Nano-enzyme-loaded multifunctional composite hydrogel as well as preparation method and application thereof

Abstract

The invention relates to nano-enzyme-loaded multifunctional composite hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. In order to solve the problem that an existing material or medicine is poor in treatment effect on infected wounds, the preparation method comprises the steps that a solution A and a solution B are prepared for standby application, Cu-TCPP-Mn nanosheets are prepared, Cu-TCPP-Mn nanodots are obtained after further treatment, the activated Cu-TCPP-Mn nanodots are added into the solution A, a mixed solution C with the concentration of 5 micrograms / mL is obtained, an activated PRP solution is added into the solution B, and the mixed solution C is mixed with the activated PRP solution to obtain a mixed solution C with the concentration of 5 micrograms / mL; mixing the mixed solution C with the mixed solution D in an isovolumetric manner, so as to obtain the CHPM composite hydrogel. The CHPM composite hydrogel disclosed by the invention can effectively relieve the oxidative stress level of acute infection wound tissues, provides a proper oxidation microenvironment for PRP to play a role in promoting angiogenesis, and accelerates healing of acute infection wounds.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nanozyme-loaded multifunctional composite hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] At present, the precise treatment of acute infectious wounds is a key topic that urgently needs to be broken through in clinical medicine. Globally, such traumas are prone to cause fatal complications such as sepsis and multiple organ dysfunction due to delayed healing, resulting in a 20%-30% increase in the mortality rate of patients, and have become a major hidden danger threatening public health. The current clinical solutions have three dilemmas: First, although systemic antibiotic administration can quickly control infections, 60%-70% of the drugs in the blood cannot be effectively concentrated on the wound surface, but instead cause systemic side effects such as liver and kidney toxicity. Second, although mechanical debridement can temporarily remove necrotic tissues, repeated operations are likely to damage newly formed granulation tissues and delay the healing process. Third, although local antibiotic preparations can increase the drug concentration at the lesion site, they disrupt the homeostasis of the wound microenvironment due to burst release, and are more likely to induce the generation of multi-drug resistant strains. Therefore, constructing a new type of biomaterial with spatiotemporally controllable drug release characteristics to achieve the synergistic regulation of antibacterial, anti-inflammatory, and repair promotion has become an important research direction to break through the 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 deepened. Hydrogels have unique advantages in wound repair management and are favored because of their excellent hydrophilicity, biocompatibility, and three-dimensional porous structure similar to the extracellular matrix. Hydrogels can optimize their properties by modifying the polymer backbone, concentration ratio, crosslinking method, etc., and can also build a delivery system by combining bioactive molecules, drugs, or cells to customize medical wound dressings for local use according to different wounds.

[0004] In this context, the emergence of nanozyme technology provides new possibilities for the treatment of acute infectious wounds. As a new type of biocatalyst, nanozymes not only inherit the catalytic characteristics of natural enzymes but also have higher stability and controllability. Copper-tetrakis(4-carboxyphenyl)porphyrin-manganese (Cu-TCPP-Mn) nanosheets are a kind of metal-organic framework (MOF) material, which mimics the cascade activity of superoxide dismutase (SOD) and catalase (CAT). In addition, it can effectively fight against bacteria and destroy biofilms, reduce wound infections, and avoid the drug resistance problems that may be caused by traditional antibiotics. Compared with natural bio-antioxidant enzymes with few sources, high preparation costs, and easy inactivation, Cu-TCPP-Mn nanozymes have excellent characteristics such as strong environmental adaptability, stable structure, and easy preparation.

[0005] Platelet-rich plasma (PRP) is a blood-derived product that has been 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, which can release a large amount of growth factors such as PDGF, TGF-β, PRP, etc. after activation. 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 usually hinders the therapeutic effect. In order to further exert the effect of PRP, we consider applying it by loading it on hydrogel materials.

[0006] Since infected wounds are often accompanied by problems such as bacterial infection, tissue necrosis, and inflammatory reactions, if not treated promptly and effectively, it may lead to the spread of infection, delayed wound healing, and even serious complications such as systemic infection. However, the therapeutic effect of single-functional materials or drugs on complex infected wounds is often limited. Integrating nanomaterials and biomaterials with different physical and chemical properties, biological activities, and drug-loading capabilities synergistically is a more reasonable means for treating infected wounds. Based on the above considerations, there is an urgent need to study a new type of biomaterial 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 the patient's hospital stay, reducing treatment costs, and improving the patient's quality of life, which has important clinical application value and broad development prospects. Summary of the Invention

[0007] Aiming at the deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a nanozyme-loaded multifunctional composite hydrogel and its preparation method and application. The OHA / CMCS hydrogel with unique self-healing properties is synthesized through the Schiff base reaction between the aldehyde group of oxidized hyaluronic acid (OHA) and the carboxymethyl group of carboxymethyl chitosan (CMCS), forming a cross-linked network with adjustable mechanical properties and good biocompatibility, which is used to treat wounds of different shapes without displacement. The effective loading and slow release of PRP provide the hydrogel with abundant growth factors, enhancing its ability to promote skin tissue regeneration; while the Cu-TCPP-Mn nanozyme mimics the activities 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, thus solving the problems existing in the background technology.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: In the first aspect, the present invention discloses a preparation method of a nanozyme-loaded multifunctional composite hydrogel, comprising the following steps: S1: Prepare solution A and solution B respectively and set aside; S2: Prepare Cu-TCPP-Mn nanosheets, and obtain Cu-TCPP-Mn nanodots after further processing them. S3: Add the Cu-TCPP-Mn nanodots obtained in step S2 into solution A to obtain a mixed solution C with a concentration of 2 - 20 μg / mL. S4: Add the activated PRP solution into solution B to obtain a mixed solution D with a concentration of 50 - 400 μg / mL. S5: Mix the mixed solution C and the mixed solution D in equal volumes to obtain the CHPM composite hydrogel.

[0009] As a further preferred implementation of the above solution, in step S1, the process of preparing solution A is as follows: S101: Dissolve hyaluronic acid in deionized water and stir to dissolve it to obtain a hyaluronic acid solution. S102: Add NaIO4 to the hyaluronic acid solution and continue stirring. After dialysis, freezing, and drying, obtain white flocculent oxidized hyaluronic acid. S103: Add deionized water to the white flocculent oxidized hyaluronic acid and stir to dissolve it to obtain solution A.

[0010] A further preferred solution is: in step S101, the concentration of solution A is 0 - 10 w / v%.

[0011] As a further preferred implementation of the above solution, in step S1, dissolve carboxymethyl chitosan in deionized water, with the volume ratio of carboxymethyl chitosan to deionized water being 0.02 - 0.06:1. After stirring and dissolving, obtain a solution B with a concentration of 1 - 20 w / v%.

[0012] As a further preferred implementation of the above solution, in step S2, the process of preparing Cu-TCPP-Mn nanosheets is as follows: S201: Weigh Cu(NO3)2 and H2TCPP in proportion, dissolve them in DMF solution, and add benzoic acid to it to form a homogeneous solution. S202: Place the obtained homogeneous solution in silicone oil at 90 °C and stir for 2 - 6 h to obtain a Cu-TCPP intermediate product. S203: Mix the Cu-TCPP intermediate product with MnCl2, heat and stir at 90 °C for 10 - 16 h, centrifuge to collect the precipitate, and wash the precipitate with absolute ethanol at least 3 times to prepare Cu-TCPP-Mn nanosheets.

[0013] A further preferred solution is: in step S2, ultrasonically treat the Cu-TCPP-Mn nanosheets for 4 h to obtain Cu-TCPP-Mn nanodots.

[0014] A further preferred solution is that in steps S3 - S4, the concentrations of the mixed solution C and the mixed solution D are 5 μg / mL and 100 μg / mL respectively.

[0015] In a second aspect, the present invention also discloses a nanozyme - loaded multifunctional composite hydrogel, which is prepared by the preparation method of the nanozyme - loaded multifunctional composite hydrogel described above.

[0016] In a third aspect, the present invention also discloses the application of the nanozyme - loaded multifunctional composite hydrogel in the preparation of drugs for acute infected wounds.

[0017] In a fourth aspect, the present invention also discloses the application of the nanozyme - loaded multifunctional composite hydrogel in the preparation of novel antibacterial materials.

[0018] Compared with the prior art, the improvements of the present invention can produce the following beneficial effects: 1. In the present invention, new functional groups (aldehyde groups) are introduced into hyaluronic acid to obtain oxidized hyaluronic acid (OHA). Based on the principle of Schiff - base reaction, the aldehyde groups of OHA and the carboxyl groups of CMCS form imine bonds, forming an OHA / CMCS hydrogel framework. By virtue of the high - efficiency reactive oxygen species scavenging biological activity and antibacterial effect of CU - TCPP - Mn nanozyme, the antibacterial ability of CMCS and the blood - vessel promoting performance of PRP, a composite bioactive material loaded with nanozyme for acute infected wounds is constructed. The antioxidant effect of CU - TCPP - Mn, the biological therapy of PRP and the wound - protecting effect of OHA / CMCS produce a synergistic effect on the treatment of acute infected wounds. Due to the dynamic Schiff - base bonding, its functionality is enhanced, and this property endows the CHPM composite hydrogel with unique self - healing characteristics, that is, it can automatically restore its shape after being damaged, and can be used for the treatment of complex wounds with different shapes and depths without displacement, showing great potential in the treatment of acute infected wounds and promoting wound healing.

[0019] 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 on the hydrogel, the limitation of the rapid release of PRP in vivo is overcome, ensuring the effective utilization of growth factors in PRP during the wound - repair process.

[0020] 3. The OHA / CMCS hydrogel framework of the present invention is constructed from OHA and CMCS. Both of these materials are natural polymers with good biocompatibility, and have good biocompatibility and biodegradability. They will not cause immune rejection reactions during skin application, and can gradually degrade along with the skin healing process, avoiding long-term retention in the body. Moreover, the OHA / CMCS hydrogel framework is self-assembled through the Schiff base reaction between CMCS and OHA. This cross-linking method endows the hydrogel with good mechanical properties and stability, as well as certain elasticity and strength, enabling it to withstand mechanical stresses such as stretching and extrusion during daily activities, and is not prone to rupture or deformation, thus ensuring its stable attachment and continuous action on the skin surface.

[0021] 4. The OHA / CMCS framework in the CHPM composite hydrogel of the present invention has good antibacterial activity and can effectively inhibit the growth of a variety of common pathogenic bacteria. CMCS itself has antibacterial properties. Its high-charge polycationic structure will have a strong electrostatic interaction with the negatively charged bacterial phospholipid membrane components, resulting in bacterial membrane damage and leakage of contents, thereby killing bacteria. In addition, the reactive 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 preventing and controlling skin infections, can reduce the incidence and severity of infections, and promote the healing of infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments of the present invention will be briefly introduced below.

[0023] Figure 1 It is a flow chart of the preparation method of the Cu-TCPP-Mn nanozyme and the CHPM composite hydrogel loaded with Cu-TCPP-Mn nanozyme for promoting the 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 the CHPM hydrogel preparation and its self-healing performance, and c represents the healing mechanism of the CHPM self-healing hydrogel for treating infected wounds.

[0024] Figure 2 It is a morphology diagram of the Cu-TCPP-Mn nanozyme provided by the present invention; wherein, a represents the SEM morphology diagram of the Cu-TCPP-Mn nanosheets, b represents the XPS spectrum of the Cu-TCPP-Mn nanodots, c represents the Cu 2p XPS spectrum, and d represents the Mn 2p XPS spectrum of the Cu-TCPP-Mn nanodots.

[0025] Figure 3Characterization diagrams of the Cu-TCPP-Mn nanozyme provided by the present invention; wherein, a shows the morphology and elemental localization of the Cu-TCPP-Mn nanosheets observed under a scanning electron microscope, b shows the TEM morphology analysis of the Cu-TCPP-Mn nanodots, c shows the particle size analysis of the Cu-TCPP-Mn nanodots, d shows the Zeta potential of the Cu-TCPP-Mn nano, and d shows the Fourier transform infrared spectrum of the Cu-TCPP-Mn nanodots.

[0026] Figure 4 Functional tests of the Cu-TCPP-Mn nanozyme provided by the present invention; wherein, a shows the ultraviolet absorption energy spectrum diagram of the Cu-TCPP-Mn nanodots, b shows the CAT-like activity of the Cu-TCPP-Mn nanodots, c shows the SOD-like activity of the Cu-TCPP-Mn nanodots, and d shows the oxygen generation amount in the CAT-like activity of the Cu-TCPP-Mn nanodots.

[0027] Figure 5 Characterization diagrams of the CHPM composite hydrogel provided by the present invention; wherein, a shows the construction of the CH hydrogel with CMCS and OHA, b shows the self-healing property of the hydrogel, c shows the injectability of the hydrogel, d shows the shear-thinning property of the hydrogel, e shows the rheological behavior of the hydrogel in the time sweep mode, f shows the rheological behavior of the hydrogel in the amplitude sweep mode, g shows the rheological behavior of the hydrogel in the alternating strain mode, h shows the swelling ratio of the hydrogel, and i shows the scanning electron microscope morphology diagram of the hydrogel.

[0028] Figure 6 of the present invention Figure 5 Magnified display diagrams of subfigures f and g of the neutron diagram.

[0029] Figure 7 Results diagrams of the cell compatibility of the CHPM composite hydrogel provided by the present invention with HUVEC, HACAT, and HSF cells; wherein, a shows the cytotoxicity of the hydrogel to HACAT, b shows the cytotoxicity of the hydrogel to HUVEC, c shows the cytotoxicity of the hydrogel to HSF, d shows the quantitative analysis of the viability of HACAT cells, e shows the quantitative analysis of the viability of HUVEC cells, and f shows the quantitative analysis of the viability of HSF cells.

[0030] Figure 8 Results diagrams of the blood compatibility under co-incubation of the CHPM composite hydrogel provided by the present invention.

[0031] Figure 9 Results diagrams of the organ compatibility under co-incubation of the CHPM composite hydrogel provided by the present invention.

[0032] Figure 10Results of the effects of the CHPM composite hydrogel provided by the present invention on HUVEC cell migration, angiogenesis, and anti-ROS; wherein, a represents the HUVEC migration experiment, b represents the angiogenesis experiment, c represents the ability of the hydrogel to scavenge ROS, d represents the quantitative statistics of the migration ratio, e represents the quantitative statistical data on the tube formation length, and f represents the quantitative statistics of ROS scavenging.

[0033] Figure 11 Results of the effects of the CHPM composite hydrogel provided by the present invention on M2 polarization of LPS-induced RAW 264.7 cells; wherein, 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.

[0034] Figure 12 Antibacterial performance of the CHPM composite hydrogel provided by the present invention against Staphylococcus aureus and Escherichia coli; wherein, a represents the plate coating experiment on Staphylococcus aureus and Escherichia coli, b represents the electron microscopy observation of the effects of the hydrogel on Staphylococcus aureus and Escherichia coli, c represents the live / dead staining experiment on Staphylococcus aureus and Escherichia coli, and d~e represent the quantitative statistics of the bacterial plate coating experiment.

[0035] Figure 13 Results of the effects of the CHPM composite hydrogel provided by the present invention on the healing of acute infected wounds in mice, with different groups being carried out on days 0, 3, 6, 9, and 12; wherein, a represents the wound healing treatment, b represents the skin animation tracing diagrams of different experimental groups, c represents the skin wound tissue sections stained with H&E, d represents the quantitative statistics of the wound healing rate, and e represents the quantitative analysis of the wound width.

[0036] Figure 14 Tissue staining analysis of the wounds by the CHPM composite hydrogel provided by the present invention; wherein, a represents the Masson staining of the skin wound tissue, b represents the Sirius red staining of the skin wound tissue, c represents the IL-6 staining of the skin wound tissue, d represents the TNF-α staining of the skin wound tissue, e represents the quantitative analysis of collagen deposition, f represents the quantitative analysis of the fibrotic area, g represents the quantitative statistics of the IL-6 expression level, and h represents the quantitative statistics of the TNF-α expression level.

[0037] Figure 15 of the present invention Figure 14 Magnified display diagrams of subgraphs e, f, g, and h. Detailed implementation manners

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention.

[0039] The present invention relates to a nanozyme-loaded multifunctional composite hydrogel, its preparation method, and application. The following will describe the present invention in detail with reference to the accompanying drawings. Figures 1 - 15 Make a detailed description of the present invention.

[0040] Experimental materials: 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 Macklin. HUVEC, HACAT, CCK-8 kit, reactive oxygen species detection kit, and cell viability / cytotoxicity staining kit were purchased from Wuhan Sevier Biotechnology Co., Ltd. The experimental water was Milli-Q grade pure water. All chemical reagents were of analytical grade and were used directly without purification.

[0041] As Figure 1 shown, a preparation method of a nanozyme-loaded multifunctional composite hydrogel provided by the present invention includes the following steps: S1: Prepare solution A and solution B respectively for later use; In this step, the preparation process of solution A is as follows: Weigh 1 g of hyaluronic acid and dissolve it in 100 mL of deionized water (100 mL, 10 mg / mL) with stirring to obtain a hyaluronic acid solution. Under dark conditions at 37 °C, add 0.536 g of oxidant NaIO4 (0.75 mL, 0.5 M) to the hyaluronic acid solution and continue stirring for 2 h. Then add 0.5 mL of ethylene glycol to terminate the reaction for 1 h to obtain a mixed solution. Dialyze the mixed solution in deionized water for 3 days using a dialysis bag (cut-off molecular weight 8000 - 14000 Da), and then freeze-dry it to obtain white flocculent oxidized hyaluronic acid (OHA). Add 5 mL of deionized water to the OHA white flocculent and stir to dissolve it to obtain solution A with a concentration of 5 w / v%. The preparation process of solution B is as follows: Dissolve carboxymethyl chitosan (CMCS) 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. After stirring and dissolving, obtain solution B with a concentration of 1 - 20 w / v%, and the preferred concentration of solution B is 4%. The hydrogel precursor solutions of 5% OHA and 2%, 4%, 6% CMCS were subjected to Schiff base reactions under gentle stirring, and hydrogels of different concentrations were formed by curing at room temperature to obtain OHA / CMCS hydrogels (hereinafter referred to as CH hydrogels); hyaluronic acid and carboxymethyl chitosan are polysaccharides of natural origin, with good biocompatibility and biodegradability, and will not cause adverse reactions when used in vivo, and can be naturally degraded after completing their functions, reducing environmental pollution.

[0042] To obtain a better concentration ratio, we explored the cross-linking system of 5% OHA and 2%, 4%, 6% CMCS and conducted relevant experiments on mechanical strength.

[0043] To ensure the formation of a uniform hydrogel, the gelling performance of CH hydrogels was observed by the vial tilting method, and their injectability was tested by extruding the hydrogels in a syringe.

[0044] After that, the self-healing performance test was carried out. Two hydrogels dyed blue and yellow respectively were cut into two pieces, and then a slight pressure was applied to make them contact to further observe their self-healing characteristics.

[0045] The rheological properties of different CH hydrogels were measured through a series of tests on a rheometer (Haake Mars40). Finally, a swelling experiment was carried out. The swelling ratio SR = (Wa− Wb) / Wb×100%, where Wb is the weight of the hydrogel at the initial drying, and Wa is the weight of the hydrogel at different times. Due to the excellent rheological properties and skin coverage ability, we finally selected 5% OHA and 4% CMCS. The hydrogel framework prepared at this concentration has good biodegradability and drug sustained-release effect.

[0046] S2: Prepare Cu-TCPP-Mn nanosheets and further process them to obtain Cu-TCPP-Mn nanodots; Dissolve 1.28 g of Cu(NO3)2 and 79 mg of H2TCPP in 10.0 ml of N, N-dimethylformamide (DMF), then add 0.8 g of benzoic acid to form a homogeneous solution. Place the obtained homogeneous solution in silicone oil at 90 °C and stir for 4 h to obtain the Cu-TCPP intermediate product. Mix the obtained Cu-TCPP intermediate product with 197 mg of MnCl2 and heat and stir at 90 °C for 12 h. Collect the precipitate by centrifugation and wash the precipitate three times with ethanol to obtain Cu-TCPP-Mn nanosheets; then, ultrasonicate the Cu-TCPP-Mn nanosheets for 4 hours to obtain activated Cu-TCPP-Mn nanodots.

[0047] S3: Take 2 mL of solution A and add the activated Cu-TCPP-Mn nanodots to it to obtain a mixed solution C with a concentration of 5 μg / mL; S4: Take 2 mL of solution B and add the activated PRP solution to it to obtain a mixed solution D with a concentration of 100 μg / mL; In this step, PRP is extracted from the blood of adult healthy volunteers. The specific process is as follows: Collect the whole blood samples of adult healthy volunteers, and separate the platelet-rich plasma (PRP) with a blood component separator (NGL-XCF 3000). The separated PRP solution is activated with CaCl2, freeze-dried and stored at -80 °C. The ethics number is No.KY20253907-1.

[0048] S5: Mix the mixed solution C and the mixed solution D in equal volumes, and then add them to a transparent glass bottle mold to obtain the CHPM composite hydrogel.

[0049] In this step, take the mixed solution C and the mixed solution D in equal volumes, so that PRP and Cu-TCPP-Mn are mixed to form OHA / CMCS / PRP@Cu-TCPP-Mn, that is, the CHPM composite hydrogel of the present invention. The microscopic morphology and characterization of the Cu-TCPP-Mn nanozyme in the present invention are as Figures 2 - 4 shown. After freeze-drying the CHPM composite hydrogel, its microscopic structure is characterized by a scanning electron microscope (SEM, Thermo Fisher Quattro S), as Figure 5 、 Figure 6 shown. It can be observed that the CHPM composite hydrogel has a porous structure, and the formation of pores helps to improve the water absorption capacity of the CHPM composite hydrogel. Quench-dry the gel sample with liquid nitrogen, perform gold spraying treatment, and observe the microscopic morphology of the sample cross-section using a scanning electron microscope.

[0050] The present invention uses the above preparation method to load Cu-TCPP-Mn and PRP into the OHA / CMCS hydrogel to prepare a multifunctional nanocomposite hydrogel (i.e., CHPM composite hydrogel). When this CHPM composite hydrogel acts on the infected wound site, the CHPM composite hydrogel slowly releases Cu-TCPP-Mn and PRP. Cu-TCPP-Mn can exert antioxidant properties, reduce the oxidative stress level at the wound site, improve the oxidative microenvironment at the wound site, which is beneficial for PRP to better play its role in promoting angiogenesis. At the same time, it also reduces cell damage and apoptosis caused by oxidative stress, thereby promoting the healing of acute infected wounds. On the other hand, through the antibacterial effects of Cu-TCPP-Mn and CMCS, bacteria can be prevented from entering the wound surface to avoid infection affecting wound healing. The CHPM composite hydrogel for treating acute infected wounds can effectively reduce the oxidative stress level of the acute infected wound tissue to protect against cell damage and apoptosis caused by oxidative stress, and play a synergistic role with PRP to provide a suitable oxidative microenvironment for PRP to play its role in promoting angiogenesis, and can further promote the healing of acute infected wounds, providing more options for clinical intensive treatment of acute infected wounds.

[0051] The following are experimental verifications of the biocompatibility of the CHPM composite hydrogel prepared by the present invention, its effects on HUVEC cell migration, angiogenesis, anti-ROS, its effects on the polarization of LPS-induced RAW 264.7 cells, its antibacterial effect, and its effect on the healing of acute infected wounds.

[0052] To verify the relevant effects of the CHPM composite hydrogel, the present invention uses the non-intervened group as the normal group (Control group), and separately prepares the OHA / CMCS hydrogel (CH group), the OHA / CMCS hydrogel loaded with PRP (CHP group), the OHA / CMCS hydrogel loaded with Cu-TCPP-Mn (CHM group), and the OHA / CMCS hydrogel loaded with Cu-TCPP-Mn and PRP (CHPM group). The base layer of all hydrogels is prepared using the OHA / CMCS (CH) hydrogel, and the leachate of the hydrogel is used for cell-related experiments.

[0053] (I) Biocompatibility of the CHPM composite hydrogel: Refer to Figures 7 - 9 As shown, biocompatibility is a key requirement in the design of biomaterials and wound dressings, and ensuring its safety and harmlessness to organisms is a necessary condition for subsequent applications. To comprehensively evaluate the biocompatibility of the hydrogel, we adopted a variety of methods and strategies, including cell experiments, hemolysis experiments, and organ compatibility tests, to ensure that the material is non-toxic.

[0054] To detect the cytotoxicity of the CHPM composite hydrogel, non-intervened HUVEC, HACAT, and HSF cells were used as the normal group. In the experimental group, the above cells were seeded into 96-well plates at a density of 2×10 4 cells per well, and the leachates of different hydrogels were added respectively. After two days, the cells were stained with Calcein-AM / PI and photographed using an inverted fluorescence microscope (Nikon, DS-Ri2, Japan). The absorbance value at 450 nm was measured by the CCK-8 method to determine the cell viability on different hydrogels and evaluate their cytocompatibility. Subsequently, a blood compatibility test was conducted. Blood was collected from the ophthalmic artery of Balb / c mice and whole blood was collected using heparinized anticoagulation tubes. The whole blood was repeatedly rinsed with 0.9% NaCl until the supernatant was clear after centrifugation. The red blood cell precipitate was diluted with PBS. The leachates of different hydrogels were incubated with the diluted red blood cell suspension at 37 °C for 4 hours, and the hemolysis of each group of red blood cell suspensions was observed. Finally, the supernatant was collected by centrifugation and the hemolysis rate was calculated using an enzyme-linked immunosorbent assay (ELISA) reader. After applying the hydrogel on the skin of Balb / c mice, its 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 group and the control group.

[0055] Based on the cell viability and death staining experiment, using the CCK-8 method and Calcein-AM / PI, the present invention confirmed that cells maintained a high level of activity in the hydrogel environment and the proportion of dead cells was extremely low. In addition, the results of the CCK-8 experiment showed that compared with the Control group, the cells treated with the hydrogel CHP and CHPM groups showed similar or even higher proliferation rates, further confirming the biocompatibility of the hydrogel, which is related to the slow release of growth factors from PRP and promotes cell proliferation. The results indicate that the CHPM composite hydrogel of the present invention has good biocompatibility, is a safe and effective biomaterial, and verifies its safety in the application of biomaterials and wound dressings.

[0056] (2) Effects of CHPM composite hydrogel on HUVEC cell migration, angiogenesis, and anti-ROS As [[ID=ll]] Figure 10 shown, to verify the effects of the CHPM composite hydrogel on angiogenesis and cell migration, the present invention conducted a cell scratch assay and an angiogenesis assay using HUVEC cells.

[0057] (1) In vitro cell scratch assay Approximately 5×10 5HUVECs were seeded and cultured until the density exceeded 90%. After the cell monolayer formed, a 200 μL sterile pipette tip was used to scratch the cells vertically along the horizontal line on the back of the well plate. The cells were gently washed three times with PBS, and then the medium containing the hydrogel leachate was added. The cells were cultured in an incubator at 37°C with 5% CO2 for 48 h. Then, the cells were observed and photographed under an inverted microscope, and the cell migration in the scratched area was recorded. Image J was used to analyze the captured images to evaluate the migration ability of HUVECs.

[0058] (2) Angiogenesis assay Matrigel stored at -20°C was thawed overnight in a 4°C refrigerator, and then evenly coated on the bottom of a 12-well culture plate with a pre-chilled pipette tip. It was solidified in an incubator at 37°C with 5% CO2 for 30 minutes. 2×10 4 HUVEC cells were seeded in each well and co-cultured with the hydrogel leachate of different experimental groups. PBS was used as a control. After incubation for 8 h, the cells were observed and stained with Calcein-AM for 20 minutes under an inverted microscope. Finally, images were taken with an inverted fluorescence microscope, and angiogenesis was analyzed using Image J software. The relative tube length was calculated based on the Control group.

[0059] The results showed that the CHPM composite hydrogel significantly enhanced the migration ability of HUVECs within 48 h. The hydrogel wound healing rate of the CHP / CHPM composite hydrogel containing PRP was 65%, while that of the Control group was 37%, indicating that it could significantly promote wound healing. The bioactive molecules and growth factors in PRP promoted neovascularization and local blood circulation, which were crucial for effective wound healing. The superior migration-promoting effect of the CHPM composite hydrogel might be due to the regulated slow release of PRP, macrophage polarization, creating a favorable environment for cell proliferation, and synergistically enhancing cell migration. Further evaluation through the angiogenesis assay on Matrigel showed that HUVECs exposed to the CHP and CHPM composite hydrogels formed more mature and complex tubular structures, indicating that the hydrogel effectively promoted vascularization and cell migration, providing a promising treatment method for wound healing.

[0060] (3) ROS scavenging ability assay HUVEC cells were seeded in a 6-well plate, approximately 5×10 5Cells were cultured for 24 hours at 37°C under 5% CO₂. The cells were first induced with Rosup for 20 min and then co-incubated with the medium containing the hydrogel leachate for 24 hours. Finally, the cells were stained with the cell-permeable fluorescent probe DCFH-DA. After incubation at 37°C for 30 minutes, the cells were washed 3 times with PBS and observed and photographed under a fluorescence microscope. The production of intracellular ROS was monitored by DCFH-DA to evaluate the anti-ROS ability of HUVEC under the action of hydrogels in each group.

[0061] The results showed that compared with the Control, the CHPM hydrogel treatment significantly reduced the intracellular ROS level, indicating its effective antioxidant properties. The CHM hydrogel also exhibited good antioxidant capacity and could significantly inhibit cellular oxidative stress. The antioxidant activity of the hydrogel was closely related to its Cu-TCPP-Mn nanozyme, probably by neutralizing excessive ROS through SOD-like activity and CAT-like activity, thereby reducing oxidative stress-induced cell damage. The ROS level in the CH group was slightly decreased compared with the Control group, suggesting that the CH hydrogel itself had a certain antioxidant stress capacity. In addition, the hydrogel increased the activity of intracellular antioxidant enzymes by releasing PRP-derived bioactive factors, thereby further reducing the ROS level. The synergistic effect of PRP and nanodots in the CHPM hydrogel showed very good antioxidant performance. The results indicated that the CHPM hydrogel mainly inhibited cellular oxidative stress depending on the antioxidant ability of Cu-TCPP-Mn.

[0062] (III) Effect of CHPM composite hydrogel on LPS-regulated RAW 264.7 cell polarization: As Figure 11 shown, to detect the effect of the CHPM composite hydrogel on macrophage regulation, the role of the CHPM composite hydrogel in macrophage polarization was verified by immunofluorescence experiments. 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.

[0063] RAW 264.7 cells were induced to polarize into the M1 type (pro-inflammatory type) after being stimulated with lipopolysaccharide for 24 hours. Subsequently, these pre-polarized cells were co-incubated with the medium containing the hydrogel leachate for 24 hours. By immunofluorescence technique, we observed the changes in M1 and M2 macrophage markers, including inducible nitric oxide synthase (iNOS) and CD206 (M2 type marker), and observed and photographed them under a confocal microscope.

[0064] The results showed that, compared with other groups, the CHPM composite hydrogel group had the strongest fluorescence intensity of CD206. The fluorescence intensity of iNOS-labeled M1 macrophages gradually decreased in different hydrogel treatment groups, with the lowest in the CHPM group. Both the CHP and CHM groups could promote the polarization of M2 macrophages, but the fluorescence intensity was weaker than that of the CHPM group. It can be seen that the CHPM composite hydrogel can significantly induce the polarization of macrophages from M1 to M2, thereby reducing oxidative stress. PRP and Cu-TCPP-Mn nanozymes synergistically promoted the transformation of macrophages from the M1 phenotype to the M2 phenotype, and the reduction of M1 macrophages alleviated excessive inflammatory damage, reduced the inflammatory response, improved the wound immune microenvironment, and promoted wound healing and repair.

[0065] (IV) Antibacterial effect of CHPM composite hydrogel: As Figure 12 shown, to detect the antibacterial properties of the CHPM composite hydrogel, the antibacterial effect of the hydrogel was evaluated by the agar plate counting method, supplemented by scanning electron microscopy and live-dead staining techniques to analyze the morphological changes of bacteria.

[0066] In this invention, two representative strains, Staphylococcus aureus and Escherichia coli, were used for the antibacterial test. The diluted bacterial suspension was mixed with the hydrogel and incubated for 12 hours. The diluted bacterial suspension was spread on the surface of the LB medium by the method of spreading with glass beads and incubated in an incubator at 37 °C for 15 h, and then taken out for colony counting. The bacterial suspension treated with the hydrogel was fixed with 2.5% glutaraldehyde solution at 4 °C for 3 hours, dehydrated with 20% - 100% ethanol solution in gradient, the sample was dried, and the morphological changes of the dried sample were observed by scanning electron microscopy. Then, according to the kit, the bacterial live-dead staining experiment was carried out: 5 μL of the concentrated bacterial suspension was dropped on a clean and oil-free glass slide, covered with a cover slip and air-dried in the dark, and observed under a fluorescence microscope. The CHPM hydrogel framework is composed of CMCS, and CMCS directly interacts with the bacterial cell wall and cell membrane, destroys its structure and inhibits bacterial growth. The Cu-TCPP-Mn nanodots are slowly released from the CHPM hydrogel, enhancing its antibacterial ability.

[0067] The CHPM composite hydrogel of the present invention exhibits strong antibacterial effects, indicating that it is the best candidate material for treating infected skin wounds. Compared with the Control group, the number of colonies in the CHM and CHPM groups, as well as the CH and CHP treatment groups, decreased significantly, indicating that the hydrogel groups have good antibacterial activities. The antibacterial effects of the hydrogels were further analyzed. The treated bacteria were collected, fixed, dehydrated step by step, and sputter-coated with gold for scanning electron microscopy observation. Compared with the Control group, both Staphylococcus aureus and Escherichia coli exposed to the hydrogels showed obvious signs of cell shrinkage and rupture, deviating significantly from their normal morphology. In addition, a live / dead bacteria staining test was performed, confirming the effective bactericidal effects of the hydrogels against Staphylococcus aureus and Escherichia coli, indicating the antibacterial effects of the CHPM composite hydrogel and providing strong experimental evidence for the development of novel antibacterial materials for clinical applications.

[0068] (V) Effects of CHPM Composite Hydrogel on the Healing of Acute Infected Wounds in Mice: Combined with Figure 13 As shown, in order to verify the effects of the CHPM composite hydrogel on wound healing, an animal model was established to evaluate the effects of the CHPM composite hydrogel on the healing of infected wounds in vivo.

[0069] First, Balb / c mice were randomly assigned to multiple experimental groups, and an infectious dorsal wound model was constructed. Skin infected wounds were created in male Balb / c mice. All animal experiments were approved by the Animal Experiment Ethics Committee of the Air Force Medical University (IACUC-20240024). The mice were anesthetized by inhaling isoflurane using a respiratory anesthesia machine, and the back skin of the mice was shaved and disinfected. A full-thickness skin wound defect with a diameter of 1 cm was made on the back of each mouse, and 100 μL of Staphylococcus aureus suspension was injected into the wound site to form an infected wound. After 24 h, when the wound was observed to be red and swollen with purulent secretions, the modeling was successful.

[0070] Then, CH, CHM, CHP, and CHPM composite hydrogels were applied to the wounds, and PBS was used as a blank control to study the effects of different hydrogels during the healing process of acute infected wounds. During this period, the dorsal wound conditions of the mice were photographed on days 0, 3, 6, 9, and 12, and the wound healing degree was recorded. After the mice were euthanized on day 12, the wound tissues were collected. The heart, liver, spleen, lung, and kidney tissues of the mice in the control group and the CHPM group were taken for organ toxicity detection, and the above tissues were fixed in 4% paraformaldehyde for subsequent experiments.

[0071] The above experimental results showed that there were still some dry scabs in the CH group on the 12th day, and the wound surface was not completely closed, but it was still better than the Control group, which was due to the protective effect of the hydrogel on the wound surface. In contrast, the wound surface of the CHPM group was smooth and there was almost no residual scab. Quantitative analysis of the change in wound area showed that compared with the Control group, the wound size in the CHPM composite hydrogel treatment group was significantly reduced, reaching more than 95% healing on the 12th day, while the healing rate of the Control group was significantly lower, about 61%.

[0072] Subsequently, hematoxylin-eosin (H&E) staining showed varying degrees of epithelialization of the wound surface. The results showed that the width of the wound surface in the CHPM group was significantly narrowed, the epidermal thickness increased, and the number of inflammatory cells decreased, showing good anti-inflammatory and repair-promoting effects, while the epidermal integrity of the Control group was the worst. This enhanced epithelialization effect was due to the continuous low-dose release of bioactive substances by the loaded PRP, which was closely related to the natural biological process of wound healing. In addition, the slow release of Cu-TCPP-Mn also had a synergistic effect on wound healing, which was due to its antioxidant and antibacterial effects reducing the inflammatory stress caused by infection and regulating macrophage polarization.

[0073] (VI) Effects of CHPM composite hydrogel on collagen deposition and inflammatory response in wounds: Combined with 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 evaluate collagen deposition during the healing process. The results showed that compared with the control group, the deposition of collagen fibers in the dermis of the CHPM hydrogel treatment group was significantly increased, and there was little presence of collagen fibers.

[0074] The inflammatory environment seriously affects whether the injury develops into a chronic or non-healing wound. An increase in the level of interleukin-6 (IL-6) will prolong the inflammatory response and hinder wound healing, while tumor necrosis factor α (TNF-α) will stimulate the proliferation and differentiation of immune cells. Immunohistochemical staining showed that compared with the control group and other groups, the expression levels of the pro-inflammatory factors IL-6 and TNF-α in the CHPM hydrogel group were significantly reduced, indicating that the CHPM hydrogel could effectively regulate the inflammatory response after trauma. In addition, chemotactic factors and active proteins present in PRP can regulate the inflammatory response, inhibit excessive inflammation, and promote wound healing, thus contributing to the overall treatment process of infected skin wounds. The synergistic effect of PRP and Cu-TCPP-Mn further enhanced the anti-inflammatory ability of the hydrogel.

[0075] Embodiments of the present invention show that the multifunctional CHPM composite hydrogel loaded with nanozymes has good effects in the treatment of acute infected wounds. The Cu-TCPP-Mn nanozyme mimics the activities of SOD and CAT, effectively neutralizing ROS. In addition, the addition of PRP enriches the growth factors in the hydrogel, significantly promoting angiogenesis and tissue regeneration. The combined action of PRP and Cu-TCPP-Mn in the hydrogel regulates macrophage polarization, modulates and alleviates the persistent inflammation of infected wounds. The hydrogel has superior water absorption capacity, self-healing property and shape memory function. Both in vitro and in vivo studies have confirmed the efficacy of the hydrogel in promoting cell proliferation, migration and differentiation, regulating the inflammatory response and accelerating wound healing. The two play a synergistic role to jointly promote the healing of acute infected wounds and can better protect acute wounds. This shows that the CHPM composite hydrogel of the present invention can be applied to the treatment of acute infected wounds and has good effects, providing more options for the clinical treatment of acute infected wounds.

[0076] The above shows and describes 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 by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a nanozyme-loaded multifunctional composite hydrogel, characterized in that, It includes the following steps: S1: Prepare solution A and solution B respectively for standby; S2: Prepare Cu-TCPP-Mn nanosheets, and obtain Cu-TCPP-Mn nanodots after further processing them; S3: Add the Cu-TCPP-Mn nanodots obtained in step S2 into solution A to obtain a mixed solution C with a concentration of 2-20 μg / mL; S4: Add the activated PRP solution into solution B to obtain a mixed solution D with a concentration of 50-400 μg / mL; S5: Mix the mixed solution C and the mixed solution D in equal volumes to obtain a CHPM composite hydrogel.

2. The preparation method of a multifunctional composite hydrogel loaded with nanozymes according to claim 1, characterized in that, In step S1, the process of preparing solution A is as follows: S101: Dissolve hyaluronic acid in deionized water and stir to dissolve it to obtain a hyaluronic acid solution; S102: Add NaIO4 to the hyaluronic acid solution and continue stirring. After dialysis, freezing, and drying, obtain a white flocculent substance, namely oxidized hyaluronic acid; S103: Add deionized water to the white flocculent oxidized hyaluronic acid and stir to dissolve it to obtain solution A.

3. The preparation method of a nanozyme-loaded multifunctional composite hydrogel according to claim 2, characterized in that, In step S103, the concentration of solution A is 0-10 w / v%.

4. The preparation method of a nanozyme-loaded multifunctional composite hydrogel according to claim 1, characterized in that, In step S1, dissolve carboxymethyl chitosan in deionized water. The volume ratio of carboxymethyl chitosan to deionized water is 0.02-0.06:

1. After stirring and dissolving, obtain a solution B with a concentration of 1-20 w / v%.

5. The preparation method of a nanozyme-loaded multifunctional composite hydrogel according to claim 1, characterized in that, In step S2, the process of preparing Cu-TCPP-Mn nanosheets is as follows: S201: Take Cu(NO3)2 and H2TCPP, and dissolve them in a DMF solution. Add benzoic acid to it to form a homogeneous solution; S202: Place the obtained homogeneous solution in methyl silicone oil at 90°C and stir for 2-6 h to obtain a Cu-TCPP intermediate product; S203: Mix the Cu-TCPP intermediate product with MnCl2, and heat and stir at 90°C for 10-16 h. After centrifugation, collect the precipitate, and wash the precipitate with anhydrous ethanol at least 3 times to prepare Cu-TCPP-Mn nanosheets.

6. The preparation method of a multifunctional composite hydrogel loaded with nanozymes according to claim 1, characterized in that, In step S2, after ultrasonic treatment of the Cu-TCPP-Mn nanosheets for 4 h, Cu-TCPP-Mn nanodots are obtained.

7. The preparation method of a nanozyme-loaded multifunctional composite hydrogel according to claim 6, wherein In step S3, the concentration of the mixed solution C is 5 μg / mL, and in step S4, the concentration of the mixed solution D is 100 μg / mL.

8. A nanozyme-loaded multifunctional composite hydrogel, characterized in that, This composite hydrogel is prepared by the preparation method of a nanozyme-loaded multifunctional composite hydrogel according to any one of claims 1-7.

9. Use of a nanozyme-loaded multifunctional composite hydrogel in the preparation of drugs for acute infected wounds.

10. Use of a nanozyme-loaded multifunctional composite hydrogel in the preparation of novel antibacterial materials.

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