Long-acting antibacterial carboxymethyl chitosan gel and preparation method and application thereof

Abstract

The present application relates to a kind of long-acting antibacterial carboxymethyl chitosan gel and its preparation method and application.Catechol functional group is introduced on the surface of carboxymethyl chitosan to obtain carboxymethyl chitosan-3,4-dihydroxybenzaldehyde;Azido group is introduced on the surface of carboxymethyl chitosan to obtain carboxymetyl chitosan-azido, dibenzocyclooctyne group is introduced on the surface of protamine to obtain protamine-dibenzocyclooctyne, carboxymethyl chitosan-protamine is synthesized by using copper-free catalytic azido alkyne cycloaddition reaction;The gel material is synthesized by using the oxidative crosslinking effect of catechol functional group and the Schiff base reaction of carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, carboxymethyl chitosan-protamine and oxidized hyaluronic acid.The carboxymethyl chitosan gel provided by the present application has long-acting antibacterial property, tissue adhesion, rapid hemostatic property and injectability, and has potential to be widely used in biomedical field.

Description

Technical Field

[0001] This invention belongs to the field of medical dressings, specifically relating to a long-lasting antibacterial carboxymethyl chitosan gel, its preparation method, and its application. Background Technology

[0002] As a vital protective organ, the skin plays an irreplaceable role in blocking the invasion of harmful external substances and maintaining the stability of the internal physiological environment. Once the skin is damaged, unprotected tissue will bleed, increasing the risk of wound infection. Without proper treatment, this can not only prolong the wound healing period but also lead to serious consequences such as amputation or even death due to bacterial infection. While traditional wound dressings offer advantages such as ease of use, low cost, and convenient production, they are less effective than hydrogel wound dressings in regulating wound moisture, providing long-term antibacterial properties, and promoting wound tissue regeneration. In recent years, various multifunctional wound dressings have been developed, but existing skin wound dressings lack adhesion, hemostasis, and long-term antibacterial properties, leading to potential bacterial infections that hinder wound healing or even worsen the wound condition. Therefore, there is an urgent need to develop a suitable wound dressing that simultaneously possesses long-lasting antibacterial properties, rapid hemostasis, and strong tissue adhesion to promote wound healing.

[0003] Carboxymethyl chitosan (CMC) is a product of carboxymethylation of chitosan. It not only has good water solubility, but also exhibits excellent biological properties, such as good biocompatibility, biodegradability and certain antibacterial activity. As a water-soluble chitosan derivative, it has a wider range of applications and has received extensive attention and in-depth research in biomedical fields such as wound repair, drug delivery and tissue engineering. However, the shortcomings of single-component carboxymethyl chitosan materials in terms of hemostasis, tissue adhesion and long-term antibacterial properties limit the application of CMC in the biomedical field. Researchers usually chemically modify CMC to improve its physicochemical properties. For example, (1) Wang Sheng, Yang Liming, Chen Jie, et al. Preparation and performance study of temperature and pH sensitive polyvinyl alcohol / carboxymethyl chitosan hydrogel [J]. Chemical World, 2006, 47(3):149-152. DOI:10.3969 / j.issn.0367-6358.2006.03.007. (2)[1]Xu Yongmei, Zhou Wei, Yin Lei, et al. Preparation of thiolated carboxymethyl chitosan microgel and its bioadhesion properties[J]. Chinese Journal of Tissue Engineering Research and Clinical Rehabilitation, 2009, 13(29):4.DOI:10.3969 / j.issn.1673-8225.2009.29.009.(3)CN110483662A A carboxymethyl chitosan crosslinked gel.

[0004] However, the antibacterial properties of modified carboxymethyl chitosan hydrogels in the prior art are generally limited, and most do not possess long-lasting antibacterial properties. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, this invention provides a method for preparing a long-lasting antibacterial carboxymethyl chitosan gel. This method improves the tissue adhesion, hemostatic properties, and antibacterial activity of carboxymethyl chitosan material by grafting catechol and protamine onto it, respectively. Subsequently, its mechanical properties are further enhanced through a Schiff base reaction with oxidized hyaluronic acid.

[0006] Another object of the present invention is to provide a carboxymethyl chitosan gel prepared by the above method, which has long-term antibacterial properties, good tissue adhesion, procoagulant properties, and rapid hemostasis.

[0007] Another object of the present invention is to provide the application of the above-mentioned carboxymethyl chitosan gel.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a long-lasting antibacterial carboxymethyl chitosan gel includes the following steps: using carboxymethyl chitosan as a raw material, introducing catechol functional groups onto its surface to obtain carboxymethyl chitosan-3,4-dihydroxybenzaldehyde; using carboxymethyl chitosan as a raw material, introducing azide groups onto its surface to obtain carboxymethyl chitosan-azide; using protamine as a raw material, introducing dibenzocyclooctynyl groups onto its surface to obtain protamine-dibenzocyclooctynyl; further utilizing a copper-free catalytic azidoyne cycloaddition (SPAAC) reaction of carboxymethyl chitosan-azide and protamine-dibenzocyclooctynyl to synthesize carboxymethyl chitosan-protamine; finally utilizing the oxidative crosslinking effect of catechol functional groups and the Schiff base reaction of carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, carboxymethyl chitosan-protamine, and oxidized hyaluronic acid to synthesize a gel material, namely the long-lasting antibacterial carboxymethyl chitosan gel.

[0010] Furthermore, the preparation steps of the oxidized hyaluronic acid are as follows: periodate is added to the hyaluronic acid solution, the mass ratio of hyaluronic acid to periodate is 1:0.25-0.5, the reaction is carried out at room temperature in the dark for 8-24 hours, quenched with ethylene glycol, dialyzed with deionized water, and then freeze-dried under vacuum to obtain oxidized hyaluronic acid.

[0011] Furthermore, the preparation steps of the carboxymethyl chitosan-3,4-dihydroxybenzaldehyde are as follows: Under nitrogen protection, a 3,4-dihydroxybenzaldehyde-ethanol solution is added to an aqueous solution of carboxymethyl chitosan, with a mass ratio of carboxymethyl chitosan to 3,4-dihydroxybenzaldehyde of 1:1.25-1.75. The mixture is heated to 50-60°C and reacted for 1-3 hours. After precipitation with anhydrous ethanol and vacuum drying, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde (DCMC) is obtained.

[0012] Furthermore, the preparation steps of the carboxymethyl chitosan-azide are as follows: carboxymethyl chitosan is dissolved in deionized water, azide-polyethylene glycol-active ester is added, and the reaction is carried out at room temperature for 12-24 hours. The mass ratio of azide-polyethylene glycol-active ester to carboxymethyl chitosan is 1:25-50. After dialysis with deionized water and vacuum freeze-drying, carboxymethyl chitosan-azide is obtained.

[0013] Further, the preparation steps of the carboxymethyl chitosan-protamine are as follows: protamine is dissolved in PBS buffer solution, dibenzocyclooctyn-polyethylene glycol-active ester is added, and the reaction is carried out at room temperature for 12-24 hours, with the mass ratio of dibenzocyclooctyn-polyethylene glycol-active ester to protamine being 1:20-40; after reacting for 12-24 hours, carboxymethyl chitosan-azide is added, and the reaction is carried out at room temperature for 12-24 hours, with the mass ratio of carboxymethyl chitosan-azide to protamine-dibenzocyclooctyn being 1:0.5-1; after dialysis with deionized water and vacuum freeze-drying, carboxymethyl chitosan-protamine (PCMC) is obtained.

[0014] Furthermore, after obtaining oxidized hyaluronic acid and carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, the further operation steps are as follows: oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, and carboxymethyl chitosan-protamine are dissolved in deionized water. The mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, and carboxymethyl chitosan-protamine is 10:2.5:1. The solutions are thoroughly mixed, and after 1-3 minutes at room temperature, a new polymeric biomedical material, namely the long-lasting antibacterial carboxymethyl chitosan gel, is formed.

[0015] Furthermore, the concentration of the solution obtained by dissolving oxidized hyaluronic acid in deionized water is 3-5 wt%.

[0016] Furthermore, the concentration of the solution obtained by dissolving carboxymethyl chitosan-3,4-dihydroxybenzaldehyde in deionized water is 5-10 wt%.

[0017] Furthermore, the concentration of the carboxymethyl chitosan-protamine solution obtained by dissolving it in deionized water was 4 wt%.

[0018] This invention is mainly achieved through the following steps:

[0019] (1) Prepare a hyaluronic acid solution, add periodate, and react in the dark for 8-24 hours; after quenching with ethylene glycol, dialyze and freeze dry to obtain oxidized hyaluronic acid;

[0020] (2) Prepare a carboxymethyl chitosan solution, then add a 3,4-dihydroxybenzaldehyde-ethanol solution, heat to 50-60℃, react for 1-3 hours under nitrogen protection, and precipitate in anhydrous ethanol solution to obtain carboxymethyl chitosan-3,4-dihydroxybenzaldehyde.

[0021] (3) Prepare a carboxymethyl chitosan solution, then add azide-polyethylene glycol-active ester, react at room temperature for 12-24 hours, dialyze and freeze dry to obtain carboxymethyl chitosan-azide;

[0022] (4) Dissolve protamine in PBS solution, then add dibenzocyclooctylene-polyethylene glycol-active ester, and react at room temperature for 12-24 h; then add carboxymethyl chitosan-azide obtained in step (3), react at room temperature for 12-24 h, and dialyze and freeze dry to obtain carboxymethyl chitosan-protamine.

[0023] (5) Dissolve the oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde and carboxymethyl chitosan-protamine obtained in the above steps in deionized water, mix the three solutions evenly in a mold, and crosslink them at room temperature for 1-3 minutes to form the long-lasting antibacterial carboxymethyl chitosan gel.

[0024] Furthermore, the dibenzocyclooctyn-polyethylene glycol-active ester is preferably at least one of dibenzocyclooctyn-tetraethylene glycol-active ester, dibenzocyclooctyn-pentaethylene glycol-active ester, or dibenzocyclooctyn-dodecylethylene glycol-active ester.

[0025] Furthermore, the periodate is sodium periodate or potassium periodate.

[0026] Furthermore, the carboxymethyl chitosan has a molecular weight of 400,000 to 1,000,000 and a degree of deacetylation of ≥80%.

[0027] Furthermore, the concentration of the 3,4-dihydroxybenzaldehyde-ethanol solution is 450-550 mg / mL.

[0028] The above-mentioned method yields a long-lasting antibacterial carboxymethyl chitosan gel that combines long-term antibacterial properties, good tissue adhesion, procoagulant properties, and rapid hemostasis, and is injectable.

[0029] The long-lasting antibacterial carboxymethyl chitosan gel can be made into freeze-dried sponges, hydrogels, and other dressings or devices for clinical use.

[0030] The long-lasting antibacterial carboxymethyl chitosan gel can be used as a hemostatic agent and for treating and preventing trauma and disease, as well as for water-based skin treatments.

[0031] The present invention has the following advantages over the prior art:

[0032] 1) This invention grafts catechol-structured compounds onto carboxymethyl chitosan, which can improve the mechanical properties, tissue adhesion, procoagulant properties, and rapid hemostasis of carboxymethyl chitosan materials.

[0033] 2) This invention grafts protamine onto carboxymethyl chitosan, which can achieve long-lasting antibacterial, procoagulant and rapid hemostatic properties of carboxymethyl chitosan materials.

[0034] 3) The carboxymethyl chitosan gel provided by this invention, which has long-lasting antibacterial properties, tissue adhesion, rapid hemostasis, and is injectable, has the potential to be widely used as a novel biomaterial in biomedical fields such as cell, tissue engineering, and drug delivery. Attached Figure Description

[0035] Figure 1 This is a composite diagram of the PCMC in Example 1.

[0036] Figure 2 This is a graph showing the long-lasting antibacterial properties of the gel. In the graph, the antibacterial rate of E bacteria refers to the antibacterial rate of Escherichia coli; and the antibacterial rate of S bacteria refers to the antibacterial rate of Staphylococcus aureus.

[0037] Figure 3 The diagram shows the compressive stress-strain profile of the gel (A) and the tissue adhesion properties (B).

[0038] Figure 4 This is a diagram showing the procoagulant properties of the gel.

[0039] Figure 5 This is a diagram showing the hemostatic properties of the gel.

[0040] Figure 2-5 The modified carboxymethyl chitosan gel dressing mentioned refers to the DCMC-OHA-PCMC gel prepared in Example 1; the ordinary carboxymethyl chitosan gel dressing is the CMC-OHA gel prepared in Comparative Example 1. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0042] The carboxymethyl chitosan used in the examples has a molecular weight of 400,000 to 1,000,000 and a degree of deacetylation of ≥80%; the molecular weight of azide-polyethylene glycol-active ester is 2,000; and the molecular weight of dibenzocyclooctylene-polyethylene glycol-active ester is 649.69.

[0043] Example 1:

[0044] (1) Synthesis of oxidized hyaluronic acid (OHA)

[0045] Weigh 1g of hyaluronic acid and add it to 100mL of deionized water to prepare a 1wt% hyaluronic acid solution. Add 0.525g of sodium periodate to the hyaluronic acid solution and react at room temperature with stirring in the dark for 24 hours. Then add 1mL of ethylene glycol and stir at room temperature for 1 hour to quench unreacted sodium periodate. Finally, dialyze the final reactant in deionized water for 3 days and freeze-dry to obtain OHA.

[0046] Weigh 400 mg of the prepared OHA and dissolve it in 10 mL of deionized water to prepare a 4% OHA solution for later use.

[0047] (2) Synthesis of carboxymethyl chitosan-3,4-dihydroxybenzaldehyde (DCMC)

[0048] Weigh 1g of carboxymethyl chitosan and add it to 20mL of deionized water to prepare a 5wt% carboxymethyl chitosan solution, which is then placed in a three-necked flask. Weigh 550mg of 3,4-dihydroxybenzaldehyde and add it to 1mL of anhydrous ethanol. Stir well to prepare a 3,4-dihydroxybenzaldehyde-ethanol solution, which is then added to the carboxymethyl chitosan solution. The reaction is carried out for 1 hour under nitrogen protection and water bath heating to 55℃. After the reaction is completed, the reaction solution is added to anhydrous ethanol solution to precipitate the product DCMC.

[0049] Weigh 100 mg of DCMC and dissolve it in 1 mL of deionized water to prepare a 10% DCMC solution for later use.

[0050] (3) Synthesis of carboxymethyl chitosan-protamine (PCMC)

[0051] First, 500 mg of carboxymethyl chitosan was weighed and dissolved in deionized water. Then, 10 mg of azide-polyethylene glycol-active ester was added, and the mixture was reacted at room temperature for 24 h. After dialyzing, the mixture was freeze-dried to obtain carboxymethyl chitosan-azide for later use.

[0052] Then, the weighed protamine was dissolved in PBS buffer to prepare a 1 wt% solution. Then, dibenzocyclooctylene-polyethylene glycol-active ester was added, and after reacting at room temperature for 24 hours, 100 mg of carboxymethyl chitosan-azide was added, and after reacting at room temperature for another 24 hours, PCMC was obtained after dialysis and lyophilization.

[0053] Weigh 40 mg of the prepared PCMC and dissolve it in 1 mL of deionized water to prepare a 4% PCMC solution for later use.

[0054] (4) Preparation of long-acting antibacterial adhesive hemostatic injectable carboxymethyl chitosan gel dressing

[0055] First, take 0.2 mL of the OHA solution prepared in step (1) into a glass bottle; then take 0.1 mL of the DCMC solution prepared in step (2) and 0.1 mL of the PCMC prepared in step (3) into a reaction flask. The mass ratio of OHA, DCMC, and PCMC is 10:2.5:1. After thorough stirring, the DCMC-OHA-PCMC pregel solution is obtained. Finally, the solution is drawn up with a medical syringe and injected into a mold. After 1-3 minutes at room temperature, cross-linking is formed, thus obtaining the injectable carboxymethyl chitosan hydrogel (DCMC-OHA-PCMC gel) with long-lasting antibacterial properties, tissue adhesion, rapid hemostasis, and other properties described in this invention.

[0056] Comparative Example 1:

[0057] Preparation of ordinary carboxymethyl chitosan gel dressing (CMC-OHA): Mix 200 μL of 4% carboxymethyl chitosan solution with 200 μL of 4% oxidized hyaluronic acid solution until homogeneous, and form CMC-OHA gel after about 5 minutes.

[0058] Application Experiment 1: Antibacterial Performance Test of Injectable Carboxymethyl Chitosan Gel for Long-Term Antibacterial Adhesion and Hemostasis

[0059] 400 μL of the DCMC-OHA-PCMC gel sample prepared in Example 1 and 400 μL of ordinary carboxymethyl chitosan gel dressing (prepared in Comparative Example 1) (CMC-OHA) were respectively mixed with Escherichia coli (E. coli). 35320 TM The antibacterial properties of DCMC-OHA-PCMC were evaluated by co-culturing DCMC and Staphylococcus aureus (ATCC6538P) for 5 days, and the agar plate counting method was used on days 1, 3, and 5. The results are shown in the figure. Figure 2 A and B in the example.

[0060] As can be seen from the figure, compared with ordinary carboxymethyl chitosan gel dressing, modified carboxymethyl chitosan gel dressing grafted with positively charged protamine can better adsorb negatively charged bacteria through electrostatic interaction, and then cause the bacteria to rupture, thereby achieving an antibacterial effect.

[0061] Application Experiment 2: Mechanical Property Testing of Injectable Carboxymethyl Chitosan Gel with Long-Term Antibacterial Adhesive Hemostasis

[0062] The DCMC-OHA-PCMC gel sample prepared in Example 1 (n=3) The hydrogel was placed on a universal testing machine (model ELF3220) and compressed at a speed of 2 mm / min, with a compression rate of 80%. Data were saved and stress-strain diagrams were plotted. A common carboxymethyl chitosan gel dressing (prepared in Comparative Example 1) was tested and compared using the same method. Results are shown below. Figure 3 Figure A in the figure shows that, compared with ordinary carboxymethyl chitosan gel dressings, modified carboxymethyl chitosan gel dressings have a denser cross-linked network, thus exhibiting superior structural stability.

[0063] Application Experiment 3: Tissue Adhesion Properties Test of Injectable Carboxymethyl Chitosan Gel for Long-Term Antibacterial Adhesion and Hemostasis

[0064] (1) The tissue adhesion strength of the gel was tested using an overlap shear test with pigskin as the substrate.

[0065] (2) Cut fresh pigskin into rectangles (50mm × 20mm), and transfer the DCMC-OHA-PCMC pre-gel solution from Example 1 to subcutaneous fat. Then place another piece of pigskin on top, with an adhesion area of ​​4cm². 2 .

[0066] (3) After pressing with a 500g weight for about 24 hours, the adhesive strength was measured with a universal testing machine at a tensile rate of 20mm / min.

[0067] Ordinary carboxymethyl chitosan gel dressing (prepared as Comparative Example 1) was tested and compared using the same method. Results are shown below. Figure 3 Figure B in the diagram.

[0068] As can be seen from the figure, compared with ordinary carboxymethyl chitosan gel dressing, the modified carboxymethyl chitosan gel dressing grafted with catechol structure has richer interaction forces with the tissue it contacts, thus exhibiting a better tissue adhesion effect.

[0069] Application Experiment 4: Procoagulant Test of Injectable Carboxymethyl Chitosan Gel for Long-Term Antibacterial Adhesive Hemostasis

[0070] (1) Place 200 μL of the DCMC-OHA-PCMC gel prepared in Example 1 into a centrifuge tube and incubate in a water bath at 37°C for 5 minutes.

[0071] (2) Add 0.5 mL of anticoagulated rabbit blood (Shanghai Mairui Chemical Technology Co., Ltd.) and 0.1 mL of CaCl2 solution (0.2 mol / L). Invert the container every 20 seconds until the blood stops flowing completely, and record the time.

[0072] Ordinary carboxymethyl chitosan gel dressing (prepared as Comparative Example 1) was tested and compared using the same method. Results are shown below. Figure 4 As can be seen from the figure, compared with ordinary carboxymethyl chitosan gel dressing, the modified carboxymethyl chitosan gel dressing grafted with positively charged protamine can better accelerate the aggregation of red blood cells in the blood through electrostatic interaction, thus exhibiting superior procoagulant properties.

[0073] Application Experiment 5: Hemostatic Performance Test of Long-Acting Antibacterial Adhesive Hemostatic Injectable Carboxymethyl Chitosan Gel

[0074] (1) The hemostatic performance of the gel was tested using a rat (Experimental Animal Center of Southern Medical University) liver hemorrhage model.

[0075] (2) After anesthetizing the rats, weighed filter paper was placed under the liver and a 4mm biopsy punch was used to make a hole to produce bleeding.

[0076] (3) Immediately inject 400 μL of DCMC-OHA-PCMC gel from Example 1 into the bleeding site. Weigh the filter paper 2 minutes later and calculate the amount of bleeding.

[0077] Ordinary carboxymethyl chitosan gel dressing (prepared as Comparative Example 1) was tested and compared using the same method. Results are shown below. Figure 5 As can be seen from the figure, compared with ordinary carboxymethyl chitosan gel dressing, the modified carboxymethyl chitosan gel dressing grafted with positively charged protamine can better aggregate red blood cells at the wound site through electrostatic interaction, thus exhibiting superior hemostatic performance.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a long-lasting antibacterial carboxymethyl chitosan gel, characterized in that, Includes the following steps: Carboxymethyl chitosan-3,4-dihydroxybenzaldehyde was prepared by introducing catechol functional groups onto its surface using carboxymethyl chitosan as a raw material; carboxymethyl chitosan-azide was prepared by introducing azido groups onto its surface using carboxymethyl chitosan as a raw material; protamine sulfate-dibenzocyclooctyne was prepared by introducing dibenzocyclooctyne groups onto its surface using protamine sulfate as a raw material; carboxymethyl chitosan-protamine sulfate was further synthesized by copper-free catalytic azidoacetylene cycloaddition reaction of carboxymethyl chitosan-azide and protamine sulfate-dibenzocyclooctyne; finally, a gel material, namely the long-lasting antibacterial carboxymethyl chitosan gel, was synthesized by utilizing the oxidative crosslinking effect of catechol functional groups and the Schiff base reaction of carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, carboxymethyl chitosan-protamine sulfate and oxidized hyaluronic acid.

2. The preparation method according to claim 1, characterized in that, The preparation steps of the oxidized hyaluronic acid are as follows: periodate is added to the hyaluronic acid solution, the mass ratio of hyaluronic acid to periodate is 1:0.25-0.5, the reaction is carried out at room temperature in the dark for 8-24 hours, quenched with ethylene glycol, dialyzed with deionized water and freeze-dried under vacuum to obtain oxidized hyaluronic acid.

3. The preparation method according to claim 1, characterized in that, The preparation steps of carboxymethyl chitosan-3,4-dihydroxybenzaldehyde are as follows: Under nitrogen protection, a 3,4-dihydroxybenzaldehyde-ethanol solution is added to an aqueous solution of carboxymethyl chitosan, with a mass ratio of carboxymethyl chitosan to 3,4-dihydroxybenzaldehyde of 1:1.25-1.

75. The mixture is heated to 50-60℃ and reacted for 1-3 hours. After precipitation with anhydrous ethanol and vacuum drying, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde is obtained.

4. The preparation method according to claim 1, characterized in that, The preparation steps of the carboxymethyl chitosan-azide are as follows: carboxymethyl chitosan is dissolved in deionized water, azide-polyethylene glycol-active ester is added, and the reaction is carried out at room temperature for 12-24 hours. The mass ratio of azide-polyethylene glycol-active ester to carboxymethyl chitosan is 1:25-50. After dialysis with deionized water, the carboxymethyl chitosan-azide is obtained by vacuum freeze-drying.

5. The preparation method according to claim 1, characterized in that, The preparation steps of the carboxymethyl chitosan-protamine are as follows: Protamine is dissolved in PBS buffer solution, and dibenzocyclooctyn-polyethylene glycol-active ester is added. The reaction is carried out at room temperature for 12-24 hours, with the mass ratio of dibenzocyclooctyn-polyethylene glycol-active ester to protamine being 1:20-40. After reacting for 12-24 hours, carboxymethyl chitosan-azide is added, and the reaction is carried out at room temperature for 12-24 hours, with the mass ratio of carboxymethyl chitosan-azide to protamine-dibenzocyclooctyn being 1:0.5-1. After dialyzing with deionized water, the carboxymethyl chitosan-protamine is obtained by vacuum freeze-drying.

6. The preparation method according to claim 1, characterized in that, After obtaining oxidized hyaluronic acid and carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, the further steps are as follows: Oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde, and carboxymethyl chitosan-protamine are dissolved in deionized water, with a mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde to carboxymethyl chitosan-protamine protein of 10: The solution of the three components was mixed thoroughly at a ratio of 2.5:1, and crosslinked at room temperature for 1-3 minutes to obtain the long-lasting antibacterial carboxymethyl chitosan gel.

7. The preparation method according to any one of claims 1-6, characterized in that, The preparation method of the long-acting antibacterial carboxymethyl chitosan gel includes the following steps: (1) Prepare a hyaluronic acid solution, add periodate, and react in the dark for 8-24 hours; after quenching with ethylene glycol, dialyze and freeze dry to obtain oxidized hyaluronic acid; (2) Prepare a carboxymethyl chitosan solution, then add a 3,4-dihydroxybenzaldehyde-ethanol solution, heat to 50-60℃, react for 1-3 hours under nitrogen protection, and precipitate in anhydrous ethanol solution to obtain carboxymethyl chitosan-3,4-dihydroxybenzaldehyde. (3) Prepare a carboxymethyl chitosan solution, then add azide-polyethylene glycol-active ester, react at room temperature for 12-24 hours, dialyze and freeze dry to obtain carboxymethyl chitosan-azide; (4) Dissolve protamine in PBS solution, then add dibenzocyclooctylene-polyethylene glycol-active ester, and react at room temperature for 12-24 h; then add carboxymethyl chitosan-azide obtained in step (3), react at room temperature for 12-24 h, and lyophilize after dialysis to obtain carboxymethyl chitosan-protamine. (5) Dissolve the prepared oxidized hyaluronic acid, carboxymethyl chitosan-3,4-dihydroxybenzaldehyde and carboxymethyl chitosan-protamine in deionized water, mix the three solutions evenly in a mold, and crosslink at room temperature for 1-3 minutes to form the long-lasting antibacterial carboxymethyl chitosan gel.

8. The preparation method according to claim 7, characterized in that, The dibenzocyclooctyne-polyethylene glycol-active ester is at least one of dibenzocyclooctyne-tetraethylene glycol-active ester, dibenzocyclooctyne-pentaethylene glycol-active ester, or dibenzocyclooctyne-dodecylethylene glycol-active ester; The periodate mentioned is sodium periodate or potassium periodate; The carboxymethyl chitosan has a molecular weight of 400,000 to 1,000,000 and a degree of deacetylation of ≥80%.

9. A long-lasting antibacterial carboxymethyl chitosan gel prepared by the method according to any one of claims 1-8.

10. The use of the long-acting antibacterial, adhesive, hemostatic, injectable carboxymethyl chitosan gel according to claim 9 in the preparation of freeze-dried sponges or hydrogels.

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