Application of hydrogel in preparation of external preparation for inhibiting excessive activation of fungal infection wound MMP-9 and promoting skin regeneration

Abstract

The invention discloses an application of hydrogel in preparation of an external preparation for treating and inhibiting excessive activation of fungal infected wound MMP-9 and promoting skin regeneration, and belongs to the technical field of biomedical materials. The invention discloses application of hydrogel to preparation of an external preparation for inhibiting excessive activation of matrix metalloproteinase-9 in fungal infection wounds, the hydrogel comprises sodium alginate and a copper ion solution, and the mass fraction of the copper ion solution is 0.01%-0.05%; the inhibition of MMP-9 over-activation and the promotion of skin regeneration are realized by down-regulating protein expression of MMP-9 in wound tissues. The copper ion hydrogel can significantly reduce the MMP-9 protein expression level within 14 days and greatly reduce continuous degradation of a matrix, so that the pathological process of fungal infection-inflammation amplification-MMP-9 activation-ECM disintegration-wound surface chronic is blocked. The application is suitable for treating human and pet skin wounds which are repeatedly ruptured due to fungal infection and are not healed for a long time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to the application of a hydrogel in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration. Background Technology

[0002] Chronic, non-healing wounds (such as diabetic foot ulcers, venous ulcers, pressure ulcers, and deep fungal infections) pose a significant challenge in clinical and veterinary practice. One of their core pathological features is an imbalanced wound microenvironment, characterized by persistent inflammation and abnormally elevated protease activity, particularly matrix metalloproteinases. 9 (MMP) 9) Overactivation and activation.

[0003] MMP 9. Under physiological conditions, MMPs participate in moderate ECM remodeling; however, in chronic wounds, their activity increases uncontrollably due to persistent infection (such as fungal infection) and inflammation. Excessive MMPs... 9. It irreversibly degrades key ECM components such as newly formed collagen (type I and type III), fibronectin, and laminin, and inactivates growth factors, leading to fragile granulation tissue, arrested epithelialization, and causing the wound to fall into a state of "synthesis". The vicious cycle of "degradation" imbalance manifests clinically as repeated ulceration and slow healing of wounds.

[0004] Currently, the mainstream treatment strategy for chronic fungal infections focuses on systemic or topical use of antifungal drugs (such as azoles and echinocandins) to eliminate the pathogens. However, many such wounds fail to heal even after the fungal burden has decreased, the root cause of which lies in MMPs. The 9-driven ECM degradation process has been activated and is running continuously. While existing conventional dressings (such as gauze, foam dressings, and alginate dressings) and some active dressings (such as silver-containing dressings) have some anti-infection or exudate absorption effects, they all lack resistance to MMPs. The specific regulatory capacity of this key destructive target, 9, is crucial. Therefore, developing a method that can precisely intervene in "infection" is essential. MMP 9 Novel treatment strategies for the "ECM disintegration" pathway are of great clinical significance for overcoming the challenges of chronic wound healing. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide the application of a hydrogel in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, thereby addressing the limitation of existing technologies in specifically targeting and inhibiting MMP-9 in fungal infections. 9. Technical problems related to over-activation.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides the application of a hydrogel in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration. The preparation method of the hydrogel includes: preparing a copper ion solution with a mass fraction of 0.01% to 0.05%, adding 0.1% to 1% sodium alginate per 100 mL of copper ion solution by mass fraction, adjusting the pH value to 4.5 to 5.5, and after defoaming and sterilization, obtaining a hydrogel that regulates the collagen ratio and reduces the risk of scarring in infected wounds.

[0007] Preferably, the hydrogel further includes hyaluronic acid, with 0.1% to 1% hyaluronic acid added per 100 mL of copper ion solution by mass fraction.

[0008] Preferably, the pH value is adjusted to 4.5-5.5 by adding an alkaline regulator.

[0009] Preferably, the alkalinity regulator is triethanolamine.

[0010] Preferably, copper gluconate is dissolved to obtain a copper ion solution with a mass fraction of 0.01% to 0.05%.

[0011] Preferably, the mass fraction of the copper ion solution is configured to be 0.05%.

[0012] Further preferred, the pH value is adjusted to 5.0~5.4.

[0013] Preferably, the pH value is adjusted to 5.3.

[0014] Preferably, the topical preparation includes a drug or a medical dressing.

[0015] Preferably, the dosage form of the topical preparation includes any one of topical dosage forms, suppositories, and injectable preparations.

[0016] Preferably, the topical preparation is used in human or companion animals for skin wounds that are chronically non-healing or have a low risk of scarring due to fungal infection.

[0017] The primary objective of this invention is to provide a novel medical application for copper ion hydrogels, namely, their use in the preparation of MMPs for specifically inhibiting fungal infections in wounds. 9. Application in over-activated topical formulations. A further object of the present invention is to, through the above-mentioned uses, block the pathological degradation of ECM, reverse the chronicity process of wounds, and ultimately promote the regeneration of high-quality skin with intact structure and good function.

[0018] The core pathological mechanism targeted by this invention (MMP-9 overactivation) is as follows: Fungal infection continuously stimulates the immune system, leading to an abnormal increase in the expression and activity of MMP-9 (matrix metalloproteinase-9) in the wound microenvironment. Excessive MMP-9 excessively degrades the extracellular matrix (ECM) and growth factors, destroying the scaffold of newly formed tissue and trapping the wound in a cycle of "inflammation-degradation-failure to heal." This invention explicitly targets the application of hydrogels to "inhibit MMP-9 overactivation," meaning that its design principle is to directly correct this specific and critical pathological imbalance, rather than simply providing antifungal or general moisturizing effects.

[0019] The hydrogel is used to prepare a topical formulation that inhibits the overactivation of matrix metalloproteinase-9 in fungal infected wounds. The hydrogel contains sodium alginate and a copper ion solution, wherein the mass fraction of the copper ion solution is 0.01% to 0.05%. The inhibition of MMP-9 overactivation and promotion of skin regeneration are achieved by downregulating the protein expression of MMP-9 in wound tissue.

[0020] The topical preparations include drugs or medical dressings.

[0021] The hydrogel can downregulate the protein expression of MMP-9 in wound tissue, reducing the relative expression level of MMP-9 / β-actin in wound tissue to below 1.0. The hydrogel can reduce the expression levels of pro-inflammatory factors TNF-α and IL-1β in wound tissue by at least 40%. The hydrogel can achieve a wound closure rate of over 90% on day 14 by blocking the pathological process of fungal infection-inflammatory response-MMP-9 overactivation-extracellular matrix degradation.

[0022] Preferably, the fungal infection includes at least one of Candida albicans, Candida parapsilosis, and Malassezia.

[0023] The hydrogel was able to increase the expression level of type I collagen in the wound tissue by at least 50% on day 14 compared with the negative control group, indicating that the application effectively promoted functional skin regeneration.

[0024] More preferably, the copper ion solution has a mass fraction of 0.05%.

[0025] A further preferred embodiment of the preparation method comprises the following specific steps: S1: Dissolve copper gluconate in water and stir until homogeneous to obtain a copper ion solution with a mass fraction of 0.01%~0.05%; S2: After adding sodium alginate to the copper ion solution, the carboxyl group (-COO-) of sodium alginate reacts with the divalent copper ions (Cu) in the copper ion solution. 2+ Coordination occurs, forming Cu-O cross-linking bonds; S3: Then add hyaluronic acid to make the hydrogel system more stable; S4: Finally, triethanolamine is added, the pH is adjusted to 4.5~5.5, air bubbles are removed by ultrasound, and after aseptic filling, a hydrogel is obtained that inhibits the overactivation of MMP-9 in infected wounds and promotes skin regeneration.

[0026] The dosage form of the topical preparation includes any one of the following: topical dosage form, suppository, and injectable preparation.

[0027] More preferably, the topical formulation is a spray gel.

[0028] The topical preparation is effective in humans or companion animals for chronic, recurrent ulceration and prolonged non-healing caused by fungal infections, often accompanied by MMPs. 9. Application in skin wounds with abnormally elevated activity in pathological conditions.

[0029] Further preferred is the use of the above-mentioned topical preparation in skin wounds caused by chronic fungal infection in human patients with underlying diseases.

[0030] More preferably, the underlying disease includes any one or more of diabetes, immunodeficiency, and peripheral vascular disease.

[0031] More preferably, the above-mentioned topical preparation is used in skin lesions caused by fungal infections, such as licking dermatitis, interdigital dermatitis, ear infections, and chronic skin ulcers in dogs or cats.

[0032] The intended use is achieved through the following mechanism: the copper ion hydrogel releases copper ions locally at the wound site, reducing the infection load through its antifungal effect, and simultaneously modulating the function of inflammatory cells and downregulating pro-inflammatory factors (such as TNF). α, IL 1β) expression, thereby reducing MMP at its source. 9. Induction of synthesis; more importantly, it can directly or indirectly significantly inhibit MMP in wound tissue. Protein expression and enzyme activity of MMP 9 9 / β The relative expression level of actin is regulated to below 1.0, preferably below 0.8, thereby effectively protecting the newly formed ECM from damage and providing a stable microenvironment for orderly collagen deposition and epithelial crawling.

[0033] The application provided by this invention, through the use of a hydrogel formed by sodium alginate and a copper ion solution with a mass fraction of 0.01%~0.05%, directly intervenes in the excessive activation of MMP-9 in fungal-infected wounds, fundamentally breaking the vicious cycle of "infection-MMP-9 overactivation-tissue damage-healing inhibition," and providing a new mechanism for promoting skin regeneration. Specifically, sodium alginate, as a natural polymer, forms a hydrophilic and breathable three-dimensional network gel scaffold, providing a moist healing environment, absorbing exudate, and acting as a sustained-release carrier to help copper ions exert a continuous and controllable effect on the wound; the key to achieving targeted function is the specific low mass fraction copper ion solution (0.01%~0.05%). Copper ions have a dual mechanism of action within this mass fraction range: firstly, their inherent antifungal activity can directly inhibit fungal growth, reducing infection stimulation from the source; secondly, copper ions can regulate cell behavior, specifically downregulating MMP-9 expression, while simultaneously promoting collagen synthesis and cross-linking. The combination of these two elements allows the hydrogel to both fight infection and precisely regulate the enzyme balance of the healing microenvironment.

[0034] By downregulating overactivated MMP-9, this hydrogel fundamentally protects the integrity of the wound basement membrane and newly formed ECM, providing a stable environment for keratinocyte migration, vascular endothelial cell proliferation, and fibroblast collagen synthesis. This allows the healing process to smoothly transition from a stage of continuous inflammatory destruction to a stage of tissue regeneration and remodeling, ultimately achieving the fundamental goal of promoting skin regeneration. This invention is the first to focus the application of copper ion hydrogels on inhibiting MMP. 9. This invention addresses a key barrier to chronic wound healing by overactivating this barrier, rather than relying solely on its antibacterial properties, and fundamentally intervenes in the core driving pathways of wound chronicity. In vitro and in vivo experiments have demonstrated that the copper ion hydrogel of this invention can significantly reduce MMPs in fungal-infected wounds. The expression level of 9 is increased, while the deposition of ECM components such as type I collagen is enhanced, forming a complete "anti-inflammatory" effect. Reduce MMP 9 Evidence chain for the role of "ECM protection".

[0035] By setting a quantitative indicator of a relative MMP-9 / β-actin expression level below 1.0, an objective and detectable molecular biological standard was provided to determine whether excessive activation was inhibited, demonstrating the precision and effectiveness of the intervention. The significant reduction in the expression levels of pro-inflammatory factors TNF-α and IL-1β indicates that, in principle, this hydrogel not only inhibits MMP-9 but also alleviates excessive inflammation upstream, synergistically creating a microenvironment conducive to regeneration. The quantitative healing target of a wound closure rate exceeding 90% on day 14 confirms the ultimate tissue repair efficacy brought about by MMP-9 inhibition.

[0036] Furthermore, the fungal infection is selected from at least one of Candida albicans, Candida parapsilosis, or Malassezia, indicating that the application has broad applicability to common pathogenic fungi, including Candida albicans, thus expanding its application scenarios.

[0037] The hydrogel, by controlling the mass fractions of copper ion solution and sodium alginate, as well as the pH value of the hydrogel, ensures stable chemical properties and the expected sustained-release characteristics of copper ions, which is the material basis for achieving the functions of inhibiting MMP-9 and promoting regeneration. This invention constructs a smart hydrogel with dual functions of antifungal properties and precise regulation of the wound microenvironment through the combination of copper ions and sodium alginate. It can inhibit the key destructive protease (MMP-9), protect the ECM, and promote granulation tissue maturation even with a low mass fraction of copper ions, and has high biocompatibility.

[0038] Furthermore, the hydrogel also contains hyaluronic acid, which further optimizes the rheological properties of the hydrogel, enhances its adhesion and retention time on the wound surface, thereby ensuring the continuous action of the active ingredients and improving the treatment effect from the perspective of dosage form.

[0039] The preparation method of the hydrogel ensures the effectiveness and safety of the product from the perspective of production process principles. Using copper gluconate as the copper source ensures good solubility and high bioavailability; adjusting to a specific pH value ensures product stability and function; degassing treatment makes the gel uniform; and aseptic treatment is a fundamental guarantee of the safety of medical products. This method has a clear principle and can stably prepare hydrogels with the aforementioned functions.

[0040] The aforementioned topical formulation, by concretizing the hydrogel product into any of the following dosage forms—topical, suppository, and injectable—significantly improves the convenience of clinical use and patient compliance, especially for irregular or difficult-to-bandage wounds, allowing the aforementioned principles to be applied in a more user-friendly manner in actual treatment. This hydrogel can reduce the expression level of MMP-9 in Candida albicans-infected wounds by more than 60% and provide a stable ECM microenvironment for cell migration and tissue regeneration, offering a novel treatment strategy for achieving a fundamental transformation of wounds from "repeated ulceration" to "stable regeneration."

[0041] The topical formulation provided by this invention precisely defines its target indication: specific refractory wounds caused by fungal infection and accompanied by abnormally elevated MMP-9 levels. This clarifies its unique clinical positioning and market value, emphasizing its therapeutic advantage against the specific pathological mechanism of MMP-9 overactivation. Its core mechanism lies in significantly reducing the protein expression level of MMP-9 in wound tissue (e.g., reducing the MMP-9 / β-actin ratio from 1.89 to 0.74), thereby blocking the vicious cycle of "fungal infection → inflammation → MMP-9 overactivation → extracellular matrix (ECM) disintegration → wound chronicity," and promoting high-quality healing.

[0042] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of a hydrogel in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration. The key to this invention is the introduction of a specific concentration (0.01%-0.05%) of copper ions. In principle, copper ions are a natural inhibitor of MMP-9, directly inhibiting the enzyme's catalytic activity by binding to zinc ions at the active site of MMP-9. This achieves "specific targeted inhibition" of the target site (MMP-9), precisely solving the problems in the prior art. Sodium alginate not only serves as a gel matrix, but its mechanism lies in its ability to cross-link with calcium ions in wound exudate, forming a physical barrier and providing a moist healing environment. More importantly, sodium alginate degradation products of specific molecular weights have been shown to regulate collagen synthesis, promoting the production of type III collagen (more similar to normal skin) while inhibiting the excessive deposition of type I collagen (associated with scarring).

[0043] Adjusting the pH to a slightly acidic level (4.5-5.5) is suitable for the wound microenvironment and can maintain the optimal valence state of copper ions (Cu). 2+ (and biological activity, to ensure its effectiveness in inhibiting MMP-9.)

[0044] Furthermore, hyaluronic acid is a high-molecular-weight thickener and adhesive. Its addition significantly improves the viscoelasticity, bioadhesion, and stability of the hydrogel, enabling it to adhere more persistently to moist, irregular infected wounds. This ensures continuous contact between copper ions and the gel matrix and the wound, thereby prolonging and enhancing the inhibitory and healing effects on MMP-9.

[0045] Furthermore, it is clearly demonstrated that the pH value is precisely controlled by adding an alkaline regulator (preferably triethanolamine). Triethanolamine provides a gentle adjustment, avoiding potential damage to the active ingredient (copper ions) and wound tissue caused by drastic local pH changes, ensuring that the final product remains stably within the most effective weakly acidic range, and guaranteeing the reliability of the core mechanism (copper ions inhibiting MMP-9).

[0046] Furthermore, copper gluconate has good water solubility and high biocompatibility, and can stably release Cu in solution. 2+ This ensures that copper ions exist in an easily usable form and are uniformly dispersed in the gel, which is the foundation for the realization of the technical solution.

[0047] Furthermore, the copper ion concentration is preferably 0.05%, and the pH is preferably 5.3. In principle, this may be the experimentally validated parameter point that achieves the optimal balance between inhibiting MMP-9 activity and cell biocompatibility. 0.05% copper ions may provide the strongest enzyme inhibitory efficacy, while pH 5.3 is closest to the acidic protective film value of healthy skin surface, which is most conducive to wound re-epithelialization and anti-infection.

[0048] Furthermore, hydrogels are defined as substances that can be used to prepare drugs or medical dressings, which broadens their product forms and application scenarios.

[0049] Furthermore, it was clarified that the formulation can be made into various dosage forms (topical, suppository, injectable), which in principle can meet the treatment needs of fungal infection wounds in different locations (body surface, cavity, subcutaneous), increasing the universality of the treatment.

[0050] Furthermore, by specifying the target population as humans or companion animals, it addresses a common clinical challenge for both humans and pets, and emphasizes its application for wounds that are slow to heal or have a low risk of scarring, directly addressing the technical problem it aims to solve. Attached Figure Description

[0051] Figure 1 The images show the morphological appearance of copper sulfate hydrogels with different mass fractions according to the present invention. Figure 2 This is a graph showing the antibacterial data of the hydrogel of this invention; Figure 3 These are macroscopic photographs of the wounds of mice in each group at different time points according to the present invention; Figure 4 The MMP of each group of wound tissues in this invention at 14 days Quantitative statistical analysis of the relative expression levels of protein 9 (bar chart); Figure 5 H&E stained sections of wound tissue from each group of the present invention at 7 and 14 days; Figure 6 This is a data graph of the pro-inflammatory factors of the present invention; Figure 7 This is a bar chart showing the quantitative statistical analysis of the relative expression levels of type I collagen in this invention. Figure 8 This is an immunohistochemical image of type I collagen from this invention. Detailed Implementation

[0052] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0053] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0054] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0055] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0056] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0057] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0058] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" represents weight percentage, "parts" represents parts by weight, and "ratio" represents weight proportion.

[0059] Example 1 A hydrogel product includes the following steps: S1: Add 0.01% copper gluconate to sterile ultrapure water to dissolve it into a copper ion solution; S2: Take 100 mL of copper ion solution and add 0.1% sodium alginate to it to make it the initial copper ion hydrogel; S3: To increase the stability of the initial copper ion hydrogel, 0.1% hyaluronic acid was added to make the hydrogel more stable. S4: In order to adjust the acidity and alkalinity of the copper ion-loaded hydrogel, triethanolamine, an alkalinity regulator, was added to the hydrogel to adjust the pH value to 4.5. S5: Finally, air bubbles are removed by ultrasound, and the product is aseptically filled to obtain a hydrogel that can be used to inhibit the overactivation of MMP-9 in fungal infected wounds and promote skin regeneration.

[0060] Example 2 A hydrogel product includes the following steps: S1: Add 0.03% copper gluconate to sterile ultrapure water to dissolve it into a copper ion solution; S2: Take 100 mL of copper ion solution and add 0.5% sodium alginate to it to make it the initial copper ion hydrogel. S3: To increase the stability of the initial copper ion hydrogel, 0.5% hyaluronic acid by mass is added to make the hydrogel more stable. S4: In order to adjust the acidity and alkalinity of the copper ion-loaded hydrogel, triethanolamine, an alkalinity regulator, was added to the hydrogel to adjust the pH value to 5.0. S5: Finally, air bubbles are removed by ultrasound, and the product is aseptically filled to obtain a hydrogel that can be used to inhibit the overactivation of MMP-9 in fungal infected wounds and promote skin regeneration.

[0061] Example 3 A hydrogel product includes the following steps: S1: Add 0.05% copper gluconate to sterile ultrapure water to dissolve it into a copper ion solution; S2: Take 100 mL of copper ion solution and add 1% sodium alginate to it to make it the initial copper ion hydrogel. S3: To increase the stability of the initial copper ion hydrogel, 1% hyaluronic acid by mass is added to make the hydrogel more stable. S4: In order to adjust the acidity and alkalinity of the copper ion-loaded hydrogel, triethanolamine, an alkalinity regulator, was added to the hydrogel to adjust the pH value to 5.5. S5: Finally, air bubbles are removed by ultrasound, and the product is aseptically filled to obtain a hydrogel that can be used to inhibit the overactivation of MMP-9 in fungal infected wounds and promote skin regeneration.

[0062] To verify the therapeutic effect of copper ion hydrogel in inhibiting MMP-9 overactivation and promoting healing in fungal infected wounds, the following experiment was conducted: 1. In vitro antifungal test: like Figure 1 As shown, three hydrogels containing copper ion solutions of different mass fractions (0.01%, 0.03%, and 0.05%) were prepared, and 2 × 10⁻⁶ hydrogels were prepared. 6 CFU / mL Candida albicans seed culture was prepared, and then hydrogels of three different mass fractions of copper ion solutions were added to the seed culture, with final copper ion mass fractions of 0.01%, 0.03%, and 0.05%, respectively. A blank control group was set up. After treatment at 30℃ and 200rpm for 3 hours, the total number of colonies was determined by gradient drop plate experiment to investigate the antibacterial effect of copper ion solution hydrogels on Candida albicans.

[0063] Result: Passed Figure 2 The results showed that the hydrogels with three different mass fractions of copper ion solutions all had significant inhibitory effects on Candida albicans, and within a certain range, the higher the mass fraction of copper ion solution, the faster the early antibacterial rate.

[0064] 2. Animal in vivo experimental verification: (1) Establishment of a chronic fungal infection wound model: Animals: Kunlun white mice, male, 8-10 weeks old.

[0065] Model creation: Two full-thickness skin defects, 1 cm in diameter, were created on the backs of mice. The wounds were immediately inoculated with Candida albicans suspension (1×10⁻⁶). 6 CFU / wound), cover with a sealing dressing for 24 hours to establish a stable infection.

[0066] (2) Grouping and processing (n=6): G1: Normal, undamaged group.

[0067] G2: Negative control group: PBS was applied to the wound daily.

[0068] G3: Positive control group (routine antifungal treatment): Apply 1% clotrimazole cream to the wound daily.

[0069] G4: Example 2 group (0.01%) G5: Example 3 group (0.03%) (Change the dressing once a day for 14 consecutive days) (3) Observation and detection indicators: Healing process: Take photos daily and calculate wound closure rate.

[0070] Tissue sample collection: Animals were euthanized on days 7 and 14, and tissues from the wound and surrounding tissues were collected.

[0071] Histopathological analysis (H&E staining): Observe and score inflammatory cell infiltration, granulation tissue thickness and maturity, and epithelial regeneration, paying particular attention to whether ECM "disintegration" occurs at the wound edge.

[0072] Key protein expression detection (immunohistochemistry / Western Blot): Detection targets: MMP-9, type I collagen.

[0073] Internal reference protein: β-actin.

[0074] Methods: Total protein was extracted from wound tissue on day 14 and subjected to Western blot analysis.

[0075] Objective: To directly verify whether the core mechanism of this invention—whether it significantly "downregulates MMP-9 protein expression" and "promotes collagen deposition"—is effective.

[0076] Results: The copper-loaded hydrogel provided by this invention, verified through in vitro and in vivo experiments, exhibits multiple synergistic regulatory effects on the healing of fungal-infected skin wounds. Its core mechanism and therapeutic effects are as follows: Figure 4 The results showed that in the infected wound model, the hydrogel group of the present invention (Example 2) significantly reduced the relative expression level of MMP-9 / β-actin in the wound tissue from approximately 1.89 to approximately 0.74. This result clearly confirms that the hydrogel of the present invention can effectively downregulate the protein expression of MMP-9 in the wound and inhibit its abnormal activation. Histopathological analysis ( Figure 5 H&E staining showed that wounds treated with the hydrogel of this invention exhibited significantly reduced inflammatory cell infiltration and more mature granulation tissue. Further molecular detection ( Figure 6 This indicates that hydrogels can significantly downregulate the expression levels of key pro-inflammatory factors TNF-α and IL-1β in the wound site, thereby inhibiting inflammation-driven healing barriers at their source. Figure 7 and 8 As shown, the hydrogel treatment of this invention significantly increased the expression and deposition of type I collagen in wound tissue, a key marker of stable extracellular matrix (ECM) reconstruction, providing a robust scaffold for wound epithelialization. Macroscopic observation of the healing process ( Figure 3The results showed that wounds treated with the hydrogel of this invention healed faster, achieving a significantly higher closure rate on day 14 than the negative control group, and the entire healing process was smooth with no recurrence of ulceration. This directly reflects that the wound has broken free from the chronic cycle and entered a stable regenerative repair track. This comprehensively demonstrates that the hydrogel of this invention effectively blocks ECM degradation by inhibiting the overactivation of MMP-9, thus promoting high-quality healing.

[0077] Example 4 This embodiment verifies the downregulation effect on the expression levels of pro-inflammatory factors: Unlike Example 1, this example aims to specifically quantify the inhibitory effect of hydrogel on the expression of pro-inflammatory factors in fungal infected wounds.

[0078] Samples were prepared according to the preparation method of Example 2 (0.03% copper ion mass fraction hydrogel), and the same animal models, grouping (G1-G5) and treatment methods as in the specification were used.

[0079] Samples were collected on day 7, and the relative mRNA expression levels of TNF-α and IL-1β in the wound tissue were detected by real-time quantitative PCR.

[0080] Results: Compared with the negative control group (G2), the mRNA expression levels of TNF-α and IL-1β in wounds treated with the 0.03% copper ion hydrogel of this invention (G5) decreased by approximately 50.2% and 48.7%, respectively. This data clearly confirms that the hydrogel of this invention can reduce the expression levels of pro-inflammatory factors by at least 40%.

[0081] Example 5 This embodiment verifies the effect of promoting wound closure rate: Unlike Example 1, this example aims to accurately determine the wound closure rate on day 14.

[0082] Samples were prepared according to the preparation method of Example 2 (0.03% copper ion mass fraction hydrogel), and the same animal models, grouping (G1-G5) and treatment methods as in the specification were used.

[0083] Record the wound area daily and calculate the wound closure rate using the formula (initial wound area - current wound area) / initial wound area × 100%.

[0084] Results: By day 14 of treatment, the wound closure rate of the negative control group (G2) was 71.5%, while the wound treated with the 0.03% copper ion hydrogel of this invention (G5) achieved a closure rate of 92.3%.

[0085] Example 6 This embodiment verifies the inhibitory effect on other fungal species: Unlike Example 1, this example aims to verify the inhibitory effect of the hydrogel of the present invention on Candida glabrata or Malassezia.

[0086] Hydrogels with copper ion mass fractions of 0.01%, 0.03%, and 0.05% were prepared according to the methods in Examples 1, 2, and 3, respectively.

[0087] Referring to the above-mentioned in vitro antifungal test method, the test strains were replaced with standard strains of Candida glabrata and Malassezia, and the antibacterial rate of each mass fraction of hydrogel was measured after 3 hours of treatment.

[0088] Results: All three mass fractions of copper ion hydrogels showed significant antibacterial effects against *Candida glabrata* and *Malassezia*, and the antibacterial effect increased with increasing copper ion concentration. Specifically, the hydrogel with a 0.05% copper ion solution showed an inhibition rate of 99.2% against *Candida glabrata* and 98.5% against *Malassezia*.

[0089] Example 7 A hydrogel product includes the following steps: S1: Add 0.03% copper gluconate to sterile ultrapure water and stir until completely dissolved to obtain a copper ion solution; S2: Take 100 mL of the above copper ion solution and add 0.5% sodium alginate by mass, stirring continuously. During this process, the carboxyl groups of sodium alginate coordinate with the divalent copper ions in the solution, gradually forming an initial copper ion-alginic acid hydrogel with a three-dimensional network structure; S3: To adjust the pH of the hydrogel system, add triethanolamine, an alkalinity regulator, to the hydrogel obtained in step S2 while stirring until the pH of the system is precisely adjusted to 5.0. S4: The pH-adjusted hydrogel is subjected to ultrasonic treatment to remove air bubbles introduced during the preparation process; S5: The degassed hydrogel is filled under aseptic conditions to obtain the finished hydrogel product used to regulate the collagen ratio and reduce the risk of infected wound scars.

[0090] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The application of a hydrogel in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that, The preparation method of the hydrogel includes: preparing a copper ion solution with a mass fraction of 0.01%~0.05%, adding 0.1%~1% sodium alginate per 100mL of copper ion solution by mass fraction, adjusting the pH value to 4.5~5.5, and after defoaming and sterilization treatment, obtaining a hydrogel that regulates the collagen ratio and reduces the risk of scarring of infected wounds.

2. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The hydrogel also includes hyaluronic acid, with 0.1% to 1% hyaluronic acid added per 100 mL of copper ion solution by mass fraction.

3. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The pH value can be adjusted to 4.5-5.5 by adding an alkaline regulator.

4. The application of the hydrogel according to claim 3 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The alkalinity regulator is triethanolamine.

5. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... Copper gluconate was dissolved to obtain a copper ion solution with a mass fraction of 0.01% to 0.05%.

6. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The mass fraction of the copper ion solution was configured to be 0.05%.

7. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... Adjust the pH to 5.

3.

8. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The topical preparations include drugs or medical dressings.

9. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The dosage form of the topical preparation includes any one of the following: topical dosage form, suppository, and injectable preparation.

10. The application of the hydrogel according to claim 1 in the preparation of a topical formulation for treating fungal infections by inhibiting MMP-9 overactivation and promoting skin regeneration, characterized in that... The topical preparation is used in humans or companion animals for skin wounds that are chronically non-healing or have a low risk of scarring due to fungal infection.

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