Modified hyaluronic acid composite gel, preparation method and application of modified hyaluronic acid composite gel in cosmetic filling

Abstract

The invention belongs to the technical field of biological materials, and relates to composite gel of modified hyaluronic acid, a preparation method of the composite gel and application of the composite gel to medical beauty. In order to obtain the HA hydrogel with low allergy rate and better biocompatibility, carboxyl on hyaluronic acid is activated by adopting a method of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid to prepare the dopamine modified hyaluronic acid HAC. According to the preparation process, the carboxylic acid group of the hyaluronic acid is easy to react with the amino group of the dopamine in an environment with the pH value of 5-6. On the basis, modified hyaluronic acid (HAC) is used as a drug delivery carrier, and the HA has high biocompatibility and degradability and also has the characteristic of eliminating active free radicals. Gel, curcumin (CUR) and a cross-linking agent (BDDE) are selected to react to form Gel-Cur-BDDE-HAC composite gel, and dodecyl acrylate is uniformly dispersed in the Gel-Cur-BDDE-HAC composite gel in the reaction process, so that the antibacterial rate and the biocompatibility of the composite gel are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials and relates to hyaluronic acid, in particular to a composite gel of modified hyaluronic acid and its application in cosmetic filling. Background Art

[0002] As an important protective barrier of the outermost layer of the human body, the skin can prevent the complex external environment from causing a certain degree of damage to the human body. When the skin is traumatized, factors such as fluid loss, infection, and inflammation will delay the skin's self-repair function, resulting in delayed wound closure. Therefore, people have developed a series of wound dressings to accelerate wound closure. Among them, hydrogel wound dressings provide a good healing environment for wound healing because of their water retention and structure similar to the extracellular matrix. Due to the unique three-dimensional network cross-linked structure of hydrogels, they can absorb a large amount of water and avoid excessive tissue exudate. Nowadays, the derivative version of hydrogel wound dressings, self-gelling powder wound dressings, can absorb blood or exudate and quickly form hydrogels. It can absorb excess body fluids and use body fluids to form gels. It can also provide higher adhesion strength for tissues to close wounds and promote healing. Natural polymers are widely used in the construction of wound dressings because of their good biocompatibility, wide resources, and low prices. Hyaluronic acid is more popular in wound dressings than other polysaccharides because of its non-immunogenicity and unique mechanism for promoting wound healing.

[0003] Hyaluronic acid (HA) is composed of N-acetylglucosamine and D-glucuronic acid and is an unsulfated anionic natural polysaccharide. HA has no immunogenicity problem and good biocompatibility, and can be used in stem cell engineering and cartilage tissue regeneration. The HA molecular chain contains abundant hydroxyl and carboxyl groups, which provide more reaction sites for HA. This also means that HA can form intermolecular or intramolecular hydrogen bonds with water or substances containing hydrophilic groups, and can also react chemically with various types of polymers to construct new systems. In terms of biological functions, HA has the advantages of regulating proteins, acting on blood vessel walls, lubricating joints, and promoting wound healing. HA is a substance that already exists in the human body, widely distributed in the extracellular matrix and various tissues, and acts by binding to specific receptors on the cell surface.

[0004] For wound repair, HA can interact with cell surface receptors, mediate cell signals, promote cell proliferation and migration, and thus accelerate tissue regeneration. Today, it has been proven that HA can inhibit platelet adhesion and aggregation and avoid thrombus formation. Due to its outstanding biological performance, research on HA-related hydrogel wound dressings is still very popular. However, HA is relatively unstable and highly biodegradable. This problem can be improved by appropriate modification of HA, such as modifying the HA molecular chain with compounds containing functional groups such as N-acetyl, carboxyl, and hydroxyl.

[0005] In order to obtain HA hydrogel with low allergy rate and better biocompatibility, the carboxyl groups on hyaluronic acid were activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid to prepare dopamine-modified hyaluronic acid HAC. According to its preparation process, the carboxyl group of hyaluronic acid is easy to react with the amino group of dopamine under the environment of pH=5. Based on this, we use modified hyaluronic acid (HAC) as a drug delivery carrier. HA has high biocompatibility and degradability, and also has the characteristics of eliminating active free radicals. Gelatin (GEL), curcumin (CUR) and crosslinker (BDDE) are selected to react to form Gel-Cur-BDDE-HAC composite gel, and dodecyl acrylate is evenly dispersed in the reaction process to improve the antibacterial rate and biocompatibility. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a composite gel of dopamine-modified hyaluronic acid.

[0007] In a first aspect, the present invention provides a method for preparing a composite gel of dopamine-modified hyaluronic acid, and the specific preparation steps are as follows: S1. Weigh 7-10 g sodium chloride, 0.2-0.5 g potassium chloride, 2.0-3.0 g potassium dihydrogen phosphate and 0.25-0.3 g potassium dihydrogen phosphate in a 500 ml beaker, add 300-400 ml deionized water, and stir with a magnetic stirrer at room temperature until completely dissolved. Transfer the mixed solution to a 1 L volumetric flask, then fill it up with deionized water to make up the volume, and finally obtain a buffer solution with a pH of 5-6. After the solution is prepared, store it in a refrigerator (4°C).

[0008] S2. Weigh 10-15 g of sodium bicarbonate and 0.1-0.3 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution A. Weigh 0.1-0.3 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution B. Take a dialysis bag of size 15 cm×10 cm and put it into pretreatment solution A, heat it to boiling on a heating furnace, take out the dialysis bag after 10 minutes and rinse it with deionized water. Then put the dialysis bag into pretreatment solution B, repeat the operation for 2-3 times or more, and finally store the cleaned dialysis bag for standby use. The pretreatment of the dialysis bag in this step can make the modified hyaluronic acid have an interpenetrating polymer network structure, which has higher mechanical properties than a single polymer network hydrogel.

[0009] S3. At room temperature, weigh 0.5-0.8g of hyaluronic acid and add it to a three-necked flask, then add 100ml of the buffer solution prepared in step S1 and a stirring magnet, stir at 60-80℃ for 6-8h with a magnetic heating stirrer until completely dissolved, then adjust the pH to 5-6 with 0.1-0.4 mol / L HCl solution, then add 0.5-0.8g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2-0.4g of dithiophenylacetic acid in sequence, and stir thoroughly until completely dissolved. Pass N2 for 30-40 minutes to remove oxygen in the three-necked flask to prevent oxidation of the more active dopamine during the reaction.

[0010] S4, add 0.5-0.8 g of dopamine hydrochloride, react for 12-14 h in an ice water bath and continuous N2 environment. After stopping the reaction, transfer the reaction product to the dialysis bag obtained in step S3, dialyze with deionized water for 3-5 days, and change the deionized water every 12-16 h to remove the residual catalyst (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid) and unreacted monomers in the solution and retain the grafted product. After freezing the dialyzed solution in a -25~-3℃ refrigerator, place it in a freeze dryer (-60℃, 20Pa) and freeze-dry for 50-60 h to obtain dopamine-modified hyaluronic acid (HAC).

[0011] S5. Pour a certain amount of gelatin (GEL) into a beaker, add an appropriate amount of deionized water to prepare a 0.06-0.08% gelatin aqueous solution, place it on a magnetic stirrer, set the temperature to 45-60°C, and stir at a speed of 1000-1100 rpm until a clear gelatin solution is obtained. Take 0.8-1g of the dopamine-modified hyaluronic acid obtained in step S4 and place it in a beaker, and add 30-40 ml of dilute hydrochloric acid solution, place it in a magnetic stirrer at 25-40°C and a speed of 800-1000 rpm, stir until a transparent hyaluronic acid solution is obtained, use 0.1-0.4 mol / L HCl solution to adjust the solution to pH = 4-5, then add 0.3-0.7 mol / L curcumin (CUR) solution to mix, and stir appropriately to obtain Gel-Cur-HAC.

[0012] S6. Dissolve 1.0-1.5 g of 1,4-butanediol diglycidyl ether (BDDE) in 50-60 ml of 1-2 mol / L sodium hydroxide solution, mix well, add 3-5 mL of Gel-Cur-HAC obtained in step S5, and react at 40-50°C for 20-40 min to form a sol. Dissolve 3-3.5 g of 12-decyl acrylate LA in 5-8 ml of ethanol, slowly add it to the sol and mix well, and continue to react for 3-5 hours until a gel is formed. Soak the gel in a phosphate buffer solution with a pH of 5-6 (take 1-2 g of Na2HPO4, 0.2-0.3 g of KH2PO4, add 1000 ml of deionized water and stir well to make a phosphate buffer solution), adjust the pH to neutral with hydrochloric acid, and then purify the gel by soaking it in deionized water for 2-3 days, and finally freeze-dry to obtain the product Gel-Cur-BDDE-HAC composite gel.

[0013] Preferably: in step S1 of the present invention, 7 g of sodium chloride, 0.2 g of potassium chloride, 2.0 g of dipotassium hydrogen phosphate and 0.25 g of potassium dihydrogen phosphate are weighed into a 500 ml beaker, 300 ml of deionized water is added, and stirred with a magnetic stirrer at room temperature until completely dissolved.

[0014] Preferably: in step S1 of the present invention, the mixed solution is transferred to a 1 L volumetric flask, and then filled with deionized water to make up the volume, and finally a buffer solution with a pH of 5 is obtained. After the solution is prepared, it is placed in a refrigerator (4°C) for storage.

[0015] Preferably: in step S2 of the present invention, 10 g of sodium bicarbonate and 0.1 g of ethylenediaminetetraacetic acid are weighed and added into a beaker containing 500 ml of deionized water and dissolved to obtain a pretreatment solution A.

[0016] Preferably: in step S2 of the present invention, 0.1-0.3 g of ethylenediaminetetraacetic acid is weighed and added into a beaker containing 500 ml of deionized water and dissolved to obtain a pretreatment solution B.

[0017] Preferably: in step S3 of the present invention, 0.5 g of hyaluronic acid is weighed and added to a three-necked flask at room temperature, and then 100 ml of the buffer solution prepared in step S1 and a stirring magnet are added, and stirred for 6 h until completely dissolved.

[0018] Preferably: In step S3 of the present invention, the pH is adjusted to 5 with HCl solution, and then 0.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 g of dithiophenylacetic acid are added in sequence, and stirred until completely dissolved. Preferably, in step S4 of the present invention, 0.5 g of dopamine hydrochloride is added, and the mixture is reacted for 12 h in an ice-water bath and under a continuous N2 atmosphere.

[0019] Preferably: in step S5 of the present invention, 0.8 g of the dopamine-modified hyaluronic acid obtained in step S4 is placed in a beaker, and 30 ml of dilute hydrochloric acid solution is added, and the mixture is placed in a magnetic stirrer at 25° C. and 800 rpm and stirred until a transparent hyaluronic acid solution is obtained.

[0020] Preferably, in step S6 of the present invention, 1.0 g of 1,4-butanediol diglycidyl ether (BDDE) is dissolved in 50 ml of 1 mol / L sodium hydroxide solution, mixed thoroughly and then 3 ml of Gel-Cur-HAC obtained in step S5 is added, and the mixture is stirred at 40° C. for a period of time to form a sol.

[0021] The benefits of the present invention are: The present invention prepares dopamine-modified hyaluronic acid HAC, effectively maintains the water storage capacity and good biocompatibility of collagen, provides a suitable environment for cell growth in tissue repair, and has a better injection filling effect.

[0022] 2. The present invention pre-treats the dialysis bag to allow the modified hyaluronic acid to have an interpenetrating polymer network structure, which has higher mechanical properties than single polymer network hydrogels.

[0023] 3. The present invention uses gelatin (GEL), curcumin (CUR) and a cross-linking agent 1,4-butanediol diglycidyl ether (BDDE) to react to form a high-strength and high-toughness Gel-Cur-BDDE-HAC composite gel based on multiple hydrogen bond interactions. Curcumin and 1,4-butanediol diglycidyl ether have a synergistic effect, which can greatly improve its antibacterial rate and biocompatibility, and can be used as an external dressing or a filling.

[0024] 4. The present invention uses 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid to activate the carboxyl groups on hyaluronic acid, so that the density increases during the cross-linking process and the surface becomes wrinkled, which is beneficial to improving the biocompatibility of the hydrogel in the body.

[0025] 5. The raw materials of the present invention are easily available and the preparation method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is the SEM image of the dopamine-modified hyaluronic acid composite gel prepared in Example 2.

[0028] Figure 2 This is the SEM image of the dopamine-modified hyaluronic acid composite gel prepared in Comparative Example 3.

[0029] Figure 3 This is the SEM image of the dopamine-modified hyaluronic acid composite gel prepared in Comparative Example 4.

[0030] Figure 4 The infrared spectra of dopamine-modified hyaluronic acid prepared in Example 2 and Comparative Examples 3 and 4 are shown.

[0031] Figure 5 The synthesis process of dopamine-modified hyaluronic acid.

[0032] Figure 6 This is the UV spectrum of dopamine-modified hyaluronic acid. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying 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.

[0034] Hyaluronic acid, gelatin, and curcumin were purchased from Shaanxi Rankang Biotechnology Co., Ltd., and 1,4-butanediol diglycidyl ether and dodecyl acrylate were purchased from Guangzhou Changhong Chemical Technology Co., Ltd. Example

[0035] S1. Weigh 7 g sodium chloride, 0.2 g potassium chloride, 2.0 g potassium dihydrogen phosphate and 0.25 g potassium dihydrogen phosphate in a 500 ml beaker, add 300 ml deionized water, and stir with a magnetic stirrer at room temperature until completely dissolved. Transfer the mixed solution to a 1 L volumetric flask, then fill it up with deionized water to make up the volume, and finally obtain a buffer solution with a pH of 5. After the solution is prepared, store it in a refrigerator (4°C).

[0036] S2. Weigh 10 g of sodium bicarbonate and 0.1 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution A. Weigh 0.1 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution B. Take a dialysis bag of size 15 cm×10 cm and put it into pretreatment solution A, heat it to boiling on a heating furnace, take out the dialysis bag after 10 minutes and rinse it with deionized water. Then put the dialysis bag into pretreatment solution B, repeat the operation more than 2 times, and finally store the cleaned dialysis bag for standby use. The pretreatment of the dialysis bag in this step can make the modified hyaluronic acid have an interpenetrating polymer network structure, which has higher mechanical properties than a single polymer network hydrogel.

[0037] S3. At room temperature, weigh 0.5 g of hyaluronic acid and add it to a three-necked flask, then add 100 ml of the buffer solution prepared in step S1 and a stirring magnet, stir at 60°C for 6 h with a magnetic heating stirrer until completely dissolved, then adjust the pH to 5 with 0.1 mol / L HCl solution, then add 0.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 g of dithiophenylacetic acid as catalysts, and stir thoroughly until completely dissolved. Pass N2 for 30 minutes to remove oxygen in the three-necked flask to prevent oxidation of the more active dopamine during the reaction.

[0038] S4, add 0.5 g of dopamine hydrochloride, react for 12 h in an ice water bath and continuous N2 environment. After stopping the reaction, transfer the reaction product into the dialysis bag obtained in step S3, dialyze with deionized water for 3 days, and change the deionized water every 12 h to remove the residual catalyst and unreacted monomers in the solution and retain the grafted product. After freezing the dialyzed solution in a -25°C refrigerator, place it in a freeze dryer (-60°C, 20Pa) and freeze-dry for 50 h to obtain dopamine-modified hyaluronic acid (HAC).

[0039] S5. Pour a certain amount of gelatin (GEL) into a beaker, add an appropriate amount of deionized water to prepare a 0.06% gelatin aqueous solution, place it on a magnetic stirrer, set the temperature to 45°C and the speed to 1000 rpm to stir until a clear gelatin solution is obtained. Take 0.8g of dopamine-modified hyaluronic acid obtained in step S4 and place it in a beaker, add 30 ml of 0.1mol / L dilute hydrochloric acid solution, place it in a magnetic stirrer at 25°C and 800 rpm, stir until a transparent hyaluronic acid solution is obtained, use 0.1 mol / LHCL solution to adjust the solution to pH=4, then add 0.3mol / L curcumin (CUR) solution to mix, stir appropriately, and obtain Gel-Cur-HAC.

[0040] S6. Dissolve 1.0 g of 1,4-butanediol diglycidyl ether (BDDE) in 50 ml of 1 mol / L sodium hydroxide solution, mix thoroughly, then add 3 ml of Gel-Cur-HAC obtained in step S5, and react at 40°C for 30 min to form a sol. Dissolve 3 g of 12-decyl acrylate LA in 5 ml of ethanol, slowly add it to the sol and mix thoroughly, and continue to react for 3 h until a gel is formed. Soak the gel in a phosphate buffer solution with a pH of 5, adjust the pH to neutral with hydrochloric acid, and then purify the gel by soaking it in deionized water for 2 days, and finally freeze-dry to obtain the product Gel-Cur-BDDE-HAC composite gel.

[0041] Comparative Example 1: Except that the pretreatment liquid A is not prepared in step S2, the remaining steps are the same as those in Example 1.

[0042] Comparative Example 2: Except that the pretreatment solution B is not prepared in step S2, the remaining steps are the same as those in Example 1.

[0043] The Gel-Cur-BDDE-HAC composite gel of Example 1 and Comparative Example 1 and Comparative Example 2 was subjected to a sensitization experiment, and the BALB / c mouse LLNA:DA method was used to test the test with reference to the document "Evaluation of the irritation and sensitization of 13 chemicals using the mouse local lymph node test (LLNA:DA)". The test substance was set at 3 different concentrations (5%, 10%, 25%), and the SI value of each test substance group was calculated. The stimulation index (SI) is the ratio of the test substance group to the solvent control group, that is, SI = the mean relative luminescence unit of the test substance group / the mean relative luminescence unit of the solvent control group. The results are shown in Table 1. Judgment criteria: Sensitization positive judgment criteria: Stimulation index SI ≥ 1.8, the test substance is sensitization positive.

[0044] ISO 10993-5 specifies procedures and guidelines to be performed on medical devices to test biocompatibility by exposing the medical device or material to a test material, extract, or indirect contact device for a period of time to determine whether it has the potential to cause cellular damage.

[0045] In addition, the test was performed according to the antimicrobial protection evaluation standard ISO-11930, and the results are shown in Table 1.

[0046] Table 1

[0047] Table 1 shows the stimulation index SI, antibacterial rate and biocompatibility of the hyaluronic acid hydrogel prepared in Example 1, Comparative Example 1 and Comparative Example 2. It is a conventional method in biotechnology to dialyze substances and purify proteins using dialysis bags made of polymer materials. It can be seen from the table that the bactericidal rate of hyaluronic acid can be greatly changed by treating the dialysis bag with different pretreatment solutions. By synergizing the two treatment solutions, the hydrogel achieves efficient bactericidal effects under mild conditions. At the same time, it can be seen from the sensitization experiment test on mice that the hyaluronic acid stimulation index SI prepared by the dialysis bag pretreated with pretreatment solutions A and B at the same time is less than 1.8, and the sensitization is negative; the hyaluronic acid stimulation index SI prepared by the dialysis bag pretreated with pretreatment solution A or B alone is greater than 1.8, and the sensitization is positive; it shows that the hyaluronic acid prepared by the dialysis bag pretreated with the treatment solution effectively reduces its allergy rate and improves biocompatibility.

[0048] Example 1

[0049] S1. Weigh 8 g sodium chloride, 0.3 g potassium chloride, 2.2 g potassium dihydrogen phosphate and 0.28 g potassium dihydrogen phosphate in a 500 ml beaker, add 320 ml deionized water, and stir with a magnetic stirrer at room temperature until completely dissolved. Transfer the mixed solution to a 1 L volumetric flask, then fill it up with deionized water to make up the volume, and finally obtain a buffer solution with a pH of 5.2. After the solution is prepared, store it in a refrigerator (4°C).

[0050] S2. Weigh 11 g of sodium bicarbonate and 0.15 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution A. Weigh 0.13 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution B. Take a dialysis bag of size 15 cm × 10 cm and put it in pretreatment solution A, heat it to boiling on a heating furnace, take out the dialysis bag after 10 minutes and rinse it with deionized water. Then put the dialysis bag into pretreatment solution B, repeat the operation more than 2 times, and finally store the cleaned dialysis bag for standby use. The pretreatment of the dialysis bag in this step can make the modified hyaluronic acid have an interpenetrating polymer network structure, which has higher mechanical properties than a single polymer network hydrogel.

[0051] S3. At room temperature, weigh 0.6 g of hyaluronic acid and add it to a three-necked flask, then add 100 ml of the buffer solution prepared in step S1 and a stirring magnet, stir at 65°C for 7 h with a magnetic heating stirrer until completely dissolved, then adjust the pH to 5.2 with 0.2 mol / L HCl solution, then add 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.29 g of dithiophenylacetic acid as catalysts, and stir thoroughly until completely dissolved. Pass N2 for 34 minutes to remove oxygen in the three-necked flask to prevent oxidation of the more active dopamine during the reaction.

[0052] S4, add 0.6 g of dopamine hydrochloride, and react for 12 hours in an ice-water bath and under a continuous N2 environment. After stopping the reaction, transfer the reaction product into the dialysis bag obtained in step S3, and dialyze with deionized water for 4 days, changing the deionized water every 13 hours, in order to remove the residual catalyst and unreacted monomers in the solution and retain the grafted product. After freezing the dialyzed solution in a -20°C refrigerator, place it in a freeze dryer (-60°C, 20Pa) and freeze-dry for 54 hours to obtain dopamine-modified hyaluronic acid (HAC).

[0053] S5. Pour a certain amount of gelatin (GEL) into a beaker, add an appropriate amount of deionized water to prepare a 0.07% gelatin aqueous solution, place it on a magnetic stirrer, set the temperature to 47°C and the speed to 1050 rpm to stir until a clear gelatin solution is obtained. Take 0.9g of dopamine-modified hyaluronic acid obtained in S4 and place it in a beaker, and add 34ml of 0.1mol / L dilute hydrochloric acid solution, place it in a magnetic stirrer at 26°C and 840 rpm, stir until a transparent hyaluronic acid solution is obtained, use 0.2 mol / L HCL to adjust the solution to PH=4.4, then add 0.4mol / L curcumin (CUR) solution to mix, stir appropriately, and obtain Gel-Cur-HAC.

[0054] S6. Dissolve 1.1 g of 1,4-butanediol diglycidyl ether (BDDE) in 55 ml of 1.4 mol / L sodium hydroxide solution, mix thoroughly, then add 3.5 mL of Gel-Cur-HAC obtained in step S5, and react at 44°C for 25 min to form a sol. Dissolve 3.2 g of 12-decyl acrylate LA in 6 ml of ethanol, slowly add it to the sol and mix thoroughly, and continue to react for 4 hours until a gel is formed. Soak the gel in a phosphate buffer solution with a pH of 5.2, adjust the pH to neutral with hydrochloric acid, and then purify the gel by soaking it in deionized water for 2 days, and finally freeze-dry to obtain the product Gel-Cur-BDDE-HAC composite gel.

[0055] Comparative Example 3: Except that the catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is not added in step S3, the remaining steps are the same as those in Example 2.

[0056] Comparative Example 4: Except that the catalyst dithiophenylacetic acid is not added in step S3, the remaining steps are the same as those in Example 2.

[0057] The Gel-Cur-BDDE-HAC composite gel of Example 2 and Comparative Examples 3 and 4 was subjected to a sensitization experiment, and the BALB / c mouse LLNA:DA method was used to test the irritation and sensitization of 13 chemicals using the mouse local lymph node test (LLNA:DA). The test substance was set at 3 different concentrations (5%, 10%, 25%), and the SI value of each test substance group was calculated. The stimulation index (SI) is the ratio of the test substance group to the solvent control group, that is, SI = the mean relative luminescence unit of the test substance group / the mean relative luminescence unit of the solvent control group. The results are shown in Table 2. Judgment criteria: Sensitization positive judgment criteria: Stimulation index SI ≥ 1.8, the test substance is sensitization positive.

[0058] ISO 10993-5 specifies procedures and guidelines to be performed on medical devices to test biocompatibility by exposing the medical device or material to a test material, extract, or indirect contact device for a period of time to determine whether it has the potential to cause cellular damage.

[0059] In addition, the test was performed according to the antimicrobial protection evaluation standard ISO-11930, and the results are shown in Table 2.

[0060] Table 2

[0061] Figure 1 This is the SEM image of the dopamine-modified hyaluronic acid composite gel prepared in Example 2. Figure 2 This is the SEM image of the dopamine-modified hyaluronic acid composite gel prepared in Comparative Example 3. Figure 3 Table 2 shows the irritation index SI, antibacterial rate and biocompatibility of the hyaluronic acid synthesized in Example 2, Comparative Example 3 and Comparative Example 4. The carboxyl groups on hyaluronic acid were activated by (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid to prepare dopamine-modified hyaluronic acid. According to the preparation process, the carboxyl groups of hyaluronic acid are easy to react with the amino groups of dopamine in the environment of pH=5-6 (such as pH 5.2). Figure 2We can clearly see that the cross-linking process increases the density and the surface is wrinkled. The SEM images without adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or dithiophenylacetic acid (Comparative Examples 3 and 4) show that the surface of hyaluronic acid is very thin, the pores are open, and some places are broken, which may be caused by some collapse or unstable gel structure during the freeze-drying process. Figure 4 The infrared spectra of dopamine-modified hyaluronic acid prepared in Example 2 and Comparative Examples 3 and 4 are shown in FIG. Figure 4 It can be seen that 1640cm -1 and 1560 cm -1 The absorption peaks of amide I and amide II on hyaluronic acid are shown in Figure 2. The absorption peaks of dopamine-modified hyaluronic acid in Example 2 are shown in Figure 3. -1 A new absorption peak appears, which is the absorption peak of v (=CH) on the benzene ring of dopamine. From Table 2, it can be seen that the catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dithiophenylacetic acid play a decisive role in the performance of hyaluronic acid. The antibacterial rate of hyaluronic acid without the addition of catalyst is low and the biocompatibility is poor.

[0062] Example 2

[0063] S1. Weigh 9g sodium chloride, 0.4g potassium chloride, 2.8g potassium dihydrogen phosphate and 0.28g potassium dihydrogen phosphate in a 500ml beaker, add 380ml deionized water, and stir with a magnetic stirrer at room temperature until completely dissolved. Transfer the mixed solution to a 1L volumetric flask, then fill it up with deionized water to make up the volume, and finally obtain a buffer solution with a pH of 5.8. After the solution is prepared, store it in a refrigerator (4℃).

[0064] S2. Weigh 14 g of sodium bicarbonate and 0.2 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution A. Weigh 0.28 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution B. Take a dialysis bag of size 15 cm × 10 cm and put it in pretreatment solution A, heat it to boiling on a heating furnace, take out the dialysis bag after 10 minutes and rinse it with deionized water. Then put the dialysis bag into pretreatment solution B, repeat the operation for more than 3 times, and finally store the cleaned dialysis bag for standby use. The pretreatment of the dialysis bag in this step can make the modified hyaluronic acid have an interpenetrating polymer network structure, which has higher mechanical properties than a single polymer network hydrogel.

[0065] S3. At room temperature, weigh 0.8 g of hyaluronic acid and add it to a three-necked flask, then add 100 ml of the buffer solution prepared in step S1 and a stirring magnet, stir with a magnetic heating stirrer at 70°C for 7 h until it is completely dissolved, then adjust the pH to 5.8 with 0.3 mol / L HCl solution, then add 0.7 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.3 g of dithiophenylacetic acid as catalysts, and stir well until it is completely dissolved. Pass N2 for 38 minutes to remove oxygen in the three-necked flask to prevent oxidation of the more active dopamine during the reaction.

[0066] S4, add 0.7 g of dopamine hydrochloride, react for 13 h in an ice water bath and continuous N2 environment. After stopping the reaction, transfer the reaction product to the dialysis bag obtained in step S3, dialyze with deionized water for 4 days, and change the deionized water every 15 h to remove the residual catalyst and unreacted monomers in the solution and retain the grafted product. After freezing the dialyzed solution in a -10°C refrigerator, put it in a freeze dryer (-60°C, 20Pa) and freeze-dry for 58 h to obtain dopamine-modified hyaluronic acid (HAC).

[0067] S5. Pour a certain amount of gelatin (GEL) into a beaker, add an appropriate amount of deionized water to prepare a 0.08% gelatin aqueous solution, place it on a magnetic stirrer, set the temperature to 55°C and the speed to 1100 rpm to stir until a clear gelatin solution is obtained. Take 0.9g of dopamine-modified hyaluronic acid obtained in S4 and place it in a beaker, and add 38 ml of 0.1mol / L dilute hydrochloric acid solution, place it in a magnetic stirrer at 38°C and 900 rpm, stir until a transparent hyaluronic acid solution is obtained, use 0.3mol / L HCL to adjust the solution to PH=4.8, then add 0.6mol / L curcumin (CUR) solution to mix, stir appropriately, and obtain Gel-Cur-HAC.

[0068] S6. Dissolve 1.4 g of 1,4-butanediol diglycidyl ether (BDDE) in 60 ml of 1.8 mol / L sodium hydroxide solution, mix thoroughly, then add 4.5 mL of Gel-Cur-HAC obtained in step S5, and react at 48°C for 35 min to form a sol. Dissolve 3.4 g of 12-decyl acrylate LA in 7 ml of ethanol, slowly add it to the sol and mix thoroughly, and continue to react for 4 hours until a gel is formed. Soak the gel in a phosphate buffer solution with a pH of 5.8, adjust the pH to neutral with hydrochloric acid, and then purify the gel by soaking it in deionized water for 3 days, and finally freeze-dry to obtain the product Gel-Cur-BDDE-HAC composite gel.

[0069] Comparative Example 5: Except that dopamine hydrochloride is not added for modification in step S5, the remaining steps are the same as those in Example 3.

[0070] Comparative Example 6: Except that 1,4-butanediol diglycidyl ether is not added for modification in step S6, the remaining steps are the same as those in Example 3.

[0071] The Gel-Cur-BDDE-HAC composite gel of Example 3 and Comparative Examples 5 and 6 was subjected to a sensitization experiment, and the BALB / c mouse LLNA:DA method was used to test the irritation and sensitization of 13 chemicals using the mouse local lymph node test (LLNA:DA). The test substance was set at 3 different concentrations (10%, 25%, 50%), and the SI value of each test substance group was calculated. The stimulation index (SI) is the ratio of the test substance group to the solvent control group, that is, SI = the mean relative luminescence unit of the test substance group / the mean relative luminescence unit of the solvent control group. The results are shown in Table 3. Judgment criteria: Sensitization positive judgment criteria: Stimulation index SI ≥ 1.8, the test substance is sensitization positive.

[0072] ISO 10993-5 specifies procedures and guidelines to be performed on medical devices to test biocompatibility by exposing the medical device or material to a test material, extract, or indirect contact device for a period of time to determine whether it has the potential to cause cellular damage.

[0073] In addition, the test was performed according to the antimicrobial protection evaluation standard ISO-11930, and the results are shown in Table 3.

[0074] Table 3

[0075] Figure 5 The synthesis process of dopamine-modified hyaluronic acid. Figure 6 Table 3 is the UV spectrum of dopamine-modified hyaluronic acid, and Table 4 is the stimulation index SI, antibacterial rate and biocompatibility of the hyaluronic acid synthesized in Example 3, Comparative Example 5 and Comparative Example 6. In order to determine whether dopamine is successfully grafted on hyaluronic acid, the dopamine-modified hyaluronic acid was firstly subjected to UV spectrum measurement. The dopamine-modified hyaluronic acid aqueous solution prepared in Example 3 was tested on a UV-visible spectrophotometer, and the results are as follows: Figure 6 As shown. A strong absorption peak appeared at 280 nm, which corresponds to the benzene ring, and no other absorption peaks appeared after 300 nm, indicating that the dopamine grafted on the hyaluronic acid was not oxidized, which can prove that dopamine was grafted on the hyaluronic acid. From Table 3, it can be seen that the addition of 1,4-butanediol diglycidyl ether for modification can greatly improve its antibacterial rate and biocompatibility.

[0076] The embodiments described above are merely descriptions of the preparation process of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solution of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a composite gel of modified hyaluronic acid, characterized in that: The specific preparation steps are as follows: S1. Weigh 7-10 g of sodium chloride, 0.2-0.5 g of potassium chloride, 2.0-3.0 g of potassium dihydrogen phosphate and 0.25-0.3 g of potassium dihydrogen phosphate into a 500 ml beaker, add 300-400 ml of deionized water, stir with a magnetic stirrer at room temperature until completely dissolved, transfer the mixed solution to a 1 L volumetric flask, and then fill it up with deionized water to make up the volume, and finally obtain a buffer solution with a pH of 5-6. After the solution is prepared, store it in a refrigerator at 4°C; S2, weigh 10-15 g of sodium bicarbonate and 0.1-0.3 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution A, weigh 0.1-0.3 g of ethylenediaminetetraacetic acid, add them to a beaker filled with 500 ml of deionized water and dissolve them to obtain pretreatment solution B, take a dialysis bag of size 15 cm×10 cm and put it into the pretreatment solution A, heat it to boiling on a heating furnace, take out the dialysis bag after 10 minutes and rinse the dialysis bag with deionized water, then put the dialysis bag into the pretreatment solution B, repeat the operation for 2-3 times or more, and finally store the cleaned dialysis bag for standby use; S3, at room temperature, weigh 0.5-0.8 g of hyaluronic acid and add it to a three-necked flask, then add 100 ml of the buffer solution prepared in step S1 and a stirring bar, stir for 6-8 h until completely dissolved, then adjust the pH to 5-6 with HCl solution, then add 0.5-0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2-0.4 g of dithiophenylacetic acid as catalysts in sequence, stir well until completely dissolved, pass N2 for 30-40 minutes to remove oxygen in the three-necked flask to prevent oxidation of the more active dopamine during the reaction; S4, add 0.5-0.8 g of dopamine hydrochloride, react in an ice water bath and continuously pass N2 for 12-14 h, after stopping the reaction, transfer the reaction product into the dialysis bag obtained in step S3, dialyze with deionized water for 3-5 days, change the deionized water every 12-16 h, freeze the dialyzed solution in a -25~-3°C refrigerator, and then freeze dry it in a freeze dryer (-60°C, 20Pa) for 50-60 h to obtain dopamine-modified hyaluronic acid; S5, pour gelatin (GEL) into a beaker, add deionized water to prepare a 0.06-0.08% gelatin aqueous solution, place it on a magnetic stirrer, set the temperature to 45-60°C, and the speed to 1000-1100 rpm to stir until a clear gelatin solution is obtained, take 0.8-1g of the dopamine-modified hyaluronic acid obtained in step S4 and place it in a beaker, and add 30-40 ml of dilute hydrochloric acid solution, place it in a magnetic stirrer at 25-40°C and a speed of 800-1000 rpm, stir until a transparent hyaluronic acid solution is obtained, use 0.1-0.4mol / L HCL to adjust the solution to pH=4-5, then add 0.3-0.7mol / L curcumin (CUR) solution to mix, and stir to obtain Gel-Cur-HAC; S6. Dissolve 1.0-1.5 g of 1,4-butanediol diglycidyl ether (BDDE) in 50-60 ml of 1-2 mol / L sodium hydroxide solution, mix well and then add 3-5 ml of Gel-Cur-HAC obtained in step S5. Stir and react at 40-50°C for 20-40 minutes to form a sol. Dissolve 3-3.5 g of dodecyl acrylate LA in 5-8 ml of ethanol and slowly add it to the sol to mix well. Continue to react for 3-5 hours until a gel is formed. Soak the gel in a phosphate buffer solution with a pH of 5-6, adjust the pH to neutral with hydrochloric acid, and then soak the gel in deionized water for 2-3 days to purify the gel. Finally, freeze-dry to obtain the product Gel-Cur-BDDE-HAC composite gel.

2. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S1, 7 g of sodium chloride, 0.2 g of potassium chloride, 2.0 g of dipotassium hydrogen phosphate and 0.25 g of potassium dihydrogen phosphate were weighed into a 500 ml beaker, 300 ml of deionized water was added, and stirred with a magnetic stirrer at room temperature until completely dissolved.

3. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S1, the mixed solution is transferred to a 1 L volumetric flask, and then filled with deionized water to make up the volume, and finally a buffer solution with a pH of 5 is obtained. After the solution is prepared, it is placed in a refrigerator and stored at 4°C.

4. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S2, 10 g of sodium bicarbonate and 0.1 g of ethylenediaminetetraacetic acid are weighed, added into a beaker containing 500 ml of deionized water and dissolved to obtain a pretreatment solution A.

5. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S2, 0.1-0.3 g of ethylenediaminetetraacetic acid is weighed and added into a beaker containing 500 ml of deionized water and dissolved to obtain a pretreatment solution B.

6. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S3, 0.5 g of hyaluronic acid is weighed and added to a three-necked flask at room temperature, and then 100 ml of the buffer solution prepared in step S1 and a stirring magnet are added, and stirred for 6 hours until completely dissolved.

7. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S3, the pH is adjusted to 5 with HCl solution, and then 0.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 g of dithiophenylacetic acid are added in sequence, and stirred until completely dissolved.

8. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S4, 0.5 g of dopamine hydrochloride was added, and the mixture was reacted for 12 h in an ice-water bath under a continuous nitrogen atmosphere.

9. The method for preparing the composite gel of modified hyaluronic acid according to claim 1, characterized in that: In the step S6, 1.0 g of 1,4-butanediol diglycidyl ether (BDDE) is dissolved in 50 ml of 1 mol / L sodium hydroxide solution, mixed thoroughly and then 3 ml of Gel-Cur-HAC obtained in step S5 is added, and stirred at 40° C. for 20-40 minutes to form a sol.

10. A composite gel of dopamine-modified hyaluronic acid obtained by the method for preparing a composite gel of modified hyaluronic acid according to any one of claims 1 to 9.

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