Preparation method of photocrosslinked hyaluronic acid / ε-polylysine antibacterial adhesive hydrogel

Abstract

The present invention discloses a method for preparing a photocrosslinked hyaluronic acid / ε-polylysine antibacterial adhesive hydrogel, the method comprising first modifying hyaluronic acid with methacrylic anhydride, introducing dopamine to modify the methacrylic anhydride-modified hyaluronic acid and mixing it evenly with ε-polylysine, then adding a photoinitiator solution and mixing evenly, and irradiating it with ultraviolet light for more than ten seconds to obtain the result. The hydrogel prepared by dopamine-modified hyaluronic acid has significantly improved adhesion to tissues compared to unmodified hyaluronic acid hydrogels, and ε-polylysine can further improve the adhesion of the hydrogel by virtue of its synergistic effect with dopamine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a photocrosslinked hyaluronic acid / ε-polylysine antibacterial hydrogel, in particular to a high-toughness photocrosslinked hyaluronic acid / ε-polylysine antibacterial hydrogel wound dressing, a preparation method thereof, and application in tissue engineering. Background Art

[0002] Hydrogels are polymers with a highly water-rich, three-dimensional network structure. By varying the crosslink density of the polymer, the pore size within the three-dimensional network can be controlled, allowing the hydrogel's water content to be programmably manipulated, maintaining a swollen but insoluble state in water. Because this three-dimensional network structure closely mimics the extracellular matrix, it offers new avenues for targeted repair and reconstruction of soft tissues, offering new insights into tissue engineering. Furthermore, because hydrogels mimicking the extracellular matrix can maintain normal cell proliferation and differentiation and possess the ability to closely conform to defect sites, they are considered ideal soft tissue replacement materials and are widely used in tissue engineering applications such as drug delivery, wound repair, and tissue engineering scaffolds. Hyaluronic acid (HA) is an anionic mucopolysaccharide composed of alternating D-glucuronic acid and N-acetylglucosamine linkages. It is found in the extracellular matrix of vertebrates, skin, vitreous humor, cartilage, and joint fluid. The physical and chemical properties of HA include hydrophilicity, antioxidant properties, fluidity, and viscoelasticity. The biological functions of hyaluronic acid are related to its molecular weight. For example, high-molecular-weight hyaluronic acid can inhibit inflammatory responses, resist angiogenesis, and inhibit scar formation; while low-molecular-weight hyaluronic acid promotes angiogenesis, causes inflammatory responses, and promotes scar formation. Due to the limited effects of endogenous hyaluronic acid, the preparation of different types of wound dressings using exogenous hyaluronic acid is of great significance for wound repair. Another natural polymer material, ε-polylysine (ε-PL), is a water-soluble polypeptide. Due to its unique amino cation, it is a natural preservative with broad antibacterial and biodegradable properties. It can be obtained through biofermentation and is considered a biomaterial with great potential in the biomedical field.

[0003] Currently, the preparation methods of hydrogels are mainly chemical cross-linking, physical cross-linking, photoinitiated polymerization, and enzymatic cross-linking reactions. However, the existing preparation process has batch instability, little research on antibacterial properties, and poor mechanical properties. Summary of the Invention

[0004] To address the above-mentioned existing technical problems, the present invention provides a tough, photocrosslinked hyaluronic acid / ε-polylysine antibacterial hydrogel, its preparation method, and application. This method crosslinks and solidifies the natural polymer hyaluronic acid / ε-polylysine through photoinitiated free radical polymerization, intermolecular hydrogen bonding, and intermolecular electrostatic attraction to form a hydrogel. This method offers a simple synthesis process, moderate mechanical strength, good biocompatibility, and broad antibacterial properties, making it widely applicable in biomedical applications such as wound and skin tissue surfaces.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preparing a photo-crosslinked hyaluronic acid / ε-polylysine antibacterial hydrogel, the method comprising the following steps:

[0007] S1: Dissolve methacrylic anhydride-modified hyaluronic acid in deionized water, add a strongly acidic cationic ion exchange resin for ion exchange (full exchange generally takes 10-14 hours, preferably 12 hours), adjust the pH to 7.01-7.06 with an aqueous solution of tetrabutylammonium hydroxide, filter, and freeze-dry the resulting filtrate to obtain HAMA-TBA;

[0008] S2: The HAMA-TBA described in step S1 is dissolved in deionized water A, an organic solvent is added, N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole hydrate are added, and the mixture is stirred and reacted for a first time under nitrogen protection (more than 50 minutes, 50 minutes to 2 hours, and 1 hour in the embodiment of the present invention). Dopamine hydrochloride, N,N-diisopropylethylamine and 4-dimethylaminopyridine are added, and the mixture is stirred and reacted for a second time under nitrogen protection for 22-48 hours (24 hours in the embodiment of the present invention). The resulting reaction solution is dialyzed in a sodium chloride aqueous solution with a pH of 5 and deionized water B (preferably dialyzed in a sodium chloride aqueous solution for one day and dialyzed in deionized water B (dialyzed in the embodiment of the present invention for 1 hour). The bag has a molecular weight cut-off of 7000Da) for 2 to 4 days, and the obtained retentate is freeze-dried to obtain dopamine-modified double-bonded hyaluronic acid; the molar ratio of N,N-diisopropylcarbodiimide (DIC) to 1-hydroxybenzotriazole hydrate (HOBt) is 1:1 to 1.5 (preferably 1:1), and the molar ratio of dopamine hydrochloride, N,N-diisopropylethylamine (DIPEA) to 4-dimethylaminopyridine (DMAP) is 1:1 to 1.5:0.5 to 0.6 (preferably 1:1:0.5); the mass ratio of HAMA-TBA, 1-hydroxybenzotriazole hydrate and dopamine hydrochloride is 1:0.85 to 1.1:1 to 2 (preferably 1:0.95:1.2);

[0009] S3: Dissolve the dopamine-modified double-bonded hyaluronic acid and photoinitiator described in step S2 in water, add an aqueous solution of ε-polylysine, and mix evenly. The resulting hydrogel prepolymer system is cured under light with a wavelength of 365-405 nm (preferably 365 nm) to obtain the photo-crosslinked hyaluronic acid / ε-polylysine antibacterial hydrogel; in the hydrogel prepolymer system, the concentration of the dopamine-modified double-bonded hyaluronic acid is 0.025 g / ml~0.04 g / ml (preferably 0.03 g / mL), and the concentration of the ε-polylysine is 0.5 g / L~3.5 g / L (preferably 2.5 g / L).

[0010] Furthermore, the mass ratio of the methacrylated hyaluronic acid to the strongly acidic cationic ion exchange resin in step S1 is 1:3-4, and in the embodiment of the present invention is 1:3.

[0011] In an embodiment of the present invention, the mass fraction of the aqueous solution of tetrabutylammonium hydroxide in step S1 is 40%. Furthermore, the methacrylic anhydride-modified hyaluronic acid in step S1 is obtained by sequentially reacting hyaluronic acid with methacrylic anhydride and then NaCl. Furthermore, the molecular weight of the hyaluronic acid is 100,000 to 200,000 Daltons, and in this embodiment of the present invention, is 100,000 Daltons.

[0012] In an embodiment of the present invention, the methacrylic anhydride-modified hyaluronic acid in step S1 is prepared according to the following method: N,N-dimethylformamide is added to a 20 mg / ml hyaluronic acid aqueous solution, methacrylic anhydride is added at 3-4° C., the first stage reaction is carried out for 0.5 h, the pH is adjusted to 8-9 (the pH is adjusted repeatedly with 1 mol / L sodium hydroxide aqueous solution until it no longer changes), the second stage reaction is carried out overnight (10-14 h), NaCl is added, and the third stage reaction is carried out for 0.5 h; the resulting reaction solution is poured into anhydrous ethanol, and after the reaction product is fully precipitated, the resulting precipitate is dissolved in water and dialyzed with deionized water as a dialyzate (3-5 days), and the resulting retentate is freeze-dried to obtain the methacrylic anhydride-modified hyaluronic acid; the molar ratio of hyaluronic acid to the methacrylic anhydride in the hyaluronic acid aqueous solution is 1 g:1.6 mL.

[0013] Furthermore, the volume ratio of the hyaluronic acid aqueous solution to N,N-dimethylformamide is 1.5-2:1, and in the embodiment of the present invention is 1.5:1.

[0014] Furthermore, the volume of the anhydrous ethanol is twice the volume of the reaction solution.

[0015] Preferably, the molecular weight of hyaluronic acid in the hyaluronic acid aqueous solution is 100,000 to 200,000 Daltons, and in the embodiment of the present invention, it is 100,000 Daltons.

[0016] Furthermore, the volume of deionized water A in step S2 is 65 ml / g to 72 ml / g, based on the mass of the HAMA-TBA, and in this embodiment is 67.5 mL / g. The letters following the deionized water A and B are simply for the purpose of distinguishing substances at different stages and for ease of description, and have no other special meaning.

[0017] In one embodiment of the present invention, the organic solvent in step S2 is N,N-dimethylformamide or N,N-dimethylacetamide (DMAC) (preferably N,N-dimethylformamide). Furthermore, the volume of the organic solvent in step S2 is 155 mL / g to 160 mL / g based on the mass of the HAMA-TBA, and in this embodiment of the present invention is 157.5 mL / g.

[0018] Preferably, the molecular weight of the ε-polylysine in step S3 is 1000 to 8000 Daltons, and in the embodiment of the present invention, it is 3500 to 5000 Daltons.

[0019] Furthermore, the curing time in step S3 is 50s to 70s. Preferably, under the condition of ultraviolet light irradiation, the adhesive can be quickly cured into glue in 50s.

[0020] The molecular weight cut-off of the dialysis bags in the above dialysis was 7000 Da.

[0021] The present invention also provides the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared by the above method. The present invention also provides the use of the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared by the above method in the field of biomedical materials.

[0022] The applications include: preparing medical wound dressings, cell culture scaffolds or cartilage tissue engineering materials.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Compared with unmodified hyaluronic acid hydrogel, the hydrogel prepared by dopamine-modified hyaluronic acid has significantly improved adhesion performance with tissues, and ε-polylysine can further improve the adhesion performance of the hydrogel by virtue of its synergistic effect with dopamine.

[0025] Photoinitiated free radical polymerization can be cured into gel according to any shape and at any position through ultraviolet light irradiation. The sample preparation and operation process are simple and no toxic products are produced. Secondly, ε-polylysine is a good antimicrobial peptide. Because the substance itself carries a positive charge and forms an electrostatic effect with the negatively charged hyaluronic acid, it further enhances the mechanical properties of the hydrogel. In addition, the addition of ε-polylysine gives the hydrogel a broad-spectrum antibacterial effect, which will have a good effect on the treatment of wounds.

[0026] The present invention adopts hyaluronic acid, the main component of biodegradable and safe and reliable cytoplasm, and ε-polylysine, a natural preservative with a broad-spectrum antibacterial effect, as the main material. Methacrylic anhydride is carried out on the hydroxyl and carboxyl groups on the hyaluronic acid main chain, and dopamine small molecular groups are chemically modified. Using simple and controllable light-triggered free radical polymerization, electrostatic adsorption between positive and negative charges, and physical crosslinking between phenolic hydroxyl groups, any damaged part can be matched, the chemical stability of the system is improved, and the mechanical properties of the system are enhanced to match the pressure-bearing performance of biological tissues such as cartilage. In addition, ε-polylysine, as a natural broad-spectrum antibacterial biomaterial, significantly improves the antibacterial properties of the hydrogel, so that bacteria can be avoided during its operation, allowing the repair and reconstruction of the defective part. At the same time, the introduction of the phenolic hydroxyl groups of the catechol structure and the introduction of ε-polylysine can synergistically enhance the adhesion between the hydrogel and the tissue, so that the gel can be permanently covered on the desired part, achieving better therapeutic effects. The hydrogel has the advantages of good biocompatibility and degradability, and has a broad application base in the fields of medical dressings, cell scaffolds, tissue engineering, etc.

[0027] This method has a mild and controllable gelation mode and a simple synthesis process. Through the polysaccharide / polypeptide system, it can better simulate the extracellular matrix and better induce tissue regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are the nuclear magnetic resonance images of HA, HAMA, and HAMA-Cat.

[0029] Figure 2 This is a physical picture of the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared by the present invention.

[0030] Figure 3 This is a test diagram of the mechanical properties of the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared in the present invention.

[0031] Figure 4 This is a comparison chart of the cytotoxicity test results of the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared in the present invention.

[0032] Figure 5 This is a comparison chart of the antibacterial performance experimental results of the hyaluronic acid / ε-polylysine antibacterial hydrogel prepared in the present invention.

[0033] Figure 6 This is a test diagram of the wet adhesion performance between the hyaluronic acid / ε-polylysine prepared by the present invention and the pig casing. DETAILED DESCRIPTION

[0034] The molecular weight of the ε-polylysine used in the following examples is 3500 to 5000 Daltons, Aladdin, CAS: 28211-04-3.

[0035] Example 1 without ε-polylysine

[0036] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA mass concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution to adjust the pH of the reaction solution to 8-9. Adjust once every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed in deionized water for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0037] (2) Dissolve 1.0 g of HAMA in 200 mL of deionized water, add 3.0 g of strongly acidic cationic ion exchange resin (DOWEX 50W*8) to carry out ion exchange overnight, then neutralize with tetrabutylammonium hydroxide solution (TBA-OH) (CAS: 2052-49-5, MERCK, 40% in water) to obtain a HAMA-TBA aqueous solution with a pH of 7.02-7.06, filter, and freeze-dry the filtrate at -60°C in a vacuum freeze dryer (PILOT2-4LD) for later use. (3) Weigh 0.4 g of HAMA-TBA and add it to 27 mL of deionized water, stir for 2 h to fully dissolve, then add 63 mL of DMF at a volume ratio of DI:DMF=3:7, take N,N-diisopropylcarbodiimide (DIC) (CAS: 7087-68-5, MERCK, ≥99.0% in water) and add 1% HAMA-TBA. The reaction solution was added with 2.5 mmol (400 μl) of water and 2.5 mmol (0.38 g) of 1-hydroxybenzotriazole hydrate (HOBt) (CAS: 123333-53-9, MERCK). The mixture was stirred and reacted uniformly under nitrogen for 1 hour. Then, dopamine hydrochloride (2.5 mmol (0.47 g)) (CAS: 62-31-7, Aladdin), N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5, Aladdin, 99%) (2.5 mmol (400 μl), and 4-dimethylaminopyridine (DMAP) (CAS: 1122-58-3, MERCK) (0.5 mmol (0.061 g)) were added. The reaction was stirred and reacted at room temperature under nitrogen for 24 hours. The product was dialyzed against a sodium chloride solution (1 M, pH = 5) acidified with concentrated hydrochloric acid using a dialysis tubing with a molecular weight cutoff of 7000 Da for 1 day, and then dialyzed against deionized water for 2 days. The resulting mixture was freeze-dried at -80°C to obtain dopamine-modified double-bonded hyaluronic acid (HAMA-Cat).

[0038] (4) 0.06 g of HAMA-Cat was added to 1 ml of an aqueous solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain a stock solution A. The stock solution A was cured by irradiating with ultraviolet light (18W 365 nm wavelength) for 60 seconds to obtain a photocrosslinked hyaluronic acid hydrogel.

[0039] Example 2

[0040] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution and adjust the pH of the reaction solution to 8-9. Adjust the pH every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed in deionized water for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0041] (2) 1.0 g of HAMA was dissolved in 200 mL of deionized water, and 3.0 g of strongly acidic cationic ion exchange resin (DOWEX 50W*8) was added for ion exchange overnight. The solution was then neutralized with tetrabutylammonium hydroxide solution (TBA-OH) (CAS: 2052-49-5, MERCK, 40% in water) to obtain a HAMA-TBA aqueous solution with a pH of 7.02-7.06. The solution was lyophilized at -60°C in a vacuum freeze dryer (PILOT2-4LD) and then used.

[0042] (3) HAMA-TBA (0.4 g) was weighed and added to 27 mL of deionized water, stirred for 2 h to fully dissolve, and then 63 mL of DMF was added according to the volume ratio of DI:DMF=3:7. N,N-diisopropylcarbodiimide (CAS: 7087-68-5, MERCK, ≥99.0% in water) was taken. To the reaction solution were added 2.5 mmol (400 μl) of (DIC) (water) and 2.5 mmol (0.38 g) of 1-hydroxybenzotriazole hydrate (HOBt) (CAS: 123333-53-9, MERCK). The mixture was stirred and reacted uniformly under nitrogen for 1 hour. Then, 2.5 mmol (0.47 g) of dopamine hydrochloride (CAS: 62-31-7, Aladdin), 2.5 mmol (400 μl) of N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5, Aladdin, 99%), and 0.5 mmol (0.061 g) of 4-dimethylaminopyridine (DMAP) (CAS: 1122-58-3, MERCK) were added. The reaction was stirred and reacted under nitrogen for 24 hours at room temperature. The hyaluronic acid was dialyzed in a sodium chloride solution (1 M, pH = 5) acidified with concentrated hydrochloric acid for 1 day using a dialysis bag with a molecular weight cut-off of 7000 Da, and then dialyzed in deionized water for 2 days. The hyaluronic acid was then freeze-dried at -80°C to obtain dopamine-modified double-bonded hyaluronic acid (HAMA-Cat).

[0043] (4) 0.06 g of HAMA-Cat was added to 1 ml of a solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain stock solution A. Subsequently, 1 ml of a 0.1% mass concentration ε-polylysine aqueous solution (stock solution B) was prepared. Stock solutions A and B were evenly mixed together and cured by ultraviolet light (18W 365 nm wavelength) for 62 seconds to obtain a photocrosslinked hyaluronic acid hydrogel.

[0044] Example 3

[0045] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution and adjust the pH of the reaction solution to 8-9. Adjust the pH every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed in deionized water for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0046] (2) 1.0 g of HAMA was dissolved in 200 mL of deionized water, and 3.0 g of strongly acidic cationic ion exchange resin (DOWEX 50W*8) (CAS: 2052-49-5, MERCK, 40% in water) was added for ion exchange overnight. The solution was then neutralized with tetrabutylammonium hydroxide solution (TBA-OH) to obtain a HAMA-TBA aqueous solution with a pH of 7.02-7.06. The solution was lyophilized at -60°C in a vacuum freeze dryer (PILOT2-4LD) and set aside.

[0047] (3) Weigh 0.4 g of HAMA-TBA and add it to 27 mL of deionized water. Stir for 2 h to fully dissolve it. Then add 63 mL of DMF according to the volume ratio of DI:DMF=3:7. Take N,N-diisopropylcarbodiimide (CAS:7087-68-5,MERCK,≥99.0%in To the reaction solution were added 2.5 mmol (400 μl) of (DIC) (water) and 2.5 mmol (0.38 g) of 1-hydroxybenzotriazole hydrate (HOBt) (CAS: 123333-53-9, MERCK). The mixture was stirred and reacted uniformly under nitrogen for 1 hour. Then, 2.5 mmol (0.47 g) of dopamine hydrochloride (CAS: 62-31-7, Aladdin), 2.5 mmol (400 μl) of N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5, Aladdin, 99%), and 0.5 mmol (0.061 g) of 4-dimethylaminopyridine (DMAP) (CAS: 1122-58-3, MERCK) were added. The reaction was stirred and reacted under nitrogen for 24 hours at room temperature. The hyaluronic acid was dialyzed in a sodium chloride solution (1 M, pH = 5) acidified with concentrated hydrochloric acid for 1 day using a dialysis bag with a molecular weight cut-off of 7000 Da, and then dialyzed in deionized water for 2 days. The hyaluronic acid was then freeze-dried at -80°C to obtain dopamine-modified double-bonded hyaluronic acid (HAMA-Cat).

[0048] (4) 0.06 g (HAMA-Cat) was added to 1 ml of a solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain a stock solution A, followed by preparing 1 ml of a 0.3% mass concentration of ε-polylysine stock solution B. The stock solutions A and B were evenly mixed together, and cured by ultraviolet light (18W 365 nm wavelength) for 63 seconds to obtain a photocrosslinked hyaluronic acid hydrogel.

[0049] Example 4

[0050] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution and adjust the pH of the reaction solution to 8-9. Adjust the pH every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0051] (2) 1.0 g of HAMA was dissolved in 200 mL of deionized water, and 3.0 g of strongly acidic cationic ion exchange resin (DOWEX 50W*8) (CAS: 2052-49-5, MERCK, 40% in water) was added for ion exchange overnight. The solution was then neutralized with tetrabutylammonium hydroxide (TBA-OH) solution to obtain a HAMA-TBA aqueous solution with a pH of 7.02-7.06. The solution was lyophilized at -60°C in a vacuum freeze dryer (PILOT2-4LD) and then used.

[0052] (3) Weigh 0.4 g of HAMA-TBA and add it into 27 mL of deionized water. Stir for 2 h to fully dissolve it. Then add 63 mL of DMF according to the volume ratio of DI:DMF=3:7. Take N,N-diisopropylcarbodiimide (DIC) (CAS: 7087-68-5, MERCK, ≥99.0% in The reaction solution was added with 2.5 mmol (400 μl) of water and 2.5 mmol (0.38 g) of 1-hydroxybenzotriazole hydrate (HOBt) (CAS: 123333-53-9, MERCK). After stirring and reacting for 1 h under nitrogen, 2.5 mmol (0.47 g) of dopamine hydrochloride (CAS: 62-31-7, Aladdin), 2.5 mmol (400 μl) of N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5, Aladdin, 99%), and 0.5 mmol (0.061 g) of 4-dimethylaminopyridine (DMAP) (CAS: 1122-58-3, MERCK) were added. The reaction was stirred at room temperature under nitrogen for 24 h. The product was dialyzed against a sodium chloride solution (1 M, pH = 5) acidified with concentrated hydrochloric acid using a dialysis tubing with a molecular weight cutoff of 7000 Da for 1 day, and then dialyzed against deionized water for 2 days. The resulting mixture was freeze-dried at -80°C to obtain dopamine-modified double-bonded hyaluronic acid (HAMA-Cat).

[0053] (4) 0.06 g of HAMA-Cat was added to 1 ml of a solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain a stock solution A. Subsequently, 1 ml of a 0.5% mass concentration of ε-polylysine stock solution B was prepared. The stock solutions A and B were evenly mixed together and cured by ultraviolet light (18W 365 nm wavelength) for 65 seconds to obtain a photocrosslinked hyaluronic acid hydrogel.

[0054] Example 5

[0055] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution and adjust the pH of the reaction solution to 8-9. Adjust the pH every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0056] (2) 1.0 g of HAMA was dissolved in 200 mL of deionized water, and 3.0 g of strongly acidic cationic ion exchange resin (DOWEX 50W*8) (CAS: 2052-49-5, MERCK, 40% in water) was added for ion exchange overnight. The solution was then neutralized with tetrabutylammonium hydroxide (TBA-OH) to obtain a HAMA-TBA aqueous solution with a pH of 7.02-7.06. The solution was lyophilized at -60°C in a vacuum freeze dryer (PILOT2-4LD) and then used.

[0057] (3) Weigh 0.4 g of HAMA-TBA and add it into 27 mL of deionized water. Stir for 2 h to fully dissolve it. Then add 63 mL of DMF according to the volume ratio of DI:DMF=3:7. Take N,N-diisopropylcarbodiimide (DIC) (CAS: 7087-68-5, MERCK, ≥99.0% in The reaction solution was added with 2.5 mmol (400 μl) of water and 2.5 mmol (0.38 g) of 1-hydroxybenzotriazole hydrate (HOBt) (CAS: 123333-53-9, MERCK). After stirring and reacting for 1 h under nitrogen, 2.5 mmol (0.47 g) of dopamine hydrochloride (CAS: 62-31-7, Aladdin), 2.5 mmol (400 μl) of N,N-diisopropylethylamine (DIPEA) (CAS: 7087-68-5, Aladdin, 99%), and 0.5 mmol (0.061 g) of 4-dimethylaminopyridine (DMAP) (CAS: 1122-58-3, MERCK) were added. The reaction was stirred at room temperature under nitrogen for 24 h. The product was dialyzed against a sodium chloride solution (1 M, pH = 5) acidified with concentrated hydrochloric acid using a dialysis tubing with a molecular weight cutoff of 7000 Da for 1 day, and then dialyzed against deionized water for 2 days. The resulting mixture was freeze-dried at -80°C to obtain dopamine-modified double-bonded hyaluronic acid (HAMA-Cat).

[0058] (4) 0.06 g of HAMA-Cat was added to 1 ml of a solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain a stock solution A. Subsequently, 1 ml of a 0.5% mass concentration of ε-polylysine stock solution B was prepared. The stock solutions A and B were evenly mixed together and cured by ultraviolet light (18W 365 nm wavelength) for 70 seconds to obtain a photocrosslinked hyaluronic acid hydrogel.

[0059] Example 6

[0060] (1) Dissolve 1.5g of hyaluronic acid (HA, molecular weight 100,000 Daltons) in 75ml of deionized water to a HA concentration of 20mg / ml. Stir thoroughly and then add 50ml of N,N-dimethylformamide (DMF) in a solution volume ratio of DI:DMF = 3:2 and mix well. Place in a condensation circulation device and cool to 4℃. Add 2.4mL of methacrylic anhydride and react at 4℃ for 0.5h. Add 1mol / L sodium hydroxide aqueous solution and adjust the pH of the reaction solution to 8-9. Adjust the pH every 0.5h-1h until the pH of the solution no longer changes with the reaction. Continue the reaction overnight. Add 0.75g of NaCl and continue the reaction for 0.5h. After the reaction is completed, pour the reaction solution into 2 times the volume of anhydrous ethanol (ethanol is twice the volume of the reaction solution). After the reaction product is fully precipitated, centrifuge to obtain a precipitate (5000 rpm, 5 minutes). The precipitated product was dissolved in deionized water, dialyzed for 3 days (the molecular weight cut-off of the dialysis bag was 7000 Da), and finally freeze-dried to obtain methacrylated hyaluronic acid (HAMA).

[0061] (2) 0.06 g of hyaluronic acid (HAMA) containing methacrylic anhydride was added to 1 ml of a solution containing 0.005 g of a photoinitiator (LAP, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, MERCK) to obtain a stock solution A. The mass concentration of HAMA-Cat in the stock solution A was 6%, and the mass concentration of LAP in the photoinitiator solution was 0.5%. Subsequently, 1 ml of a 0.1% mass concentration of ε-polylysine stock solution B was prepared. The stock solutions A and B were evenly mixed together, and cured by ultraviolet light (18W 365 nm wavelength) for 62 seconds to obtain a photocrosslinked hyaluronic acid hydrogel, which was recorded as HAP.

[0062] Example 7: Mechanical performance evaluation

[0063] The hydrogel sample (10 mm in diameter, 5 mm in height) was subjected to compression testing using a high and low temperature dual-column testing machine (Instron 5966, America). The compression strain was set to 95% and the compression speed was 10 mm / min. The test was stopped when the sample completely broke. Figure 3 As shown in the figure, with the addition of ε-polylysine, the mechanical properties of the hydrogel will increase from the initial 58KPa to 180KPa. The improvement in mechanical properties is mainly attributed to the electrostatic adsorption between the positive and negative charges of hyaluronic acid and ε-polylysine.

[0064] Example 8: Cytotoxicity evaluation experiment

[0065] The biocompatibility of the hydrogel was evaluated by the indirect contact method (sample extract) and the direct contact method. The indirect contact method was as follows: L929 cells were seeded in a 24-well culture plate (5×10 4cells) and then treated with different hydrogel extracts (hydrogel: DMEM mass ratio of 1:5) for 1, 2, and 3 days. Subsequently, 10 μL of CCK-8 solution was added to the cells in each well. After incubation for 2 hours, the absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = As / Ac×100% As and Ac are the absorbances of the experimental group and the control group, respectively. The results are shown in Figure 2. Figure 4 As shown in the experimental results, the cell viability of all experimental groups showed no cytotoxicity compared with the control group, indicating that the prepared hydrogel has good biocompatibility

[0066] Example 9: Evaluation of the antibacterial properties of hydrogels

[0067] First, the hydrogel samples with different polylysine contents were soaked in 75% ethanol for 2 hours for thorough disinfection, and then the alcohol in the hydrogel was washed with sterile PBS. The sterilized hydrogel samples were placed in a 12-well plate and 2 mL of Staphylococcus aureus (S.aureus) or Escherichia coli (E.coil) solution was added. The optical density (OD600nm) of the two bacterial solutions was 0.05 and 0.1, respectively. The 12-well plate was placed in a constant temperature shaker at 37°C and 120 rpm and cultured for 24 hours, then washed with PBS, and then stained with 20 μL of Live / Dead Back Light Kit stain. After fixation and washing, the number of live and dead bacteria was observed under a fluorescent inverted microscope. The blue color represents the number of live bacteria, and the red color represents the number of dead bacteria. The experimental results are as follows: Figure 5 As shown in the figure, as the content of ε-polylysine gradually increases, the antibacterial effect becomes better and better. When the mass concentration reaches 0.7%, the antibacterial rate of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil) reaches nearly 100%.

[0068] Example 10: Shear adhesion experiment of hydrogel

[0069] The shear adhesion strength of the hydrogel was evaluated in accordance with ASTM standards (F2255-05 method) using a high-low temperature dual-column testing machine (Instron 5966, America). 3.0 cm*1.0 cm strips of hog casings purchased from the market were cut and supported on a glass slide. The hydrogel precursors prepared according to Examples 1, 2, and 6 and the hydrogel precursors with the same concentration of HAMA were applied to the wet casing surface, with the size of the applied hydrogel being 1.0 cm*1.0 cm. The hydrogel-coated portions of the two hog casing strips were overlapped, with the strips facing opposite directions. The strips were then irradiated and cured using an ultraviolet lamp (18W 365 nm wavelength). The prepared samples were placed in a high-low temperature dual-column testing machine (Instron 5966, America) in the fixture, the force direction is the long axis direction of the sample, the moving speed of the fixture is 5mm / min, and the tensile force is continuously applied until the adhesive part of the hydrogel is broken. The maximum load F (N) of the sample shear failure is obtained. Each set of data is measured three times and the average value is taken. The experimental results are shown in Figure 6 As shown, the adhesion forces of the HAMA and HAP hydrogels prepared in Example 6 were only 113.5 kPa and 118 kPa, respectively, with little difference. The adhesion force of the HAMA-Cat hydrogel prepared in Example 1 was 345 kPa, and the adhesion force of the HAMA-Cat and ε-polylysine hydrogel components prepared in Example 2 could reach 553.26 kPa, indicating that the ε-polylysine introduced into the system can synergize with the dopamine-grafted hyaluronic acid to enhance the adhesion effect of the gel.

[0070] In summary, we have provided a hyaluronic acid / ε-polylysine that simulates the construction of extracellular matrix through light-initiated free radical polymerization, electrostatic complexation and hydrogen bonding. It has excellent mechanical properties, good cell compatibility, good adhesion properties, and certain broad-spectrum antibacterial properties. It is expected that the application and research and development of hyaluronic acid / ε-polylysine for encapsulating cells in tissue engineering scaffolds and wound dressings in the future will provide new ideas for the development of alternative biomedical materials.

Claims

1. A method for preparing a photocrosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel, characterized in that The method comprises the following steps: S1: dissolving methacrylic anhydride-modified hyaluronic acid in deionized water, adding a strongly acidic cation exchange resin for ion exchange, adjusting the pH to 7.01-7.06 with an aqueous solution of tetrabutylammonium hydroxide, filtering, and freeze-drying the resulting filtrate to obtain HAMA-TBA; S2: The HAMA-TBA described in step S1 was dissolved in deionized water A, and an organic solvent was added. N, N-diisopropylcarbodiimide and 1-hydroxybenzotriazole hydrate were added, and the mixture was stirred for a first time under nitrogen protection to fully react. Dopamine hydrochloride, N, N-diisopropylethylamine and 4-dimethylaminopyridine were added, and the mixture was stirred for a second time under nitrogen protection for 22-48 hours. The reaction solution was dialyzed against a sodium chloride aqueous solution with a pH of 5 and deionized water B in turn, and the cut-off was obtained. The retained liquid is freeze-dried to obtain dopamine-modified double-bonded hyaluronic acid; the molar ratio of N,N-diisopropylcarbodiimide to 1-hydroxybenzotriazole hydrate is 1:1-1.5, the molar ratio of dopamine hydrochloride, N,N-diisopropylethylamine and 4-dimethylaminopyridine is 1:1-1.5:0.5-0.6; the mass ratio of HAMA-TBA, 1-hydroxybenzotriazole hydrate and dopamine hydrochloride is 1:0.85-1.1:1-2; S3: dissolving the dopamine-modified double-bonded hyaluronic acid and the photoinitiator described in step S2 in water, adding an aqueous solution of ɛ-polylysine, and mixing uniformly. The resulting hydrogel prepolymer system is cured under light with a wavelength of 365-405 nm to obtain the photocrosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel; In the hydrogel prepolymerization system, the concentration of the dopamine-modified double-bonded hyaluronic acid is 0.025 g / ml to 0.04 g / ml, and the concentration of the ɛ-polylysine is 0.5 g / L to 3.5 g / L.

2. The method for preparing the photocrosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 1, wherein: The mass ratio of the methacrylic anhydride-modified hyaluronic acid to the strongly acidic cation exchange resin in step S1 is 1:3-4.

3. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 1, wherein: The methacrylic anhydride-modified hyaluronic acid in step S1 is obtained by sequentially reacting hyaluronic acid with methacrylic anhydride and NaCl.

4. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 3, wherein: The molecular weight of the hyaluronic acid is 100,000 to 200,000 Daltons.

5. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 3, wherein: The methacrylic anhydride-modified hyaluronic acid in step S1 is prepared according to the following method: N,N-dimethylformamide is added to a 20 mg / ml hyaluronic acid aqueous solution, methacrylic anhydride is added at 3-4°C, the first stage of the reaction is carried out for 0.5 h, the pH is adjusted to 8-9, the second stage of the reaction is carried out overnight, NaCl is added, and the third stage of the reaction is carried out for 0.5 h; the resulting reaction solution is poured into anhydrous ethanol, and after the reaction product is fully precipitated, the resulting precipitate is dissolved in water and dialyzed with deionized water as a dialyzate. The resulting retentate is freeze-dried to obtain the methacrylic anhydride-modified hyaluronic acid; the molar ratio of hyaluronic acid to methacrylic anhydride in the hyaluronic acid aqueous solution is 1 g:1.6 mL.

6. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 5, wherein: The volume ratio of the hyaluronic acid aqueous solution to N,N-dimethylformamide is 1.5-2:

1.

7. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 1, wherein: The volume of the deionized water A in step S2 is 65 ml / g to 72 ml / g based on the mass of the HAMA-TBA.

8. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 1, wherein: The organic solvent in step S2 is N,N-dimethylformamide or N,N-dimethylacetamide, and the volume of the organic solvent is 155 mL / g to 160 mL / g based on the mass of the HAMA-TBA.

9. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to claim 1, wherein: The molecular weight of the ɛ-polylysine described in step S3 is 1000 to 8000 Daltons.

10. The method for preparing the photo-crosslinked hyaluronic acid / ɛ-polylysine antibacterial hydrogel according to any one of claims 1 to 9, characterized in that: The curing time in step S3 is 50s to 70s.

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