Preparation method and application of zinc ion cross-linked hydrogel

Abstract

The invention relates to the field of biomedical materials, in particular to a novel zinc ion cross-linked hydrogel, which is prepared by the following steps: preparing a hyaluronic acid solution with the oxidation degree of 40%, then preparing a chitosan solution, violently stirring and mixing the two solutions according to the volume ratio of 2: 8 under the acidic condition, and standing and reacting at 37 DEG C for 3-5 minutes. The material is constructed based on chemical crosslinking of oxidized hyaluronic acid and chitosan, zinc ions are introduced to form a metal coordination network, and the material is endowed with good structural stability, drug loading performance and biological activity. The hydrogel can effectively load and slowly release antibacterial and antifungal drugs, and has good local tissue compatibility while inhibiting pathogenic microorganisms. Experiments on cell and tissue levels prove that the hydrogel can induce activation of a mitochondrial autophagy pathway, further regulate oxidative stress and improve mitochondrial membrane potential, so that the hydrogel shows enhanced tissue repair and anti-recurrence effects in urinary system infection treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, specifically a hydrogel formed by crosslinking oxidized hyaluronic acid and chitosan, with zinc ions added to enhance its antimicrobial activity and structural stability. This hydrogel exhibits both bacterial and antifungal properties and can activate mitochondrial function in bladder and kidney tissue, demonstrating significant efficacy in treating urinary tract infections and other urinary system diseases. Background Art

[0002] Urinary tract infection is one of the most common infectious diseases in clinical practice and is widely seen in women, the elderly, and immunosuppressed people. Its main pathogens are Gram-negative rods, especially Escherichia coli. In recent years, with the widespread use of broad-spectrum antibiotics and the prolonged indwelling of urinary catheters, the proportion of opportunistic fungal infections such as Candida albicans in urinary tract infections has increased significantly, and the incidence of mixed bacterial infections has also gradually increased. The current treatment of UTIs is mainly based on systemic antibiotics or antifungal drugs. However, in the clinical scenario of bacterial-fungal co-infection, a single targeted strategy is difficult to achieve comprehensive control, which can easily lead to repeated treatment and prolonged infection. At the same time, the abuse of antibiotics has led to the widespread spread of drug-resistant strains, becoming a key factor affecting efficacy. The above challenges highlight the urgent need to develop an efficient treatment system that has both antibacterial and antifungal capabilities and adapts to the complex infectious microecology to improve the breadth and durability of clinical treatment.

[0003] Regarding material carriers, while some traditional natural or synthetic polymers can achieve sustained drug release, they often present challenges such as high immunogenicity, poor biocompatibility, or the irritation of metabolic degradation products during tissue contact. These factors can activate the innate immune response, leading to a significant upregulation of proinflammatory cytokines (such as interleukin-6, tumor necrosis factor-α, and interleukin-1β), thereby disrupting the tissue's original repair microenvironment. More critically, in the urinary system, particularly in high-energy-consuming tissues such as renal tubular epithelial cells and bladder wall smooth muscle cells, these inflammatory responses and some antibiotic metabolites can damage mitochondria, core organs of energy metabolism. Studies have shown that inflammation-related oxidative stress can trigger changes in mitochondrial membrane permeability, loss of membrane potential, and inactivation of respiratory chain complexes, ultimately leading to impaired ATP synthesis and activation of apoptotic signaling. For example, in patients with recurrent pyelonephritis or chronic cystitis, recurrent local tissue infections and drug stimulation contribute to mitochondrial dysfunction, a fundamental obstacle to effective tissue repair. If the infection and metabolic functions cannot be regulated synchronously, simple antibacterial treatment can often only temporarily relieve symptoms and has poor long-term efficacy.

[0004] Compared with existing injectable hydrogels based on natural polymers, the current technologies have certain limitations in the following aspects: It is difficult to strike a balance between injectability and gel-forming properties: when traditional natural polymers such as hyaluronic acid and chitosan are used alone, it is difficult to ensure good injection fluidity while quickly forming in situ in the body; some systems that require ultraviolet light or high-temperature cross-linking have great irritation to tissues, limiting their clinical application.

[0005] Insufficient biocompatibility data or potential irritation: Although some synthetic polymer hydrogels have good mechanical properties, their monomers or degradation products may be potentially toxic to tissues, and most have not been systematically verified for their biosafety in urinary tract system models.

[0006] Short residence time in the body and limited window of action: Conventional hydrogel materials degrade rapidly in the body, resulting in a short drug release cycle, which makes it difficult to cover the drug action duration required for infection treatment (usually 7 days or more).

[0007] Therefore, in the treatment of urinary tract infections, the development of a novel biomaterial that can inhibit microorganisms while modulating inflammatory responses and promoting mitochondrial function recovery has important clinical application value. This not only helps to improve the thoroughness of anti-infection treatment, but also has the potential to break the vicious cycle of traditional treatment, "antibacterial-inflammation-recurrence," and provide a new treatment strategy for chronic and recurrent urinary tract infections. Summary of the Invention

[0008] The present invention provides a novel zinc ion cross-linked hydrogel, which is constructed based on the chemical cross-linking of oxidized hyaluronic acid and chitosan, and introduces zinc ions to form a metal coordination network, giving the material good structural stability, drug loading performance and biological activity.

[0009] To achieve the above object, the technical solution adopted by the present invention is: a method for preparing a zinc ion cross-linked hydrogel, comprising the following steps: (1) Preparation of 40% hyaluronic acid solution: Sodium hyaluronate was weighed and dissolved in distilled water to prepare a solution with a concentration of 20 mg / mL. Sodium periodate was slowly added at a molar ratio of 1:0.4. The solution was allowed to react in the dark for 4 hours. The solution was then dialyzed using a 3 kilodalton dialysis bag for 48 hours. The solution was freeze-dried to obtain oxidized hyaluronic acid powder. The oxidized hyaluronic acid powder was dissolved in distilled water to prepare a solution with a concentration of 20 mg / mL. Zinc chloride with a concentration of 10 mg / mL was added to obtain a zinc chloride concentration of 20 μl / mL in the final hydrogel. (2) Preparation of chitosan solution: Dissolve chitosan in distilled water to a concentration of 20 mg / mL; (3) Cross-linking reaction: The two solutions prepared in step (1) and step (2) were mixed vigorously with stirring at a volume ratio of 2:8 under acidic conditions, and allowed to react at 37°C for 3-5 minutes.

[0010] Furthermore, the acidic condition is pH 5.5-6.0.

[0011] The zinc ion cross-linked hydrogel prepared by the present invention can be used to prepare drugs for treating urinary tract infections. During the hydrogel construction process, an antibacterial drug (such as levofloxacin) or an antifungal drug (such as fluconazole) is pre-dissolved in an oxidized hyaluronic acid solution and fully mixed to achieve drug loading.

[0012] In the invention, the aldehyde groups (–CHO) on the OHA molecular chain react with the primary amine groups (–NH2) on the NOCC segments through a Schiff base reaction, forming imine bonds (C=N), thereby achieving covalent crosslinking between the molecules. This crosslinking mechanism gives the resulting hydrogel a well-defined three-dimensional network structure and high chemical stability.

[0013] Zinc ions coordinate with amino groups (–NH₂) in NOCC molecules and some carboxyl groups (–COO⁻) or residual aldehyde groups (–CHO) in the OHA segments, further stabilizing the three-dimensional network structure through non-covalent interactions. This metal coordination cross-linking mechanism not only enhances the mechanical strength and molding stability of the gel, but also imparts superior dilution resistance and environmental adaptability, providing key support for its application in fluid environments such as the urinary system.

[0014] The beneficial technical effects of the present invention are: The oxidized hyaluronic acid-chitosan composite hydrogel provided by the present invention has the following advantages: (1) Excellent injectability and gel stability: The hydrogel exhibits good shear thinning properties in rheological performance tests, making it easy to administer drugs by injection in a minimally invasive manner. At the same time, it can quickly restore viscoelasticity in the in vivo environment to form a stable three-dimensional structure, which is conducive to the in situ formation and retention of the material in the target organ.

[0015] (2) Structurally controllable three-dimensional network micromorphology: Transmission electron microscopy and scanning electron microscopy analysis showed that the hydrogel formed a uniformly distributed porous three-dimensional network structure, effectively improving the drug loading capacity and providing a stable diffusion channel for the drug, making it suitable for the construction of a sustained-release system. Electron microscopy observations before and after drug loading showed that its microporous structure remained intact and the pore size distribution did not change significantly, indicating that it has good mechanical stability and structural retention ability, and can adapt to various drug encapsulation requirements without causing structural collapse or fluctuations in release performance.

[0016] (3) The material composition is clear and the cross-linking reaction is fully controllable: Infrared spectroscopy analysis shows that the cross-linking reaction between oxidized hyaluronic acid and chitosan is carried out through a reversible Schiff base bond. The functional group changes of each component in the system are clear, the reaction conditions are mild and easy to control, and it is suitable for large-scale preparation.

[0017] (5) Good biocompatibility and safety: In vitro experiments using urinary tract epithelial cells as a model showed that the hydrogel did not inhibit cell migration in the scratch test and did not induce significant apoptosis in the flow cytometry test, indicating that it had no obvious toxic reaction at the cellular level and had good biosafety. The main raw materials are hyaluronic acid and chitosan derivatives from natural sources, which can be biodegraded into non-toxic products in the body, reducing long-term safety risks and meeting the material use standards for clinical transformation.

[0018] (6) Stable performance in vivo and long-lasting drug efficacy: In vivo imaging of small animals showed that the hydrogel could remain stable in the target tissue for more than 7 days after injection. Urine drug concentration monitoring showed that it still maintained a level higher than the minimum inhibitory concentration on the 7th day, indicating that it has long-term controlled-release drug delivery function.

[0019] The present invention provides a novel zinc ion-crosslinked hydrogel constructed by chemically crosslinking oxidized hyaluronic acid and chitosan. Zinc ions are introduced to form a metal coordination network, endowing the material with excellent structural stability, drug-loading properties, and bioactivity. This hydrogel can effectively load and sustainably release antibacterial and antifungal drugs (such as levofloxacin and fluconazole), inhibiting pathogenic microorganisms while exhibiting good local tissue compatibility. Experimental validation at the cellular and tissue levels has demonstrated that this hydrogel can induce activation of the mitochondrial autophagy pathway, thereby modulating oxidative stress and improving mitochondrial membrane potential, thereby demonstrating enhanced tissue repair and anti-recurrence effects in the treatment of urinary tract infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the technical roadmap of the hydrogel preparation method in Example 1.

[0022] Figure 2 This is the rheological test result of the blank hydrogel in Example 1.

[0023] Figure 3 This is the infrared analysis result of the oxidized hyaluronic acid in Example 1.

[0024] Figure 4 This is the infrared analysis result of chitosan in Example 1.

[0025] Figure 5 This is the electron microscopy result of the hydrogel in Example 1.

[0026] Figure 6 This is the in vivo imaging of small animals obtained from the in vivo hydrogel experiment in Example 1.

[0027] Figure 7 Example 1 is a diagram showing the results of a scratch test on urinary tract epithelial cells treated with hydrogel.

[0028] Figure 8 The figure is a representative result of the Western blotting experiment in Example 2.

[0029] Figure 9 It is a representative diagram of the results of the mouse urine spot experiment in Application Example 1.

[0030] Figure 10 This is an electron microscopic image of mouse kidney and bladder tissue in Application Example 1.

[0031] Figure 11 This is a typical electron micrograph of kidney tissue after treatment with the hydrogel of Example 2, with a local magnified image on the right. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1

[0034] like Figure 1 As shown, a method for preparing a zinc ion cross-linked hydrogel comprises the following steps: (1) Preparation of 40% hyaluronic acid solution: Sodium hyaluronate was weighed and dissolved in distilled water to prepare a solution with a concentration of 20 mg / mL. Sodium periodate was slowly added at a molar ratio of 1:0.4. The solution was allowed to react in the dark for 4 hours. The solution was then dialyzed using a 3 kilodalton dialysis bag for 48 hours. The solution was freeze-dried to obtain oxidized hyaluronic acid powder. The oxidized hyaluronic acid powder was dissolved in distilled water to prepare a solution of 20 mg / mL. A zinc chloride solution with a solubility of 10 mg / mL was added to obtain a zinc chloride concentration of 20 μl / mL in the final hydrogel. (2) Preparation of chitosan solution: Dissolve chitosan in distilled water to a concentration of 20 mg / mL; (3) Cross-linking reaction: The two solutions prepared in step (1) and step (2) were mixed vigorously with stirring at a volume ratio of 2:8 under acidic conditions, and allowed to react at 37°C for 3-5 minutes.

[0035] During the hydrogel construction process, antibacterial drugs (such as levofloxacin) or antifungal drugs (such as fluconazole) are pre-dissolved in the oxidized hyaluronic acid solution and fully mixed to achieve drug loading.

[0036] The experimental verification is as follows: Experimental Grouping: 6-8 week old female C57BL / 6 mice (weighing 18-22 g) were used. All animal experiments were conducted in an SPF-grade facility in accordance with the relevant regulations approved by the Laboratory Animal Ethics Committee. Mice were anesthetized with isoflurane inhalation (3% induction, 1.5-2% maintenance) to ensure sedation and painlessness throughout the experimental procedures.

[0037] The experimental animals were divided into four groups: 1. Blank control group: No infection model was established, and only an equal volume of phosphate buffer was injected.

[0038] 2. Infection model group: establish a cystitis model by infusing phosphate buffered saline without drugs and gel.

[0039] 3. Free drug group: Based on the infection model, free antibiotics (such as levofloxacin solution) were instilled into the bladder.

[0040] 4. Hydrogel treatment group: Based on the infection model, composite cross-linked hydrogel loaded with levofloxacin was perfused.

[0041] Use common bacterial strains (such as Escherichia coli, 1×10 7 Colony forming units per ml; Candida albicans, 1×10 6 A bacterial / fungal cystitis model was established by instilling 20 μL of infection suspension (CFU / mL) into the bladder through the urethra. Therapeutic instillation was performed 24 hours later.

[0042] Under inhalation anesthesia, a lubricated polyethylene-10 tubing was slowly inserted through the urethra into the bladder. After emptying the residual urine, the pre-prepared drug solution or hydrogel precursor solution (40-50 μL) was slowly instilled at a rate of approximately 2 μL / s. The catheter was retained for 15 minutes after instillation and then slowly removed. To prevent leakage, the mouse was positioned supine for 10 minutes to ensure in situ cross-linking and formation of the hydrogel within the bladder cavity.

[0043] Verify the results like Figure 2 As shown, rheological experiments showed that the hydrogel had good initial fluidity and injection properties, and the gradually enhanced support after injection helped to stably form in the bladder and maintain structural integrity.

[0044] Infrared spectroscopy confirmed that oxidized hyaluronic acid ( Figure 3 ) and chitosan ( Figure 4) and the changes in characteristic absorption peaks support the successful construction of the material skeleton, and no other impurity peaks are observed, indicating that the chemical structure of the system is clear and stable.

[0045] like Figure 5 As shown, electron microscopy observation shows that the hydrogel has a uniform porous three-dimensional network structure, which is conducive to drug loading and sustained release; after the addition of drugs, its three-dimensional porous structure remains stable and the porosity does not change significantly.

[0046] The results of small animal in vivo imaging experiments are as follows Figure 6 As shown, in vivo imaging of small animals and in vitro drug release concentration analysis verified that the hydrogel can exist stably in vivo and still maintain effective antibacterial efficacy on the 7th day.

[0047] Using human urothelial cells as a model, the effect of hydrogel on cell migration ability was evaluated by in vitro scratch healing assay. Figure 7 As shown in the figure, the material did not significantly inhibit cell migration; at the same time, Annexin V-FITC / PI double staining flow cytometry was used to detect the cell apoptosis rate. The results showed that there was no significant difference in the early and late apoptosis ratios between the hydrogel-treated group and the control group, verifying that it has good biocompatibility and low toxicity at the cellular level.

[0048] Example 2

[0049] Transcriptome analysis of bladder tissues in the normal group, infection group, and drug-loaded hydrogel treatment group showed that the drug-loaded hydrogel treatment group exhibited significant improvements in multiple mitochondrial function-related pathways, especially in the enrichment analysis of important biological processes such as mitochondrial protein complexes, respiratory chains, and electron transport.

[0050] Specifically, gene ontology enrichment analysis revealed that the drug-loaded hydrogel treatment group significantly upregulated the expression of genes related to cellular energy metabolism, such as mitochondrial small / large subunits, mitochondrial protein complexes, etc., suggesting that this treatment method helps to improve cellular energy production and metabolic processes. Kyoto Encyclopedia of Genes and Genomes enrichment analysis further showed that the mitochondrial biogenesis pathway was enriched in the treatment group, emphasizing the potential role of drug-loaded hydrogels in cell repair and anti-oxidative stress. Reaction pathway analysis also confirmed that drug-loaded hydrogel treatment enhanced pathways related to extracellular matrix organization and collagen formation, further proving that it can promote bladder tissue repair and restore function. The enrichment of pathways such as the cellular respiratory chain and mitochondrial translation also strongly supports its important role in the restoration of mitochondrial function.

[0051] These findings indicate that the drug-loaded hydrogel not only has antibacterial effects, but can also effectively improve mitochondrial dysfunction caused by bacterial infection and further promote tissue repair.

[0052] To verify the results of transcriptome analysis, we further detected the expression levels of key proteins related to mitochondrial function and autophagy. Figure 8 Compared with the infection group, the drug-loaded hydrogel-treated group showed upregulation of the mitochondrial membrane protein mitochondrial outer membrane translocation protein 20, suggesting improved mitochondrial structure and function. Simultaneously, expression of the autophagy marker autophagy receptor protein P62 decreased, while autophagy-related gene 55 was significantly upregulated, indicating enhanced autophagic fluidity. Furthermore, levels of phosphatase and tensin homolog-inducing kinase and parkin were elevated in the treatment group, suggesting activation of the mitophagy pathway, which helps clear infection-induced mitochondrial damage.

[0053] Example 3

[0054] This embodiment provides a drug-loaded hydrogel with synergistic antibacterial and cell-protective functions, taking the delivery of levofloxacin as an example, which alleviates tissue damage associated with bacterial urinary tract infection.

[0055] 1. Specific preparation steps of drug-loaded (levofloxacin) hydrogel: (1) Preparation of oxidized hyaluronic acid (oxidation degree 40%) Weigh 2.0 g of sodium hyaluronate and dissolve it in 200 mL of distilled water. Slowly add sodium periodate to maintain a molar ratio of 1:0.4 (i.e., 0.4 mol of sodium periodate per 1 mol of sugar unit). Incubate in the dark for 4 hours. Then, dialyze using a 3 kDa dialysis tubing for 48 hours and freeze-dry to obtain oxidized hyaluronic acid powder.

[0056] (2) Drug-loaded oxidized hyaluronic acid solution: Dissolve oxidized hyaluronic acid in distilled water to a concentration of 20 mg / mL, and add levofloxacin (0.1 mg / mL) and 10 mg / mL zinc chloride (20 μl). (3) Chitosan solution: Chitosan was dissolved in distilled water to a concentration of 20 mg / mL without adding drugs or metal ions.

[0057] (4) The two solutions were mixed in a volume ratio of 2:8 (oxidized hyaluronic acid: chitosan), and after being fully shaken, a translucent injectable hydrogel was formed at 37°C. The gelation time was about 3-5 minutes.

[0058] 2. Animal Experiment Verification Urine spot test results Figure 9 As shown, the hydrogel drug-loaded group showed a decrease in the number of urine spots and an increase in the area of ​​single spots at the beginning of treatment, suggesting that it can effectively relieve the symptoms of frequent urination and improve bladder emptying function.

[0059] The urine white blood cell count results showed that the bacterial infection group decreased significantly on the third day after the drug-loaded hydrogel intervention, and the decrease was better than that in the free drug group, indicating that inflammation control was more rapid and effective.

[0060] In the bacterial urinary tract infection model, the serum interleukin-6, tumor necrosis factor-α and C-reactive protein levels in the drug-loaded hydrogel group were significantly lower than those in the free drug group, and the inhibitory effect lasted until the observation endpoint, indicating that it has a stable regulatory effect on systemic inflammatory response.

[0061] In a bacterial infection model, immunohistochemistry was used to examine the expression levels of interleukin-6 and tumor necrosis factor-α in bladder and kidney tissues. The results showed that the drug-loaded hydrogel group significantly reduced the expression of inflammatory factors in both tissues compared to the free drug group, with a greater degree of inhibition in the bladder, indicating a stronger local anti-inflammatory regulatory effect on the primary infection site.

[0062] The electron microscopy results are as follows Figure 10 As shown, the normal group showed intact renal pelvic and bladder epithelial structures, dense cell connections, and normal mitochondrial morphology. After infection, nuclear condensation, mitochondrial structural disorder, and inflammatory infiltration were observed. The free drug group only partially improved shedding, while mitochondrial damage remained significant. The drug-loaded hydrogel group showed reduced mitochondrial damage and significantly enhanced autophagic clearance and lysosomal maturation, suggesting its superiority in alleviating cellular stress damage.

[0063] Application Example 4

[0064] This embodiment provides an injectable oxidized hyaluronic acid / chitosan hydrogel synergistically loaded with the antifungal drug fluconazole and zinc ions, which is used to alleviate fungal urinary tract infections caused by Candida albicans.

[0065] 1. The specific preparation method includes: (1) Oxidized hyaluronic acid and chitosan solutions were prepared at a concentration of 20 mg / mL respectively.

[0066] (2) Fluconazole was dissolved in the oxidized hyaluronic acid solution to a final concentration of 0.2 mg / mL; zinc chloride was added at the same time to make its concentration in the final hydrogel 2 μg / mL.

[0067] (3) Mix the two substances in a volume ratio of 2:8 (oxidized hyaluronic acid:chitosan) and form a stable injectable hydrogel at 37°C with a gelation time of about 3 minutes.

[0068] 2. Verification method A Candida albicans infection model was established in C57BL / 6 mice by intravesical injection. The intervention group was injected with fluconazole-zinc ion hydrogel, while the control group was given free fluconazole solution or drug-free blank gel.

[0069] After the infection model was established, urine was collected regularly and colony counts were performed to assess antifungal efficacy. The results showed that the drug-loaded hydrogel group significantly reduced the number of Candida albicans in urine early in treatment, and the reduction persisted until the end of the observation period, outperforming the free fluconazole group, indicating a more stable and long-lasting antifungal effect in vivo. This result was consistent with the in vitro zone of inhibition results.

[0070] The immunohistochemical detection results of interleukin-6 and tumor necrosis factor in bladder and kidney tissues showed that the expression level of inflammatory factors in the drug-loaded hydrogel group was significantly lower than that in the free drug group, indicating that this form of administration can effectively alleviate local inflammatory responses related to infection.

[0071] Transmission electron microscopy results are as follows Figure 11 As shown, the bladder and renal pelvis tissues in the infection group showed significant nuclear condensation, mitochondrial morphology disruption, and accumulation of autophagosomes. After treatment with the drug-loaded hydrogel, cellular structural integrity improved, mitochondrial morphology normalized, and autophagy-related structures increased, suggesting enhanced tissue repair and cellular homeostasis recovery.

[0072] The hydrogel delivery method effectively slowed down the weight loss trend of mice during infection intervention and improved their overall survival rate, further supporting the stability and safety of its therapeutic effect.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing zinc ion cross-linked hydrogel, characterized in that the steps include: (1) Preparation of 40% hyaluronic acid solution: Sodium hyaluronate was weighed and dissolved in distilled water to prepare a solution with a concentration of 20 mg / mL. Sodium periodate was slowly added at a molar ratio of 1:0.

4. The solution was allowed to react in the dark for 4 hours. The solution was then dialyzed using a 3 kDa dialysis bag for 48 hours. The solution was freeze-dried to obtain oxidized hyaluronic acid powder. The oxidized hyaluronic acid powder was dissolved in distilled water to prepare a solution with a concentration of 20 mg / mL. Zinc chloride with a concentration of 10 mg / mL was added to obtain a zinc chloride concentration of 20 μl / mL in the final hydrogel. (2) Preparation of chitosan solution: Dissolve chitosan in distilled water to a concentration of 20 mg / mL; (3) Cross-linking reaction: The two solutions prepared in step (1) and step (2) were mixed vigorously with stirring at a volume ratio of 2:8 under acidic conditions, and allowed to react at 37°C for 3-5 minutes.

2. The method according to claim 1, characterized in that The acidic condition is pH 5.5-6.

0.

3. The zinc ion cross-linked hydrogel prepared according to the method of claim 1.

4. Use of the zinc ion cross-linked hydrogel according to claim 3 in preparing a medicament for treating urinary tract infection.

5. The use according to claim 4, wherein during the hydrogel construction process, antibacterial drugs or antifungal drugs are selected and dissolved in the oxidized hyaluronic acid solution to achieve drug loading.

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