Nanozyme-encapsulated multifunctional composite hydrogel, preparation method and application thereof

Abstract

The application is a kind of nanoscale enzyme multifunctional composite hydrogel and its preparation method and application, belonging to the field of biological medicine technology. In view of the problem that the existing materials or drugs have poor treatment effect on infected wound, the application comprises the following steps: preparing solution A and solution B respectively, standby, preparing Cu-TCPP-Mn nanosheet, obtaining Cu-TCPP-Mn nanodot after further treatment, adding the activated Cu-TCPP-Mn nanodot into solution A to obtain mixed solution C with a concentration of 5 mu g / mL, adding the activated PRP solution into solution B to obtain mixed solution D with a concentration of 100 mu g / mL, and mixing the mixed solution C and the mixed solution D in equal volume to obtain CHPM composite hydrogel. The CHPM composite hydrogel can effectively reduce the oxidative stress level of acute infected wound tissue, provide a suitable oxidative microenvironment for PRP to play a role in promoting angiogenesis, and accelerate the healing of acute infected wound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a multifunctional composite hydrogel carrying nanozymes, its preparation method, and its application. Background Technology

[0002] Currently, precise treatment of acute infected wounds is a critical issue that urgently needs to be addressed in clinical medicine. Globally, such wounds, due to their prolonged healing time, are prone to fatal complications such as sepsis and multiple organ dysfunction, increasing patient mortality by 20%-30% and posing a significant threat to public health. Current clinical protocols face three major challenges: First, while systemic antibiotic administration can rapidly control infection, 60%-70% of the drug in the blood cannot effectively accumulate at the wound site, leading to systemic side effects such as liver and kidney toxicity. Second, while mechanical debridement can temporarily remove necrotic tissue, repeated procedures can damage newly formed granulation tissue, delaying the healing process. Third, while local antibiotic preparations increase drug concentration at the lesion site, their explosive release disrupts the wound microenvironment homeostasis, making it easier to induce the development of multidrug-resistant strains. Therefore, constructing novel biomaterials with spatiotemporally controllable drug release characteristics to achieve synergistic regulation of antibacterial, anti-inflammatory, and repair-promoting effects has become an important research direction for overcoming existing treatment bottlenecks.

[0003] Since Winter proposed the theory of moist wound healing in 1962, basic, translational, and clinical research on the wound repair microenvironment has been continuously deepening. Hydrogels, due to their excellent hydrophilicity, biocompatibility, and three-dimensional porous structure resembling the extracellular matrix, have gained considerable favor in wound repair management due to their unique advantages. The performance of hydrogels can be optimized by modifying the polymer backbone, concentration ratio, and cross-linking methods. They can also be combined with bioactive molecules, drugs, or cells to construct delivery systems, allowing for the creation of customized medical wound dressings for local application based on different wound types.

[0004] Against this backdrop, the emergence of nanozyme technology offers new possibilities for the treatment of acutely infected wounds. As a novel biocatalyst, nanozymes not only inherit the catalytic properties of natural enzymes but also possess higher stability and controllability. Copper-tetra(4-carboxyphenyl)porphyrin-manganese (Cu-TCPP-Mn) nanosheets are metal-organic framework (MOF) materials that mimic the cascade activity of superoxide dismutase (SOD) and catalase (CAT). Furthermore, they effectively combat bacteria and disrupt biofilms, reducing wound infection and avoiding the drug resistance problems that may arise from traditional antibiotics. Compared to natural antioxidant enzymes, which are scarce, costly to prepare, and easily inactivated, Cu-TCPP-Mn nanozymes exhibit superior characteristics such as strong environmental adaptability, structural stability, and ease of preparation.

[0005] Platelet-rich plasma (PRP) is a blood-derived product widely used in many clinical fields such as orthopedics and dentistry. PRP can be derived from the patient's own blood, thus avoiding immune rejection and reducing the risk of infectious diseases. PRP contains a high concentration of platelets, and upon activation, it can release a large number of growth factors, such as PDGF, TGF-β, and PRP itself. These growth factors can promote cell proliferation and differentiation, epithelialization, fibroblast proliferation, angiogenesis, and macrophage polarization. However, the transient release of growth factors or proteins in PRP often hinders therapeutic efficacy. To further enhance the effects of PRP, we considered loading it onto hydrogel materials for application.

[0006] Infected wounds are often accompanied by bacterial infection, tissue necrosis, and inflammatory reactions. If not treated promptly and effectively, they can lead to infection spread, delayed wound healing, and even serious complications such as systemic infection. However, single-function materials or drugs often have limited effectiveness in treating complex infected wounds. Synergistically integrating nanomaterials and biomaterials with different physicochemical properties, bioactivity, and drug-carrying capacities is a more rational approach to treating infected wounds. Based on these considerations, there is an urgent need to research novel biomaterials that can stably, economically, efficiently, and conveniently promote the healing of infected wounds, effectively control infection, promote tissue repair, and accelerate wound healing, thereby reducing hospital stays, lowering treatment costs, and improving patients' quality of life. This has significant clinical application value and broad development prospects. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, the present invention aims to provide a multifunctional composite hydrogel loaded with nanozymes, its preparation method, and its applications. An OHA / CMCS hydrogel with unique self-healing properties is synthesized through a Schiff base reaction between the aldehyde group of oxidized hyaluronic acid (OHA) and the carboxymethyl group of carboxymethyl chitosan (CMCS). This forms a cross-linked network with adjustable mechanical properties and good biocompatibility, suitable for treating wounds of different shapes without displacement. The effective loading and slow release of PRP provides the hydrogel with abundant growth factors, enhancing its ability to promote skin tissue regeneration. Meanwhile, the Cu-TCPP-Mn nanozyme mimics the activity of SOD and CAT, effectively scavenging reactive oxygen species (ROS). The synergistic effect of PRP and Cu-TCPP-Mn within the hydrogel framework regulates macrophage polarization and reduces the level of persistent inflammation in infected wounds, thereby solving the problems existing in the background technology.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention discloses a method for preparing a multifunctional composite hydrogel carrying nanozymes, comprising the following steps:

[0010] S1: Prepare solutions A and B separately for later use;

[0011] S2: Prepare Cu-TCPP-Mn nanosheets, and further process them to obtain Cu-TCPP-Mn nanodots;

[0012] S3: Add the Cu-TCPP-Mn nanodots obtained in step S2 to solution A to obtain a mixed solution C with a concentration of 2~20μg / mL;

[0013] S4: Add the activated PRP solution to solution B to obtain a mixed solution D with a concentration of 50~400μg / mL;

[0014] S5: Mixing solution C and solution D in equal volumes yields CHPM composite hydrogel.

[0015] As a further preferred embodiment of the above scheme, the process of preparing solution A in step S1 is as follows:

[0016] S101: Dissolve hyaluronic acid in deionized water and stir to dissolve, to obtain a hyaluronic acid solution;

[0017] S102: Add NaIO4 to the hyaluronic acid solution and continue stirring. After dialysis, freezing and drying, a white flocculent substance of oxidized hyaluronic acid is obtained.

[0018] S103: Add deionized water to the white flocculent oxidized hyaluronic acid and stir to dissolve, to obtain solution A.

[0019] A further preferred embodiment is that, in step S101, the concentration of solution A is 0~10 w / v.

[0020] As a further preferred embodiment of the above scheme, in step S1, carboxymethyl chitosan is dissolved in deionized water, and the volume ratio of carboxymethyl chitosan to deionized water is 0.02~0.06:1. After stirring and dissolving, a solution B with a concentration of 1~20 w / v% is obtained.

[0021] As a further preferred embodiment of the above scheme, the process of preparing Cu-TCPP-Mn nanosheets in step S2 is as follows:

[0022] S201: Weigh out Cu(NO3)2 and H2TCPP in proportion, dissolve them in DMF solution, and add benzoic acid to form a homogeneous solution;

[0023] S202: The obtained homogeneous solution was placed in methyl silicone oil at 90℃ and stirred for 2~6h to obtain Cu-TCPP intermediate product;

[0024] S203: The Cu-TCPP intermediate product was mixed with MnCl2 and heated and stirred at 90℃ for 10-16 h. After centrifugation, the precipitate was collected and washed with anhydrous ethanol at least 3 times to obtain Cu-TCPP-Mn nanosheets.

[0025] A further preferred method is as follows: In step S2, Cu-TCPP-Mn nanosheets are ultrasonically treated for 4 hours to obtain Cu-TCPP-Mn nanodots.

[0026] A further preferred embodiment is that, in steps S3-S4, the concentrations of mixed solution C and mixed solution D are 5 μg / mL and 100 μg / mL, respectively.

[0027] Secondly, the present invention also discloses a nanoenzyme-loaded multifunctional composite hydrogel, which is prepared by the above-mentioned method for preparing a nanoenzyme-loaded multifunctional composite hydrogel.

[0028] Thirdly, the present invention also discloses the application of a nanoenzyme-loaded multifunctional composite hydrogel in the preparation of drugs for acute infected wounds.

[0029] Fourthly, this invention also discloses the application of a nanoenzyme-loaded multifunctional composite hydrogel in the preparation of novel antibacterial materials.

[0030] Compared with the prior art, the improvements of the present invention can produce the following beneficial effects:

[0031] 1. This invention introduces a novel functional group (aldehyde group) into hyaluronic acid to obtain oxidized hyaluronic acid (OHA). Based on the Schiff base reaction principle, the aldehyde group of OHA and the carboxyl group of CMCS form an imine bond, forming an OHA / CMCS hydrogel framework. Utilizing the efficient scavenging of reactive oxygen species and antibacterial activity of CU-TCPP-Mn nanozymes, the antibacterial ability of CMCS, and the pro-angiogenic properties of PRP, a composite bioactive material loaded with nanozymes for acute infected wounds is constructed. The synergistic effect of CU-TCPP-Mn's antioxidant activity, PRP's biotherapy, and OHA / CMCS's wound protection in treating acute infected wounds is achieved. Due to the dynamic Schiff base bonding enhancing its functionality, this characteristic endows the CHPM composite hydrogel with unique self-healing properties, meaning it can automatically recover its shape after injury. It can be used to treat complex wounds of different shapes and depths without displacement, demonstrating great potential in treating acute infected wounds and promoting wound healing.

[0032] 2. The PRP-based CHPM composite hydrogel of the present invention is very suitable for promoting the healing of infected wounds and accelerating the healing of acute and chronic wounds. When PRP is loaded onto the hydrogel, it overcomes the limitation of rapid release of PRP in vivo and ensures that the growth factors in PRP are effectively utilized in the wound repair process.

[0033] 3. This invention constructs an OHA / CMCS hydrogel framework from OHA and CMCS. Both materials are biocompatible natural polymers with good biocompatibility and biodegradability. They do not cause immune rejection reactions in skin applications and gradually degrade during the skin healing process, avoiding long-term retention in the body. Furthermore, the OHA / CMCS hydrogel framework is formed through self-assembly via a Schiff base reaction between CMCS and OHA. This cross-linking method endows the hydrogel with good mechanical properties and stability, as well as a certain degree of elasticity and strength, enabling it to withstand mechanical stresses such as stretching and compression during daily activities. It is not prone to cracking or deformation, thus ensuring stable adhesion and continuous effect on the skin surface.

[0034] 4. The OHA / CMCS framework in the CHPM composite hydrogel of this invention exhibits good antibacterial activity, effectively inhibiting the growth of various common pathogenic bacteria. CMCS itself possesses antibacterial properties; its highly charged multi-cationic structure interacts strongly with the negatively charged bacterial phospholipid membrane components, leading to bacterial membrane damage and leakage of contents, thereby killing bacteria. Furthermore, the active aldehyde groups in OHA can also react with the amino groups on the bacterial surface, further enhancing the antibacterial effect of the hydrogel. This antibacterial property is of great significance for the prevention and control of skin infections, reducing the incidence and severity of infections and promoting wound healing. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0036] Figure 1 This is a flowchart of the preparation method of Cu-TCPP-Mn nanozyme and CHPM composite hydrogel carrying Cu-TCPP-Mn nanozyme to promote accelerated healing of infected wounds provided by the present invention; wherein, a represents the synthesis of Cu-TCPP-Mn nanodots, b represents the synthesis of CHPM hydrogel formulation and its self-healing properties, and c represents the healing mechanism of CHPM self-healing hydrogel in treating infected wounds.

[0037] Figure 2 These are morphological images of the Cu-TCPP-Mn nanozyme provided by the present invention; wherein, a represents the SEM morphology image of Cu-TCPP-Mn nanosheets, b represents the XPS spectrum of Cu-TCPP-Mn nanodots, c represents the Cu 2p XPS spectrum, and d represents the Mn 2p XPS spectrum of Cu-TCPP-Mn nanodots.

[0038] Figure 3This is a characterization diagram of the Cu-TCPP-Mn nanozyme provided by the present invention; where a represents the morphology and elemental localization of Cu-TCPP-Mn nanosheets observed under a scanning electron microscope, b represents the TEM morphology analysis of Cu-TCPP-Mn nanodots, c represents the particle size analysis of Cu-TCPP-Mn nanodots, d represents the Zeta potential of Cu-TCPP-Mn nanodots, and e represents the Fourier transform infrared spectrum of Cu-TCPP-Mn nanodots.

[0039] Figure 4 This invention provides a functional test of Cu-TCPP-Mn nanozymes; where a represents the UV absorption spectrum of Cu-TCPP-Mn nanodots, b represents the CAT-like activity of Cu-TCPP-Mn nanodots, c represents the SOD-like activity of Cu-TCPP-Mn nanodots, and d represents the amount of oxygen generated in the CAT-like activity of Cu-TCPP-Mn nanodots.

[0040] Figure 5 These are characterization images of the CHPM composite hydrogel provided by this invention; where a represents the CH hydrogel constructed from CMCS and OHA, b represents the self-healing property of the hydrogel, c represents the injectability of the hydrogel, d represents the shear thinning property of the hydrogel, e represents the rheological behavior of the hydrogel in time-scanning mode, f represents the rheological behavior of the hydrogel in amplitude-scanning mode, g represents the rheological behavior of the hydrogel in alternating strain mode, h represents the expansion rate of the hydrogel, and i represents the scanning electron microscope morphology of the hydrogel.

[0041] Figure 6 This is the invention Figure 5 Enlarged view of neutron diagrams f and g.

[0042] Figure 7 This is a graph showing the cell compatibility results of the CHPM composite hydrogel provided by this invention on HUVEC, HACAT, and HSF cells; where a represents the cytotoxicity of the hydrogel to HACAT, b represents the cytotoxicity of the hydrogel to HUVEC, c represents the cytotoxicity of the hydrogel to HSF, d represents the quantitative analysis of HACAT cell viability, e represents the quantitative analysis of HUVEC cell viability, and f represents the quantitative analysis of HSF cell viability.

[0043] Figure 8 This is a graph showing the blood compatibility results under co-incubation with the CHPM composite hydrogel provided by this invention.

[0044] Figure 9 This is a diagram showing the organ compatibility results under co-incubation with CHPM composite hydrogel provided by the present invention.

[0045] Figure 10The figure shows the effects of the CHPM composite hydrogel provided by this invention on HUVEC cell migration, angiogenesis, and ROS resistance. In the figure, a represents the HUVEC migration experiment, b represents the angiogenesis experiment, c represents the ROS scavenging ability of the hydrogel, d represents the quantitative statistics of the migration ratio, e represents the quantitative statistics of the tube formation length, and f represents the quantitative statistics of ROS scavenging.

[0046] Figure 11 The figure shows the effect of the CHPM composite hydrogel provided by this invention on LPS-induced M2 polarization of RAW 264.7 cells; where a represents the immunofluorescence images of iNOS and CD206 in RAW264.7 cells treated with different hydrogels, iNOS (green), CD206 (red) and DAPI (blue), b represents the relative quantitative expression level of iNOS, and c represents the relative quantitative expression level of CD206 detected.

[0047] Figure 12 This is a graph showing the antibacterial properties of the CHPM composite hydrogel provided by this invention against Staphylococcus aureus and Escherichia coli; where a represents the plate coating experiment against Staphylococcus aureus and Escherichia coli, b represents the effect of the hydrogel on Staphylococcus aureus and Escherichia coli observed by electron microscopy, c represents the live / dead staining experiment against Staphylococcus aureus and Escherichia coli, and d~e represent the quantitative statistics of the bacterial plate coating experiment.

[0048] Figure 13 The figure shows the effect of the CHPM composite hydrogel provided by this invention on the healing of acute infected wounds in mice. Different groups were treated at 0, 3, 6, 9, and 12 days. Among them, a represents wound healing treatment, b represents skin animation tracer images of different experimental groups, c represents H&E stained skin wound tissue sections, d represents quantitative statistics of wound healing rate, and e represents quantitative analysis of wound width.

[0049] Figure 14 This invention provides a tissue staining analysis of wounds using CHPM composite hydrogel; where a represents Masson staining of skin wound tissue, b represents Sirius red staining of skin wound tissue, c represents IL-6 staining of skin wound tissue, d represents TNF-α staining of skin wound tissue, e represents quantitative analysis of collagen deposition, f represents quantitative analysis of fibrotic areas, g represents quantitative statistics of IL-6 expression, and h represents quantitative statistics of TNF-α expression.

[0050] Figure 15 This is the invention Figure 14 Enlarged view of neutron diagrams e, f, g, and h. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0052] This invention relates to a multifunctional composite hydrogel carrying nanozymes, its preparation method, and its applications. The following is a detailed description in conjunction with the appendix. Figures 1-15 The present invention will be described in detail.

[0053] Experimental materials:

[0054] Hyaluronic acid, carboxymethyl chitosan, sodium periodate, H2TCPP, copper nitrate trihydrate, N,N-dimethylformamide (DMF, 99.8%), manganese chloride tetrahydrate, benzoic acid, and ethylene glycol were purchased from Shanghai Maclean's. HUVEC, HACAT, CCK-8 assay kits, reactive oxygen species detection kits, and cell viability / death staining kits were purchased from Wuhan Saiwei Biotechnology Co., Ltd. Milli-Q purified water was used in the experiments, and all chemical reagents were analytical grade and used directly without purification.

[0055] like Figure 1 As shown, the present invention provides a method for preparing a multifunctional composite hydrogel carrying nanozymes, comprising the following steps:

[0056] S1: Prepare solutions A and B separately for later use;

[0057] In this step, the preparation process of solution A is as follows: 1g of hyaluronic acid is weighed and dissolved in 100mL of deionized water (100mL, 10mg / mL) and stirred to obtain a hyaluronic acid solution. Under the condition of 37℃ in the dark, 0.536g of oxidant NaIO4 (0.75mL, 0.5M) is added to the hyaluronic acid solution and stirred for 2h. 0.5mL of ethylene glycol is added to terminate the reaction for 1h to obtain a mixed solution. The mixed solution is dialyzed in deionized water for 3 days using a dialysis bag (molecular weight cutoff 8000~14000Da). After freeze-drying, oxidized hyaluronic acid (OHA) white flocculent precipitate is obtained. 5mL of deionized water is added to the OHA white flocculent precipitate and stirred to dissolve to obtain solution A with a concentration of 5 w / v%.

[0058] The preparation process of solution B is as follows: carboxymethyl chitosan (CMCS) is dissolved in 5 mL of deionized water, the volume ratio of carboxymethyl chitosan to deionized water is 0.02~0.06:1, preferably 0.05:1, and after stirring and dissolving, a solution B with a concentration of 1~20 w / v% is obtained, and the preferred concentration of solution B is 4%;

[0059] 5% OHA was mixed with hydrogel precursor solutions of 2%, 4%, and 6% CMCS under gentle stirring. The hydrogels in each group underwent a Schiff base reaction and solidified at room temperature to form OHA / CMCS hydrogels of different concentrations (hereinafter referred to as CH hydrogels). Hyaluronic acid and carboxymethyl chitosan are polysaccharides of natural origin with good biocompatibility and biodegradability. They do not cause adverse reactions when used in vivo and can naturally degrade after completing their functions, reducing environmental pollution.

[0060] To obtain a better concentration ratio, we explored crosslinking systems of 5% OHA with 2%, 4%, and 6% CMCS, and conducted relevant experiments on mechanical strength.

[0061] To ensure the formation of a uniform hydrogel, the gelation properties of the CH hydrogel were observed using the vial tilting method, and its injectability was tested by squeezing the hydrogel into a syringe.

[0062] The self-healing properties were then tested. Two pieces of hydrogel, dyed blue and yellow respectively, were cut into two pieces and then subjected to slight pressure to bring them into contact, in order to further observe their self-healing characteristics.

[0063] The rheological properties of different CH hydrogels were measured through a series of experiments using a Haake Mars40 rheometer. Finally, a swelling experiment was conducted, with the swelling rate SR = (Wa - Wb) / Wb × 100%, where Wb is the initial weight of the hydrogel after drying, and Wa is the weight of the hydrogel at different times. Due to their excellent rheological properties and skin coverage, we ultimately selected 5% OHA and 4% CMCS. The hydrogel framework prepared at these concentrations exhibited good biodegradability and drug sustained-release effects.

[0064] S2: Prepare Cu-TCPP-Mn nanosheets, and further process them to obtain Cu-TCPP-Mn nanodots;

[0065] 1.28 g Cu(NO3)2 and 79 mg H2TCPP were dissolved in 10.0 ml N,N-dimethylformamide (DMF), and then 0.8 g benzoic acid was added to form a homogeneous solution. The resulting homogeneous solution was placed in methyl silicone oil at 90 °C and stirred for 4 h to obtain Cu-TCPP intermediate. The obtained Cu-TCPP intermediate was mixed with 197 mg MnCl2 and heated and stirred at 90 °C for 12 h. The precipitate was collected by centrifugation and washed three times with ethanol to obtain Cu-TCPP-Mn nanosheets. Then, the Cu-TCPP-Mn nanosheets were sonicated for 4 h to obtain activated Cu-TCPP-Mn nanodots.

[0066] S3: Take 2 mL of solution A and add activated Cu-TCPP-Mn nanodots to it to obtain a mixed solution C with a concentration of 5 μg / mL;

[0067] S4: Take 2 mL of solution B and add activated PRP solution to it to obtain a mixed solution D with a concentration of 100 μg / mL;

[0068] In this step, PRP is extracted from the blood of healthy adult volunteers. The specific process is as follows: whole blood samples are collected from healthy adult volunteers, and high-concentration platelet-rich plasma (PRP) is separated using a blood component separator (NGL-XCF 3000). The separated PRP solution is activated with CaCl2, freeze-dried, and stored at -80°C. The ethics code is No. KY20253907-1.

[0069] S5: Mix equal volumes of mixed solution C and mixed solution D, and then add the mixture into a transparent glass bottle mold to obtain CHPM composite hydrogel.

[0070] In this step, mixed solution C and an equal volume of mixed solution D are taken to mix PRP and Cu-TCPP-Mn to form OHA / CMCS / PRP@Cu-TCPP-Mn, which is the CHPM composite hydrogel of this invention. The microstructure and characterization of the Cu-TCPP-Mn nanozyme of this invention are as follows: Figures 2-4 As shown. After freeze-drying, the microstructure of the CHPM composite hydrogel was characterized by scanning electron microscopy (SEM, Thermo Fisher Quattro S), as shown. Figure 5 , Figure 6 As shown, the porous structure of the CHPM composite hydrogel can be observed, and the formation of pores helps to improve the water absorption capacity of the CHPM composite hydrogel. The gel sample was dried by liquid nitrogen quenching, sputtered with gold, and the microstructure of the sample cross-section was observed using scanning electron microscopy.

[0071] This invention utilizes the above-mentioned preparation method to load Cu-TCPP-Mn and PRP into an OHA / CMCS hydrogel to prepare a multifunctional nanocomposite hydrogel (i.e., CHPM composite hydrogel). When this CHPM composite hydrogel is applied to an infected wound site, it slowly releases Cu-TCPP-Mn and PRP. Cu-TCPP-Mn exerts antioxidant properties, reducing oxidative stress levels at the wound site and improving the oxidative microenvironment, which is beneficial for PRP to better promote angiogenesis. It also reduces cell damage and apoptosis caused by oxidative stress, thereby promoting the healing of acute infected wounds. On the other hand, Cu-TCPP-Mn and CMCS exert antibacterial effects, preventing bacteria from entering the wound and avoiding infection that could hinder wound healing. CHPM composite hydrogel treatment of acute infected wounds can effectively reduce oxidative stress levels in the wound tissue, protecting against oxidative stress-induced cell damage and apoptosis. Furthermore, it works synergistically with PRP, providing a suitable oxidative microenvironment for PRP to exert its pro-angiogenic effect, further promoting the healing of acute infected wounds and providing more options for intensive clinical treatment of acute infected wounds.

[0072] The following are experimental verifications of the biocompatibility of the CHPM composite hydrogel prepared in this invention, its effects on HUVEC cell migration, angiogenesis, and anti-ROS, its effects on LPS-induced RAW 264.7 cell polarization, its antibacterial effect, and its effects on the healing of acute infected wounds.

[0073] To verify the effects of the CHPM composite hydrogel, the present invention used the untreated group as the control group and prepared OHA / CMCS hydrogels (CH group), OHA / CMCS hydrogels loaded with PRP (CHP group), OHA / CMCS hydrogels loaded with Cu-TCPP-Mn (CHM group), and OHA / CMCS hydrogels loaded with Cu-TCPP-Mn and PRP (CHPM group). The base layer of all hydrogels was prepared using OHA / CMCS (CH) hydrogels, and cell-related experiments were performed using the hydrogel leachate.

[0074] (I) Biocompatibility of CHPM composite hydrogel:

[0075] Reference Figures 7-9 As shown, biocompatibility is a critical requirement in the design of biomaterials and wound dressings, and ensuring their safety and harmlessness to organisms is a prerequisite for subsequent applications. To comprehensively evaluate the biocompatibility of hydrogels, we employed a variety of methods and strategies, including cell experiments, hemolysis experiments, and organ compatibility tests, to ensure that the material is non-toxic.

[0076] To investigate the effect of CHPM composite hydrogel on cytotoxicity, untreated HUVEC, HACAT, and HSF cells were used as the normal group. In the experimental group, these cells were seeded in 96-well plates at a density of 2 × 10⁶ cells / well. 4 Cells were extracted with different groups of hydrogel extracts. Two days later, they were stained with Calcein-AM / PI and photographed using an inverted fluorescence microscope (Nikon, DS-Ri2, Japan). The absorbance at 450 nm was measured using the CCK-8 assay to determine cell viability on different groups of hydrogels and assess cell compatibility. Next, a blood compatibility test was performed. Blood was collected from the ophthalmic artery of Balb / c mice using heparinized anticoagulant tubes. The whole blood was repeatedly washed with 0.9% NaCl until the supernatant was clear after centrifugation. The red blood cell pellet was diluted with PBS. The extracts from different groups of hydrogels and the diluted red blood cell suspensions were incubated at 37°C for 4 hours. Hemolysis of the red blood cell suspensions was observed. Finally, the supernatant was collected by centrifugation, and the hemolysis rate was calculated using a microplate reader. After applying the hydrogels to the skin of Balb / c mice, organ compatibility was demonstrated by H&E staining. No signs of organ toxicity were found, and H&E staining showed no significant difference between the hydrogel-treated groups and the control group.

[0077] This invention, based on cell viability staining experiments, utilizes the CCK-8 assay and Calcein-AM / PI to confirm that cells maintained high levels of activity and exhibited an extremely low proportion of dead cells in the hydrogel environment. Furthermore, the CCK-8 results showed that, compared to the control group, cells treated with hydrogel CHP and CHPM exhibited similar or even higher proliferation rates, further confirming the biocompatibility of the hydrogel. This is related to the sustained-release of growth factors by PRP, which promotes cell proliferation. The results demonstrate that the CHPM composite hydrogel of this invention possesses excellent biocompatibility and is a safe and effective biomaterial, validating its safety in biomaterial and wound dressing applications.

[0078] (II) Effects of CHPM composite hydrogel on HUVEC cell migration, angiogenesis, and ROS resistance:

[0079] like Figure 10 As shown, in order to verify the effects of CHPM composite hydrogel on angiogenesis and cell migration, this invention used HUVEC cells to conduct cell scratch experiments and angiogenesis experiments.

[0080] (1) In vitro cell scratch assay

[0081] Inoculate approximately 5 × 10⁵ cells into a six-well plate. 5HUVECs were cultured until their density exceeded 90%. After a cell monolayer formed, a 200 μL sterile pipette tip was used to make a horizontal scratch perpendicular to the back of the well plate. The cells were then gently washed three times with PBS, followed by the addition of culture medium containing hydrogel extract. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h. Cell migration in the scratched areas was observed and photographed under an inverted microscope. ImageJ was used to analyze the images to assess the migration ability of HUVECs.

[0082] (2) Angiogenesis experiment

[0083] Melt the substrate gel stored at -20°C overnight in a 4°C freezer, then apply it evenly to the bottom of a 12-well plate using a pre-cooled pipette tip, and cure it in a 37°C, 5% CO2 incubator for 30 minutes. Inoculate 2 × 10⁶ cells / well per well. 4 HUVEC cells were co-cultured with hydrogel extracts from different experimental groups, with PBS as a control. After 8 hours of incubation, the cells were observed and stained with Calcein-AM under an inverted microscope for 20 minutes. Finally, the cells were imaged using an inverted fluorescence microscope, and angiogenesis was analyzed using ImageJ software. The relative tube length was calculated with the Control group as the benchmark.

[0084] The results showed that the CHPM composite hydrogel significantly enhanced the migration ability of HUVECs within 48 hours. The wound healing rate of the PRP-containing CHP / CHPM composite hydrogel was 65%, compared to 37% in the control group, demonstrating a significant promotion of healing. The bioactive molecules and growth factors in PRP promote angiogenesis and local blood circulation, which are crucial for effective wound healing. The superior migration-promoting effect of the CHPM composite hydrogel may be due to the regulation of slow PRP release and macrophage polarization, creating a favorable environment for cell proliferation and synergistically enhancing cell migration. Further evaluation using angiogenesis assays on the matrix gel showed that HUVECs exposed to the CHP and CHPM composite hydrogel formed more mature and complex tubular structures, indicating that the hydrogel effectively promotes vascularization and cell migration, providing a promising therapeutic approach for wound healing.

[0085] (3) ROS scavenging ability experiment

[0086] HUVEC cells were seeded into 6-well plates, approximately 5 × 10⁶ cells per well. 5Cells were cultured at 37°C and 5% CO2 for 24 hours. Cells were induced with Rosup for 20 min, then co-incubated with culture medium containing hydrogel extract for 24 hours. Finally, the cells were stained with the cell-permeable fluorescent probe DCFH-DA, incubated at 37°C for 30 min, washed three times with PBS, and observed and photographed using a fluorescence microscope. Intracellular ROS production was monitored using DCFH-DA to evaluate the ROS resistance of HUVECs under hydrogel treatment in each group.

[0087] The results showed that, compared with the control group, CHPM hydrogel treatment significantly reduced intracellular ROS levels, demonstrating its effective antioxidant properties. CHPM hydrogel also exhibited good antioxidant capacity, significantly inhibiting cellular oxidative stress. The antioxidant activity of the hydrogel was closely related to its Cu-TCPP-Mn nanozyme, possibly neutralizing excess ROS through SOD-like and CAT-like activities, thereby mitigating oxidative stress-induced cell damage. The CH group showed a slight decrease in reactive oxygen species levels compared to the control group, indicating that the CH hydrogel itself possesses a certain antioxidant capacity. Furthermore, the hydrogel further reduced ROS levels by releasing PRP-derived bioactive factors to enhance the activity of intracellular antioxidant enzymes. The synergistic effect of PRP and nanodots in the CHPM hydrogel demonstrated excellent antioxidant performance. These results indicate that the CHPM hydrogel mainly relies on the antioxidant capacity of Cu-TCPP-Mn to inhibit cellular oxidative stress.

[0088] (III) Effects of CHPM composite hydrogel on LPS-regulated RAW 264.7 cell polarization:

[0089] like Figure 11 As shown, in order to detect the effect of CHPM composite hydrogel on macrophage regulation, the role of CHPM composite hydrogel in macrophage polarization was verified by immunofluorescence experiment. Macrophages play a key role in maintaining immune homeostasis and promoting wound healing. They are highly plastic and can induce, mediate or inhibit inflammatory responses.

[0090] RAW 264.7 cells were induced to polarize to the M1 (pro-inflammatory) type after lipopolysaccharide stimulation for 24 hours. These pre-polarized cells were then co-incubated with culture medium containing hydrogel extract for 24 hours. Changes in markers of M1 and M2 macrophages, including inducible nitric oxide synthase (iNOS) and CD206 (an M2 marker), were observed using immunofluorescence and photographed using confocal microscopy.

[0091] The results showed that the CD206 fluorescence intensity was strongest in the CHPM composite hydrogel group compared to other groups. The fluorescence intensity of iNOS-labeled M1 macrophages gradually decreased in different hydrogel treatment groups, with the lowest intensity observed in the CHPM group. Both the CHP and CHM groups promoted M2 macrophage polarization, but their fluorescence intensity was weaker than that of the CHPM group. Therefore, the CHPM composite hydrogel can significantly induce macrophage polarization from M1 to M2, thereby alleviating oxidative stress. PRP and Cu-TCPP-Mn nanozymes synergistically promote the transformation of macrophages from the M1 to the M2 phenotype. The reduction of M1 macrophages alleviates excessive inflammatory damage, reduces the inflammatory response, improves the wound immune microenvironment, and promotes wound healing and repair.

[0092] (iv) Antibacterial effect of CHPM composite hydrogel:

[0093] like Figure 12 As shown, in order to detect the antibacterial properties of CHPM composite hydrogel, the antibacterial effect of the hydrogel was evaluated by agar plate counting method, and the morphological changes of bacteria were analyzed by scanning electron microscopy and live-dead staining techniques.

[0094] In this invention, two representative strains, Staphylococcus aureus and Escherichia coli, were used for antibacterial testing. The diluted bacterial suspension was mixed with the hydrogel and incubated for 12 hours. The diluted bacterial suspension was then spread onto LB agar using a glass bead spreader and incubated at 37°C for 15 hours before colony counting. The hydrogel-treated bacterial suspension was fixed with 2.5% glutaraldehyde solution at 4°C for 3 hours, followed by gradient dehydration with 20%–100% ethanol solutions. The samples were then dried, and the morphological changes were observed using a scanning electron microscope. Bacterial viability staining was then performed according to the kit: 5 μL of concentrated bacterial suspension was dropped onto a clean, oil-free glass slide, covered with a coverslip, and air-dried in the dark, then observed under a fluorescence microscope. The CHPM hydrogel framework is composed of CMCSs, which directly interact with the bacterial cell wall and cell membrane, disrupting their structure and inhibiting bacterial growth. Cu-TCPP-Mn nanodots are slowly released from the CHPM hydrogel, enhancing its antibacterial ability.

[0095] The CHPM composite hydrogel of this invention exhibits strong antibacterial activity, indicating it is an optimal candidate material for treating infected skin wounds. Compared with the Control group, the number of bacterial colonies in the CHM and CHPM groups, as well as the CH and CHP treatment groups, was significantly reduced, demonstrating the good antibacterial activity of the hydrogel. Further analysis of the hydrogel's antibacterial effect was conducted. Treated bacteria were collected, fixed, dehydrated in a gradient, and sputter-coated with gold for scanning electron microscopy. Compared with the Control group, Staphylococcus aureus and Escherichia coli exposed to the hydrogel showed obvious signs of cell shrinkage and rupture, significantly deviating from their normal morphology. Furthermore, a live / dead bacterial staining test confirmed the effective bactericidal effect of the hydrogel against Staphylococcus aureus and Escherichia coli, demonstrating the antibacterial effect of the CHPM composite hydrogel and providing strong experimental evidence for the development of novel antibacterial materials for clinical application.

[0096] (V) Effects of CHPM composite hydrogel on the healing of acutely infected wounds in mice:

[0097] Combination Figure 13 As shown, in order to verify the effect of CHPM composite hydrogel on wound healing, an animal model was established to evaluate the effect of CHPM composite hydrogel on the healing of infected wounds in vivo.

[0098] First, Balb / c mice were randomly assigned to multiple experimental groups, and an infectious dorsal wound model was constructed. Male Balb / c mice were used for skin infection wound modeling. All animal experiments were approved by the Animal Experiment Ethics Committee of Air Force Medical University (IACUC-20240024). Mice were anesthetized with isoflurane inhalation using a respiratory anesthesia machine. The mice were shaved and the dorsal skin was disinfected. A full-thickness skin defect with a diameter of 1 cm was created on the back of each mouse, and 100 μL of Staphylococcus aureus suspension was injected into the wound site to form an infectious wound. The model was considered successful when redness and swelling of the wound and purulent discharge were observed 24 hours later.

[0099] Then, composite hydrogels of CH, CHM, CHP, and CHPM were applied to the wound, with PBS used as a blank control, to study the role of different hydrogels in the healing process of acute infected wounds. The back wounds of mice were photographed on days 0, 3, 6, 9, and 12, and the degree of wound healing was recorded. Mice were euthanized on day 12, and wound tissue was collected. Heart, liver, spleen, lung, and kidney tissues from the control and CHPM groups were taken for organ toxicity testing. These tissues were fixed in 4% paraformaldehyde for subsequent experiments.

[0100] The above experimental results show that after 12 days, the CH group still had some dry scabs and incomplete wound closure, but it was still better than the Control group, which is attributed to the protective effect of the hydrogel on the wound. In contrast, the CHPM group had smooth wounds with almost no residual scabs. Quantitative analysis of wound area changes showed that compared with the Control group, the wound size of the CHPM composite hydrogel treatment group was significantly reduced, reaching more than 95% healing by day 12, while the healing rate of the Control group was significantly lower, at approximately 61%.

[0101] Subsequently, hematoxylin and eosin (H&E) staining revealed varying degrees of epithelialization in the wound. The results showed that the CHPM group exhibited significantly narrower wound width, increased epidermal thickness, and reduced inflammatory cells, demonstrating good anti-inflammatory and pro-repair effects, while the Control group showed the worst epidermal integrity. This enhanced epithelialization effect is attributed to the sustained low-dose release of bioactive substances from the loaded PRP, which is closely related to the natural biological processes of wound healing. Furthermore, the slow release of Cu-TCPP-Mn also had a synergistic effect on wound healing, due to its antioxidant and antibacterial properties reducing infection-induced inflammatory stress and regulating macrophage polarization.

[0102] (vi) Effects of CHPM composite hydrogel on collagen deposition and inflammatory response in wounds:

[0103] Combination Figures 14-15 As shown, collagen is an important component of the dermal extracellular matrix and plays a crucial role in wound healing and scar formation. Masson staining and Sirius red staining were used to assess collagen deposition during the healing process. The results showed that, compared with the control group, the CHPM hydrogel treatment group had significantly increased collagen fiber deposition in the dermis, although the presence of collagen fibers was minimal.

[0104] The inflammatory environment significantly influences whether an injury develops into a chronic or non-healing wound. Elevated levels of interleukin-6 (IL-6) prolong the inflammatory response and hinder wound healing, while tumor necrosis factor-α (TNF-α) stimulates the proliferation and differentiation of immune cells. Immunohistochemical staining showed that, compared with the control group and other groups, the expression levels of pro-inflammatory factors IL-6 and TNF-α were significantly reduced in the CHPM hydrogel group, indicating that CHPM hydrogel can effectively regulate the inflammatory response after trauma. Furthermore, the chemokines and active proteins present in PRP can regulate the inflammatory response, inhibit excessive inflammation, and promote wound healing, thereby contributing to the overall treatment process of infected skin wounds. The synergistic effect of PRP and Cu-TCPP-Mn further enhances the anti-inflammatory capacity of the hydrogel.

[0105] This invention demonstrates that the multifunctional CHPM composite hydrogel loaded with nanozymes exhibits good efficacy in the treatment of acute infected wounds. The Cu-TCPP-Mn nanozyme mimics the activity of SOD and CAT, effectively neutralizing ROS. Furthermore, the addition of PRP enriches the hydrogel with growth factors, significantly promoting angiogenesis and tissue regeneration. The combined effect of PRP and Cu-TCPP-Mn in the hydrogel regulates macrophage polarization, modulating and alleviating persistent inflammation in infected wounds. The hydrogel possesses superior water absorption capacity, self-healing properties, and shape memory function. In vitro and in vivo studies have confirmed its efficacy in promoting cell proliferation, migration, and differentiation, regulating inflammatory responses, and accelerating wound healing. The two components work synergistically to promote the healing of acute infected wounds, providing better protection for these wounds. Therefore, this invention's CHPM composite hydrogel can be applied to the treatment of acute infected wounds with good results, offering more options for the clinical treatment of acute infected wounds.

[0106] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a multifunctional composite hydrogel carrying nanozymes, characterized in that, Includes the following steps: S1: Prepare solutions A and B separately for later use; S2: Prepare Cu-TCPP-Mn nanosheets, and further process them to obtain Cu-TCPP-Mn nanodots; S3: Add the Cu-TCPP-Mn nanodots obtained in step S2 to solution A to obtain a mixed solution C with a concentration of 2~20μg / mL; S4: Add the activated PRP solution to solution B to obtain a mixed solution D with a concentration of 50~400μg / mL; S5: After mixing equal volumes of mixed solution C and mixed solution D, a composite hydrogel is obtained; In step S1, the process of preparing solution A is as follows: S101: Dissolve hyaluronic acid in deionized water and stir to dissolve, to obtain a hyaluronic acid solution; S102: Add NaIO4 to the hyaluronic acid solution and continue stirring. After dialysis, freezing and drying, a white flocculent substance is obtained, namely oxidized hyaluronic acid. S103: Add deionized water to the white flocculent oxidized hyaluronic acid and stir to dissolve, thus obtaining solution A; In step S1, carboxymethyl chitosan is dissolved in deionized water at a volume ratio of 0.02 to 0.06:

1. After stirring and dissolving, a solution B with a concentration of 1 to 20 w / v% is obtained. In step S2, the process for preparing Cu-TCPP-Mn nanosheets is as follows: S201: Take Cu(NO3)2 and H2TCPP, dissolve them in DMF solution, and add benzoic acid to form a homogeneous solution; S202: The obtained homogeneous solution was placed in methyl silicone oil at 90℃ and stirred for 2-6 hours to obtain Cu-TCPP intermediate product; S203: Mix Cu-TCPP intermediate with MnCl2 and heat and stir at 90℃ for 10~16h. After centrifugation, collect the precipitate and wash the precipitate with anhydrous ethanol at least 3 times to obtain Cu-TCPP-Mn nanosheets. In step S2, Cu-TCPP-Mn nanosheets are ultrasonically treated for 4 hours to obtain Cu-TCPP-Mn nanodots.

2. The method for preparing a multifunctional composite hydrogel carrying nanozymes according to claim 1, characterized in that, In step S103, the concentration of solution A is 5~10 w / v.

3. The method for preparing a nanoenzyme-loaded multifunctional composite hydrogel according to claim 1, characterized in that, In step S3, the concentration of mixed solution C is 5 μg / mL, and in step S4, the concentration of mixed solution D is 100 μg / mL.

4. A multifunctional composite hydrogel carrying nanozymes, characterized in that, The composite hydrogel is prepared by any one of the preparation methods of the nanozyme-loaded multifunctional composite hydrogel according to any one of claims 1-3.

5. The application of the nanozyme-loaded multifunctional composite hydrogel as described in claim 4 in the preparation of drugs for acute infected wounds.

6. The application of the nanoenzyme-loaded multifunctional composite hydrogel as described in claim 4 in the preparation of novel antibacterial materials.

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