antibacterial properties.

[0021] 4. The modified hyaluronic acid prepared by the present invention has a good crosslinking degree.

[0022] 5. The modified hyaluronic acid prepared by the present invention has excellent lubricity, antibacterial property, non-allergic property and high biocompatibility.

[0023] 6. The preparation process of the present invention is simple in operation and the product price is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a schematic structural diagram of the hyaluronic acid prepared by the present invention.

[0026] Figure 2 It is a TEM (transmission electron microscope) image of the modified hyaluronic acid prepared in Example 4 of the present invention.

[0027] Figure 3 It is a dark-field TEM (transmission electron microscope) image and a surface scanning distribution map of the modified hyaluronic acid prepared in Example 4 of the present invention.

[0028] Figure 4 It is an infrared spectrum diagram of the modified hyaluronic acid prepared in Example 4 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the following further details the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the content of the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] S1. Dissolve 28 g of D-glucuronic acid in 280 mL of deionized water, and dissolve 32 g of N-acetylglucosamine in 296 mL of deionized water. Mix the two solutions, then add 3 g of glucuronyltransferase, 5 g of N,N'-dicyclohexylcarbodiimide (DCC), 2.1 g of terephthalic acid, and 4 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Sonicate for 25 min at a temperature of 33 °C, then continue to add 1 M hydrochloric acid to adjust the pH value of the system to 4. Continue stirring for 23 min, control the reaction temperature at 68 °C, and react for 26 h. Dialyze the reaction solution with a dialysis bag for 35 h to remove unreacted monomers and by-products. Further purify hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities. Then, make the purified solution into a powder by freeze-drying to obtain hyaluronic acid powder. In this step, hydrochloric acid is used to promote the polymerization reaction of monomers. Terephthalic acid not only participates in the reaction but also plays a role in adjusting the pH value. Glucuronyltransferase is used to catalyze the biosynthesis reaction. N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used to promote the esterification or amidation reaction in the reaction.

[0032] S2. Prepare antibacterial nanoparticles: Take 2.5 g of silver acetylacetonate, 1.1 g of zinc sulfate, 1.7 g of copper acetylacetonate, 1.8 g of sodium dodecylbenzenesulfonate (LAS), and 5 g of reduced graphene oxide and dissolve them in 18 mL of ethylene glycol and 55 mL of distilled water. After stirring for 25 min, transfer it to a microwave reaction kettle, set the microwave power to 300 W, the temperature to 185 °C, and the reaction time to 35 min. After the reaction is completed, obtain antibacterial nanoparticles after centrifugation, washing, and drying. In this step, taking advantage of the characteristic of rapid heat release of microwave, silver, zinc, and copper are rapidly and evenly attached to the lamellar reduced graphene oxide, increasing the antibacterial property of the material. In addition, the antibacterial effects of silver, zinc, and copper combined with the advantages of reduced graphene oxide also have better effects in stimulating collagen regeneration.

[0033] S3. Dissolve 23 g of the hyaluronic acid prepared in step S1 in 138 mL of deionized water, add 15 g of stearic acid, react at 48 °C for 3 h, then add 5 g of 1,4-butanediol diglycidyl ether (BDDGE), 6 g of polyethylene glycol (PEG) and 1 g of ammonium persulfate (APS). After stirring evenly, add 0.9 g of the antibacterial nanoparticles prepared in step S2. Expose this solution to a γ-ray radiation source (Co-60), continue to stir while ultrasonically treating for 40 min, and control the irradiation dose to be 10 kGy. After the reaction ends, remove the unreacted by-products by dialysis, and make the purified modified hyaluronic acid solution into a powder by spray drying method, which is the modified hyaluronic acid powder. The purpose of adding stearic acid in this step is to improve the hydrophobicity and stability of hyaluronic acid. The purpose of adding 1,4-butanediol diglycidyl ether is to improve the mechanical strength and stability of hyaluronic acid and form a gel or hydrogel. The purpose of adding polyethylene glycol is to improve the new antioxidant property of hyaluronic acid. The purpose of irradiation in this step is to improve the crosslinking degree and stability of hyaluronic acid. The in-situ addition of antibacterial nanoparticles can make them better fuse in the modified hyaluronic acid, enhance the antibacterial performance of the material, and combined with the moisturizing property of hyaluronic acid, it can promote the regeneration of skin cells. In addition, the modified hyaluronic acid prepared in this step has good moisturizing performance. When used to prepare a gynecological gel, it can provide a lasting lubricating and moisturizing effect, improve vaginal comfort, and relieve symptoms such as vaginal dryness, dyspareunia, and menopause.

[0034] S4. Take 25 g of carbomer (C 3 H 4 O 2 ) n Dissolve it in 303 mL of water to form a gel matrix solution; add 18 g of the modified hyaluronic acid powder prepared in step S3 into the above gel matrix solution, stir evenly to ensure that the hyaluronic acid is completely dissolved; then add 3 g of metronidazole, 1 g of clotrimazole, 0.8 g of sodium benzoate and 3 g of vitamin C into the gel matrix solution. After ultrasonic stirring evenly, add 60 mL of glycerol, and use citric acid to adjust the pH value of the gel to 4.5; finally, fill the prepared material into an appropriate container, seal and package to ensure sterility, and the gynecological gel can be obtained. The control of the pH value in this step makes it close to the natural pH value of the vagina. The role of adding vitamin C is to promote the synthesis of collagen, accelerate tissue repair, have antioxidant property, and enhance immunity; the purpose of adding sodium benzoate is to ensure the stability of the gel. Combined with the modified hyaluronic acid prepared in step S3, the gynecological gel prepared by the present invention has the effects of antibacterial and promoting collagen regeneration functions, and has broad application prospects in vaginal lubrication, antibacterial treatment and tissue repair.

[0035] Comparative Example 1: Except that 2.5 g of silver acetylacetonate is not added in step S2, the other steps are the same as those in Example 1.

[0036] Comparative Example 2: Except that 1.1 g of zinc sulfate is not added in step S2, the remaining steps are the same as those in Example 1.

[0037] Comparative Example 3: Except that 1.7 g of cupric acetylacetonate is not added in step S2, the remaining steps are the same as those in Example 1.

[0038] Comparative Example 4: Except that 0.9 g of the antibacterial nanoparticles prepared in step S2 is not added in step S3, the remaining steps are the same as those in Example 1.

[0039] The antibacterial nanoparticles prepared in step S2 of the present invention were formulated into suspensions with concentrations of 18% and 8%, and the prepared solutions were stored in a refrigerator, with the refrigerator temperature controlled at 3°C. During the experiment, the gavage administration method was selected, and different concentrations of drugs were gavaged to the experimental subjects in equal volumes. The subjects selected in the present invention were 27 rats, with a body weight of 100 - 110 g and a rat age of 4 - 8 weeks. The specific operation method was as follows: ① Group the experimental mice. Select 27 mice to conduct an acute toxicity test. Divide them into a control group, experimental group 1, and experimental group 2 according to the random grouping method. The control group was not medicated, experimental group 1 was given a suspension of antibacterial nanoparticles with a concentration of 18%, and experimental group 2 was given a suspension of antibacterial nanoparticles with a concentration of 8%. ② Fast and water-restrict the three groups of rats for 18 h. ③ The experimental groups fed the rats with a solution containing antibacterial nanoparticles in the gavage administration method. The control group was fed with negative ion water, and the volume fed was the same as the drug volume of the experimental groups. ④ Conduct observations. On the same day, not only observe the appearance and behavioral activities of the rats, but also record their respiration, secretions, defecation, etc. All rats were fed for 7 days and their body weights were weighed. In addition, record the death time and toxic and side reactions of the rats. ⑤ After the experiment, observe the pathological changes of the rat organ tissues, and complete the calculation and record of the maximum tolerated dose. In experiment 1 of the present invention, gavage administration was carried out according to the requirements of the maximum concentration and maximum volume. The drug concentrations and solvents administered were 18% / 8% and 0.2 mL / 20 g, respectively. After continuous observation for 7 days, the appearance and behavioral activities of the rats were normal. At the same time, there were no significant differences in water intake, food intake, respiration, secretions, and defecation compared with usual. There were no deaths among the experimental rats. During continuous weighing, the body weights of the rats changed. Before administration, the body weights of the control group rats were (98.05 ± 1.14) g, the body weights of the experimental group 1 rats were (97.65 ± 1.65) g, and the body weights of the experimental group 2 rats were (96.85 ± 2.01) g. After a 12-week acute toxicity test, the average body weights of the control group, experimental group 1, and experimental group 2 rats were (183.65 ± 20.37) g, (196.32 ± 17.6) g, and (189.52 ± 20.3) g, respectively, and the differences were not statistically significant. It can be seen that the antibacterial nanoparticles prepared in the present invention are relatively safe. Combining the anatomical results in the later stage of the experiment, it can be known that after using the antibacterial nanoparticles prepared in the present invention at the maximum dose, there were no obvious differences in the heart, liver, kidney and other organs of the rats. It can be seen that the antibacterial nanoparticles prepared in the present invention are non-toxic and can be applied in gynecological gels.

[0040] Experiments were carried out according to the technical solutions of Example 1 and Comparative Examples 1 - 4. 28 mice were randomly sampled and tested according to the antibacterial protection evaluation standard ISO - 11930. The results are shown in Table 1.

[0041] Table 1

[0042] Test items Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Antibacterial rate (%) 96.26±0.07 78.35±0.09 83.21±0.12 79.17±0.14 64.41±0.17

[0043] Table 1 shows the statistics of the allergy rates and antibacterial rates of the modified hyaluronic acid synthesized in Example 1 and Comparative Examples 1-4. It can be seen from Table 1 that compared with the hyaluronic acid prepared in Comparative Examples 1-4, the antibacterial rate of the modified hyaluronic acid added with the antibacterial nanometer particles prepared in the present invention in Example 1 can reach 96.26%, and it has relatively excellent biocompatibility. By comparing Example 1 with Comparative Examples 1-3, it can be known that there is a synergistic effect among the three metal ions of silver, zinc and copper in the antibacterial nanometer particles, and none of them can be missing. By utilizing the characteristic of rapid heat release of microwave in the present invention, silver, zinc and copper are rapidly and uniformly attached to the lamellar reduced graphene oxide, increasing the antibacterial property of the material, and combining with the advantages of reduced graphene oxide, it also has a better effect in stimulating collagen regeneration. In Comparative Example 4, the antibacterial nanometer particles prepared in step S2 with a weight of 0.9 g are not added, and the antibacterial property is greatly reduced. It shows that in the process of preparing the antibacterial gynecological gel, it is crucial to add the antibacterial nanometer particles of lamellar reduced graphene oxide uniformly attached with silver, zinc and copper.

[0044] Example 2

[0045] S1. Dissolve 36 g of D-glucuronic acid in 320 mL of deionized water, dissolve 43 g of N-acetylglucosamine in 338 mL of deionized water, mix the two solutions, then add 5 g of glucuronic acid transferase, 8 g of N,N'-dicyclohexylcarbodiimide (DCC), 3.3 g of terephthalic acid and 6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), perform ultrasonic treatment at a temperature of 45 °C for 30 min, then continue to add 3 M hydrochloric acid to adjust the pH value of the system to 6, continue to stir for 32 min, control the reaction temperature at 88 °C, react for 45 h, dialyze the reaction solution with a dialysis bag for 40 h to remove unreacted monomers and by-products, further purify the hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities, and then make the purified solution into a powder by freeze-drying to obtain hyaluronic acid powder. Hydrochloric acid in this step is used to promote the polymerization reaction of monomers. Terephthalic acid can not only participate in the reaction but also play a role in adjusting the pH value. Glucuronic acid transferase is used to catalyze the biosynthesis reaction. N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used to promote the esterification or amidation reaction in the reaction.

[0046] S2. Preparation of antibacterial nanoparticles: Take 2.9 g of silver acetylacetonate, 1.8 g of zinc sulfate, 2.3 g of copper acetylacetonate, 2.4 g of sodium dodecylbenzenesulfonate (LAS), and 7 g of reduced graphene oxide and dissolve them in 35 mL of ethylene glycol and 62 mL of distilled water. After stirring for 45 min, transfer the solution to a microwave reactor. Set the microwave power to 600 W, the temperature to 215 °C, and the reaction time to 40 min. After the reaction is completed, centrifuge, wash, and dry to obtain antibacterial nanoparticles. In this step, taking advantage of the characteristic of rapid heat release of microwave, silver, zinc, and copper are rapidly and uniformly attached to the lamellar reduced graphene oxide, increasing the antibacterial property of the material. In addition, the antibacterial effects of silver, zinc, and copper combined with the advantages of reduced graphene oxide also have better effects in stimulating collagen regeneration.

[0047] S3. Dissolve 35 g of the hyaluronic acid prepared in step S1 in 185 mL of deionized water, add 28 g of stearic acid, and react at 56 °C for 5 h. Then add 9 g of 1,4-butanediol diglycidyl ether (BDDGE), 8 g of polyethylene glycol (PEG), and 3 g of ammonium persulfate (APS). After stirring evenly, add 1.6 g of the antibacterial nanoparticles prepared in step S2. Expose this solution to a γ-ray radiation source (Co-60), continue to stir while ultrasonically agitating for 50 min, and control the irradiation dose to 50 kGy. After the reaction is completed, remove the unreacted by-products by dialysis. The purified modified hyaluronic acid solution is made into powder by spray drying, which is the modified hyaluronic acid powder. The purpose of adding stearic acid in this step is to improve the hydrophobicity and stability of hyaluronic acid. The purpose of adding 1,4-butanediol diglycidyl ether is to improve the mechanical strength and stability of hyaluronic acid and form a gel or hydrogel. The purpose of adding polyethylene glycol is to improve the new antioxidant property of hyaluronic acid. The purpose of irradiation in this step is to improve the crosslinking degree and stability of hyaluronic acid. The in-situ addition of antibacterial nanoparticles can make them better integrated into the modified hyaluronic acid, enhancing the antibacterial performance of the material. Combining with the moisturizing property of hyaluronic acid can promote the regeneration of skin cells. In addition, the modified hyaluronic acid prepared in this step has good moisturizing performance and can provide long-lasting lubrication and moisturizing effects when used to prepare gynecological gels, improving vaginal comfort and alleviating symptoms such as vaginal dryness, dyspareunia, and menopause.

[0048] S4. Take 42 g of carbomer (C 3 H 4 O 2 ) nDissolve it in 348 mL of water to form a gel matrix solution; add 31 g of the modified hyaluronic acid powder prepared in step S3 into the above gel matrix solution, stir evenly to ensure complete dissolution of hyaluronic acid; then add 5 g of metronidazole, 3 g of clotrimazole, 1.2 g of potassium sorbate and 6 g of vitamin C into the gel matrix solution, ultrasonically stir evenly and then add 80 mL of glycerol, use citric acid to adjust the pH value of the gel to 5.5; finally, fill the prepared material into a suitable container, seal and package to ensure sterility, then the gynecological gel can be obtained. The control of the pH value in this step makes it close to the natural pH value of the vagina. The function of adding vitamin C is to promote the synthesis of collagen, accelerate tissue repair, have antioxidant effect and enhance immunity; the purpose of adding potassium sorbate is to ensure the stability of the gel. Combining with the modified hyaluronic acid prepared in step S3, the gynecological gel prepared by the present invention has the effects of bacteriostasis and promoting collagen regeneration, and has broad application prospects in vaginal lubrication, antibacterial treatment and tissue repair.

[0049] Comparative Example 5: Except that 3.3 g of terephthalic acid is not added in step S1, the other steps are the same as those in Example 2.

[0050] Comparative Example 6: Except that 8 g of polyethylene glycol is not added in step S3, the other steps are the same as those in Example 2.

[0051] Perform sensitization experiments on the gynecological gels prepared in Example 2 and Comparative Examples 5-6. Use the BALB / c mouse LLNA:DA method and test according to the reference "Evaluation of the irritation and sensitization of 13 chemicals using the local lymph node assay in mice (LLNA:DA)". Weigh 5 g (Group A), 10 g (Group B), and 15 g (Group C) of the gynecological gels prepared in Example 2 and Comparative Examples 5-6 and soak them in 50 mL of physiological saline for 24 h. Take the soaking solution as the test substance and calculate the SI value of each test substance group. The stimulation index (SI) is the ratio of the test substance group to the solvent control group, that is: SI = mean relative light unit of the test substance group / mean relative light unit of the solvent control group. The results are shown in Table 2. Judgment criterion: Sensitization positive judgment criterion: Stimulation index SI ≥ 1.8, and the test substance is sensitization positive.

[0052] Table 2

[0053]

[0054] As can be seen from Table 2, compared with the modified hyaluronic acid prepared in Comparative Examples 5 and 6, the modified hyaluronic acid prepared in Example 2 has a lower allergy rate. This indicates that in addition to the role of terephthalic acid in adjusting the pH value in Step S1 and the improvement of the new antioxidant property of hyaluronic acid by adding polyethylene glycol in Step S3 in the present invention, the two work synergistically to enhance the anti-allergy performance of hyaluronic acid, making the modified hyaluronic acid more suitable for application in gynecological gels.

[0055] Example 3

[0056] S1. Dissolve 32 g of D-glucuronic acid in 305 mL of deionized water, dissolve 39 g of N-acetylglucosamine in 312 mL of deionized water, mix the two solutions, then add 4 g of glucuronic acid transferase, 6 g of N,N'-dicyclohexylcarbodiimide (DCC), 2.8 g of terephthalic acid, and 5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Ultrasonically treat for 28 min at a temperature of 40°C, then continue to add 2 M hydrochloric acid to adjust the pH value of the system to 5, continue stirring for 28 min, control the reaction temperature at 78°C, and react for 36 h. Dialyze the reaction solution through a dialysis bag for 38 h to remove unreacted monomers and by-products, further purify the hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities, and then make the purified solution into a powder by freeze-drying to obtain hyaluronic acid powder. In this step, hydrochloric acid is used to promote the polymerization reaction of monomers. Terephthalic acid can not only participate in the reaction but also play a role in adjusting the pH value. Glucuronic acid transferase is used to catalyze the biosynthesis reaction, and N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used to promote the esterification or amidation reaction in the reaction.

[0057] S2. Prepare antibacterial nanoparticles: Take 2.7 g of silver acetylacetonate, 1.6 g of zinc sulfate, 1.9 g of copper acetylacetonate, 1.9 g of sodium dodecylbenzenesulfonate (LAS), and 6 g of reduced graphene oxide and dissolve them in 22 mL of ethylene glycol and 58 mL of distilled water. After stirring for 37 min, transfer to a microwave reaction kettle, set the microwave power to 400 W, the temperature to 198°C, and the reaction time to 38 min. After the reaction is completed, obtain antibacterial nanoparticles after centrifugation, washing, and drying. In this step, taking advantage of the characteristic of rapid heat release of microwave, silver, zinc, and copper are quickly and evenly attached to the lamellar reduced graphene oxide, increasing the antibacterial property of the material. In addition, the antibacterial effects of silver, zinc, and copper combined with the advantages of reduced graphene oxide also have better effects in stimulating collagen regeneration.

[0058] S3. Dissolve 31 g of the hyaluronic acid prepared in step S1 in 165 mL of deionized water, add 22 g of stearic acid, react at 52 °C for 4 h, then add 8 g of 1,4-butanediol diglycidyl ether (BDDGE), 7 g of polyethylene glycol (PEG) and 2 g of ammonium persulfate (APS). After stirring evenly, add 1.4 g of the antibacterial nanoparticles prepared in step S2. Expose this solution to a γ-ray radiation source (Co-60), continue to stir while ultrasonically treating for 45 min, and control the irradiation dose to be 30 kGy. After the reaction ends, remove the unreacted by-products by dialysis, and make the purified modified hyaluronic acid solution into powder by spray drying to obtain modified hyaluronic acid powder. The purpose of adding stearic acid in this step is to improve the hydrophobicity and stability of hyaluronic acid. The purpose of adding 1,4-butanediol diglycidyl ether is to improve the mechanical strength and stability of hyaluronic acid and form a gel or hydrogel. The purpose of adding polyethylene glycol is to improve the new antioxidant property of hyaluronic acid. The purpose of irradiation in this step is to improve the crosslinking degree and stability of hyaluronic acid. The in-situ addition of antibacterial nanoparticles can enable them to better fuse in the modified hyaluronic acid, enhance the antibacterial performance of the material, and promote the regeneration of skin cells in combination with the moisturizing property of hyaluronic acid. In addition, the modified hyaluronic acid prepared in this step has good moisturizing performance, and can provide a lasting lubricating and moisturizing effect when used to prepare a gynecological gel, improving vaginal comfort and relieving symptoms such as vaginal dryness, dyspareunia, and menopause.

[0059] S4. Take 32 g of carbomer (C 3 H 4 O 2 ) n and dissolve it in 312 mL of water to form a gel matrix solution; add 29 g of the modified hyaluronic acid powder prepared in step S3 to the above gel matrix solution, stir evenly to ensure that the hyaluronic acid is completely dissolved; then add 4 g of metronidazole, 2 g of clotrimazole, 1.1 g of potassium sorbate and 4 g of vitamin C to the gel matrix solution. After ultrasonically stirring evenly, add 75 mL of glycerol, and use citric acid to adjust the pH value of the gel to 4.5; finally, fill the prepared material into a suitable container, seal and package it to ensure sterility, and then the gynecological gel can be obtained. The control of the pH value in this step makes it close to the natural pH value of the vagina. The role of adding vitamin C is to promote the synthesis of collagen, accelerate tissue repair, have antioxidant properties, and enhance immunity; the purpose of adding potassium sorbate is to ensure the stability of the gel. Combining with the modified hyaluronic acid prepared in step S3, the gynecological gel prepared by the present invention has the effects of antibacterial and promoting collagen regeneration, and has broad application prospects in vaginal lubrication, antibacterial treatment and tissue repair.

[0060] Comparative Example 7: Except that 1.4 g of the antibacterial nanoparticles prepared in step S2 is not added in step S3, the other steps are the same as those in Example 3.

[0061] Comparative Example 8: Except that 2 g of ammonium persulfate (APS) was not added in step S3, the remaining steps were the same as those in Example 3.

[0062] Comparative Example 9: Except that in step S3, it was not irradiated with a γ-ray radiation source (Co-60), the remaining steps were the same as those in Example 3.

[0063] The wettability of the material was tested using an SDC-350 contact angle tester (Shengding Co., Ltd.).

[0064] Table 3

[0065] Group Example 3 Comparative Example 7 Comparative Example 8 Comparative Example 9 Contact angle 33.8±1.4 13.3±1.2 18.6±0.7 19.4.3±1.2

[0066] Table 3 shows the contact angle test data of the modified hyaluronic acid prepared in Example 3 and Comparative Examples 7-9 with water. Analyzing the data in Table 3, it can be seen that hyaluronic acid is a hydrophilic material. However, too strong hydrophilicity will bring some limitations to the application of hyaluronic acid. Therefore, the modified hyaluronic acid prepared in the present invention improves the state of too strong hydrophilicity of hyaluronic acid. By adding antibacterial nanoparticles, the contact angle between the modified hyaluronic acid and water is increased, and the problem of too strong hydrophilicity is improved. Comparing Example 3 with Comparative Examples 7 and 8, it can be seen that there is a certain synergistic effect between the antibacterial nanoparticles and ammonium persulfate (APS). Adding both at the same time can improve the wettability of hyaluronic acid. Comparing Example 3 with Comparative Example 9, it can be seen that irradiating the material with a γ-ray radiation source (Co-60) can also improve the disadvantage of too strong hydrophilicity of the hyaluronic acid in the material.

[0067] Example 4

[0068] S1. Dissolve 31 g of D-glucuronic acid in 299 mL of deionized water, and dissolve 39 g of N-acetylglucosamine in 311 mL of deionized water. Mix the two solutions, then add 4 g of glucuronyltransferase, 6 g of N,N'-dicyclohexylcarbodiimide (DCC), 2.9 g of terephthalic acid, and 5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Sonicate for 28 min at a temperature of 41 °C, then continue to add 2 M hydrochloric acid to adjust the pH value of the system to 5. Continue stirring for 29 min, control the reaction temperature at 78 °C, and react for 33 h. Dialyze the reaction solution through a dialysis bag for 38 h to remove unreacted monomers and by-products. Further purify hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities. Then, obtain hyaluronic acid powder by freeze-drying the purified solution. In this step, hydrochloric acid is used to promote the polymerization reaction of monomers. Terephthalic acid not only participates in the reaction but also plays a role in adjusting the pH value. Glucuronyltransferase is used to catalyze the biosynthesis reaction. N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are used to promote the esterification or amidation reaction in the reaction.

[0069] S2. Prepare antibacterial nanoparticles: Take 2.8 g of silver acetylacetonate, 1.6 g of zinc sulfate, 1.9 g of copper acetylacetonate, 2.1 g of sodium dodecylbenzenesulfonate (LAS), and 6 g of reduced graphene oxide and dissolve them in 22 mL of ethylene glycol and 59 mL of distilled water. After stirring for 38 min, transfer to a microwave reaction kettle, set the microwave power to 500 W, the temperature to 205 °C, and the reaction time to 37 min. After the reaction is completed, obtain antibacterial nanoparticles after centrifugation, washing, and drying. In this step, taking advantage of the characteristic of rapid heat release of microwave, silver, zinc, and copper are quickly and evenly attached to the lamellar reduced graphene oxide to increase the antibacterial property of the material. In addition, the antibacterial effects of silver, zinc, and copper combined with the advantages of reduced graphene oxide also have better effects in stimulating collagen regeneration.

[0070] S3. Dissolve 31 g of the hyaluronic acid prepared in step S1 in 165 mL of deionized water, add 24 g of stearic acid, react at 51 °C for 4.2 h, then add 8 g of 1,4-butanediol diglycidyl ether (BDDGE), 7 g of polyethylene glycol (PEG), and 2.6 g of ammonium persulfate (APS). After stirring evenly, add 1.5 g of the antibacterial nanoparticles prepared in step S2. Expose this solution to a γ-ray radiation source (Co-60), continue to stir while ultrasonically treating for 41 min, and control the irradiation dose to be 40 kGy. After the reaction ends, remove the unreacted by-products by dialysis. The purified modified hyaluronic acid solution is made into powder by spray drying, which is the modified hyaluronic acid powder. The purpose of adding stearic acid in this step is to improve the hydrophobicity and stability of hyaluronic acid. The purpose of adding 1,4-butanediol diglycidyl ether is to improve the mechanical strength and stability of hyaluronic acid and form a gel or hydrogel. The purpose of adding polyethylene glycol is to improve the new antioxidant property of hyaluronic acid. The purpose of irradiation in this step is to improve the crosslinking degree and stability of hyaluronic acid. The in-situ addition of antibacterial nanoparticles can make them better integrated into the modified hyaluronic acid, enhancing the antibacterial performance of the material. Combining with the moisturizing property of hyaluronic acid can promote the regeneration of skin cells. In addition, the modified hyaluronic acid prepared in this step has good moisturizing performance and can provide a long-lasting lubricating and moisturizing effect when used to prepare a gynecological gel, improving vaginal comfort and relieving symptoms such as vaginal dryness, dyspareunia, and menopause.

[0071] S4. Take 38 g of carbomer (C 3 H 4 O 2 ) n Dissolve it in 321 mL of water to form a gel matrix solution; add 26 g of the modified hyaluronic acid powder prepared in step S3 into the above gel matrix solution, stir evenly to ensure that the hyaluronic acid is completely dissolved; then add 4 g of metronidazole, 1.9 g of clotrimazole, 0.9 g of potassium sorbate, and 5 g of vitamin C into the gel matrix solution. After ultrasonic stirring evenly, add 68 mL of glycerol, and use citric acid to adjust the pH value of the gel to 5.5; finally, fill the prepared material into an appropriate container, seal and package it to ensure sterility, and the gynecological gel can be obtained. The control of the pH value in this step makes it close to the natural pH value of the vagina. The role of adding vitamin C is to promote the synthesis of collagen, accelerate tissue repair, have antioxidant properties, and enhance immunity; the purpose of adding potassium sorbate is to ensure the stability of the gel. Combining with the modified hyaluronic acid prepared in step S3, the gynecological gel prepared by the present invention has the effects of antibacterial and promoting collagen regeneration functions and has broad application prospects in vaginal lubrication, antibacterial treatment, and tissue repair.

[0072] Chondrocyte-gel experiment: Take 50 μL with a density of 5×10 6Chondrocyte suspension at / mL was inoculated into a 24-well culture plate containing the gels prepared in each of the above examples and comparative examples (5 replicates were set for each group). In this experiment, the surface of the tissue culture plate was used as the positive control group. The surface of the tissue culture plate (Tissue culture plate surface, TCPS) was treated to have appropriate hydrophilic properties, which is more conducive to cell adhesion and proliferation and is usually used as the positive control group. After culturing each cell group for 21 days, a part of the cell-gel complex was taken out, washed with PBS solution, the complex was cut into pieces, 1 mL of papain digestion solution (10 μg / ml, containing 0.4 g of papain, 0.032 g of cysteine, 1.5 g of disodium hydrogen phosphate, 0.088 g of EDTA, and made up to 50 mL with PBS) was added, and it was placed in a constant temperature shaker and digested at 65 °C for 20 h, pipetted evenly, centrifuged to take the supernatant, and used as the sample to be tested. Quantitative detection of collagen: The total collagen content was detected by detecting the content of hydroxyproline in the sample. Take 50 μL of the sample to be tested and add it to a clean cryotube, add 50 μL of 1 M hydrochloric acid solution according to a volume ratio of 1:1, tighten the tube cap, hydrolyze at 120 °C for 40 min, then take out the cryotube, add 50 μL of chloramine-T solution (ice acetic acid-citrate buffer solution with a pH of 6.5 containing 13.6 mg / mL of chloramine-T and 26 v / v% isopropanol) to it, mix well, react at room temperature for 15 min, then add 50 mL of 2,2-dimethylolbutyric acid (abbreviated as p-DMBA) solution (prepared by dissolving 0.7 g of p-DMBA in 3.5 ml of n-propanol and then adding 1.5 ml of perchloric acid and mixing well), react at 37 °C for 30 min, and measure its absorbance value at 550 nm using an enzyme-linked immunosorbent assay reader. A standard curve was drawn using 4-hydroxyproline as the standard. Quantitative detection of osteocalcin (BGP): The content of BGP in the sample to be tested was detected using a BGP enzyme-linked immunosorbent assay kit (Shanghai XinYu Biotechnology Co., Ltd.). The experimental data were statistically sorted using Excel 2013 and SPSS 22.0 statistical software, and each data was measured multiple times.

[0073] Table 4

[0074]

[0075]

[0076] Table 3 lists the contents of collagen and osteocalcin (BGP) in the chondrocyte suspension after the culture experiment of the cell-gel complex of the gynecological gels prepared in Examples 1-4 and Comparative Examples 1-5. As can be seen from Table 3, chondrocytes secreted collagen on the gynecological gels prepared in Examples 1-4 and Comparative Examples 1-5. And after 21 days of culture, the collagen content in the chondrocyte suspension corresponding to Examples 1-4 was significantly higher than that of Comparative Examples 1-5. The increase in collagen content indicates that the chondrocytes have a differentiation trend and have normal differentiation function on the gynecological gel. Osteocalcin (BGP) is produced and secreted by osteoblasts and is an effective marker for bone turnover during the coupling of resorption and formation. Its content reflects the bone metabolic state. As can be seen from the results of Table 4, the BGP content in the cell suspension after the gel-chondrocyte experiment corresponding to Examples 1-4 was significantly higher than that of the comparative examples, indicating that the chondrocytes on the gel had a greater osteogenic trend and higher growth and differentiation activity. Figure 2 This is the TEM (transmission electron microscope) image of the modified hyaluronic acid prepared in Example 4 of the present invention. As can be seen from the figure, there are many small black dots on the lamellar graphene, which are presumably formed by the aggregation of small silver, zinc, and copper metal particles on the graphene. Figure 3 This is the dark-field TEM (transmission electron microscope) image and surface scan distribution map of the modified hyaluronic acid prepared in Example 4 of the present invention. As can be seen from the figure, Ag, Zn, and Cu elements are evenly distributed on the lamellar reduced graphene oxide. The three metal elements quickly and evenly adhere to the lamellar reduced graphene oxide, increasing the antibacterial property of the material, and combining the advantages of reduced graphene oxide has a better effect in stimulating collagen regeneration. Figure 4 This is the infrared spectrum of the modified hyaluronic acid prepared in Example 4 of the present invention. From Figure 4 it can be seen that the appearance of the stretching vibration peak of the ester carbonyl group (C=O) at the 1723 cm -1 position indicates the completion of the esterification reaction.

[0077] The above-described embodiments merely describe the preparation process of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A modified hyaluronic acid, characterized in that: The specific preparation steps are as follows: S1. Dissolve 28-36 g of D-glucuronic acid in 280-320 mL of deionized water, dissolve 32-43 g of N-acetylglucosamine in 296-338 mL of deionized water, mix the two solutions, then add 3-5 g of glucuronyl transferase, 5-8 g of N,N'-dicyclohexylcarbodiimide, 2.1-3.3 g of terephthalic acid and 4-6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and ultrasonically treat at 33-45 °C for 25-30 min, then continue to add 1-3 M hydrochloric acid, adjust the pH value of the system to 4-6, continue stirring for 23-32 min, control the reaction temperature to 68-88 °C, react for 26-45 h, and then dialyze the reaction solution with a dialysis bag for 35-40 h, removing unreacted monomers and by-products, further purifying the hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities, and then freeze-drying the purified solution to obtain hyaluronic acid powder; S2, preparation of antibacterial nanoparticles: 2.5-2.9 g of silver acetylacetonate, 1.1-1.8 g of zinc sulfate, 1.7-2.3 g of copper acetylacetonate, 1.8-2.4 g of sodium dodecylbenzene sulfonate and 5-7 g of reduced graphene oxide were dissolved in 18-35 mL of ethylene glycol and 55-62 mL of distilled water, stirred for 25-45 min, transferred to a microwave reactor, set the microwave power to 300-600 W, the temperature to 185-215 ° C, the reaction time to 35-40 min, after the reaction, centrifuged, washed and dried to obtain antibacterial nanoparticles; S3. Dissolve 23-35 g of the hyaluronic acid prepared in step S1 in 138-185 mL of deionized water, add 15-28 g of stearic acid, and react at 48-56 °C for 3-5 h, then add 5-9 g of 1,4-butanediol diglycidyl ether, 6-8 g of polyethylene glycol and 1-3 g of ammonium persulfate, stir evenly, then add 0.9-1.6 g of the antibacterial nanoparticles prepared in step S2, expose the solution to a gamma-ray radiation source Co-60, continue stirring while ultrasonicating for 40-50 min, and control the irradiation dose to 10-50 kGy; after the reaction is completed, remove the unreacted by-products by dialysis, and prepare the modified hyaluronic acid solution into powder by spray drying, i.e., the modified hyaluronic acid powder.

2. A modified hyaluronic acid according to claim 1, characterized in that: In the step S1, 28 g of D-glucuronic acid is dissolved in 280 mL of deionized water, 32 g of N-acetylglucosamine is dissolved in 296 mL of deionized water, the two solutions are mixed, and then 3 g of glucuronyl transferase, 5 g of N,N'-dicyclohexylcarbodiimide, 2.1 g of terephthalic acid and 4 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added, and ultrasonic treatment is performed at a temperature of 33°C for 25 minutes, and then 1 M hydrochloric acid is added to adjust the pH value of the system to 4, and stirring is continued for 23 minutes. The reaction temperature is controlled to 68°C and the reaction is carried out for 26 hours. The reaction solution is dialyzed with a dialysis bag for 35 hours to remove unreacted monomers and by-products, and the hyaluronic acid is further purified by an ultrafiltration membrane to remove macromolecular impurities. The purified solution is then freeze-dried to form a powder.

3. A modified hyaluronic acid according to claim 1 or 2, characterized in that: In step S1, 36 g of D-glucuronic acid is dissolved in 320 mL of deionized water, and 43 g of N-acetylglucosamine is dissolved in 338 mL of deionized water. The two solutions are mixed, and then 5 g of glucuronyl transferase, 8 g of N,N'-dicyclohexylcarbodiimide, 3.3 g of terephthalic acid and 6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added, and ultrasonic treatment is performed at a temperature of 45°C for 30 minutes. Then, 3 M hydrochloric acid is added to adjust the pH value of the system to 6, and stirring is continued for 32 minutes. The reaction temperature is controlled to 88°C and the reaction is carried out for 45 hours. The reaction solution is dialyzed with a dialysis bag for 40 hours to remove unreacted monomers and by-products, and the hyaluronic acid is further purified by an ultrafiltration membrane to remove macromolecular impurities. Then, the purified solution is freeze-dried to prepare a powder.

4. A modified hyaluronic acid according to claim 1, characterized in that: In the step S2, 2.5 g of silver acetylacetonate, 1.1 g of zinc sulfate, 1.7 g of copper acetylacetonate, 1.8 g of sodium dodecylbenzene sulfonate and 5 g of reduced graphene oxide are dissolved in 18 mL of ethylene glycol and 55 mL of distilled water, stirred for 25 min, and then transferred to a microwave reactor, and the microwave power was set to 300 W, the temperature was set to 185 ° C, and the reaction time was set to 35 min. After the reaction, the antibacterial nanoparticles were obtained by centrifugation, washing and drying.

5. A modified hyaluronic acid according to claim 1 or 3, characterized in that: In the step S2, 2.9 g of silver acetylacetonate, 1.8 g of zinc sulfate, 2.3 g of copper acetylacetonate, 2.4 g of sodium dodecylbenzene sulfonate and 7 g of reduced graphene oxide are dissolved in 35 mL of ethylene glycol and 62 mL of distilled water, stirred for 45 min, and then transferred to a microwave reactor, the microwave power is set to 600 W, the temperature is set to 215 ° C, the reaction time is set to 40 min, and after the reaction is completed, centrifugation, washing and drying are performed to obtain antibacterial nanoparticles.

6. The modified hyaluronic acid according to claim 1, characterized in that: In the step S3, 23 g of the hyaluronic acid prepared in step S1 is dissolved in 138 mL of deionized water, 15 g of stearic acid is added, and the mixture is reacted at 48° C. for 3 h, and then 5 g of 1,4-butanediol diglycidyl ether, 6 g of polyethylene glycol and 1 g of ammonium persulfate are added. After stirring evenly, 0.9 g of the antibacterial nanoparticles prepared in step S2 are added, and the solution is exposed to a gamma-ray radiation source Co-60, and the mixture is continuously stirred while being ultrasonicated for 40 min, and the irradiation dose is controlled to be 10 kGy. After the reaction is completed, the unreacted by-products are removed by dialysis, and the purified modified hyaluronic acid solution is prepared into a powder by spray drying, i.e., the modified hyaluronic acid powder.

7. A modified hyaluronic acid according to claim 1 or 5, characterized in that: In the step S3, 35 g of the hyaluronic acid prepared in step S1 is dissolved in 185 mL of deionized water, 28 g of stearic acid is added, and the mixture is reacted at 56° C. for 5 h, and then 9 g of 1,4-butanediol diglycidyl ether, 8 g of polyethylene glycol and 3 g of ammonium persulfate are added. After stirring evenly, 1.6 g of the antibacterial nanoparticles prepared in step S2 are added, and the solution is exposed to a gamma-ray radiation source Co-60, and the mixture is continuously stirred while being ultrasonicated for 50 min, and the irradiation dose is controlled to be 50 kGy. After the reaction is completed, the unreacted by-products are removed by dialysis, and the purified modified hyaluronic acid solution is prepared into a powder by spray drying, i.e., the modified hyaluronic acid powder.

8. Application of modified hyaluronic acid in gynecological gel, characterized in that: The specific steps are as follows: S1. Dissolve 28-36 g of D-glucuronic acid in 280-320 mL of deionized water, dissolve 32-43 g of N-acetylglucosamine in 296-338 mL of deionized water, mix the two solutions, then add 3-5 g of glucuronyl transferase, 5-8 g of N,N'-dicyclohexylcarbodiimide, 2.1-3.3 g of terephthalic acid and 4-6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and treat with ultrasound at 33-45 °C for 25-30 min, then continue to add 1-3 M hydrochloric acid, adjust the pH value of the system to 4-6, continue stirring for 23-32 min, control the reaction temperature to 68-88 °C, react for 26-45 h, and dialyze the reaction solution with a dialysis bag for 35-40 h, removing unreacted monomers and by-products, further purifying the hyaluronic acid through an ultrafiltration membrane to remove macromolecular impurities, and then freeze-drying the purified solution to obtain hyaluronic acid powder; S2, preparation of antibacterial nanoparticles: 2.5-2.9 g of silver acetylacetonate, 1.1-1.8 g of zinc sulfate, 1.7-2.3 g of copper acetylacetonate, 1.8-2.4 g of sodium dodecylbenzene sulfonate and 5-7 g of reduced graphene oxide were dissolved in 18-35 mL of ethylene glycol and 55-62 mL of distilled water, stirred for 25-45 min, transferred to a microwave reactor, set the microwave power to 300-600 W, the temperature to 185-215 ° C, the reaction time to 35-40 min, after the reaction, centrifuged, washed and dried to obtain antibacterial nanoparticles; S3, dissolving 23-35 g of the hyaluronic acid prepared in step S1 in 138-185 mL of deionized water, adding 15-28 g of stearic acid, reacting at 48-56° C. for 3-5 h, then adding 5-9 g of 1,4-butanediol diglycidyl ether, 6-8 g of polyethylene glycol and 1-3 g of ammonium persulfate, stirring evenly, adding 0.9-1.6 g of the antibacterial nanoparticles prepared in step S2, exposing the solution to a gamma-ray radiation source Co-60, continuing to stir while ultrasonicating for 40-50 min, and controlling the irradiation dose to 10-50 kGy; after the reaction is completed, removing unreacted by-products by dialysis, and preparing the purified modified hyaluronic acid solution into powder by spray drying, i.e., the modified hyaluronic acid powder; S4, take 25-42 g of carbomer and dissolve it in 303-348 mL of water to form a gel matrix solution; add 18-31 g of the modified hyaluronic acid powder prepared in step S3 to the above gel matrix solution, stir evenly to ensure that the hyaluronic acid is completely dissolved; then add 3-5 g of metronidazole, 1-3 g of clotrimazole, 0.8-1.2 g of sodium benzoate or potassium sorbate and 3-6 g of vitamin C to the gel matrix solution, stir evenly under ultrasonication, then add 60-80 mL of glycerol, and use citric acid to adjust the pH value of the gel to 4.5-5.5; finally, fill the prepared materials into appropriate containers, seal and package to ensure sterility, and the gynecological gel can be obtained.

9. The use of a modified hyaluronic acid in gynecological gel according to claim 7, characterized in that: In the step S4, 25 g of carbomer (C3H4O2) is dissolved in 303 mL of water to form a gel matrix solution; 18 g of the modified hyaluronic acid powder prepared in step S3 is added to the above gel matrix solution and stirred evenly to ensure that the hyaluronic acid is completely dissolved; then 3 g of metronidazole, 1 g of clotrimazole, 0.8 g of potassium sorbate and 3 g of vitamin C are added to the gel matrix solution, ultrasonically stirred evenly, 60 mL of glycerol is added, and citric acid is used to adjust the pH value of the gel to 4.5; finally, the prepared materials are filled into appropriate containers, sealed and packaged to ensure sterility, and the gynecological gel can be obtained.

10. Use of a modified hyaluronic acid in gynecological gel according to claim 7 or 8, characterized in that: In the step S4, 42 g of carbomer (C3H4O2) is dissolved in 348 mL of water to form a gel matrix solution; 31 g of the modified hyaluronic acid powder prepared in step S3 is added to the above gel matrix solution and stirred evenly to ensure that the hyaluronic acid is completely dissolved; then 5 g of metronidazole, 3 g of clotrimazole, 1.2 g of sodium benzoate and 6 g of vitamin C are added to the gel matrix solution, ultrasonically stirred evenly, 80 mL of glycerol is added, and citric acid is used to adjust the pH value of the gel to 5.5; finally, the prepared materials are filled into appropriate containers, sealed and packaged to ensure sterility, and the gynecological gel can be obtained.

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