A hyaluronic acid hydrogel containing natural fatty acids for treating acne and a preparation method thereof

Abstract

The present invention provides a hyaluronic acid hydrogel for treating acne containing natural fatty acids and a preparation method thereof. First, a hyaluronic acid-cystamine polymer is synthesized by a condensation reaction of a carboxyl group and an amino group, and then a cross-linking agent and a fatty acid are added to obtain a hyaluronic acid hydrogel containing natural fatty acids by a cross-linking reaction. The hydrogel has good biocompatibility and degradability, and its preparation method is simple, low-cost and easy to implement. Propionibacterium acnes is eliminated by introducing antibacterial molecules, and no serious toxic side effects are produced, and it has a great application prospect in improving acne treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of hyaluronic acid gel preparation, and specifically relates to the synthesis of high molecular polymer and a gel preparation method thereof. Background Art

[0002] Acne is a common chronic inflammatory disease of the hair follicles and sebaceous glands in adolescents. Many adolescents suffer from acne, and the psychological and social aspects of the patients are greatly affected. The occurrence of acne is closely related to factors such as excessive secretion of sebum, blockage of the hair follicles and sebaceous gland ducts, bacterial infection and inflammation. Excessive secretion of sebum by the sebaceous glands causes blockage of pores, promotes the proliferation of Gram-positive anaerobic bacteria Propionibacterium acnes, and triggers skin inflammation to form acne. Therefore, many studies are to treat inflammatory acne by eliminating P. acnes (Yang DR, Pornpattananangku D l, Nakatsuji T, Chan M, Carson D, Huang CM, Zhang L F. The antimicrobial activity of liposomal lauricacids against Propionibacterium acnes [J]. Biomaterials. 2009, 30 (30): 6035-6040). Due to the large-scale reproduction of Propionibacterium acnes, skin cells are stimulated to secrete excessive reactive oxygen free radicals, causing skin oxidative stress to eliminate Propionibacterium acnes. Although high concentrations of reactive oxygen free radicals eliminate Propionibacterium acnes, they also destroy the skin tissue structure and form acne marks (scars). Recently, many studies on acne treatments that resist free radicals and inhibit oxidation have been reported, all of which have shown good anti-inflammatory activity. However, there are fewer reports on studies that have both antibacterial and anti-free radical properties, and their therapeutic effects are far from satisfactory, and no in vivo experimental evaluation has been performed. Therefore, it is urgent to develop a synergistic treatment method that can kill Propionibacterium acnes and eliminate oxygen free radicals.

[0003] Lauric acid, a natural product that can be extracted from coconut oil and milk, is also a component of human sebum and has been found to have excellent anti-Propionibacterium acnes therapeutic effects without causing side effects. However, the above-mentioned longer carbon chain fatty acids are not easy to use directly due to their poor hydrophilicity and low skin absorption rate. They need to be coated with a carrier to achieve antibacterial treatment. Currently, the literature reports that the commonly used carriers for encapsulating fatty acids are micelles or liposomes (Pornpattananangkul D, Fu V, Thamphiwatana S, Zhang L, Chen M, Vecchio J, Gao WW, Huang CM, Zhang L F. In vivo treatment of Propionibacterium acnes infection with liposomal lauric acids [J]. Advanced Healthcare Materials. 2013, 2 (10): 1322-1328). The hydrophilicity of the carrier increases the absorption of fatty acids by the skin. The fatty acids enter the skin and use the glycerol secreted by the bacteria to form monolaurin with lauric acid, destroying the bacterial structure and achieving antibacterial therapeutic effects. Hyaluronic acid not only acts as a carrier of antibacterial agents, but can also effectively eliminate active free radicals. In an inflammatory environment with highly active oxygen free radicals, hyaluronic acid gel can achieve a controlled release of fatty acids, which then exerts an antibacterial effect, achieving synergistic antibacterial and anti-free radical anti-acne treatment. The residual hyaluronic acid also has the function of absorbing water and moisturizing, further improving the skin effect after acne is cured.

[0004] The design of the hyaluronic acid-based drug delivery system effectively combines antibacterial and anti-free radical to synergistically treat acne. The selected materials are low-cost, the synthesis and preparation schemes are simple and feasible, and the drug use method is convenient and effective. It has great application prospects in improving acne treatment. Summary of the invention

[0005] The present invention aims to disclose a hyaluronic acid hydrogel containing natural fatty acids for treating acne and a preparation method thereof, wherein the gel is formed by cross-linking reaction of hyaluronic acid with good biocompatibility and degradability and a cross-linking agent. The preparation method of the gel is simple, low-cost and easy to implement, and eliminates Propionibacterium acnes by introducing antibacterial molecules without causing serious toxic side effects, and has great application prospects in improving acne treatment.

[0006] The present invention is achieved through the following technical scheme: a hyaluronic acid-cystamine polymer is synthesized through a condensation reaction of a carboxyl group and an amino group, a cross-linking reaction is then carried out with a cross-linking agent, and a fatty acid is encapsulated therein to prepare a gel.

[0007] A hyaluronic acid hydrogel containing natural fatty acids for treating acne and a preparation method thereof, characterized in that the preparation method comprises the following steps:

[0008] The synthesis method of the hyaluronic acid-cystamine can be prepared by the literature method (Li SZ, Pei MJ, Wan TT, Yang HJ, Gu SJ, Tao Y Z. Self-healing hyaluronic acid hydrogels based on dynamic Schiff base linkages as biomaterials. Carbohydrate Polymers. 2020, 250 (15): 116922), and the specific synthesis method is as follows:

[0009] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate (1-3): (0.3-1), prepare a solution with a mass fraction of 1-10%, add it to a round-bottom flask, stir it at room temperature for 5-12 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10: (3-8): (1-6): (1-15), prepare a solution with a mass fraction of 13-29%, add it to a round-bottom flask, stir it at room temperature for 12-24 hours, dialyze it with deionized water for 36-48 hours after the reaction, and obtain the target polymer after freeze-drying.

[0010] The structural formula of the hyaluronic acid-cystamine is as follows:

[0011]

[0012] Among them, n=890-1457, m=2-8, that is, the molecular weight is 800000-1300000, and the grafting rate of cystamine is 13-45%.

[0013] A cross-linking agent with a mass fraction of 5 to 24% is dissolved in 0.25mM sodium hydroxide, and a hyaluronic acid-cystamine with a mass fraction of 72 to 94% is added. After reacting at 40 to 50°C for 1 to 2 hours, a sol is formed; a fatty acid with a mass fraction of 1 to 5% is dissolved in 200 μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 2 to 5 hours until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is (216 to 323): (1000 to 4000): (50 to 60).

[0014] The fatty acid is one of lauric acid, palmitic acid, palmitoleic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid or arachidonic acid.

[0015] The crosslinking agent is one of 1,4-butanediol diglycidyl ether, polyethylene glycol, polyvinyl alcohol or divinyl sulfone.

[0016] The organic solvent is one of ethanol, dimethyl sulfoxide or methanol.

[0017] Gel performance tests include antibacterial test, biocompatibility test, rheological property test, swelling property test and in vitro degradation property test.

[0018] Antimicrobial testing: 600 = 1 bacteria were added to different test tubes containing BHI medium, and then blank control group (PBS) and gel of different concentrations were added. The mixed solution was cultured overnight at 37°C. The bacterial survival rate was calculated by the number of colonies by plate spreading method.

[0019] Biocompatibility test: After the cells were co-cultured with the gel extract at 37°C for 24 hours, the diluted CCK8 was added to a 96-well plate and cultured in an incubator for 1.5 hours. The absorbance of the solution was measured at a wavelength of 450 nm using an ELISA reader. We used eight independent experiments to show its average and standard error.

[0020] Rheological property test: The gel was placed on a sample stage, the strain value was set to 1%, the frequency was varied from 0.01 to 100 Hz, and the values ​​of loss modulus (G″) and storage modulus (G′) were collected.

[0021] Swelling performance test: Weigh a certain mass of gel W0 and soak it in PBS with pH = 7.4. Take out the gel at different time periods, absorb the excess water on the surface with filter paper, and weigh the mass of the gel W1. We use 3 parallel groups to show its error and average value. The swelling rate of the gel is calculated using the following formula:

[0022] Swelling rate (%) = W1-W0 / W0*100

[0023] In vitro degradation performance test: A certain amount of freeze-dried gel was immersed in a PBS solution containing H2O2 (0mM or 1mM). The supernatant in the glass bottle was removed within the same period of time, and the gel was washed with deionized water and then dried in an oven at 50°C. The remaining weight of the gel was measured using a balance. We used 3 parallel groups to show its error and average value. The degradation rate of the gel is expressed as the weight ratio (%) of the remaining gel to the original gel:

[0024] Degradation rate (%) = weight of remaining gel (W1) / weight of original gel (W0) * 100

[0025] The present invention uses sodium hyaluronate, cystamine and lauric acid as raw materials, synthesizes hyaluronic acid-cystamine polymer through condensation reaction of carboxyl and amino groups, and then undergoes cross-linking reaction with a cross-linking agent and encapsulates fatty acids therein to prepare gel. Sodium hyaluronate gives the gel good biocompatibility; hydrophobic fatty acids give the gel good antibacterial properties.

[0026] The beneficial effects of the present invention are as follows: (1) the prepared hyaluronic acid-cystamine embeds fatty acids therein through cross-linking, which can have a good antibacterial effect and eliminate active oxygen, thereby improving the skin effect; (2) the prepared gel has a good degradation rate and can delay the release of fatty acids to achieve a better antibacterial effect; (3) the gel preparation method is simple and can be mass-produced, with practical application value; (4) the prepared gel will not produce serious toxic side effects and has great application prospects in improving acne treatment. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with implementation cases. The following implementation cases are further illustrations of the present invention and do not limit the scope of application of the present invention.

[0028] Example 1

[0029] (1) Synthesis of hyaluronic acid-cystamine:

[0030] Weigh the drugs according to the mass ratio of IRN-77 hydrogen cation exchange resin and sodium hyaluronate at 3:1, prepare a solution with a mass fraction of 10%, add it to a round-bottom flask, stir it at room temperature for 10 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:8:6:8, prepare a solution with a mass fraction of 24%, add it to a round-bottom flask, stir it at room temperature for 24 hours, dialyze it with deionized water for 48 hours after the reaction, and obtain the target polymer after freeze-drying.

[0031] The structural formula of the hyaluronic acid-cystamine is as follows:

[0032]

[0033] Wherein, n=1200, m=3, that is, the molecular weight is 1000000, and the grafting rate of cystamine is 33%.

[0034] (2) Preparation of gel:

[0035] A cross-linking agent with a mass fraction of 10% is dissolved in 0.25mM sodium hydroxide, and hyaluronic acid-cystamine with a mass fraction of 88% is added. After reacting for 2 hours at 42°C, a sol is formed; a fatty acid with a mass fraction of 2% is dissolved in 200μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 2 hours until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 216:2000:50.

[0036] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 95%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 85.12%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 80min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 14 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 14 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0037] Example 2

[0038] (1) Synthesis of hyaluronic acid-cystamine:

[0039] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate at 3:1, prepare a solution with a mass fraction of 10%, add it to a round-bottom flask, stir it at room temperature for 12 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:8:6:15, prepare a solution with a mass fraction of 29%, add it to a round-bottom flask, stir it at room temperature for 24 hours, dialyze it with deionized water for 48 hours after the reaction, and obtain the target polymer after freeze-drying.

[0040] The structural formula of the hyaluronic acid-cystamine is as follows:

[0041]

[0042] Wherein, n=1345, m=2, that is, the molecular weight is 1200000, and the grafting rate of cystamine is 45%.

[0043] (2) Preparation of gel:

[0044] A cross-linking agent with a mass fraction of 5% is dissolved in 0.25mM sodium hydroxide, and a hyaluronic acid-cystamine with a mass fraction of 94% is added. After reacting at 50°C for 1h, a sol is formed; a fatty acid with a mass fraction of 1% is dissolved in 200μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 5h until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, and the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 216:4000:60.

[0045] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 98%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 90.12%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 60min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 10 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 10 days and 7 days respectively, indicating that the degradation of the gel depends on ROS.

[0046] Example 3

[0047] (1) Synthesis of hyaluronic acid-cystamine:

[0048] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate at 3:0.9, prepare a solution with a mass fraction of 9%, add it to a round-bottom flask, stir it at room temperature for 10 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:8:6:12, prepare a solution with a mass fraction of 26%, add it to a round-bottom flask, stir it at room temperature for 20 hours, dialyze it with deionized water for 48 hours after the reaction, and obtain the target polymer after freeze-drying.

[0049] The structural formula of the hyaluronic acid-cystamine is as follows:

[0050]

[0051] Wherein, n=1457, m=2.5, that is, the molecular weight is 1300000, and the grafting rate of cystamine is 40%.

[0052] (2) Preparation of gel:

[0053] A cross-linking agent with a mass fraction of 7% is dissolved in 0.25mM sodium hydroxide, and a hyaluronic acid-cystamine with a mass fraction of 91% is added. After reacting at 47°C for 1h, a sol is formed; a fatty acid with a mass fraction of 1% is dissolved in 200μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 4h until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, and the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 323:4000:50.

[0054] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 97%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 87.64%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 60min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 10 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 10 days and 7 days respectively, indicating that the degradation of the gel depends on ROS.

[0055] Example 4

[0056] (1) Synthesis of hyaluronic acid-cystamine:

[0057] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate of 2.7:0.8, prepare a solution with a mass fraction of 8%, add it to a round-bottom flask, stir it at room temperature for 10 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:7:5:8, prepare a solution with a mass fraction of 23%, add it to a round-bottom flask, stir it at room temperature for 18 hours, dialyze it with deionized water for 40 hours after the reaction, and obtain the target polymer after freeze-drying.

[0058] The structural formula of the hyaluronic acid-cystamine is as follows:

[0059]

[0060] Wherein, n=1200, m=3, that is, the molecular weight is 1000000, and the grafting rate of cystamine is 30%.

[0061] (2) Preparation of gel:

[0062] A cross-linking agent with a mass fraction of 12% is dissolved in 0.25mM sodium hydroxide, and hyaluronic acid-cystamine with a mass fraction of 85% is added. After reacting at 40°C for 1 hour, a sol is formed; a fatty acid with a mass fraction of 3% is dissolved in 200μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 3 hours until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 216:1500:50.

[0063] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 94%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 83.27%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 80min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 14 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 14 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0064] Example 5

[0065] (1) Synthesis of hyaluronic acid-cystamine:

[0066] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate of 2.4:0.7, prepare a solution with a mass fraction of 7%, add it to a round-bottom flask, stir it at room temperature for 10 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine dihydrochloride according to the mass ratio of 10:6:4:9, prepare a solution with a mass fraction of 22%, add it to a round-bottom flask, stir it at room temperature for 18 hours, dialyze it with deionized water for 40 hours after the reaction, and obtain the target polymer after freeze-drying.

[0067] The structural formula of the hyaluronic acid-cystamine is as follows:

[0068]

[0069] Wherein, n=1000, m=4, that is, the molecular weight is 900000, and the grafting rate of cystamine is 24%.

[0070] (2) Preparation of gel:

[0071] A cross-linking agent with a mass fraction of 17% is dissolved in 0.25mM sodium hydroxide, and hyaluronic acid-cystamine with a mass fraction of 80% is added. After reacting for 2 hours at 45°C, a sol is formed; a fatty acid with a mass fraction of 5% is dissolved in 200μL of an organic solvent, slowly added to the sol and fully mixed, and the reaction is continued for 3 hours until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 323:1500:60.

[0072] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 91%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 81.36%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 100min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 18 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 18 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0073] Example 6

[0074] (1) Synthesis of hyaluronic acid-cystamine:

[0075] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate of 1.8:0.5, prepare a solution with a mass fraction of 5%, add it to a round-bottom flask, stir it at room temperature for 8 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine dihydrochloride according to the mass ratio of 10:3:1:8, prepare a solution with a mass fraction of 18%, add it to a round-bottom flask, stir it at room temperature for 18 hours, after the reaction is completed, dialyze it with deionized water for 36 hours, and obtain the target polymer after freeze-drying.

[0076] The structural formula of the hyaluronic acid-cystamine is as follows:

[0077]

[0078] Wherein, n=10000, m=6, that is, the molecular weight is 900000, and the grafting rate of cystamine is 18%.

[0079] (2) Preparation of gel:

[0080] A cross-linking agent with a mass fraction of 23% is dissolved in 0.25mM sodium hydroxide, and a hyaluronic acid-cystamine with a mass fraction of 72% is added. After reacting for 2 hours at 40°C, a sol is formed; a fatty acid with a mass fraction of 4% is dissolved in 200μL of an organic solvent, slowly added to the sol and mixed thoroughly, and the reaction is continued for 2 hours until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 323:1000:60.

[0081] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 89%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 62.5μg / mL, the survival rate of L929 cells reached 87.32%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 120min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 18 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 18 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0082] Example 7

[0083] (1) Synthesis of hyaluronic acid-cystamine:

[0084] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate at 1:0.3, prepare a solution with a mass fraction of 3%, add it to a round-bottom flask, stir it at room temperature for 6 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:4:2:5, prepare a solution with a mass fraction of 17%, add it to a round-bottom flask, stir it at room temperature for 12 hours, dialyze it with deionized water for 36 hours after the reaction, and obtain the target polymer after freeze-drying.

[0085] The structural formula of the hyaluronic acid-cystamine is as follows:

[0086]

[0087] Wherein, n=890, m=8, that is, the molecular weight is 800000, and the grafting rate of cystamine is 12%.

[0088] (2) Preparation of gel:

[0089] A cross-linking agent with a mass fraction of 17% is dissolved in 0.25mM sodium hydroxide, and hyaluronic acid-cystamine with a mass fraction of 80% is added. After reacting at 43°C for 2h, a sol is formed; a fatty acid with a mass fraction of 4% is dissolved in 200μL of an organic solvent, slowly added to the sol and mixed thoroughly, and the reaction is continued for 3h until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 216:1000:50.

[0090] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 91%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 80.72%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 100min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 18 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 18 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0091] Example 8

[0092] (1) Synthesis of hyaluronic acid-cystamine:

[0093] Weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate at 1.5:0.4, prepare a solution with a mass fraction of 4%, add it to a round-bottom flask, stir it at room temperature for 6 hours, and freeze-dry it after the reaction to obtain hyaluronic acid. Weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10:5:3:3, prepare a solution with a mass fraction of 17%, add it to a round-bottom flask, stir it at room temperature for 12 hours, dialyze it with deionized water for 40 hours after the reaction, and obtain the target polymer after freeze-drying.

[0094] The structural formula of the hyaluronic acid-cystamine is as follows:

[0095]

[0096] Wherein, n=1230, m=7, that is, the molecular weight is 1100000, and the grafting rate of cystamine is 15%.

[0097] (2) Preparation of gel:

[0098] A cross-linking agent with a mass fraction of 14% is dissolved in 0.25mM sodium hydroxide, and hyaluronic acid-cystamine with a mass fraction of 84% is added. After reacting at 46°C for 2h, a sol is formed; a fatty acid with a mass fraction of 2% is dissolved in 200μL of an organic solvent, slowly added to the sol and mixed thoroughly, and the reaction is continued for 4h until a gel is formed. The gel is soaked in a phosphate buffer solution with a pH of 7.4, the pH is adjusted to neutral with hydrochloric acid, and then soaked in deionized water to purify the gel, and finally freeze-dried to obtain the product. The mass ratio of the cross-linking agent to hyaluronic acid-cystamine to fatty acid is 323:2000:60.

[0099] When the minimum inhibitory concentration of the gel was 31.25μg / mL, its antibacterial effect reached 93%. After the cytotoxicity experiment, when the concentration of cHA-Cys@LA was 125μg / mL, the survival rate of L929 cells reached 81.32%. After the rheological test, it was shown that the gel formed a relatively stable three-dimensional network structure, behaved as a viscoelastic solid, and had a certain shear resistance. After the swelling test, the gel reached swelling equilibrium after 80min, indicating that the prepared gel had a certain cross-linking density and LA could be slowly released to achieve effective antibacterial effects. After the in vitro degradation experiment, after 14 days, the gel was completely degraded and dissolved in PBS. The degradation time of the gel in PBS containing 1mM and 10mM H2O2 was 14 days and 10 days, respectively, indicating that the degradation of the gel depends on ROS.

[0100] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for preparing a hyaluronic acid hydrogel containing natural fatty acids for treating acne, characterized in that: The preparation method comprises the following steps: 1) Synthesis of hyaluronic acid-cystamine: weigh the drugs according to the mass ratio of IRN-77 hydrogen type cation exchange resin and sodium hyaluronate (1-3): (0.3-1), prepare a solution with a mass fraction of 1-10%, add it to a round-bottom flask, stir it at room temperature for 5-12 hours, and after the reaction is completed, freeze-dry to obtain hyaluronic acid; weigh hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cystamine according to the mass ratio of 10: (3-8): (1-6): (1-15), prepare a solution with a mass fraction of 13-29%, add it to a round-bottom flask, stir it at room temperature for 12-24 hours, after the reaction is completed, dialyze it with deionized water for 36-48 hours, and obtain the target polymer after freeze-drying; The structural formula of the hyaluronic acid-cystamine is as follows: Wherein, n=890-1457, m=2-8, i.e., the molecular weight is 800000-1300000, and the grafting rate of cystamine is 13-45%; 2) dissolving a cross-linking agent with a mass fraction of 5 to 24% in 0.25 mM sodium hydroxide, adding hyaluronic acid-cystamine with a mass fraction of 72 to 94%, and reacting at 40 to 50° C. for a period of time to form a sol; dissolving a fatty acid with a mass fraction of 1 to 5% in 200 μL of an organic solvent, slowly adding it to the sol and mixing it thoroughly, and continuing to react for 2 to 5 hours until a gel is formed; soaking the gel in a phosphate buffer solution with a pH of 7.4, adjusting the pH to neutral with hydrochloric acid, and then soaking it in deionized water to purify the gel, and finally freeze-drying to obtain a product; the mass ratio of the cross-linking agent to hyaluronic acid-cystamine to the fatty acid is (216 to 323): (1000 to 4000): (50 to 60); the fatty acid is lauric acid.

2. The method for preparing a hyaluronic acid hydrogel containing natural fatty acids for treating acne according to claim 1, characterized in that: The crosslinking agent is 1,4-butanediol diglycidyl ether.

3. The method for preparing a hyaluronic acid hydrogel containing natural fatty acids for treating acne according to claim 1, characterized in that: The organic solvent is one of ethanol, dimethyl sulfoxide or methanol.

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