Preparation method of high-viscosity photo-crosslinking sodium hyaluronate hydrogel

Abstract

The invention relates to the field of biomedical polymer materials, and discloses a preparation method of high-viscosity photo-crosslinking sodium hyaluronate hydrogel, which comprises the following steps: dissolving sodium hyaluronate in deionized water / N, N-dimethylformamide to obtain a sodium hyaluronate solution; mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester / N, N-dimethylformamide solution to obtain a pre-formed sodium hyaluronate glue solution; composite antibacterial microspheres and a photoinitiator are added into the preformed sodium hyaluronate glue solution, a photo-crosslinking reaction is performed, conjugated diene glycidyl ester is prepared from raw materials including n-butyllithium, ethyl acetate, 2, 3-dibromo-1-propylene, vinyl magnesium chloride, sodium hydroxide and glycidol, and the conjugated diene glycidyl ester is prepared from raw materials including sodium hyaluronate, sodium hyaluronate and sodium hyaluronate. According to the preparation method disclosed by the invention, the modification reaction and photo-crosslinking time is shortened through the grafting of conjugated diene glycidyl ester, the viscosity of the hydrogel is greatly improved, and the antibacterial property of the hydrogel is improved through the addition of the composite antibacterial microspheres.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical polymer materials, and particularly relates to a method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel. Background Art

[0002] Sodium hyaluronate is a biomacromolecule polysaccharide. As the sodium salt of hyaluronic acid, its chemical structure is composed of disaccharides composed of D-glucuronic acid and N-acetylglucosamine. In addition to its moisturizing and skin repairing effects, sodium hyaluronate also exhibits good biocompatibility and biodegradability, so it is also used in plastic surgery such as facial filling. In addition, sodium hyaluronate can also act as a tissue lubricant. It can form a viscoelastic solution in water. Its viscosity provides mechanical protection for tissues, and its elasticity helps absorb external forces to provide buffering for tissues. Its characteristics make sodium hyaluronate an ideal biological lubricant.

[0003] Sodium hyaluronate molecular chains contain a large number of hydroxyl groups. Currently, chemical cross-linking methods are mostly used to prepare sodium hyaluronate hydrogels, such as the use of cross-linking agents such as butanediol glycidyl ether or divinyl sulfone. However, these cross-linking agents have disadvantages such as low antibacterial activity, poor adhesion and low mechanical properties. At the same time, these cross-linking agents have certain biological toxicity, and the cross-linking time is long. The resulting hydrogel is also easily degraded in an alkaline environment. Therefore, it is urgent to develop a more environmentally friendly and efficient cross-linking method for hyaluronic acid (sodium).

[0004] Photocrosslinking has the advantages of fast speed and low toxicity. The Chinese patent with application number CN202010688035.4 provides an acrylate-modified hyaluronic acid (sodium) and its synthesis method and application. The chloroacrylic acid modification method used requires a long reaction time and cannot efficiently prepare sodium hyaluronate hydrogel, which is related to the low reactivity of the modifier used. Summary of the invention

[0005] In order to solve the deficiencies mentioned in the above background technology, the purpose of the present invention is to provide a method for preparing a high-viscosity photo-cross-linked sodium hyaluronate hydrogel, by grafting conjugated diene glycidyl ester on the sodium hyaluronate molecular chain, increasing the double bond content on the molecular chain, shortening the modification reaction time and photo-cross-linking time, and greatly improving the viscosity of the hydrogel. In addition, by adding composite antibacterial microspheres, the antibacterial properties of the hydrogel are greatly improved.

[0006] The purpose of the present invention can be achieved through the following technical solutions: A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve sodium hyaluronate in a mixed solution of deionized water and N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8-9 with sodium bicarbonate; S2, dissolving the conjugated diene glycidyl ester in N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution evenly, and then stirring and reacting at 40-60° C. for 4-8 hours. After the reaction is completed, adjusting the pH value of the system to neutral with a hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4, adding composite antibacterial microspheres and a photoinitiator to the preformed sodium hyaluronate gel and mixing them evenly, then pouring them into a mold, placing them in a UV box for photocrosslinking reaction, and preparing a high-viscosity photocrosslinked sodium hyaluronate hydrogel; The conjugated diene glycidyl ester is prepared by using n-butyl lithium and ethyl acetate to prepare product A, which is then reacted with 2,3-dibromo-1-propylene to obtain product B, which is further reacted with vinyl magnesium chloride to prepare product C, and sodium hydroxide-methanol solution is added to generate product D, which is finally prepared by esterification reaction with glycidol; The composite antibacterial microspheres are prepared by using a modified dialdehyde sodium alginate solution as an aqueous phase, mixing it with a polylactic acid-glycolic acid copolymer-dichloromethane solution to obtain a W1 / O emulsion, then mixing it with a chitosan-polyvinyl alcohol solution to obtain a W1 / O / W2 emulsion, and finally using sodium hyaluronate as a material through an emulsion method; the modified dialdehyde sodium alginate solution is prepared by using dialdehyde sodium alginate oxidized by sodium periodate to react with ε-polylysine to undergo a Schiff base reaction.

[0007] Preferably, in step S1, the volume ratio of deionized water to N,N-dimethylformamide is 1:3-5; and the mass percentage concentration of the sodium hyaluronate solution is 1.5-2.5wt%.

[0008] Preferably, the mass percentage concentration of the conjugated diene glycidyl ester solution in step S2 is 1-5wt%.

[0009] Preferably, in step S4, the mass percentage concentration of the photoinitiator in the preformed sodium hyaluronate gel is 0.1-0.5wt%.

[0010] Preferably, in the photo-crosslinking reaction in step S4, the ultraviolet light power is 50-200 W, and the irradiation time is 5-15 min.

[0011] Preferably, the synthetic route of the conjugated diene glycidyl ester is as follows: ; The preparation method comprises the following steps: (1) Place n-butyl lithium-n-hexane solution, ethyl acetate and tetrahydrofuran in a reactor, place at -80 to -75°C under a nitrogen atmosphere, and stir to react for 0.5 to 1.5 hours to obtain product A; (2) Add 2,3-dibromo-1-propylene to product A and stir the reaction for 8-12 hours to obtain product B; (3) Product B was dissolved in tetrahydrofuran, vinyl magnesium chloride was added, and the mixture was stirred for 4 to 6 hours to obtain product C; (4) Product C was dissolved in a sodium hydroxide-methanol solution, and the reaction was stirred for 8-12 hours. Then, the pH value of the system was adjusted to 5-6 with dilute hydrochloric acid to obtain product D; (5) Product D, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and glycidol are mixed and stirred for 8-12 hours to prepare conjugated diene glycidyl ester.

[0012] Preferably, the method for preparing the composite antibacterial microspheres comprises the following steps: ① Disperse sodium alginate in ethanol solution, then add sodium periodate, and place in room temperature in the dark for 5-7 hours. After the reaction is completed, add ethylene glycol to terminate the reaction, then transfer to a dialysis bag for dialysis to remove periodate, and finally freeze-dry to prepare dialdehyde sodium alginate; ② Dissolve dialdehyde sodium alginate in deionized water to obtain a dialdehyde sodium alginate solution, then add ε-polylysine, stir and react at 35-45° C. for 2-4 hours to prepare a modified dialdehyde sodium alginate solution; ③ Dissolve the polylactic acid-glycolic acid copolymer in dichloromethane to obtain a polylactic acid-glycolic acid copolymer solution, stir and mix the modified dialdehyde sodium alginate solution and the polylactic acid-glycolic acid copolymer solution, and obtain a W1 / O emulsion by ultrasonication; ④ Dissolve chitosan in glacial acetic acid to obtain a chitosan solution, and mix the chitosan solution and a polyvinyl alcohol aqueous solution with a mass concentration of 2.5-5% to obtain a chitosan-polyvinyl alcohol solution; ⑤ Evenly mix the W1 / O emulsion and the chitosan-polyvinyl alcohol solution to obtain a W1 / O / W2 emulsion; ⑥ Evenly mix the sodium hyaluronate aqueous solution and the polyvinyl alcohol aqueous solution with a mass concentration of 0.3-0.5% to obtain a sodium hyaluronate-polyvinyl alcohol solution; ⑦ Mix the W1 / O / W2 emulsion and sodium hyaluronate-polyvinyl alcohol solution evenly, stir mechanically at 750-850 r / min for 1-2 h, then stir at 900-1050 r / min for 20-40 min at 40-50 °C, then add a 4.5-7% sodium tripolyphosphate aqueous solution, continue stirring to react for 1-2 h, let stand overnight after the reaction is completed, and prepare composite antibacterial microspheres after centrifugation, washing and drying.

[0013] Preferably, in step ②, the mass ratio of dialdehyde sodium alginate to ε-polylysine is 1:0.1-0.2.

[0014] Preferably, the mass percentage concentration of the polylactic acid-glycolic acid copolymer solution is 5-15wt%; the mass percentage concentration of the chitosan solution is 0.5-2wt%.

[0015] Preferably, the volume ratio of the modified dialdehyde sodium alginate solution to the polylactic acid-glycolic acid copolymer solution is 1:4-7; the volume ratio of the W1 / O emulsion to the chitosan-polyvinyl alcohol solution is 1:4-5; the volume ratio of the W1 / O / W2 emulsion to the sodium hyaluronate-polyvinyl alcohol solution is 1:5-7.

[0016] Beneficial effects of the present invention: The photo-crosslinked sodium hyaluronate hydrogel prepared by the present invention is modified by a novel conjugated diene glycidyl ester, which shortens the photo-crosslinking time during the preparation process and can effectively increase the viscosity of the hydrogel. Therefore, the present invention provides an efficient method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel.

[0017] The invention utilizes sodium periodate to oxidize the ortho-hydroxyl groups of sodium alginate molecules into aldehyde groups, then reacts dialdehyde sodium alginate with ε-polylysine to obtain a modified dialdehyde sodium alginate solution by Schiff base reaction, and takes the modified dialdehyde sodium alginate solution as an aqueous phase, mixes it with a polylactic acid-glycolic acid copolymer solution to obtain a W1 / O emulsion, and then evenly mixes it with a chitosan-polyvinyl alcohol solution to obtain a W1 / O / W2 emulsion, and finally takes sodium hyaluronate as a material to prepare a composite antibacterial microsphere material with a multilayer structure by an emulsion method, wherein the sodium alginate has good sphering property and safety, ε-polylysine is a cationic polypeptide with significant antibacterial ability, which has good heat resistance and a wide antibacterial spectrum, the polylactic acid-glycolic acid copolymer has good adsorption stability and mechanical strength, chitosan is a bioactive polymer with good bioadhesion and good biocompatibility, the sodium hyaluronate is negatively charged, and the chitosan is positively charged, and the two can attract each other through electrostatic force to form an ionic bond, thereby enhancing the tensile stress of the composite material. The present invention adds composite antibacterial microspheres into the photo-cross-linked sodium hyaluronate hydrogel, which can achieve the slow release of antibacterial substances and has excellent antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a schematic diagram of the reaction of modifying sodium hyaluronate with conjugated diene glycidyl ester of the present invention; Figure 2 It is the infrared spectrum of sodium hyaluronate modified with conjugated diene glycidyl ester in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 A method for preparing a conjugated diene glycidyl ester comprises the following steps: (1) 10 mL of 2.5 mol / L n-butyl lithium-n-hexane solution, 2.5 mL of ethyl acetate and 40 mL of tetrahydrofuran were placed in a reactor, and stirred at -78 °C for 1 h under a nitrogen atmosphere to prepare product A; (2) Add 2.7 mL of 2,3-dibromo-1-propylene to product A and stir the reaction for 12 h to obtain product B; (3) Product B was dissolved in 50 mL of tetrahydrofuran, 2.3 g of vinyl magnesium chloride was added, and the mixture was stirred for 6 h to obtain product C; (4) Product C was dissolved in 3.5 mL of 7.5 mol / L sodium hydroxide-methanol solution, stirred for 12 h, and then the pH value of the system was adjusted to 5 with dilute hydrochloric acid to prepare product D; (5) Product D, 0.5 g of dicyclohexylcarbodiimide, 0.2 g of 4-dimethylaminopyridine, and 1.9 g of glycidol were mixed and stirred for 12 h to prepare conjugated diene glycidyl ester.

[0022] Example 2 A method for preparing composite antibacterial microspheres comprises the following steps: ① Take 6g of sodium alginate and disperse it in 120mL of 50% ethanol solution, then add 2.2g of sodium periodate, and place it at room temperature in the dark for 6h. After the reaction is completed, add 5mL of ethylene glycol to terminate the reaction, then transfer it to a dialysis bag for dialysis to remove periodate, and finally freeze-dry it to prepare dialdehyde sodium alginate; ② Take 10g of dialdehyde sodium alginate and dissolve it in 50mL of deionized water to obtain a dialdehyde sodium alginate solution, then add 2g of ε-polylysine, stir and react at 40°C for 4h to prepare a modified dialdehyde sodium alginate solution; ③ Dissolve polylactic acid-glycolic acid copolymer in dichloromethane to prepare a polylactic acid-glycolic acid copolymer solution with a mass percentage concentration of 10wt%, stir and mix the modified dialdehyde sodium alginate solution and the polylactic acid-glycolic acid copolymer solution in a volume ratio of 1:5, and obtain a W1 / O emulsion by ultrasonication; ④ Dissolve chitosan in glacial acetic acid to prepare a chitosan solution with a mass percentage concentration of 1.5wt%, and mix the chitosan solution and a polyvinyl alcohol aqueous solution with a mass concentration of 3% in a volume ratio of 1:1 to obtain a chitosan-polyvinyl alcohol solution; ⑤ Mix the W1 / O emulsion and chitosan-polyvinyl alcohol solution in a volume ratio of 1:4 to obtain a W1 / O / W2 emulsion; ⑥ Mix a sodium hyaluronate aqueous solution with a mass concentration of 2.5% and a polyvinyl alcohol aqueous solution with a mass concentration of 0.4% in a volume ratio of 1:1 to obtain a sodium hyaluronate-polyvinyl alcohol solution; ⑦ Mix the W1 / O / W2 emulsion and sodium hyaluronate-polyvinyl alcohol solution in a volume ratio of 1:5, stir mechanically at 800 r / min for 1 h, then stir at 1000 r / min for 30 min at 45 °C, then add a 5.5% sodium tripolyphosphate aqueous solution, continue stirring and react for 2 h, let stand overnight after the reaction is completed, and prepare composite antibacterial microspheres after centrifugation, washing and drying.

[0023] Example 3 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 1.5 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution evenly, and then stirring and reacting at 40° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 0.6 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 50 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0024] Example 4 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution evenly, and then stirring and reacting at 40° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.4 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 50 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0025] Example 5 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 6 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution evenly, and then stirring and reacting at 40° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.6 g of the composite antibacterial microspheres prepared in Example 2 and 3.2 g of photoinitiator 2959 to the pre-formed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 50 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0026] Example 6 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 50 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0027] Example 7 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 8 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 50 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0028] Example 8 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 100 W and the irradiation time is 5 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0029] Example 9 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the pre-formed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 100 W and the irradiation time is 10 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0030] Example 10 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 0.2 g of the composite antibacterial microspheres prepared in Example 2 and 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly. Then pour the mixture into a mold and place the mold in a UV box for photocrosslinking reaction. The UV light power is 200 W and the irradiation time is 10 min to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0031] Comparative Example 1 A method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel comprises the following steps: S1. Dissolve 10 g of sodium hyaluronate in a mixed solution of 100 mL of deionized water and 400 mL of N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8 with sodium bicarbonate; S2, taking 3 g of the conjugated diene glycidyl ester prepared in Example 1 and dissolving it in 100 mL of N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution, and then stirring and reacting at 50° C. for 4 hours. After the reaction is completed, adjusting the pH value of the system to neutral with 1 mol / L hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4. Add 1.8 g of photoinitiator 2959 to the preformed sodium hyaluronate gel and mix evenly, then pour into a mold and place in a UV box for photocrosslinking reaction, wherein the UV light power is 200 W and the irradiation time is 10 min, to prepare a high-viscosity photocrosslinked sodium hyaluronate hydrogel.

[0032] Performance Testing The photo-crosslinked sodium hyaluronate hydrogel prepared in Example 3-10 was subjected to dynamic viscosity testing, and the data results are shown in Table 1.

[0033] Antibacterial performance test: Prepare physiological saline and LB culture medium, and use physiological saline to dilute Staphylococcus aureus culture to a concentration of 10 5 CFU / mL. Weigh 50 mg of the photo-crosslinked sodium hyaluronate hydrogel sample prepared in Example 3-10, sterilize with ultraviolet light, and place it in a 50 mL conical flask. Add 1 mL of LB culture medium and 0.2 mL of Staphylococcus aureus culture solution. Place the sample and the culture solution in a shaker for co-culture (37°C, 120 r / min). Take 100 μL of the mixed solution at a set time point, inoculate the plate by the culture medium pouring method, and calculate the antibacterial rate after 24 hours. The data results are shown in Table 1.

[0034] Table 1 Dynamic viscosity and antibacterial properties of photocrosslinked sodium hyaluronate hydrogels

[0035] From the data results in Table 1, it can be seen that the photo-crosslinked sodium hyaluronate hydrogel prepared in Examples 3-10 of the present invention has a good antibacterial effect. After modification with conjugated diene glycidyl ester, the photo-crosslinking time in the preparation process can be shortened, and the viscosity of the hydrogel can be effectively improved. In Comparative Example 1, no composite antibacterial microspheres were added, and the antibacterial rate of Staphylococcus aureus measured was significantly lower than that of Examples 3-10, indicating that the addition of composite antibacterial microspheres can greatly improve the antibacterial properties of the photo-crosslinked sodium hyaluronate hydrogel.

[0036] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0037] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a high-viscosity photocrosslinked sodium hyaluronate hydrogel, characterized in that: The following steps are involved: S1. Dissolve sodium hyaluronate in a mixed solution of deionized water and N,N-dimethylformamide to obtain a sodium hyaluronate solution, and adjust the pH value of the sodium hyaluronate solution to 8-9 with sodium bicarbonate; S2, dissolving the conjugated diene glycidyl ester in N,N-dimethylformamide to obtain a conjugated diene glycidyl ester solution; S3, stirring and mixing the sodium hyaluronate solution and the conjugated diene glycidyl ester solution evenly, and then stirring and reacting at 40-60° C. for 4-8 hours. After the reaction is completed, adjusting the pH value of the system to neutral with a hydrochloric acid solution to prepare a preformed sodium hyaluronate gel; S4, adding composite antibacterial microspheres and a photoinitiator to the preformed sodium hyaluronate gel and mixing them evenly, then pouring them into a mold, placing them in a UV box for photocrosslinking reaction, and preparing a high-viscosity photocrosslinked sodium hyaluronate hydrogel; The conjugated diene glycidyl ester is prepared by using n-butyl lithium and ethyl acetate to prepare product A, which is then reacted with 2,3-dibromo-1-propylene to obtain product B, which is further reacted with vinyl magnesium chloride to prepare product C, and sodium hydroxide-methanol solution is added to generate product D, which is finally prepared by esterification reaction with glycidol; The composite antibacterial microspheres are prepared by using a modified dialdehyde sodium alginate solution as an aqueous phase, mixing it with a polylactic acid-glycolic acid copolymer-dichloromethane solution to obtain a W1 / O emulsion, then mixing it with a chitosan-polyvinyl alcohol solution to obtain a W1 / O / W2 emulsion, and finally using sodium hyaluronate as a material through an emulsion method; the modified dialdehyde sodium alginate solution is prepared by using dialdehyde sodium alginate oxidized by sodium periodate to react with ε-polylysine to undergo a Schiff base reaction.

2. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: In step S1, the volume ratio of deionized water to N,N-dimethylformamide is 1:3-5; the mass percentage concentration of the sodium hyaluronate solution is 1.5-2.5wt%.

3. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: The mass percentage concentration of the conjugated diene glycidyl ester solution in step S2 is 1-5wt%.

4. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: In step S4, the mass percentage concentration of the photoinitiator in the preformed sodium hyaluronate gel is 0.1-0.5wt%.

5. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: In the photo-crosslinking reaction in step S4, the ultraviolet light power is 50-200 W, and the irradiation time is 5-15 min.

6. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: The synthetic route of the conjugated diene glycidyl ester is as follows: ； The preparation method comprises the following steps: (1) Place n-butyl lithium-n-hexane solution, ethyl acetate and tetrahydrofuran in a reactor, place at -80 to -75°C under a nitrogen atmosphere, and stir to react for 0.5 to 1.5 hours to obtain product A; (2) Add 2,3-dibromo-1-propylene to product A and stir the reaction for 8-12 hours to obtain product B; (3) Product B was dissolved in tetrahydrofuran, vinyl magnesium chloride was added, and the mixture was stirred for 4 to 6 hours to obtain product C; (4) Product C was dissolved in a sodium hydroxide-methanol solution, and the reaction was stirred for 8-12 hours. Then, the pH value of the system was adjusted to 5-6 with dilute hydrochloric acid to obtain product D; (5) Product D, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and glycidol are mixed and stirred for 8-12 hours to prepare conjugated diene glycidyl ester.

7. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 1, characterized in that: The preparation method of the composite antibacterial microspheres comprises the following steps: ① Disperse sodium alginate in ethanol solution, then add sodium periodate, and place in room temperature in the dark for 5-7 hours. After the reaction is completed, add ethylene glycol to terminate the reaction, then transfer to a dialysis bag for dialysis to remove periodate, and finally freeze-dry to prepare dialdehyde sodium alginate; ② Dissolve dialdehyde sodium alginate in deionized water to obtain a dialdehyde sodium alginate solution, then add ε-polylysine, stir and react at 35-45° C. for 2-4 hours to prepare a modified dialdehyde sodium alginate solution; ③ Dissolve the polylactic acid-glycolic acid copolymer in dichloromethane to obtain a polylactic acid-glycolic acid copolymer solution, stir and mix the modified dialdehyde sodium alginate solution and the polylactic acid-glycolic acid copolymer solution, and obtain a W1 / O emulsion by ultrasonication; ④ Dissolve chitosan in glacial acetic acid to obtain a chitosan solution, and mix the chitosan solution and a polyvinyl alcohol aqueous solution with a mass concentration of 2.5-5% to obtain a chitosan-polyvinyl alcohol solution; ⑤ Evenly mix the W1 / O emulsion and the chitosan-polyvinyl alcohol solution to obtain a W1 / O / W2 emulsion; ⑥ Evenly mix the sodium hyaluronate aqueous solution and the polyvinyl alcohol aqueous solution with a mass concentration of 0.3-0.5% to obtain a sodium hyaluronate-polyvinyl alcohol solution; ⑦ Mix the W1 / O / W2 emulsion and sodium hyaluronate-polyvinyl alcohol solution evenly, stir mechanically at 750-850 r / min for 1-2 h, then stir at 900-1050 r / min for 20-40 min at 40-50 °C, then add a 4.5-7% sodium tripolyphosphate aqueous solution, continue stirring to react for 1-2 h, let stand overnight after the reaction is completed, and prepare composite antibacterial microspheres after centrifugation, washing and drying.

8. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 7, characterized in that: In the step ②, the mass ratio of dialdehyde sodium alginate to ε-polylysine is 1:0.1-0.

2.

9. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 7, characterized in that: The mass percentage concentration of the polylactic acid-glycolic acid copolymer solution is 5-15wt%; the mass percentage concentration of the chitosan solution is 0.5-2wt%.

10. The method for preparing a high-viscosity photo-crosslinked sodium hyaluronate hydrogel according to claim 7, characterized in that: The volume ratio of the modified dialdehyde sodium alginate solution to the polylactic acid-glycolic acid copolymer solution is 1:4-7; the volume ratio of the W1 / O emulsion to the chitosan-polyvinyl alcohol solution is 1:4-5; the volume ratio of the W1 / O / W2 emulsion to the sodium hyaluronate-polyvinyl alcohol solution is 1:5-7.

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