Drug delivery system for promoting healing of diabetic wound as well as preparation method and application of drug delivery system

Abstract

The invention relates to the field of biological medicine, in particular to a drug delivery system for promoting healing of diabetic wounds and a preparation method and application of the drug delivery system. A more intelligent and more efficient drug delivery system is created, the limitation that traditional injectable hydrogel can only serve as a physical barrier is overcome, multiple benefits brought by natural active ingredients such as P1 are fully utilized, and meanwhile the drug delivery system has the following advantages that 1, the drug delivery system has excellent injection performance and in-situ forming capacity; 2, a drug release mechanism capable of intelligently responding to pH change is achieved; 3, a strong immune microenvironment regulation effect is achieved; 4, antibacterial and antioxidant properties are achieved; 5, the dependence on expensive factors and cell therapy can be reduced; 6, the mechanical property and the biocompatibility can be enhanced; and 7, the clinical application process is simplified. In conclusion, the drug delivery system provided by the invention shows huge potential in the aspect of management of chronic wounds such as diabetic ulcer and the like due to the unique composition and technical advantages of the drug delivery system.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug delivery system for promoting diabetic wound healing, and a preparation method and application thereof. Background Art

[0002] With the dramatic increase in diabetes cases worldwide, chronic wounds, as a common complication, have attracted great attention. Studies have shown that a hyperglycemic environment leads to microvascular damage, nerve damage, and impaired immune function, which significantly weaken the ability of wound healing and lead to persistent, non-healing chronic ulcers. In addition, excessive reactive oxygen species (ROS) are the main obstacle to diabetic wound healing. These ROS cause permanent and irreversible oxidative stress to cell tissues and promote the proliferation of M1 phenotype macrophages, thereby exacerbating the inflammatory response. Furthermore, exposed wounds are particularly susceptible to bacterial infection, which can aggravate hypoxia and inflammation and worsen wound conditions. Therefore, the development of a wound dressing with immunomodulatory function and multifunctional (with ROS scavenging, antibacterial and anti-inflammatory capabilities) has important practical significance in the clinical management of chronic diabetic wounds. This new dressing not only needs to provide a physical barrier, but also needs to actively participate in the treatment process to improve the wound healing environment and combat the above-mentioned adverse factors. Specifically, the ideal diabetic wound dressing should be able to remove excess ROS and reduce the damage to cell tissues caused by oxidative stress; provide effective antibacterial protection to prevent the occurrence and development of bacterial infections; reduce inflammatory responses and promote wound healing by regulating the immune system; support the formation of new blood vessels to ensure that the wound area receives adequate supply of oxygen and nutrients.

[0003] At present, although traditional dressing materials such as gauze and cotton can seal wounds and show certain absorption properties for exudates, they are difficult to fix, have poor air permeability, and are prone to bacterial growth, all of which are obviously not conducive to the healing of diabetic wounds. Traditional dressings cannot keep the wound moist and delay wound healing; wound granulation tissue easily grows into the mesh of the dressing, and it is easy to adhere to the wound when changing the dressing, damaging the new granulation tissue. In contrast, hydrogel dressings, as a new generation of wound dressings, have been widely used in modern medicine. Due to its porous structure that promotes oxygen exchange, excellent moisture retention and biocompatibility, hydrogel dressings provide a moist environment for wounds that is conducive to healing. In addition, hydrogel dressings have the following advantages: 1. Promote oxygen exchange: The porous structure of hydrogels allows oxygen to penetrate into the wound site, which is essential for supporting cell metabolism and promoting healing; 2. Excellent moisturizing ability: Maintaining a moist environment around the wound helps prevent scab formation, reduce pain and accelerate the healing process; 3. Good biocompatibility: Hydrogels are usually made of biomaterials, are mild and non-irritating to the skin, and reduce the risk of allergies or adverse reactions; 4. Strong adaptability: Some types of hydrogels can fit irregularly shaped wound surfaces to ensure full coverage and support; 5. Antibacterial properties: Some hydrogels contain antibacterial ingredients or have antibacterial properties, which can help prevent infection. For example, compared with traditional dressings, new chitosan-based dressings not only have good biocompatibility and coagulability, but also enhance their antibacterial effects by adding special substances (such as oxidized carboxymethyl cellulose). In addition, multifunctional hydrogel dressings can be loaded with active substances and change the composition and structure of hydrogels to give them stronger tissue adhesion, antibacterial and anti-inflammatory abilities, thereby more effectively promoting the healing of chronic wounds.

[0004] Injectable hydrogels have been praised by many researchers for their self-healing properties, flexibility, and ability to form in situ. They can achieve closure and repair of various irregular wounds. Such hydrogels can be injected into the body through a needle and quickly solidify at the target location to form a shape that fits the local anatomical structure, providing support for the wound and promoting the healing process. However, despite the high technical complexity and innovation of injectable hydrogel dressings, their intrinsic capabilities are basically limited to passively providing a physical barrier and absorbing wound exudate, and lack the function of active therapeutic intervention at the wound site. However, the therapeutic effect of these hydrogel dressings depends largely on the active substances they encapsulate. For example, although the introduction of protein factors, cell therapy, and anti-inflammatory drugs has achieved certain results, the large-scale application of these methods is challenged due to the unstable release and inactivation of active ingredients, coupled with high costs. In addition, in order to enhance the therapeutic effect of hydrogels, researchers are also exploring the addition of natural products with specific biological activities. However, a dressing that can effectively promote diabetic wound healing has not yet been found. Summary of the invention

[0005] The purpose of the present invention is to provide a drug delivery system for promoting diabetic wound healing and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a drug delivery system for promoting diabetic wound healing, wherein the drug delivery system comprises oxidized dextran, methacrylylated gelatin and drug-loaded UiO-66-NH wrapped by oxidized chondroitin sulfate. 2 Nanoparticles; the drug-loaded UiO-66-NH 2 The drug loaded in the nanoparticles is Paris polyphylla saponin l;

[0008] The drug-loaded UiO-66-NH 2 The preparation method of the nanoparticles comprises the steps of: 2 The nanoparticles are mixed to obtain the drug-loaded UiO-66-NH 2 Nanoparticle steps.

[0009] Preferably, the drug-loaded UiO-66-NH 2 The preparation method of the nanoparticles comprises the steps of: 2 and Paris polyphylla saponin 1 to obtain the drug-loaded UiO-66-NH 2 Nanoparticle steps.

[0010] The present invention provides a method for preparing the above-mentioned drug delivery system, comprising the following steps:

[0011] Oxidized chondroitin sulfate solution and drug-loaded UiO-66-NH 2 The nanoparticles are mixed and dissolved in water to obtain the drug-loaded UiO-66-NH 2 Nanoparticle solution; the drug-loaded UiO-66-NH 2 The drug loaded in the nanoparticles is Paris polyphylla saponin l;

[0012] The drug-loaded UiO-66-NH 2 The nanoparticle solution, the oxidized dextran solution and the methacrylylated gelatin solution are mixed to obtain the drug delivery system.

[0013] Preferably, the drug-loaded UiO-66-NH 2 The preparation method of the nanoparticles comprises the steps of: 2 and Paris polyphylla saponin 1 to obtain the drug-loaded UiO-66-NH 2 Nanoparticle steps.

[0014] Preferably, the UiO-66-NH 2 The mass ratio of the polyphylla saponin 1 to the polyphylla saponin 1 is 2:1;

[0015] Preferably, the oxidized chondroitin sulfate in the oxidized chondroitin sulfate solution and the drug-loaded UiO-66-NH 2 The mass ratio of the nanoparticles was 2:1.

[0016] Preferably, the final concentration of oxidized dextran in the drug delivery system is 4%, the final concentration of methacrylylated gelatin is 15%, and the drug-loaded UiO-66-NH 2 The final concentration of nanoparticles was 0.2%.

[0017] The present invention provides application of the above-mentioned drug delivery system in preparing a product for promoting diabetic wound healing.

[0018] Preferably, the product comprises a drug; and the drug comprises a hydrogel excipient.

[0019] Further preferably, the hydrogel excipient comprises an injectable hydrogel dressing.

[0020] The present invention has developed an innovative injectable hydrogel dressing based on an amino-functionalized zirconium-based metal organic framework (UiO-66-NH 2, hereinafter referred to as MOF). This new hydrogel dressing can reshape the microenvironment of wound tissue and accelerate the healing process of chronic diabetic wounds. In order to maintain the activity of the loaded drug and enhance its sustained release effect, UiO-66-NH 2 The nanospheres were loaded with Paris polyphylla saponin (Pl) through hydrogen bonding and electrostatic adsorption, and then encapsulated by oxidized chondroitin sulfate (OCS) as a "gatekeeper" through Schiff base reaction ( Figure 1 A in the figure) to obtain OCS@MOF@Pl nanoparticles. OCS@MOF@Pl nanoparticles are combined with oxidized dextran (oxidized dextran, DEXO) and methacryloyl gelatin (GelMA) to produce elastic hydrogels (GelDE-OCS@MOF@Pl) in situ through Schiff base reaction and free radical polymerization process ( Figure 1 B). In this composite structure, OCS@MOF@Pl nanoparticles are anchored in the GelDE hydrogel network through Schiff base reaction and hydrogen bonding interaction, which not only enhances the toughness of the composite network hydrogel, but also improves the sustained release efficiency of the loaded drug. The hydrogel exhibits excellent injectability and the ability to form in situ at the wound site, which can seal wounds of various irregular shapes. In addition, GelDE-OCS@MOF@Pl hydrogel also has the ability to regulate the release of Pl according to changes in environmental pH, which can effectively eliminate bacteria, neutralize reactive oxygen species, reduce inflammatory responses, and promote the polarization of M1 macrophages to M2 phenotypes, thereby accelerating the process of wound re-epithelialization and angiogenesis ( Figure 1 C in the figure). This injectable nanocomposite hydrogel accelerates wound healing by regulating the immune environment, resisting bacterial infection, resisting oxidative stress, and reducing inflammatory response, showing great potential in the management of chronic diabetic wounds. Moreover, MOFs materials themselves can be used as ideal carriers for drug delivery due to their high specific surface area and porous structure, especially when sustained release of drugs is required. For example, amino acid-functionalized zirconium-based metal organic framework UiO-66-NH 2 After being covered by molecular imprinting polymer, it is used as an adsorbent, showing good selectivity and reusability. In this study, MOF not only serves as a drug carrier, but its unique physicochemical properties also give the hydrogel additional functional properties, such as better mechanical strength and sensitive response to environmental changes. It can be seen that the new injectable hydrogel dressing proposed in the present invention combines the amino-functionalized zirconium-based metal organic framework UiO-66-NH 2Together with Paris polyphylla saponin 1, it reshapes the microenvironment of wound tissue and accelerates the healing of chronic diabetic wounds. This hydrogel can not only form closure in situ on the wound surface, but also has the ability to rationally adjust drug release according to changes in environmental pH, thereby effectively eradicating bacteria, eliminating reactive oxygen species, inhibiting inflammatory responses, and accelerating epithelialization and angiogenesis.

[0021] The present invention provides a product for promoting diabetic wound healing, wherein the product comprises the above-mentioned drug delivery system.

[0022] The present invention discloses the following technical effects:

[0023] The present invention creates a smarter and more efficient drug delivery system that overcomes the limitation of traditional injectable hydrogels as physical barriers only and fully utilizes the multiple benefits of natural active ingredients like Pl, with the following advantages:

[0024] 1. Excellent injection performance and in-situ forming ability: The drug delivery system (GelDE-OCS@MOF@Pl hydrogel) has excellent injection properties and can be formed in situ on the wound surface, suitable for closing and repairing various irregularly shaped wounds. This feature allows it to be more accurately applied to difficult or complex wound sites without surgical incision.

[0025] 2. Drug release mechanism with intelligent response to pH changes: GelDE-OCS@MOF@Pl hydrogel can intelligently regulate the release of Paris polyphylla saponin l(Pl), effectively removing bacteria, neutralizing reactive oxygen species, and inhibiting inflammatory responses according to changes in environmental pH. This pH responsiveness helps to better adapt to different stages of wound healing and provide sustained and appropriate therapeutic effects.

[0026] 3. It has a strong immune microenvironment regulation effect: through the synergistic effect of its components, it can promote the transformation of M1 macrophages to M2 macrophages, thereby accelerating the process of wound re-epithelialization and angiogenesis. This not only promotes the speed of wound healing, but also improves the quality of healing and reduces the risk of scar formation.

[0027] 4. Antibacterial and antioxidant properties: UiO-66-NH 2 The combination of nanoparticles and PI endows GelDE-OCS@MOF@Pl hydrogel with excellent antibacterial ability and the ability to fight oxidative stress, which is particularly critical for chronic infections common in diabetic patients.

[0028] 5. Reduce dependence on expensive factors and cell therapy: Traditional methods often require the use of expensive cytokines, cell therapy or other drugs, which may be accompanied by toxic side effects. The hydrogel of the present invention relies on the characteristics of the material itself to achieve the purpose of treatment, reducing costs and improving safety.

[0029] 6. Enhanced mechanical properties and biocompatibility: Due to the use of oxidized dextran (DEXO), methacryloyl gelatin (GelMA) and oxidized chondroitin sulfate (OCS), the re-epithelialized hydrogel exhibits good mechanical strength and flexibility while maintaining a high degree of biocompatibility and degradability, which is beneficial to tissue regeneration.

[0030] 7. Simplified clinical application process: As a ready-to-use product, GelDE-OCS@MOF@Pl hydrogel can be directly injected into the wound, which simplifies the clinical operation steps, improves treatment efficiency, and provides doctors with a more convenient option.

[0031] In summary, the GelDE-OCS@MOF@Pl hydrogel provided by the present invention has shown great potential in the management of chronic wounds such as diabetic ulcers due to its unique composition and technical advantages, and is expected to become an important breakthrough in the field of wound care in the future.

[0032] The present invention also provides the application of GelDE-OCS@MOF@Pl hydrogel in diabetic wound healing, and verifies its physicochemical properties, mechanical properties, biological activity, and in vivo and in vitro therapeutic effects through a series of experiments. The specific experimental results are as follows:

[0033] 1. Material properties and design advantages: (1) Multi-component synergistic effect: GelDE-OCS@MOF@Pl hydrogel combines methacryloyl gelatin (GelMA), oxidized dextran (DEXO), and UiO-66-NH loaded with Paris polysaccharide l wrapped with oxidized chondroitin sulfate. 2 The hydrogel is composed of multiple components, such as OCS@MOF@Pl and nanoparticles (OCS@MOF@Pl), each of which has unique functions and forms a multifunctional platform after compounding. For example, GelMA provides good biocompatibility and cell adhesion; DEXO enhances the mechanical strength of the hydrogel; and OCS@MOF@Pl gives pH-responsive drug release ability. (2) Intelligent response mechanism: GelDE-OCS@MOF@Pl hydrogel can intelligently adjust the drug release rate under different pH environments, especially accelerating release under acidic conditions, which is particularly effective in dealing with the inflammatory microenvironment commonly found in chronic wounds. This feature not only improves the treatment efficiency, but also reduces unnecessary side effects.

[0034] 2. Evaluation of biological activity: (1) Antioxidant properties: DPPH and ABTS free radical scavenging experiments showed that GelDE-OCS@MOF@Pl hydrogel and its components have significant antioxidant capacity and can effectively combat excessive oxidative stress caused by hyperglycemia. This helps protect cells from damage and promote the regeneration of healthy tissues. (2) Antibacterial properties: Inhibition zone experiments showed that the hydrogel exhibited a strong inhibitory effect on common pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), which is crucial for preventing and controlling infections in diabetic patients. (3) Macrophage polarization: Immunohistochemical staining and quantitative analysis revealed the advantages of GelDE-OCS@MOF@Pl hydrogel in regulating macrophage polarization, especially promoting the conversion from the pro-inflammatory M1 phenotype to the anti-inflammatory and reparative M2 phenotype. This transformation is of great significance for alleviating local inflammation, accelerating epithelialization and angiogenesis.

[0035] 3. In vivo experimental results: (1) Wound healing progress: Through in vivo evaluation of the full-thickness skin defect model of diabetic rats, it was found that GelDE-OCS@MOF@Pl hydrogel significantly accelerated the speed of wound closure and shortened the healing time. Histological evaluation further confirmed the increase in new blood vessel formation and collagen deposition, indicating that the hydrogel promoted a healthy wound remodeling process. (2) Regulation of inflammatory response: Analysis of the expression levels of IL-6 and IL-10 showed that GelDE-OCS@MOF@Pl hydrogel effectively reduced the expression of inflammatory markers and promoted the wound to quickly enter the repair stage. This finding emphasizes the important role of this hydrogel in regulating the inflammatory response and provides a new strategy for improving the healing of chronic wounds.

[0036] 4. Clinical application prospects: GelDE-OCS@MOF@Pl hydrogel can be used to formulate personalized treatment plans. Based on the excellent physicochemical properties, biological activity and in vivo efficacy of GelDE-OCS@MOF@Pl hydrogel, GelDE-OCS@MOF@Pl hydrogel is expected to become a new type of personalized treatment tool suitable for different types of chronic wounds, especially those accompanied by severe inflammation or difficult to heal. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The preparation process and structure of OCS@MOF@Pl nanoparticles and GelDE1-OCS@MOF@Pl hydrogel are schematically shown; wherein, A) is a schematic diagram of the preparation process of OCS@MOF@Pl nanoparticles; B) is a schematic diagram of the preparation process and structure of GelDE1-OCS@MOF@Pl hydrogel; C) is the function of GelDE1-OCS@MOF@Pl hydrogel;

[0038] Figure 2 For OCS and CS 1 H MNR spectrum (A) and FTIR spectrum (B);

[0039] Figure 3 TEM images (A), particle size distribution statistics (B), XRD spectra (C), nitrogen fixation adsorption-desorption isotherms (D), and pore size distribution (E) of MOF, MOF@PI, MOF@Pl, and OCS@MOF@Pl;

[0040] Figure 4 For GelMA and Gel 1 HMNR spectrum (A) and FTIR spectrum (B);

[0041] Figure 5 For DEXO and DEX 1 H MNR spectrum (A) and FTIR spectrum (B);

[0042] Figure 6 Demonstration of the injectable properties of GelDE hydrogel;

[0043] Figure 7 The performance test of GelDE1-OCS@MOF@Pl hydrogel; A is the tensile stress-strain curve of GelDE and GelDE-OCS@MOF@PI in the absence of UV post-crosslinking agent; B is the tensile stress-strain curve of GelDE-OCS@MOF@Pl hydrogel and GelDE after UV secondary crosslinking; C is the elastic modulus of the tensile elastic modulus of GelDE and GelDE-OCS@MOF@P hydrogel; D is the compressive stress-strain curve of GelDE and GelDE OCS@MOF@Pl hydrogel without UV post-crosslinking; E is the compressive stress-strain curve of GelDE and GelDE-OCS@MOF@Pl hydrogel. Compressive stress-strain curve of GelDE-OCS@MOF@Pl hydrogel after cross-linking by ultraviolet radiation under secondary impact; F is the compressive elastic modulus of GelDE and GelDE GelDE-OCS@MOF@Pl hydrogel; G is the cyclic tensile test curve of GelDE-OCS@MOF@Pl hydrogel; H is the compressive stress curve of GelDE-OCS@MOF@Pl hydrogel; I is the swelling rate of GelDE and GelDE-OCS@MOF@Pl hydrogel; J is the scanning electron microscope image of GelDE and GelDE-OCS@MOF@PI hydrogel;

[0044] Figure 8The antioxidant, pH response and antibacterial effects of GelDE1-OCS@MOF@Pl hydrogel; A and B are the effects of GelMA, GelDE, OCS@MOF@Pl and GelDE1-OCS@MOF@Pl hydrogel in scavenging DPPH free radicals; C and D are the effects of GelMA, GelDE, OCS@MOF@Pl and GelDE-OCS@MOF@Pl in clearing ABTS free radicals; E is the level of Pl released from GelDE-OCS@MOF@Pl hydrogel in pH solution of 7.4, 6.2 and 5.2 respectively; F is the proportion of bacterial survival rate; G is the photos of bacterial colonies of Escherichia coli and Staphylococcus aureus in different groups; Negative is negative ion and Positive is positive ion;

[0045] Fig. 9 The figure shows the biocompatibility evaluation and angiogenesis-promoting properties of different hydrogels; A is the cell viability of human umbilical vein endothelial cells after treatment with OCS@MOF@Pl at different concentrations (0 to 120 μg / mL); B is the cell viability of different hydrogels at different times; C is the live / dead staining image after treatment with GelDE, OCS@MOF@Pl or GelDE-OCS@MOF@PL hydrogels, lasting for 1, 3 and 5 days; D is the ratio of human umbilical vein endothelial cells after cell expansion treated with different hydrogels; E is a cytoskeleton staining photo after treatment with different hydrogels for 5 days; F and G are the migration results after treatment with different hydrogels; H is the tubular formation image of human umbilical vein endothelial cells cultured in vitro at 0h, 24 and 48h after treatment with different hydrogels; I is the tubular formation image when co-cultured with GelDE;

[0046] Fig.10 Evaluation of antioxidant effect and induction of macrophage polarization; A is the ROS level in RAW264.7 cells after treatment with different hydrogels; B is the immunofluorescence image after treatment with different hydrogels, green represents CD86, and red represents CD206; C is the expression level of CD86 and CD206 in RAW264.7 cells detected by Flow cytometry;

[0047] Fig.11 Evaluation of wound healing in diabetic patients; A is a flowchart of wound treatment in diabetic rats; B is a picture of rat wounds; C is the wound healing rate after treatment with different hydrogels; D is an H&E staining picture; E is the epidermal thickness; F is the number of hair follicles; G is the Masson staining picture of wound tissue on the 7th and 14th days, and H is the collagen deposition after treatment with different hydrogels;

[0048] Fig.12The expression analysis of IL-6 and IL-10 and the investigation results of angiogenesis indicators; A is the immunohistochemical staining of different hydrogels; B is the quantitative statistical results of IL-6 of different hydrogels; C is the immunohistochemical staining of different hydrogels; D is the quantitative statistical results of IL-10 of different hydrogels; E is the immunofluorescence staining image of a-SMA and CD31 of postoperative wound tissue, and CD3l and a-SMA are stained with dyes of different colors (blue, red and green), respectively; F is the relative expression of CD3l; G is the relative expression of a-SMA. DETAILED DESCRIPTION

[0049] Polyphyllin (Pl), derived from the extract of Paris spp., has multiple mechanisms such as anti-inflammatory, pro-angiogenesis, anti-oxidation, promotion of cell proliferation and repair, and antibacterial, and plays an important role in accelerating wound healing. Specifically, Pl, as a bioactive ingredient extracted from plants, can promote multiple key steps in the wound healing process without affecting the environment. First, the anti-inflammatory effect of Pl can reduce the inflammatory response around the wound and reduce the degree of tissue damage; secondly, Pl can stimulate the formation of new blood vessels to ensure that the wound area has sufficient oxygen and nutrient supply; thirdly, by promoting cell proliferation and migration, Pl helps to accelerate the epithelialization process, that is, the speed at which new skin tissue covers the wound surface; finally, its antibacterial properties can also prevent or reduce the incidence of infection.

[0050] Unless otherwise specified, “%” in the examples refers to mass concentration percentage.

[0051] Example 1

[0052] 1. Preparation of hydrogel

[0053] 1.1. Components and proportions of hydrogel

[0054] GelDE-OCS@MOF@Pl hydrogel dressing is a complex nanocomposite material, the components and their proportions are as follows:

[0055] Oxidized dextran solution (DEXO solution): As part of the skeleton structure of the hydrogel network, it provides good biocompatibility and adjustable mechanical properties. The concentration commonly used is 2% (w / v).

[0056] Methacryloyl gelatin solution (GelMA solution): It imparts bioactivity and cell adhesion to the hydrogel and is also one of the key components for forming a stable three-dimensional network. The conventional concentration is set at 10% (w / v).

[0057] UiO-66-NH 2 Nanoparticles (OCS@MOF@Pl): These nanoparticles are the core functional units of the entire system. They can not only intelligently respond to changes in environmental pH to regulate drug release, but also effectively eliminate bacteria, remove reactive oxygen species and inhibit inflammatory responses. During the preparation process, 0.2g of OCS@MOF@Pl nanoparticles were dissolved in 40mL of deionized water to obtain an OCS@MOF@Pl nanoparticle solution, which was mixed with the above two macromolecular solutions to obtain GelDE-OCS@MOF@Pl hydrogel, in which the final concentration of oxidized dextran in GelDE-OCS@MOF@Pl hydrogel was 4%, the final concentration of methacryloyl gelatin was 15%, and the drug-loaded UiO-66-NH 2O 2 was wrapped by oxidized chondroitin sulfate. 2 The final concentration of nanoparticles was 0.2%.

[0058] Specifically, when preparing GelDE-OCS@MOF@Pl hydrogel, the solution containing OCS@MOF@Pl nanoparticles is first mixed with the pre-prepared DEXO solution and GelMA solution in a certain proportion (for example, a volume ratio of 1:1 or adjusted according to the experimental design). In order to ensure that all components are evenly dispersed and fully fused, the mixture needs to be stirred continuously under sterile conditions. In addition, in order to start the polymerization process, an appropriate amount of photoinitiator (such as LAP) is added to the mixture to a final concentration of approximately 0.5% (w / v). The mixture is then transformed into an elastic three-dimensional network structure, namely GelDE-OCS@MOF@Pl hydrogel, by UV irradiation or chemical cross-linking.

[0059] 1.2 Key steps and technical parameters in the manufacturing method (synthesis and characterization of OCS, DEXO and GelMA and synthesis of GelDE)

[0060] 1.2.1 Synthesis of Oxidized Chondroitin Sulfate (OCS)

[0061] Material preparation: Dissolve 1 g of chondroitin sulfate (CS) powder in 100 mL of deionized water and add 0.27 g of sodium periodate (NaIO 4 ) was dissolved in 5 mL of deionized water.

[0062] Reaction process: NaIO 4 The solution was added to the CS solution, and the mixture was stirred at 600 rpm for up to 2 h at room temperature and protected from light.

[0063] Termination of the reaction: Add 1 mL of ethylene glycol to the mixture to terminate the reaction of sodium periodate to form an OCS solution.

[0064] Purification and drying: The OCS solution was dialyzed in a dialysis bag with a molecular weight cutoff of 3.5 kDa for 5 days and then freeze-dried.

[0065] Storage: The obtained OCS product should be stored in a dry environment and kept at room temperature.

[0066] 1.2.2 Synthesis of oxidized dextran (DEXO)

[0067] Material preparation: Dextran (DEX, 30 mg / mL) and sodium periodate were dissolved in deionized water to obtain dextran solution and sodium periodate solution respectively.

[0068] Reaction conditions: Mix the two solutions in a ratio of 4:10 between the glucose in the dextran solution and the sodium periodate in the sodium periodate solution, and stir in the dark for 16 h.

[0069] Purification and drying: The final product DEXO was obtained by dialyzing for 5 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa and then freeze-drying.

[0070] Characterization: Proton NMR spectroscopy ( 1 Its chemical structure was analyzed by HNMR and Fourier transform infrared spectroscopy (FTIR).

[0071] 1.2.3 Synthesis of methacryloyl gelatin (GelMA)

[0072] Material preparation: Dissolve 10 g of gelatin (Gel) in 100 mL of carbonate buffer (pH 9), swell at room temperature for 1 h, and then transfer to a 60°C constant temperature water bath until completely dissolved.

[0073] Methacrylation reaction: 0.8 mL of methacrylic anhydride (MA) was added dropwise at a rate of 0.8 mL / min during stirring, and the reaction time was 3 h.

[0074] Purification and drying: The liquid after the reaction was diluted with 400 mL of deionized water and dialyzed using a dialysis bag with a molecular weight cutoff of 10 kD for 1 week, with deionized water replaced four times a day. Finally, the GelMA sample was obtained by centrifugation, freezing (-80°C) and freeze-drying.

[0075] Storage: Finished GelMA should be stored in a dry cabinet.

[0076] Characterization: By 1 HNMR and FTIR analyses confirmed its chemical structure, ensuring that GelMA with characteristic peaks was successfully synthesized.

[0077] These detailed synthetic steps ensure the correct preparation of OCS, DEXO, and GelMA, while the characterization steps are used to verify the chemical properties and structural integrity of the synthesized products.

[0078] 1.2.4 Synthesis of GelDE

[0079] The GelMA deionized water solution and the DEXO deionized water solution were mixed to obtain the GelDE deionized water solution (GelDE).

[0080] 2. Preparation and characterization of OCS@MOF@Pl nanoparticles

[0081] 2.1 UiO-66-NH 2 Preparation of (MOF) nanocrystals

[0082] Material preparation: Weigh 75 mg of zirconium oxide chloride (ZrOCl 2 8H 2 O), 50 mg of p-aminobenzoic acid and 1.25 g of benzoic acid, and added them to a reaction vessel with a polytetrafluoroethylene liner.

[0083] Dissolution and reaction: Add 5 mL of N,N-dimethylformamide (DMF), stir until all the powder is completely dissolved, and then heat at 120°C for 24 h.

[0084] Post-treatment: After the reaction was completed, the product was washed alternately with anhydrous ethanol and DMF three times, and then dried at 37 °C to obtain MOF nanoparticles.

[0085] 2.2 Preparation of MOF@Pl composite

[0086] Material mixing: 0.2 g of the MOF obtained above, 0.1 g of Paris polyphylla saponin 1 (P1) and 0.1 g of auxiliary substance (dimethyl sulfoxide) were dissolved in anhydrous ethanol.

[0087] Loading and separation: After the mixture was stirred at 100 rpm for 4 h, the precipitate was collected by centrifugation and dried at 37 °C to obtain the MOF@Pl nanocomposite.

[0088] 2.3 Preparation of OCS@MOF@Pl nanoparticles

[0089] Material preparation: Take 0.2 g of the previously prepared OCS and dissolve it in 40 mL of water to obtain an OCS solution.

[0090] Encapsulation process: 0.1 g MOF@Pl nanocomposite was added to the OCS solution and mixed using a vortex oscillator for 30 min.

[0091] Purification and drying: The samples were washed three times with deionized water to remove unbound components and finally dried at 37 °C to obtain the final product, OCS@MOF@Pl nanoparticles.

[0092] 2.4 Characterization methods

[0093] Microstructure morphology observation: Transmission electron microscopy (TEM) was used to observe the microstructure morphology of the prepared MOF, MOF@Pl and OCS@MOF@Pl nanoparticles.

[0094] Porosity determination: The porosity of the nanoparticles was characterized by BET testing.

[0095] Crystal structure analysis: X-ray diffraction (XRD) technology was used to determine the crystal structure of the nanoparticles to ensure that their crystal structure was consistent with that reported in relevant literature.

[0096] Size distribution statistics: The particle size distribution of nanoparticles was measured using transmission electron microscopy images combined with statistical methods, confirming that the particle size was mainly concentrated in the range of 190-290nm and the morphology was close to spherical.

[0097] 3. Preparation method and steps of GelDE-OCS@MOF@Pl hydrogel.

[0098] 3.1. Prepare materials

[0099] OCS@MOF@Pl nanoparticles: composite nanoparticles synthesized in the above steps.

[0100] Oxidized dextran (DEXO) solution: used to provide a part of the skeleton structure of the hydrogel network.

[0101] Methacryloyl gelatin (GelMA) solution: serves as another part of the skeleton and endows the hydrogel with biocompatibility and cell adhesion.

[0102] 3.2 Dissolving and mixing

[0103] 0.2 g of OCS@MOF@Pl nanoparticles were dissolved in 40 mL of deionized water and a vortex oscillator was used to ensure uniform dispersion to obtain a solution containing OCS@MOF@Pl nanoparticles.

[0104] Meanwhile, prepare appropriate amounts of DEXO solution and GelMA solution. The specific concentrations are determined according to the experimental design, usually 10% (w / v) GelMA and 2% (w / v) DEXO.

[0105] The DEXO solution and the GelMA solution are heated to appropriate temperatures (eg, 37° C.) respectively to ensure that all components are completely dissolved and form a uniform solution.

[0106] 3.3 Prepolymer mixing

[0107] Under sterile conditions, the solution containing OCS@MOF@Pl nanoparticles, the DEXO solution, and the GelMA solution are mixed together in a predetermined ratio, such as a volume ratio of 1:1 or adjusted according to actual needs. During the mixing process, continuous stirring is maintained to ensure that the components are fully integrated while avoiding the introduction of too many bubbles.

[0108] 3.4. Initiation of polymerization

[0109] Add a photoinitiator (lithium phenolic acid) or other suitable chemical crosslinking agent to the mixed solution, and the final concentration is about 0.5% (w / v). The present invention uses I2959.

[0110] If light curing is used, the mixture is placed under a UV lamp for a period of time (such as 5 minutes) to start the free radical polymerization process; if chemical cross-linking is used, it is directly placed at room temperature to wait for the cross-linking to be completed.

[0111] 3.5. Hydrogel Formation

[0112] After UV irradiation or chemical cross-linking, the mixture gradually transforms into an elastic three-dimensional network structure, namely GelDE-OCS@MOF@Pl hydrogel.

[0113] At this point, the GelDE-OCS@MOF@Pl hydrogel can maintain fluidity in the syringe, and can be injected into the wound site and quickly solidified in situ.

[0114] 3.6 Characterization and Application

[0115] The physical and chemical properties of the formed hydrogel were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR).

[0116] Test the mechanical strength, swelling properties, degradation behavior, and drug release characteristics of the hydrogel.

[0117] 4. Mechanical testing methods and steps of GelDE-OCS@MOF@Pl hydrogel dressing

[0118] 4.1 Sample preparation

[0119] Solution preparation: First, deionized water solutions containing GelMA and DEXO were prepared in a predetermined ratio.

[0120] Mixing components: mixing a GelMA deionized water solution and a DEXO deionized water solution to obtain a GelDE deionized water solution (GelDE);

[0121] The solution containing OCS@MOF@Pl nanoparticles and the photoinitiator I2959 were added to the DEXO deionized water solution to form Solution B. Then Solution A (GelDE deionized water solution) was evenly mixed with Solution B to obtain GelDE-OCS@MOF@Pl hydrogel. The final concentration of oxidized dextran in GelDE-OCS@MOF@Pl hydrogel was 4% (W / V), the final concentration of methacryloyl gelatin was 15% (W / V), and the drug-loaded UiO-66-NH 2 The final concentration of nanoparticles was 0.2% (W / V), and the final concentration of photoinitiator I2959 was 0.25% (W / V).

[0122] 4.2 Gelation treatment

[0123] Initial cross-linking: The mixed liquid begins the gelation process through the Schiff base reaction to form a preliminary hydrogel structure.

[0124] Secondary crosslinking: In order to enhance the mechanical strength of the hydrogel, ultraviolet light (UV, 365m, 50mW / cm 2 ) was irradiated for 5 min for photocuring to complete the secondary cross-linking and finally obtain the GelDE-OCS@MOF@Pl hydrogel sample.

[0125] 4.3. Sample forming

[0126] Tensile test samples: For the tensile test, the hydrogel needs to be processed into a dumbbell shape with specific dimensions of 50 mm in length, 4 mm in width, and 2 mm in thickness.

[0127] Compression test specimens: The specimens used for compression test were molded into cylindrical shape with a diameter of 10 mm and a height of 6 mm.

[0128] 4.4 Mechanical properties test

[0129] Equipment preparation: Use a universal testing machine from Shimadzu Corporation of Japan and equip it with a 1kN load sensor for testing.

[0130] Tensile test: A dumbbell-shaped hydrogel sample was fixed between the tensile clamps. The test speed was set at 10 mm / min, and the stress-strain curve of the hydrogel was recorded until it broke.

[0131] Compression test: The cylindrical hydrogel sample was placed in a compression fixture. The compression speed was set at 2 mm / min, and the pressure change of the hydrogel during the compression process was recorded.

[0132] 4.5 Cyclic loading test

[0133] Cyclic tensile test: GelDE-OCS@MOF@Pl hydrogel was subjected to 30 cycles of cyclic tensile test at a strain ratio of 40%.

[0134] Cyclic compression test: 30 cycles of compression test with a compression ratio of 40% were carried out under the same conditions to evaluate the stability and recovery ability of the material under repeated deformation.

[0135] 4.6 Data Analysis

[0136] Comparative analysis: Comparison of the mechanical properties between GelDE without photocuring and GelDE-OCS@MOF@Pl hydrogel after photocuring.

[0137] Stability evaluation: Observe and record the changes in the mechanical properties of the hydrogel before and after cyclic loading, especially whether its key parameters such as elastic modulus, tensile strength, and compressive strength remain unchanged or improve.

[0138] The mechanical test aims to fully understand the mechanical behavior of GelDE-OCS@MOF@Pl hydrogel dressing, so as to verify its applicability and reliability as a medical material. Through a rigorous testing process, it can be ensured that the hydrogel has sufficient mechanical strength and flexibility to meet the needs of practical applications, especially long-term support in dynamic wound environments.

[0139] 5. Methods and steps of microstructure and swelling characteristics experiment

[0140] 5.1 Microstructure Analysis

[0141] 5.1.1 Sample preparation

[0142] Freezing treatment: The prepared GelDE and GelDE-OCS@MOF@Pl hydrogel samples were placed in an ultra-low temperature freezer at -80 °C overnight.

[0143] Dehydration: The frozen samples were dehydrated using a cryogenic freeze dryer.

[0144] Fracture surface preparation: The dried samples were placed in liquid nitrogen and cracked to obtain fresh fracture surfaces.

[0145] Gold plating: Use a gold sprayer to sputter gold to metalize the fracture surface to increase conductivity for easy observation under a scanning electron microscope (SEM). Finally, the microstructural characteristics of the fracture interface are detected under a scanning electron microscope.

[0146] 5.1.2 Observation and Analysis

[0147] SEM observation: Using a German-made ZEISS sigma500 scanning electron microscope, the microstructure images of the hydrogel were taken at different magnifications, with special attention paid to the morphological characteristics of its fracture interface. By comparing the SEM images of the two hydrogels, the difference in their internal structures can be intuitively seen.

[0148] 5.2 Evaluation of swelling characteristics

[0149] 5.2.1 Sample pretreatment

[0150] Initial mass measurement: GelDE and GelDE-OCS@MOF@Pl hydrogels were immersed in phosphate-buffered saline (PBS, pH 7.4, 0.1 M) and their swelling properties were evaluated at 37 °C. Before the swelling test, the initial mass (Wi) of the hydrogel and the mass after swelling 564 equilibrium (Wf) were recorded. The SR ratio of the hydrogel sample was determined using Equation (1) for the soaking process.

[0151] Swelling equilibrium: Immerse the hydrogel sample completely in a container filled with PBS and keep the temperature at 37°C. Allow the sample to fully absorb water until it reaches a swelling equilibrium state. This step usually takes a certain amount of time, depending on the type of hydrogel and environmental conditions.

[0152] 5.2.1 Data Collection

[0153] Final mass measurement: After confirming that the hydrogel had reached swelling equilibrium, the sample was taken out of PBS again and the excess liquid on the surface was gently removed, and then its wet weight (Wf) was quickly weighed.

[0154] 5.2.1. Calculation of swelling ratio

[0155] Calculation formula: The swelling ratio (SR) of the hydrogel is calculated according to formula (1), which reflects the ability of the material to absorb water:

[0156]

[0157] These detailed experimental methods and steps ensure an accurate understanding of the microstructure and swelling properties of GelDE and GelDE-OCS@MOF@Pl hydrogels. Through careful sample handling and rigorous testing procedures, important information about the two hydrogels can be obtained, including their internal structural characteristics, pore distribution, and response behavior to the surrounding environment. This is crucial for evaluating the application potential of new hydrogels as biomedical materials.

[0158] 6. Antioxidant performance test

[0159] The methods and steps of the antioxidant performance test in this study mainly focused on evaluating the free radical scavenging ability of GelDE-OCS@MOF@Pl hydrogel and its components, including the scavenging experiments of two free radicals: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt).

[0160] 6.1. DPPH free radical scavenging experiment

[0161] 6.1.1 Reagent preparation

[0162] Preparation of DPPH ethanol solution: Dissolve DPPH in anhydrous ethanol to prepare a solution with a concentration of 0.1 mM. Prepare fresh solution before each use.

[0163] 6.1.2 Experimental operation

[0164] Sample treatment: Take appropriate amounts of different materials (such as pure GelMA, GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel) and add them into test tubes containing 3 mL of DPPH ethanol solution.

[0165] Incubation process: Incubate the mixture in the dark for 30 min to ensure adequate reaction.

[0166] Absorbance measurement: The absorbance value of each group of samples was measured at a wavelength of 517 nm using a UV-visible spectrophotometer.

[0167] Calculate the clearance rate: Calculate the DPPH free radical clearance rate according to formula (2):

[0168]

[0169] Among them, A 0 is the absorbance of the control group containing only DPPH ethanol solution, A t It is the absorbance of the experimental group (i.e. the mixture after adding the test material).

[0170] 6.2 ABTS free radical scavenging experiment

[0171] 6.2.1. Reagent preparation

[0172] Generate ABTS cation radicals: Mix 7 mM ABTS solution with 4.95 mM potassium persulfate (K 2 S 2 O 8 ) solution was mixed in equal volumes and oxidized at room temperature in the dark for 12 h. It was then diluted with PBS to 5% of the original concentration and used as the working solution.

[0173] 6.2.2 Experimental operation

[0174] Sample treatment: Add 3 mL of pre-prepared ABTS working solution to each group of materials.

[0175] Incubation process: Similarly, the mixture was incubated in a dark environment for 30 minutes.

[0176] Absorbance measurement: The absorbance changes of each group of samples were detected at a wavelength of 734 nm using a UV-visible spectrophotometer.

[0177] Calculate the clearance rate: Calculate the ABTS free radical clearance rate according to formula (3):

[0178]

[0179] Among them, A 0 represents the absorbance of the control group containing only ABTS working solution, while A t It refers to the absorbance of the experimental group.

[0180] 6.3 Data Analysis

[0181] The DPPH and ABTS free radical scavenging efficiencies of different materials were compared to analyze the differences in their antioxidant properties. In particular, attention was paid to the significant advantages of OCS@MOF@Pl nanoparticles over other materials, and the slightly lower but still effective scavenging effect of GelDE-OCS@MOF@Pl hydrogel compared to individual nanoparticles, indicating that the hydrogel can gradually release active ingredients, thereby continuously exerting an antioxidant effect. The above experimental design under strictly controlled conditions allows the effective evaluation of the antioxidant properties of the developed hydrogel and its components, which is crucial for understanding its role in combating the common oxidative stress in diabetic wound healing.

[0182] 7. Drug release performance

[0183] The method and steps for evaluating the drug release performance of GelDE-OCS@MOF@Pl hydrogel are as follows:

[0184] 7.1 Test preparation

[0185] 7.1.1 Sample preparation

[0186] First, GelDE-OCS@MOF@Pl hydrogel was prepared according to the method described previously. The hydrogel was encapsulated in a dialysis bag with a molecular weight cutoff of 10,000 to ensure that the hydrogel was completely encapsulated and leak-free.

[0187] 7.1.2. Medium selection

[0188] In order to simulate the drug release behavior under different pH environments in vivo, phosphate buffered saline (PBS) solutions with pH values ​​of 7.4, 6.2, and 5.2 were prepared as release media, respectively. These pH values ​​correspond to normal physiological conditions (pH 7.4), mildly acidic inflammatory microenvironment (pH 6.2), and more acidic wound sites (pH 5.2).

[0189] 7.2 Drug release experiment

[0190] 7.2.1. Soaking and sampling

[0191] The dialysis bag containing the hydrogel was immersed in the above three PBS solutions with different pH values, and three parallel samples were set under each condition. The system was placed in a 37°C constant temperature shaker to maintain a constant temperature and promote uniform diffusion.

[0192] 7.2.2. Timed Sampling

[0193] At predetermined time intervals (e.g., 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, etc.), a certain volume (e.g., 1 mL) of release medium is taken out from each container and immediately supplemented with the same volume of fresh PBS solution to maintain the total volume unchanged.

[0194] 7.2.3 Absorbance measurement

[0195] The absorbance of each sample was measured at a wavelength of 408 nm using a UV-Vis spectrophotometer, which was determined based on the standard absorption peak of the loaded drug Paris polyphylla saponin 1 (P1).

[0196] 7.2.4. Calculate the release ratio

[0197] According to the standard curve, the measured absorbance is converted into the corresponding drug concentration, and the cumulative release percentage is further calculated. The formula is shown in formula (4):

[0198]

[0199] Among them, C i is the drug concentration at time point i, V is the volume of each sample, W is the total amount of drug initially loaded into the hydrogel, and C 0 is the initial drug concentration.

[0200] 7.3 Data Analysis and Model Fitting

[0201] Kinetic model fitting: In order to better understand the drug release mechanism, the obtained data were fitted with zero-order, first-order, Higuchi equation and Korsmeyer-Peppas equation. This helps to determine whether the drug release follows simple diffusion, dissolution or other complex mechanisms.

[0202] 7.4. Interpretation of results

[0203] By comparing the changes in drug release rates under different pH environments, it can be concluded that lower pH values ​​accelerate drug release because acidic conditions promote the dissociation of OCS on the surface of MOF materials (through the breaking of Schiff base bonds) and the degradation of the MOF material itself, thereby accelerating the release rate of encapsulated drugs.

[0204] In summary, this series of detailed drug release performance testing methods and steps can comprehensively evaluate the drug release characteristics of GelDE-OCS@MOF@Pl hydrogel under different pH conditions, providing an important basis for optimizing its therapeutic effect.

[0205] 8. Antibacterial test

[0206] The antibacterial test was designed to evaluate the antibacterial effects of GelMA, GelDE, OCS@MOF@Pl and GelDE-OCS@MOF@Pl hydrogels against two common pathogens, Escherichia coli (Escherichia coli, E.coli) and Staphylococcus aureus (S.aureus), while the control group was a control group without intervention. The following are the detailed experimental methods and steps:

[0207] 8.1. Material preparation

[0208] Test materials: including GelMA, GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel.

[0209] Bacterial culture medium: Use LB liquid medium to culture E. coli and S. aureus and ensure that they are in the logarithmic growth phase.

[0210] 8.2 Preparation of bacterial suspension

[0211] Activate bacteria: Pick a single colony from the frozen strain into 5 mL of LB liquid medium and culture it overnight in a shaker at 37°C.

[0212] Adjust the concentration: Dilute the bacterial suspension after overnight culture with fresh LB liquid medium to an appropriate concentration (e.g. OD 600is about 0.1), ensuring that each sample contains approximately 1×10 8 CFU / mL of bacteria to obtain E. coli suspension or S. aureus suspension.

[0213] 8.3 Co-cultivation process

[0214] Material processing: The four different types of hydrogel materials mentioned above were cut into small pieces or made into shapes suitable for co-culture and placed in sterile 96-well plates respectively.

[0215] Add bacteria: Add 100 μL of the prepared E. coli suspension or S. aureus suspension to each well so that each material is in contact with both bacteria.

[0216] Incubation conditions: The 96-well plate was placed in a 37°C incubator and incubated for 8 h to allow for adequate interaction.

[0217] 8.4. Result determination

[0218] Method 1: Plate count method

[0219] Sampling: After incubation, take out 100 μL of bacterial suspension from each well.

[0220] Dilution and spreading: After diluting the suspension in appropriate multiples, take a certain amount and spread it evenly on the LB agar plate.

[0221] Culture and counting: Place the plate in a 37°C incubator and continue culturing for 24 hours, then count the number of colonies (CFU) on each plate.

[0222] Survival rate calculation: According to formula (5), determine the effect of each group of materials on bacterial growth;

[0223]

[0224] Method 2: Microscope observation

[0225] Fixation and staining: If further confirmation is required, the remaining bacterial suspension can be centrifuged to collect the precipitate, washed twice with PBS, and then subjected to Gram staining or other appropriate staining methods.

[0226] Microscopic examination: Directly observe the morphological changes and death of bacteria using an optical microscope or a fluorescence microscope.

[0227] 8.5 Data Analysis

[0228] The antibacterial effects of different materials were compared, with particular attention paid to the advantages of GelDE-OCS@MOF@Pl hydrogel over other materials, which may be attributed to the drug components carried by the OCS@MOF@Pl nanoparticles contained therein and their unique structural properties.

[0229] The analysis results should take into account possible differences, such as material surface properties, released active substances, etc., and explain the antibacterial performance in conjunction with subsequent biocompatibility assessments. Through the above antibacterial experimental design under strictly controlled conditions, the antibacterial ability of the prepared hydrogel and its components can be effectively evaluated, which is crucial for understanding its role in combating diabetic wound infection.

[0230] 9. Biocompatibility assessment

[0231] The methods and steps of the biocompatibility evaluation test are aimed at comprehensively evaluating the effects of GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel on human cells, while taking the treatment without any intervention as the control. The following are the detailed experimental methods and steps:

[0232] 9.1. Material preparation

[0233] Test materials: including GelDE, OCS@MOF@Pl nanoparticles, and GelDE-OCS@MOF@Pl hydrogel.

[0234] Cell lines: Human umbilical vein endothelial cells (HUVECs) were chosen as model cells because they play an important role in angiogenesis and are a common indicator for evaluating the safety of biomaterials.

[0235] 9.2 Cell Culture

[0236] Basic conditions: Endothelial Cell Growth Medium (EGM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used as the culture medium, and the cells were cultured at 37°C and 5% CO. 2 Maintain cell growth under the conditions.

[0237] 9.3. Cytotoxicity test of OCS@MOF@Pl nanoparticles

[0238] 9.3.1 Cell treatment

[0239] Concentration gradient setting: OCS@MOF@Pl nanoparticles were prepared into different concentrations (0, 20, 40, 60, 80, 100 and 120 μg / mL) to investigate the dose-dependent cellular response.

[0240] 9.3.2 Co-cultivation

[0241] HUVECs in the logarithmic growth phase were seeded into 96-well plates, with approximately 5 × 10 3 After the cells attached to the wall, the above nanoparticle solutions of different concentrations were added and incubated for 24 h.

[0242] 9.3.3. Vitality Detection

[0243] The cell viability was measured using the CCK-8 method (Cell Counting Kit-8 (CCK-8) kit). The old culture medium was removed, 10 μL of CCK-8 solution was added to each well, and then 100 μL of fresh culture medium was added. After incubation for 2 h, the absorbance value was read at a wavelength of 450 nm.

[0244] 9.3.4 Data Analysis

[0245] The cell viability was calculated based on the standard curve, and a trend graph showing changes in cell viability versus nanoparticle concentration was drawn to determine the optimal safe concentration range.

[0246] 9.4. Hydrogel Cell Compatibility Test

[0247] 9.4.1 Short-term co-culture

[0248] Sample preparation: GelDE, OCS@MOF@Pl nanoparticles, and GelDE-OCS@MOF@Pl hydrogels were cut into small pieces suitable for placing into a 24-well plate.

[0249] Cell seeding: Inoculate an appropriate amount of HUVECs (about 1×10 4 / well), which were contacted with the hydrogel and incubated together for 24h and 48h.

[0250] Viability and proliferation assessment: CCK-8 method was used to measure cell viability again. Live / Dead staining experiment was also performed to observe the ratio of live cells (green) and dead cells (red) under fluorescence microscopy to intuitively show the effect of hydrogel on cell health.

[0251] 9.4.2. Long-term co-culture and morphological observation

[0252] Continuous monitoring: For some samples, the co-culture time was extended to 5 days, with daily culture medium changes without changing the hydrogel position.

[0253] Fixation and staining: At the end of the third day, cells were fixed with 4% paraformaldehyde, then permeabilized with 0.1% TritonX-100, and nonspecific binding sites were blocked with bovine serum albumin (BSA).

[0254] F-actin and nuclear staining: Rhodamine-conjugated phalloidin was used to stain F-actin fibers in the cytoskeleton, and DAPI was used to stain the cell nucleus for subsequent confocal laser scanning microscopy (CLSM) observation.

[0255] 9.4.3 Hemolysis test

[0256] Blood collection: 1.5 mL of blood was drawn from the ophthalmic venous plexus of the experimental mice, mixed with 3.5 mL of normal saline, and centrifuged to obtain a pure red blood cell suspension.

[0257] Hemolysis test: 100 μL of red blood cell suspension was mixed with 900 μL of NS solution of different concentrations containing the required components, incubated at 37°C for 1 hour, and then centrifuged to obtain the supernatant to measure the absorbance to evaluate whether the material causes hemolysis.

[0258] 9.4.4 Data Analysis

[0259] The cell viability, proliferation ability and morphological characteristics of different materials were compared, with particular attention paid to the advantages or differences of GelDE-OCS@MOF@Pl hydrogel compared with other materials.

[0260] The analysis results should take into account factors such as the surface properties of the material and the active substances released, and comprehensively interpret the performance of biocompatibility in combination with the evaluation of other aspects such as antibacterial properties. These detailed biocompatibility evaluation methods and steps ensure a comprehensive understanding of the safety and applicability of new hydrogel materials, and provide an important scientific basis for their application in clinical treatment.

[0261] 10. Cell migration assay

[0262] The cell migration experiment was designed to evaluate the effects of GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel on the migration ability of human umbilical vein endothelial cells (HUVECs). The following are the detailed experimental methods and steps:

[0263] 10.1 Experimental Preparation

[0264] 10.1.1. Material preparation

[0265] Ensure that all reagents and consumables are sterile, including 24-well plates, PBS buffer, cell culture medium (containing 10% fetal bovine serum), CCK-8 kit, crystal violet staining solution, etc.

[0266] 10.1.2 Cell Culture

[0267] Endothelial Cell Growth Medium (EGM) was used as the culture medium and the cells were cultured at 37°C and 5% CO 2 The growth of HUVECs was maintained under the optimal conditions to keep them in a good and healthy state.

[0268] 10.1.3 Cell inoculation and pretreatment

[0269] Cell seeding: HUVECs in logarithmic growth phase were plated at 4×10 5 The cells were seeded into 24-well plates at a density of 10 cells / mL and an appropriate amount of complete culture medium was added.

[0270] Adhesion and stabilization: Allow the cells to adhere and grow stably in the incubator for 24 hours to ensure the formation of a uniform and dense monolayer of cells.

[0271] Wound streaking: Use a 200μL sterile plastic pipette tip to gently streak along the center of the bottom of the well to create a "wound" of uniform width, simulating the injured area in the body.

[0272] Wash and remove floating cells: Wash gently three times with PBS to remove cells that are not attached to the wall or have fallen off due to the streaking operation, ensuring that the subsequent observation is the true migration behavior.

[0273] 10.1.4. Material Addition and Co-cultivation

[0274] Material treatment: According to the experimental design, serum-free culture medium containing GelDE, OCS@MOF@Pl nanoparticles or GelDE-OCS@MOF@Pl hydrogel components was added to different wells, and three replicate wells were set for each condition.

[0275] Control group setting: A control group containing only serum-free culture medium was also set up to compare the differences in effects among the treatment groups.

[0276] 10.1.5 Migration process monitoring

[0277] Initial image recording: Immediately after streaking, an image of the “wound” area of ​​each well was taken using an inverted microscope as S 0 (initial scratch area) reference point.

[0278] Time series imaging: The 24-well plate was placed back into the incubator for a further 48 h, and taken out and photographed at regular intervals (e.g., 12 h, 24 h, 48 h) during this period to record the process of cell migration and covering the wound surface.

[0279] 10.2 Data Analysis

[0280] Final image acquisition: After 48 h, take an image of each well again and mark it as St (the scratch area after th).

[0281] Calculation of migration rate: The scratch area change at each time point was measured using image analysis software, and then the cell migration ratio (MR) was calculated using formula (6):

[0282]

[0283] Among them, S 0 is the initial scratch area, S t = is the scratch area after th.

[0284] 10.3 Statistical Analysis

[0285] The differences in cell migration rates between different treatment groups were compared and appropriate statistical methods (such as ANOVA analysis of variance) were used to determine the significance level.

[0286] 10.4 Results

[0287] Make a bar graph or line graph to visually display the improvement of cell migration efficiency in each treatment group compared with the control group.

[0288] 10.5 Conclusion

[0289] Based on the experimental data, whether GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogels promoted the migration of HUVECs was discussed, and its potential mechanism was explored.

[0290] Through the above detailed methods and steps, the effects of novel hydrogel materials on cell migration behavior can be systematically evaluated, which is crucial for understanding the mechanism of action of these materials in promoting wound healing.

[0291] 11. Tube Formation Analysis

[0292] The tube formation assay was designed to evaluate the effects of GelDE, OCS@MOF@Pl nanoparticles, and GelDE-OCS@MOF@Pl hydrogels on the angiogenesis of human umbilical vein endothelial cells (HUVECs). The following are the detailed experimental methods and steps:

[0293] 11.1 Experimental Preparation

[0294] 11.1.1. Material preparation

[0295] Ensure that all reagents and consumables are sterile, including 96-well plates, Matrigel matrix, PBS buffer, cell culture medium (containing 10% fetal bovine serum), CCK-8 kit, etc.

[0296] 11.1.2 Matrigel coating

[0297] Melt Matrigel matrix on ice. Add 10 μL Matrigel to each well and quickly cover the entire bottom of the well to ensure even distribution. Place the 96-well plate in a 37°C incubator for 30 minutes to allow Matrigel to solidify into a gel-like matrix.

[0298] 11.1.3 Cell inoculation and pretreatment

[0299] Cell seeding: HUVECs in logarithmic growth phase were resuspended in serum-free medium at a density of 5,000 cells per well, gently mixed and added dropwise to the solidified Matrigel surface.

[0300] Initial attachment: Incubate cells at 37°C and 5% CO 2 Incubate for 1-2 h under low temperature conditions to allow sufficient time for the cells to attach and begin to spread.

[0301] Remove old medium: Carefully aspirate the medium containing the suspended non-adherent cells, avoiding disturbing the already attached cells.

[0302] 11.1.4. Material Addition and Co-cultivation

[0303] Material treatment: According to the experimental design, serum-free culture medium containing GelDE, OCS@MOF@Pl nanoparticles or GelDE-OCS@MOF@Pl hydrogel components was added to different wells, and three replicate wells were set for each condition.

[0304] Control group setting: A control group containing only serum-free culture medium was also set up to compare the differences in effects among the treatment groups.

[0305] 11.1.5. Tube Formation Process Monitoring

[0306] Real-time observation: Using an inverted microscope, starting from 3 hours after the addition of the material, observe and record the formation of the tubular structure at regular intervals (for example, once every 3 hours).

[0307] Image capture: At selected time points (e.g., 3h, 6h, 9h, 12h), a camera system was used to capture images of the tubular network in each well for subsequent quantitative analysis.

[0308] 11.2 Data Analysis

[0309] To calculate tube formation parameters, use image analysis software (such as ImageJ) to measure the following metrics:

[0310] Total tube length: the cumulative length of all formed tubular structures;

[0311] Number of nodes: defined as where three or more tubes meet;

[0312] Number of rings: The number of completely closed tubular structures.

[0313] 11.2 Statistical Analysis

[0314] The differences in the above parameters between different treatment groups were compared and appropriate statistical methods (such as ANOVA analysis of variance) were used to determine the significance level.

[0315] 11.4 Results

[0316] Make a bar graph or line graph to visually display the improvement of tube formation efficiency in each treatment group relative to the control group.

[0317] 11.5 Conclusion

[0318] Based on the experimental data, whether GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogels promote the angiogenesis of HUVECs and explore its potential mechanism.

[0319] Through the above detailed methods and steps, the effects of new hydrogel materials on angiogenesis behavior can be systematically evaluated, which is crucial for understanding the mechanism of action of these materials in promoting wound healing. This experiment not only provides important information about the biological activity of the material, but also provides a scientific basis for the development of new therapeutic strategies.

[0320] 12. Assessment of intracellular antioxidant capacity

[0321] The intracellular antioxidant capacity test was designed to detect the effects of GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel on the antioxidant defense system of human umbilical vein endothelial cells (HUVECs). The following are the detailed experimental methods and steps:

[0322] 12.1 Experimental Preparation

[0323] 12.1.1. Material preparation

[0324] Ensure that all reagents and consumables are sterile, including 6-well plates, PBS buffer, cell culture medium (containing 10% fetal bovine serum), 2',7'-dichlorofluorescein diacetate (DCFH-DA), hydrogen peroxide (H 2 O 2 ) solution, etc.

[0325] 12.1.2 Cell Culture

[0326] Endothelial Cell Growth Medium (EGM) was used as the culture medium and the cells were cultured at 37°C and 5% CO 2 The growth of HUVECs was maintained under the optimal conditions to keep them in a good and healthy state.

[0327] 12.1.3 Cell inoculation and pretreatment

[0328] Cell seeding: HUVECs in logarithmic growth phase were plated at 2×10 5 The cells were seeded into 6-well plates at a density of 10 cells / well and an appropriate amount of complete culture medium was added.

[0329] Adhesion and stabilization: Allow the cells to adhere and grow stably in the incubator for 24 hours to ensure the formation of a uniform and dense monolayer of cells.

[0330] 12.1.4. Induced Reactive Oxygen Species (ROS) Generation

[0331] H 2 O 2 Treatment: After washing the cells twice with PBS, add 100 μM H 2 O 2 Fresh medium was added and incubated for 2 h to induce ROS generation in cells.

[0332] Wash to remove H 2 O 2 : Wash gently three times with PBS to remove residual H 2 O 2 , ensuring that subsequent experiments only reflect the intrinsic antioxidant response of the cells.

[0333] 12.1.5. Material Addition and Co-cultivation

[0334] Material treatment: According to the experimental design, serum-free culture medium containing GelDE, OCS@MOF@Pl nanoparticles or GelDE-OCS@MOF@Pl hydrogel components was added to different wells, and three replicate wells were set for each condition.

[0335] Control group setting: A control group containing only serum-free culture medium was also set up to compare the differences in effects among the treatment groups.

[0336] 12.2 Antioxidant capacity monitoring

[0337] DCFH-DA staining: At 6 h after material treatment, the cells were washed twice again with PBS, and then DCFH-DA staining solution with a final concentration of 10 μM was added and incubated in the dark for 30 min.

[0338] Stop staining: Wash three times with PBS to stop the staining process and remove as much dye as possible that has not entered the cells.

[0339] 12.3 Data Analysis

[0340] Fluorescence imaging: An inverted fluorescence microscope was used to observe and record the fluorescence intensity in each well. DCFH-DA would emit green fluorescence after being oxidized by intracellular ROS, and its intensity reflected the intracellular ROS level.

[0341] Quantitative analysis: The fluorescence intensity was measured by image analysis software (such as ImageJ) and the average fluorescence unit (AU) was calculated to quantify the intracellular ROS content.

[0342] 12.4 Statistical Analysis

[0343] The fluorescence intensity differences between different treatment groups were compared and the significance level was determined using appropriate statistical methods (such as ANOVA analysis of variance).

[0344] 12.5 Results

[0345] Make a bar graph or line graph to visually display the changes in intracellular ROS levels in each treatment group relative to the control group.

[0346] 12.6 Conclusion

[0347] Based on the experimental data, whether GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogels enhance the antioxidant capacity of HUVECs is discussed, and its potential mechanism is explored.

[0348] Through the above detailed methods and steps, the effects of new hydrogel materials on the intracellular antioxidant defense system can be systematically evaluated, which is crucial for understanding the role of these materials in combating the common oxidative stress in diabetic wound healing. This experiment not only provides important information about the biological activity of the material, but also provides a scientific basis for the development of new treatment strategies.

[0349] 13. In vitro assessment of macrophage polarization

[0350] The methods and procedures for evaluating macrophage polarization in vitro were designed to investigate the effects of GelDE, OCS@MOF@Pl nanoparticles, and GelDE-OCS@MOF@Pl hydrogels on the conversion of RAW 264.7 macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. The following are the detailed experimental methods and procedures:

[0351] 13.1 Experimental Preparation

[0352] Ensure that all reagents and consumables are sterile, including 96-well plates, PBS buffer, cell culture medium (containing 10% fetal bovine serum), LPS (lipopolysaccharide), CD86 / CD206 antibodies, DAPI staining solution, PE-CD86, APC-CD206, FITC-F4 / 80 fluorescent-labeled antibodies, etc.

[0353] 13.2 Cell Culture

[0354] The cells were cultured in RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin) at 37°C and 5% CO 2 The growth of RAW 264.7 macrophages was maintained under the following conditions, keeping them in a good healthy state.

[0355] 13.3 Cell seeding and pretreatment

[0356] Cell seeding: RAW264.7 cells in logarithmic growth phase were plated at 1×10 5 The cells were seeded into 96-well plates at a density of 10 cells / mL and an appropriate amount of complete culture medium was added.

[0357] Adhesion and stabilization: Allow the cells to adhere and grow stably in the incubator for 24 hours to ensure the formation of a uniform and dense monolayer of cells.

[0358] 13.4 M1 polarization induction

[0359] LPS stimulation: After washing the cells gently twice with PBS, fresh culture medium containing LPS at a final concentration of 10 μg / mL was added to each well and incubated for 24 h to induce the formation of M1 macrophages.

[0360] Wash to remove LPS: Gently wash three times with PBS to remove residual LPS to ensure that subsequent experiments only reflect the effect of the material on the conversion of M1 to M2.

[0361] 13.5. Material Addition and Co-cultivation

[0362] Material treatment: According to the experimental design, serum-free culture medium containing GelDE, OCS@MOF@Pl nanoparticles or GelDE-OCS@MOF@Pl hydrogel components was added to different wells, and three replicate wells were set for each condition.

[0363] Control group setting: A control group containing only PBS was also set up to compare the differences in effects among the treatment groups.

[0364] 13.6. Fixation and Permeabilization

[0365] Fixing cells: 24 hours after material treatment, wash the cells again twice with PBS, then add 4% paraformaldehyde and fix them at room temperature for 15 minutes.

[0366] Permeabilization treatment: After washing with PBS for 3 times, add PBS solution containing 0.5% TritonX-100 and incubate at room temperature for 10 minutes to increase the permeability of the cell membrane.

[0367] Block nonspecific binding sites: Block with PBS solution containing 10% BSA at room temperature for 30 minutes.

[0368] 13.7 Immunofluorescence staining

[0369] Primary antibody incubation: Add primary antibody against CD86 (M1 marker) or CD206 (M2 marker) and incubate overnight at 4°C.

[0370] Secondary antibody incubation: After washing three times with PBS, add the corresponding fluorescent-labeled secondary antibody and incubate at room temperature in the dark for 30 min.

[0371] Nuclear staining: Use DAPI to stain the cell nucleus and incubate in the dark for 5 min.

[0372] Sealing and imaging: The slides were sealed with antifade mounting medium, and then the fluorescence intensity in each well was observed and recorded using an inverted fluorescence microscope.

[0373] 13.8 Flow Cytometry Analysis

[0374] Cell collection: The RAW 264.7 cells treated as above were digested with trypsin to prepare a single cell suspension.

[0375] Fluorescent labeling: Add PE-CD86, APC-CD206 and FITC-F4 / 80 fluorescent labeled antibodies and incubate at room temperature in the dark for 30 minutes.

[0376] Flow cytometry: The expression levels of CD86 and CD206 were measured by flow cytometry to quantitatively analyze the changes in the proportion of M1 and M2 macrophages.

[0377] 13.9 Data Analysis

[0378] Image quantitative analysis: Use image analysis software (such as ImageJ) to measure the fluorescence intensity and calculate the average fluorescence unit (AU) to quantify the expression of CD86 and CD206.

[0379] Statistical analysis: Compare the differences in fluorescence intensity and flow cytometry results between different treatment groups, and use appropriate statistical methods (such as ANOVA analysis of variance) to determine the significance level.

[0380] 13.10. Results presentation

[0381] Make a bar graph or line graph to visually display the changes in the M1 / M2 ratio of each treatment group relative to the control group.

[0382] 13.11 Conclusion

[0383] Based on the experimental data, we discussed whether GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogels promoted the conversion of RAW 264.7 macrophages from M1 to M2 phenotype, and explored its potential mechanism.

[0384] Through the above detailed methods and steps, the effects of novel hydrogel materials on macrophage polarization behavior can be systematically evaluated, which is crucial for understanding the role of these materials in regulating immune responses during wound healing. This experiment not only provides important information about the biological activity of the material, but also provides a scientific basis for the development of new therapeutic strategies.

[0385] 14. Wound healing experiment in vivo

[0386] The methods and procedures for in vivo wound healing evaluation experiments were designed to investigate the effects of GelDE, OCS@MOF@Pl nanoparticles, and GelDE-OCS@MOF@Pl hydrogels on wound healing in a full-thickness skin defect model in diabetic rats. The following are the detailed experimental methods and procedures:

[0387] 14.1 Experimental Preparation

[0388] 14.1.1 Animal preparation:

[0389] Select healthy male Sprague-Dawley (SD) rats weighing between 200-250 g and ensure that all operations comply with the guidelines of the National Research Council's Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Shenzhen Lingfu Biotechnology Co., Ltd. (Permit No.: TOP-IACUC-2024-0065).

[0390] 14.1.2. Establishment of diabetes model:

[0391] After fasting for 12 h, the rats were intraperitoneally injected with streptozotocin (STZ, final concentration 55 mg / kg body weight) dissolved in citrate buffer (pH 4.4, 50 mM) to induce diabetes.

[0392] Fasting blood glucose levels were measured on the 3rd and 7th days after injection, and rats with blood glucose concentrations exceeding 16.7 mmol / L were used as the standard for successful establishment of the diabetes model.

[0393] 14.1.3. Surgery and wound creation:

[0394] Anesthesia: Rats were put into a deep sleep state using isoflurane inhalation anesthesia.

[0395] Surgical procedures under sterile conditions: The back of the rat was shaved and disinfected with iodine. Two circular full-thickness skin defects with a diameter of 8 mm were made in the center of the back using a sterile surgical blade, ensuring that each wound penetrated the dermis to the muscle tissue but did not damage the deeper structures.

[0396] 14.2 Material Application

[0397] Grouping and treatment: The rats were randomly divided into four groups, each containing at least 6 individuals: control group (PBS treatment only), GelDE group, OCS@MOF@Pl group, and GelDE-OCS@MOF@Pl group.

[0398] Immediately apply the corresponding material evenly to each wound surface and press gently to promote its contact with the wound surface; for the control group, only apply an equal amount of PBS solution.

[0399] 14.3 Observation and Recording

[0400] Monitoring of wound healing process:

[0401] Observe and take photos of the wound every day, and record changes in wound appearance.

[0402] Use image analysis software (such as ImageJ) to measure the wound area and calculate the daily healing rate. The formula is shown in formula (7):

[0403]

[0404] The time required for wound closure was recorded until complete epithelialization.

[0405] 14.4 Histological evaluation

[0406] Sampling and fixation: At specific time points (e.g., 7th and 14th days after surgery), some rats were killed and skin samples covering the entire wound area were taken out. The samples were immediately fixed in 4% paraformaldehyde fixative for 24 hours, then dehydrated, transparentized, and wax-embedded.

[0407] Sectioning and staining: Paraffin sections with a thickness of 5 μm were prepared and routine hematoxylin-eosin (H&E) staining was performed to evaluate the inflammatory cell infiltration, epithelial regeneration, and hair follicle regeneration of the wound tissue.

[0408] Masson's trichrome staining was performed to observe the deposition and arrangement of collagen fibers.

[0409] 14.5 Data Analysis

[0410] Compare the wound healing rates, histological characteristics and immunohistochemical results between different treatment groups, and use appropriate statistical methods (such as ANOVA analysis of variance) to determine the significance level. Analyze the healing progress trend of each group at different time points and explore the specific effects of the materials on each stage of wound healing (hemostasis, inflammation, proliferation, and remodeling).

[0411] 14.6. Results presentation

[0412] Make a bar graph or line graph to visually show the improvement of wound healing efficiency of each treatment group compared with the control group. Provide typical H&E and Masson staining pictures to show the microstructural characteristics of wound tissue under different treatment conditions.

[0413] 14.7 Conclusion

[0414] Based on the experimental data, this paper discusses whether GelDE, OCS@MOF@Pl nanoparticles and GelDE-OCS@MOF@Pl hydrogel promote skin wound healing in diabetic rats, and explores its potential mechanisms, including but not limited to reducing inflammatory response, promoting epithelialization and angiogenesis, and enhancing collagen deposition.

[0415] Through the above detailed methods and steps, the effects of new hydrogel materials on wound healing behavior in diabetic rats can be systematically evaluated, which is crucial for understanding the mechanism of action of these materials in the clinical treatment of chronic wounds. This experiment not only provides important information about the biological activity of the materials, but also provides a scientific basis for the development of new treatment strategies.

[0416] 15. Relevant experimental data and test results

[0417] 1. OCS and CS 1 H MNR and FTIR spectra

[0418] Prior to the synthesis of OCS@MOF@Pl nanoparticles, OCS was oxidized using sodium periodate. 1 H MNR and FTIR spectra are shown in Figure 2 As shown. The results showed that compared with CS, the OCS spectrum showed a new peak at 8.25ppm, which is a characteristic peak indicating the -CHO group. In addition, the FTIR spectrum of OCS has a new absorption peak at 1730cm mainly due to the stretching vibration characteristics of the C=0 bond of the acetaldehyde group. 'H NMR and FTIR results jointly confirmed the successful synthesis of organic compounds. Subsequently, Ui0-66-NH, (MOF), MOF@Pl and OCS@MOF@PI nanoparticles were continuously synthesized by hydrothermal method and blending method.

[0419] 2. Preparation and characterization of MOF, MOF@PI, MOF@Pl and OCS@MOF@Pl

[0420] MOF nanocrystals exhibit a well-ordered morphology, assuming a cubic octahedral structure, with particle sizes ranging mainly from 140 nm to 250 nm ( Figure 1 A and B in the figure). In contrast, the nanocrystals of MOF@Pl showed a slight increase in particle size indicating successful loading of Pl. After further encapsulation by OCS, the OCS@MOF@Pl nanocrystals further increased in size, mainly in the range of 190-290nm, and their morphology was close to spherical, indicating that MOF@Pl was successfully encapsulated by OCS. In order to evaluate whether the loading of Pl and the encapsulation of OCS changed the crystal structure of MOF, the crystal structure of OCS@MOF@Pl was analyzed by powder X-ray diffraction. The results showed that the crystal structures of MOF@Pl and OCS@MOF@Pl were consistent with those reported in related studies ( Figure 1 In addition, the nitrogen adsorption test results showed that the total pore volume and pore size were significantly reduced, and the pore size of OCS@MOF@Pl nanoparticles was reduced compared with MOF@Pl nanoparticles, which indicated that OCS effectively encapsulated MOF@Pl nanoparticles as a "gatekeeper" ( Figure 1 D and E in ).

[0421] 2.1 Morphological characteristics of MOF, MOF@PI, MOF@Pl and OCS@MOF@Pl

[0422] Transmission electron microscopy (TEM) images show that MOF nanocrystals present a well-ordered morphology with a cubic octahedral structure, and the particle size is mainly distributed between 140nm and 250nm ( Figure 3 A and B in Figure 1). With the loading of Pl, MOF@Pl nanocrystals showed a slightly increased particle size, indicating the successful loading of Pl onto MOF. After further encapsulation by OCS, the size of OCS@MOF@Pl nanoparticles increased again, mainly concentrated in the range of 190nm to 290nm, and the morphology was closer to spherical, which confirmed the successful encapsulation of MOF@Pl by OCS.

[0423] 2.2 Crystal structure analysis of MOF, MOF@PI, MOF@Pl and OCS@MOF@Pl

[0424] Powder X-ray diffraction (XRD) spectra were used to evaluate whether loading Pl and OCS encapsulation changed the crystal structure of MOF. The results showed that the crystal structures of MOF@Pl and OCS@MOF@Pl were consistent with those reported in the literature ( Figure 1C), indicating that despite the composite treatment, the basic crystal form of MOF has not changed significantly.

[0425] 2.3 Pore characteristics of MOF, MOF@PI, MOF@Pl and OCS@MOF@Pl

[0426] The specific surface area and pore structure of the nanoparticles (nitrogen adsorption-desorption isotherm (nitrogen fixation adsorption-desorption isotherm) and pore size distribution) were evaluated by nitrogen adsorption test. The test results showed that the total pore volume and average pore size of OCS@MOF@Pl nanoparticles were significantly reduced compared with MOF@Pl nanoparticles ( Figure 3 This phenomenon proves that OCS, as a “gating” substance, effectively blocks the pores of MOF@Pl nanoparticles and limits the internal space.

[0427] 3. GelMA and Gel 1 H MNR and FTIR spectra

[0428] Before the synthesis of GelDE and GelDE-OCS@MOF@Pl hydrogels, the preliminary synthesis of GelMA and DEXO was carried out. 1 The HNMR results are as follows Figure 4 As shown in A in FIG. At the chemical shifts of 5.4 and 5.7 ppm, GelMA shows two additional proton peaks compared to Gel, which can be mainly attributed to the characteristic peaks of protons on the C=C bond of methacrylic acid, which confirms the successful synthesis of GelMA. In addition, as Figure 4 As shown in B, GelMA exhibits characteristic bands of the Gel scaffold, including amide I, amide II, and amide III, demonstrating that GelMA retains the chemical properties of Gel.

[0429] 4. DEXO and DEX 1 HMNR spectrum (A) and FTIR spectrum (B);

[0430] In addition, compared with DEX, DEXO’s 1 The HNMR spectrum showed a new peak in the chemical shift range of 8.2-8.5 ppm, which was mainly attributed to the characteristic peak generated by the proton on the aldehyde group ( Figure 5 A). The infrared spectrum of DEXO shows a new characteristic absorption peak at 1730 cm -1 (aldehyde group)( Figure 5 B), which is significantly different from the DEX curve, thus confirming the successful synthesis of DEXO.

[0431] 5. Demonstration of the injectable properties of GelDE hydrogel

[0432] GelMA or DEXO are highly soluble in water alone; however, when they are combined by syringe mixing, they undergo a rapid Schiff base reaction, resulting in the formation of a water-insoluble gel, namely GelDE hydrogel ( Figure 6 ).

[0433] 6. Performance testing of GelDE-OCS@MOF@Pl hydrogel

[0434] However, the GelDE or GelDE-OCS@MOF@Pl hydrogels formed by Schiff base reaction alone are characterized by being soft and mechanically weak, with their tensile elastic moduli measured as 2.48 ± 0.49 kPa and 6.45 ± 0.71 kPa, respectively, and their compressive elastic moduli measured as 0.49 ± 0.08 kPa and 1.67 ± 0.2 kPa, respectively. Figure 7 AF in). After exposure to UV light for secondary cross-linking, the mechanical properties of GelDE and GelDE-OCS@MOF@Pl hydrogels have been significantly enhanced. Moreover, the introduction of OCS@MOF@Pl nanoparticles has significantly improved the mechanical properties of GelDE-OCS@MOF@Pl hydrogels over those of GelDE hydrogels. This improvement may be attributed to the Schiff base reaction or the hydrogen bonding interaction between the OCS@MOF@Pl nanoparticles and the GelMA or DEXO macromolecules, thereby further enhancing the toughness of the hydrogel network. In addition, after undergoing 30 cycles of cyclic tensile and compression tests, the tensile and compressive strengths of the GelDE-OCS@MOF@Pl hydrogels remained significantly unchanged, indicating the excellent mechanical stability of the GelDE-OCS@MOF@Pl hydrogels ( Figure 7 G and H in this paper). This advanced hydrogel dressing, known for its excellent flexibility and mechanical stability, creates a strong barrier for the wound, which is particularly beneficial for wounds during exercise, ensuring long-term and effective support for the repair process. Swelling is an important property of hydrogels. Figure 7 As shown in Figure 1, the swelling ratios of GelDE and GelDE-OCS@MOF@Pl hydrogels are 173.55±5.41% and 156.07±7.97%, respectively. GelDE and GelDE-OCS@MOF@Pl hydrogels have the best swelling ratio, which can cleverly absorb wound exudate and maintain a clean and moist environment for the wound. In addition, when these hydrogels swell, the volume changes are minimal, ensuring that no undue pressure is applied to the wound and that they do not spontaneously fall off the wound. At the same time, SEM images show that compared with GelDE hydrogel, GelDE-OCS@MOF@Pl hydrogel has a more ordered and dense pore structure ( Figure 7J). This rich microporous structure promotes enhanced cell adhesion and the transport of nutrients and oxygen, thereby establishing a physical environment that is conducive to wound healing.

[0435] 7. Antioxidant properties of hydrogel

[0436] Diabetic patients, due to factors such as hyperglycemia and insulin resistance, are more susceptible to oxidative stress, which may lead to cell damage, aggravate inflammatory responses, inhibit angiogenesis, and cause delayed wound healing or even the formation of chronic wounds. Therefore, therapeutic strategies targeting oxidative stress, such as the administration of antioxidants, represent a reflex approach to promote diabetic wound healing. Subsequently, the antioxidant performance of GelDE-OCS@MOF@PL hydrogels was evaluated by DPPH scavenging method and ABTS scavenging method. Figure 8 As shown in the AD, both GelMA and GelDE hydrogels have significant scavenging ability for DPPH. In contrast, OCS@MOF@Pl nanoparticles exhibited significant free radical scavenging effects with a scavenging efficiency of 64.69±2.82%, with DPPH radicals accounting for 58.14±1.57% and ABTS radicals accounting for 58.14±1.57%. PL polymers contain many 13 phenolic hydroxyl groups, which endow them with special antioxidant properties and, by extension, endow OCS@MOF@Pl nanoparticles with excellent antioxidant capacity. In addition, the antioxidant properties of GelDE-OCS@MOF@Pl hydrogels were slightly reduced compared with those of CS@MOF@Pl nanoparticles. This phenomenon, which can be attributed to the dual encapsulation of Pl by hydrogels and nanoparticles, highlights that GelDE-OCS@MOF@Pl hydrogels gradually release Pl at the wound site, thereby continuously scavenging ROS and promoting rapid wound healing.

[0437] 8. In vitro drug release and antibacterial properties

[0438] In vitro drug release behavior is a key property of tissue repair materials, and the wound pH environment is variable, which can induce different drug release profiles. Figure 8As shown in Figure E, the release rate of Pl exhibits a rapid initial stage within the first 8 h, after which it gradually slows down. In addition, the decrease in solution pH is associated with an increased release rate of Pl. Specifically, OCS@MOF@Pl hydrogel was 44.30±3.21% at pH 74, pH 6.2, and pH 5.2, and 52.69±4.18% and 67.5±1.6% after 96 h, respectively. This phenomenon arises in an acidic environment to catalyze the dissociation of OCS on the surface of MO materials (via the cleavage of Schiff base bonds) and subsequently MOF materials, accelerating the release of encapsulated drugs. In addition, the release behavior of Pl was carefully evaluated using zero-order, first-order, Higuchi, and Korsmeyer-Peppas kinetic models. S5A-S5D, 14. The R' values ​​of the Korsmeyer-Peppas and Higuchi models were significantly higher than those at pH 5.2 and pH 6.2, while the results of the other models were close to 1. This indicates that the hydrogel loaded with Pl contains multiple release mechanisms, including free diffusion and carrier dissolution. Skin injuries are open wounds that are constantly facing bacterial invasion, which greatly impairs the healing process. Therefore, antimicrobial activity refers to the basic property of wound dressings. Antimicrobial results ( Figure 8 F and G in Figure 2 indicate that the survival rates of OCS@MOF@Pl and GelDE-OCS@MOF@Pl against Escherichia coli and Staphylococcus aureus were both less than 40% when compared with the control group (Control). More importantly, as Pl was slowly released from GelDE-OCS@MOF@Pl hydrogel, it promoted the continuous inhibition of bacterial growth on the wound surface, thus playing a solid role as a guardian of the wound healing process.

[0439] 9. Biocompatibility evaluation of hydrogels

[0440] Good cytocompatibility is a prerequisite for wound dressings to promote wound healing. Due to the ultra-micro size of nanomaterials, they can be easily taken up and accumulated by cells, posing a potential risk. After 24 hours of co-culture of OCS@MOF@Pl with human umbilical vein endothelial cells, the cytotoxicity index was 0.40l. The survival rate of umbilical vein endothelial cells first increased and then decreased with increasing concentration (from 0 to 120μg / mL). When the concentration reached 60μg / mL, the survival rate of human umbilical vein endothelial cells peaked at 120.65±2.7%. When the OCS@MOF@Pl nanoparticles were increased to 100μg / mL, the cell viability index of HUVECs remained strong, remaining at 104.45+2.38% ( Fig. 9In addition, GelDE, OCS@MOF@Pl and GelDE-OCS@MOF@P hydrogels were used to culture human umbilical vein endothelial cells for five consecutive days to further investigate the cell compatibility of these hydrogels (wound healing materials). CCK-8 test results ( Fig. 9 B) and live / dead cell staining images ( Fig. 9 C) shows that GelDE, OCS@MOF@PI, GelDE-OCS@MOF@Pl hydrogels continuously promote cell proliferation and show significant cell compatibility. In addition, the hemolysis test results show that GelDE, OCS@MOF@Pl. and GelDE-OCS@MOF@P show the lowest hemolysis rate (<0.16). Therefore, this experiment confirms the excellent biocompatibility of these materials. In addition, GelDE, OCS@MOF@Pl and GelDE-OCS@MOF@Pl hydrogels improve the adhesion ability of cells ( Fig. 9 (D and E) Compared with the control group, the cell spreading ratio of GelDE-OCS@MOF@Pl hydrogel reached 1.54 ± 0.1. This enhanced function helps in the epidermalization of skin wounds and accelerates the healing process.

[0441] 10. Angiogenic properties

[0442] Angiogenesis within damaged tissue is another key component affecting wounds. The effects of different materials on angiogenesis were studied. Cell migration and tube formation analysis were performed using HUVEC. Cell migration in OCS@MOF@Pl and GelDE-OCS@MOF@Pl treatments significantly exceeded that of the control group. Fig. 9 As described in F and G in Figure 3. In addition, GelDE, OCS@MOF@Pl, and GelDE-OCS@MOF@Pl hydrogels all showed the ability to promote HUVECs to form tubular structures, with each subsequent formulation showing an enhanced effect. Notably, GelDE-OCS@MOF@Pl hydrogel-treated HUVECs formed the most comprehensive and extensive vascular networks, both in terms of structural integrity and numerical abundance ( Fig. 9 H and I in Figure 3). The results highlighted that the gradual release of nanoparticles and PI promoted the formation of blood vessels, which began to supply oxygen and nutrients to the wound site, thus accelerating the healing process.

[0443] 11. Evaluation of antioxidant effects and induction of macrophage polarization

[0444] In diabetic ulcers, bacterial infection or inflammatory response usually produces a large amount of ROS, which aggravates the inflammatory response, causes cell damage, hinders angiogenesis, and significantly delays wound healing. Therefore, the development of biomedical materials with ROS removal is of great significance in promoting wound healing. The results show that ( Fig.10 A) Compared with the PBS group and the GelDE group, cells treated with OCS@MOF@Pl and GelDE-OCS@MOF@Pl showed lower levels of ROS. This indicates that OCS@MOF@Pl nanoparticles endow the hydrogel with excellent antioxidant properties, effectively scavenging intracellular ROS and thus protecting cells from damage caused by oxidative stress.

[0445] Macrophages are a type of innate immune cell that plays a key role in the regulation of diabetic wounds and tissue repair. The abnormal function of M macrophages (promoting inflammatory responses) transforms the phenotype of M2 macrophages (involved in immune regulation and tissue remodeling), exacerbating inflammation and delaying wound healing. Fig.10 As shown in B, compared with the normal group, macrophages in the PBS group showed significant green fluorescence (CD86) expression, indicating the successful conversion of M0 phenotype macrophages into M1 macrophages by LPS induction. In OCS@MOF@Pl and GelDEOCS@MOF@Pl treatments, the green fluorescence gradually weakened, while the red fluorescence (CD206) was significantly enhanced, marking the successful polarization of GelDE on the effect of macrophages from M1 phenotype to M2 phenotype. In OCS@MOF@PI treatment and GelDE-OCS@MOF@Pl treatment, the green fluorescence was significantly weaker and the red fluorescence was significantly enhanced, indicating a special anti-inflammatory effect. Subsequently, the results were analyzed by flow cytometry, and they were consistent with the results of cell fluorescence staining ( Fig.10 C). Notably, the number of CD86 in macrophages after treatment with OCS@MOF@P and GelDE-OCS@MOF@Pl was 9.18% and 3.07%, respectively, while the expression of CD206 was 39.4% and 40.5%, respectively. These findings suggest that the combination of GelDE and OCS@MOF@PL endows GelDE-OCS@MOF@Pl hydrogel with significant anti-inflammatory properties, actively catalyzing the phenotype transition of macrophages from M1 to M2. This provides a solid theoretical basis for reducing wound inflammation and accelerating wound healing.

[0446] 12. Evaluation of wound healing in diabetic patients

[0447] like Fig.11As shown in Figure A, the investigation explored the benefits of wound healing. The effects of different hydrogels on full-thickness wound repair in diabetic mice were studied for 14 days. As the treatment time increased, the wound area in the control group was significantly reduced. The wound area in the GelDE.OCS@MOF@Pl and GelDE-OCS@MOF@Pl groups was significantly reduced. As the wound healing rate gradually increased in each group ( Fig.11 B). The wound healing rates of the control group and GelDE group were slower, with severe wounds remaining even on the 14th day after injury, and the healing rates were 60.09±2.92% and 73.9±1.83%, respectively. In sharp contrast, GelDE-OCS@MOF@PL hydrogel showed the fastest acceleration in wound repair, reaching a healing rate of 94.04±2.08% on the 14th day after wound healing ( Fig.11 C) GelDE-OCS@MOF@Pl hydrogels are known for their outstanding biocompatibility. While continuously and gradually releasing drugs, they create a physical barrier for the wound, thereby improving the wound microenvironment and accelerating the wound healing process. In contrast, the application of OCS@MOF@Pl nanoparticles alone in wound repair poses a challenge: rapid drug release may disrupt the healing trajectory. And the limited residence time of nanoparticles on the wound surface hinders their full potential, as they can easily lose activity before they can achieve their maximum efficacy.

[0448] The effects of different hydrogels on the repair process were analyzed by histology to more comprehensively evaluate the wound healing process. H&E staining results of skin wound tissue showed that by the 7th day of wound healing, the wound area of ​​the control group, GelDE group, and OCS@MOF@PL group was significantly increased ( Fig.11 D in the figure). The high infiltration of inflammatory cells, the reduced number of regenerated hair follicles, and the low degree of re-epithelialization indicate poor wound tissue repair, while GelDE-OCS@MOF@PL hydrogel has a good wound tissue repair effect. In contrast, wounds treated with GelDE-OCS@MOF@PL hydrogel showed a significant reduction in wound area after 7 days, and the regenerated hair follicles and epidermal thickness reached a peak 14 days after the wound healing experiment. The epidermalization of the wound tissue in the GelDE-OCS@MOF@PL group was completely completed and restored to its typical morphology, while the control group and GelDE group still had significant expansibility in the wound area, characterized by an increase in inflammatory cells, indicating a reduced degree of wound repair ( Fig.11 E and F in the figure). The results of Masson staining are as follows Fig.11As shown in G and H in Figure 3. The results showed that on the 7th day after wound healing, collagen was significantly scarce and unevenly distributed in the wound tissue of the control group, GelDE group, and OCS@MOF@Pl group. In contrast, the collagen distribution in the GelDE-OCS@MOF@PL group was more uniform, and interstitial edema was not obvious; after healing on the 14th day after injury, collagen deposition increased in all groups. It is worth noting that the GelDE-OCS@MOF@PL group showed the most abundant and best organized collagen deposition. These histological findings are consistent with the visual observation results of wound repair. It shows that GelDE-OCS@MOF@Pl hydrogel effectively reduces wound inflammatory response, promotes epidermal and hair follicle regeneration, promotes collagen deposition, and ultimately has a positive effect on wound repair.

[0449] 13. Expression analysis of IL-6 and IL-10

[0450] The stages of wound healing can usually be divided into hemostasis, inflammation, proliferation and remodeling. Inflammation significantly impedes tissue response. Immunohistochemical staining and quantitative analysis of L-6 and L-10 showed ( Fig.12 AD), the expression of IL-6 and IL-10 was the highest in the control group a few days after wound repair, indicating that the wound was still in the inflammatory stage. However, the OCS@MOF@Pl group and the GelDE-OCS@MOF@Pl group decreased successively, indicating that the wounds of diabetic patients were transitioning from the inflammatory stage to the repair stage. Among them, in the GelDE-OCS@MOF@Pl group, the expression of IL-6 and IL-10 in breast cancer was the lowest, indicating that the GelDE-OCS@MOF@Pl hydrogel has an excellent anti-inflammatory effect in wound repair, and actively promotes macrophages to the M2 phenotype, accelerating the wound repair process.

[0451] 14. Angiogenesis Indicators

[0452] Angiogenesis is an important indicator of wound healing. The wounds were treated with GelDE, OCS@MOF@Pl and GelDE-OCS@MOF@Pl for 7 times, and CD31 (green) and a-SMA (red) marker staining were depicted in the wound sites of different treatment groups by immunofluorescence. During the wound healing stage, GelDE-OCS@MOF@Pl hydrogels can contract and move actin stress fibers, which can be produced in large quantities through the expression of α-smooth muscle actin by myofibroblasts, thereby enabling the remodeling and healing of damaged tissues. Immunofluorescence images and semi-quantitative fluorescence analysis ( Fig.12E and G in the figure show that the expression of CD31 and a-SMA in other groups was significantly higher than that in the control group. In the GelDE group, OCS@MOF@P group 1 and GelDE-OCS@MOF@Pl group, the fluorescence intensity of CD31 and a-SMA gradually increased, and the expression levels of CD31 and a-SMA in the GelDE-OCS@MOF@Pl group were the highest, indicating that the wounds treated with GelDE-OCS@MOF@Pl hydrogel had the most extensive vascular network. This enhanced angiogenesis effect promotes the supply of oxygen and nutrients to the injured area, thereby accelerating the wound healing process. GelDE-OCS@MOF@Pl hydrogel can provide the best microenvironment for wound tissue and gradually release active substances that promote the recruitment of myofibroblasts and angiogenesis, ultimately accelerating the healing of diabetic wounds.

[0453] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A drug delivery system for promoting diabetic wound healing, characterized in that: The drug delivery system comprises oxidized dextran, methacryloyl gelatin and drug-loaded UiO-66-NH2 nanoparticles wrapped by oxidized chondroitin sulfate; the drug loaded by the drug-loaded UiO-66-NH2 nanoparticles is Paris polyphylla saponin 1; The method for preparing the drug-loaded UiO-66-NH2 nanoparticles wrapped by oxidized chondroitin sulfate comprises the steps of mixing oxidized chondroitin sulfate and drug-loaded UiO-66-NH2 nanoparticles to obtain the drug-loaded UiO-66-NH2 nanoparticles wrapped by oxidized chondroitin sulfate.

2. The drug delivery system according to claim 1, characterized in that The preparation method of the drug-loaded UiO-66-NH2 nanoparticles comprises the steps of mixing UiO-66-NH2 and Paris polyphylla saponin 1 to obtain the drug-loaded UiO-66-NH2 nanoparticles.

3. The method for preparing the drug delivery system according to claim 1, characterized in that: The following steps are involved: The oxidized chondroitin sulfate solution and the drug-loaded UiO-66-NH2 nanoparticles are mixed and dissolved in water to obtain a solution containing the drug-loaded UiO-66-NH2 nanoparticles wrapped by the oxidized chondroitin sulfate; the drug loaded by the drug-loaded UiO-66-NH2 nanoparticles is Paris polyphylla saponin 1; The solution containing the drug-loaded UiO-66-NH2 nanoparticles wrapped by oxidized chondroitin sulfate, the oxidized dextran solution and the methacrylylated gelatin solution are mixed to obtain the drug delivery system.

4. The preparation method according to claim 3, characterized in that: The preparation method of the drug-loaded UiO-66-NH2 nanoparticles comprises the steps of mixing UiO-66-NH2 and Paris polyphylla saponin 1 to obtain the drug-loaded UiO-66-NH2 nanoparticles.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the UiO-66-NH2 to the Paris polyphylla saponin 1 is 2:

1.

6. The preparation method according to claim 3, characterized in that: The mass ratio of the oxidized chondroitin sulfate and the drug-loaded UiO-66-NH2 nanoparticles in the oxidized chondroitin sulfate solution is 2:

1.

7. The preparation method according to claim 3, characterized in that: In the drug delivery system, the final concentration of oxidized dextran is 4%, the final concentration of methacryloyl gelatin is 15%, and the final concentration of drug-loaded UiO-66-NH2 nanoparticles wrapped by oxidized chondroitin sulfate is 0.2%.

8. Use of the drug delivery system according to claim 1 or 2 in the preparation of a product for promoting diabetic wound healing.

9. The use according to claim 8, characterized in that: The product includes a drug; the drug includes a hydrogel excipient.

10. A product for promoting diabetic wound healing, characterized in that: The product comprises the drug delivery system according to claim 1 or 2.

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