Multifunctional hydrogel based on delivery of CeO2 / Cu-coated Au-Pt nano enzyme as well as preparation method and application of multifunctional hydrogel

Abstract

The invention discloses multifunctional hydrogel based on delivery of CeO2 / Cu-coated Au-Pt nano-enzyme as well as a preparation method and application of the multifunctional hydrogel, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: under a water bath heating condition, sequentially adding gallic acid modified quaternized chitosan and 4-arm polyvinyl alcohol benzaldehyde into deionized water to obtain a hydrogel precursor solution; mixing the CeO2 / Cu-coated Au-Pt nano-enzyme with the hydrogel precursor solution to obtain a precursor hydrogel solution containing the nano-enzyme; and carrying out vortex treatment on the precursor hydrogel solution containing the nano-enzyme, and carrying out Schiff base crosslinking to form a QPCCAP multifunctional hydrogel network, so as to prepare the multifunctional hydrogel based on the delivery of the CeO2 / Cu-coated Au-Pt nano-enzyme. The artificial nano-enzyme hydrogel platform with multi-enzyme cascade reaction activity, developed by the invention, is helpful for solving a complex microenvironment in which hyperglycemia, high ROS and serious hypoxia of a diabetic wound surface are mutually staggered, improving wound angiogenesis and blood supply reconstruction of a diabetic patient, and is used for treating a chronic diabetic wound and accelerating wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus, characterized by the failure of the body's glucose regulation mechanisms, is a significant disease affecting the endocrine and metabolic systems. As one of the most serious complications, improper care of diabetic ulcers directly leads to increased amputation and mortality rates, posing a major challenge to global healthcare systems. Normal skin wound healing typically follows an interconnected and progressively ordered physiological process, comprising four main stages: hemostasis, inflammation, proliferation, and remodeling. For example, local hyperglycemia at the wound site leads to bacterial growth and recurrent infections, while the accumulation of large amounts of glycosylated products triggers the production of pro-inflammatory cytokines, exacerbating oxidative stress damage and inflammatory responses in the wound. The increased inflammation, in turn, increases the accumulation of reactive oxygen species (ROS) at the wound site, forming a harmful ROS-inflammatory cycle that severely hinders tissue regeneration. Therefore, there is an urgent need to establish an intelligent regulatory system that can adapt to each stage of diabetic wound healing and rationally coordinate various complex factors to achieve the remodeling of the wound microenvironment. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, the present invention aims to provide a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, its preparation method and application. By loading metal nanozymes onto the multifunctional hydrogel, continuous delivery to diabetic wounds can be achieved, and the hydrogel and nanozyme can work together to solve multiple problems in chronic wounds, such as infection, bleeding and continuous oxidative stress.

[0004] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, comprising the following steps: Under water bath heating conditions, gallic acid-modified quaternized chitosan and 4-arm polyvinyl alcohol benzaldehyde were added sequentially to deionized water to obtain a hydrogel precursor solution. A precursor hydrogel solution containing nanozyme was obtained by mixing CeO2 / Cu@Au-Pt nanozyme and hydrogel precursor solution. A precursor hydrogel solution containing nanozymes was subjected to vortex treatment to crosslink with Schiff bases to form a QPCCAP multifunctional hydrogel network, thus obtaining a multifunctional hydrogel based on the delivery of CeO2 / Cu@Au-Pt nanozymes.

[0005] In one embodiment, the temperature of the water bath heating condition is 50~80°C.

[0006] In one embodiment, the mass ratio of gallic acid-modified quaternized chitosan to 4-arm polyvinyl alcohol benzaldehyde is (18~20):(80~100).

[0007] In one embodiment, the concentration of CeO2 / Cu@Au-Pt nanozyme in the precursor hydrogel solution containing nanozyme is 100~400 μg / mL.

[0008] In one embodiment, the ratio of CeO2 / Cu@Au-Pt nanozyme to hydrogel precursor solution is 0.2g~0.8g:2mL.

[0009] In one embodiment, the preparation method of the CeO2 / Cu@Au-Pt nanozyme is as follows: An aqueous solution of Ce(NO3)3•6H2O was added to ethylene glycol and stirred to carry out the first reaction, yielding CeO2 nanoparticles; Cu(NO3)2•4H2O was added to CeO2 nanoparticles and stirred to carry out the second reaction. Then, precipitation, centrifugation, washing and freeze-drying were performed sequentially to obtain CeO2 / Cu nanoparticles with hollow structure. CeO2 / Cu nanoparticles were dissolved in deionized water and wrapped in tin foil to protect them from light. Then, HAuCl4 solution and H2PtCl6 solution were added. The pH of the reaction solution was adjusted to the set value with NaOH, and then NaBH4 solution was added. After stirring at room temperature, the mixture was centrifuged, washed and freeze-dried to obtain CeO2 / Cu@Au-Pt nanozyme.

[0010] In one embodiment, the concentration of the Ce(NO3)3•6H2O aqueous solution is 0.5 M, and the volume ratio of the Ce(NO3)3•6H2O aqueous solution to ethylene glycol is 1:30. The mass ratio of Cu(NO3)2•4H2O and CeO2 nanoparticles is 57.85 mg: 172.11 mg; The ratio of CeO2 / Cu nanoparticles to deionized water is 1 mg: 1 mL; The concentrations of the HAuCl4 solution and the H2PtCl6 solution are 20 mM, and the volume ratio of the HAuCl4 solution to the H2PtCl6 solution is 1:1. The set pH value is 10; the concentration of the NaBH4 solution is 0.1 M, and the NaBH4 solution includes 1% sodium citrate and 0.5% citric acid.

[0011] In one embodiment, the temperature of the first reaction is 180°C and the reaction time is 12 hours; the temperature of the second reaction is 180°C and the reaction time is 4.5 hours.

[0012] In one embodiment, the process parameters of the vortex treatment are as follows: rotation speed 200 r, time 12 h, and oscillation mode continuous.

[0013] The present invention also provides a method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, and the multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery is prepared.

[0014] The present invention also provides the application of a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery in the preparation of wound dressings for the treatment of diabetes.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozymes. The method involves sequentially mixing gallic acid-grafted quaternized chitosan (QCSG) and 4-arm polyvinyl alcohol benzaldehyde (4-arm PEG-BA or PEG-CHO) in deionized water under water bath heating to obtain a hydrogel precursor solution. The monodisperse CeO2 / Cu@Au-Pt nanozymes are then mixed with the hydrogel precursor solution to obtain a precursor hydrogel solution containing the nanozymes. A multifunctional hydrogel network loaded with CeO2 / Cu@Au-Pt nanozymes is formed through vortex treatment involving Schiff base crosslinking, hydrogen bonding, and π-π stacking, resulting in a multifunctional hydrogel (referred to as "QPCCAP"). Hydrogels are used as wound dressings to slowly release CeO2 / Cu@Au-Pt nanozymes. CeO2 / Cu@Au-Pt exhibits highly efficient GOX and POD enzyme activities, achieving potent bactericidal effects under NIR photothermal irradiation, adapting to the high-glycemic microenvironment of diabetic wounds. The quaternized chitosan in the hydrogel enhances its antibacterial effect. Through the multiple actions of the QPCCAP hydrogel, ROS clearance is enhanced, wound angiogenesis is promoted, and the severe oxidative stress-inflammation cycle in the diabetic wound microenvironment is disrupted, enabling programmed management of chronic diabetic wound healing. Specifically, in the preparation method, gallic acid-modified quaternized chitosan is mixed with PEG-CHO to obtain a hydrogel precursor solution. PEG-CHO containing catechol groups imparts significant antioxidant and adhesive capabilities to the hydrogel dressing. The multi-arm structure of PEG-CHO contains abundant aldehyde groups and dynamically covalently cross-linked Schiff base structures formed between these aldehyde groups and amino groups on the tissue surface. This interaction promotes good wet tissue adhesion and is independent of the liquid environment. Furthermore, the catechol groups of GA exhibit high affinity for various nucleophiles of peptides and proteins on tissue surfaces, enabling the formation of hydrogen bonds, cation-π interactions, π-π interactions, and Michael addition reactions. Additionally, the positively charged quaternary ammonium salt groups from QCS also promote electrostatic interactions between the hydrogel and the tissue surface, collectively endowing the hydrogel with excellent wet tissue adhesion properties. This broad adhesion to target tissues provides the hydrogel with the potential for extensive application in practical medical wound healing. The hydrogel's excellent injectability and superior adhesive properties help it adapt to irregular wound sites and provide a physical barrier, keeping bacteria out and moisture in, achieving hemostasis through physical closure. Furthermore, the electrostatic interaction between the negative charge on the CeO2 / Cu@Au-Pt nanozyme surface and the positive charge of QCSG allows for stable and uniform loading into the hydrogel network, overcoming the obstacles to sustained nanozyme delivery and enabling continuous nanozyme delivery.

[0016] Furthermore, the hydrogel described in this invention is a dressing repair system for promoting diabetic wound healing by delivering CeO2 / Cu@Au-Pt nanozymes. When CeO2 / Cu@Au-Pt nanozymes are simultaneously introduced into the dressing, the hydrogel's hardness reaches 11.4 kPa, which is beneficial for skin wound repair. Simultaneously, the hydrogel possesses the ability to regulate and precisely release therapeutic substances and can promote wound healing by simultaneously loading a large number of bioactive compounds into the hydrogel network. It can achieve long-term release and improve cell behavior. During the preparation of the hydrogel, the CeO2 / Cu@Au-Pt nanozymes are encapsulated within the gel network, giving the hydrogel excellent ROS scavenging ability, which contributes to wound healing. In summary, the QPCCAP hydrogel has the potential to reduce excessive oxidative stress and protect cells in the microenvironment of diabetic wounds.

[0017] Furthermore, the synergistic antibacterial effect of CeO2 / Cu@Au-Pt nanozymes and QCS is the reason for the improved antibacterial efficiency. The strong electrostatic interaction between the positive charge of QCS and the negatively charged bacterial phospholipid membrane components leads to bacterial death through membrane decomposition. The potential mechanisms by which CeO2 / Cu@Au-Pt nanozymes limit microbial growth are as follows: First, the hydrogel captures bacteria through the charge interaction of its numerous antibacterial groups (quaternary ammonium salts, gallic acid). Next, using endogenous high sugars as "sacrificial agents," it utilizes GOx activity to catalyze the production of H2O2, while the resulting pH decrease inhibits bacterial colonization. Subsequently, H2O2 is converted to more efficient •OH through POD-like activity. This further initiates the GPx reaction program, disrupting the bacterial antioxidant defense mechanism, damaging the bacterial cell membrane, leading to membrane structure disorder and leakage of membrane components, ultimately promoting bacterial death. Importantly, the hydrogel can respond to NIR stimulation to enhance the above cascade reaction, combining with the photothermal effect of PTT to achieve highly efficient sterilization. The combination of all these properties significantly improves the antibacterial activity of the hydrogel. QPCCAP hydrogel adhesive is a promising wound-closing hemostatic barrier because it possesses the natural hemostatic properties of chitosan, positively charged quaternary ammonium groups, and excellent adhesion, thus synergistically enhancing hemostatic activity. The encapsulation of CeO2 / Cu@Au-Pt nanozymes further accelerates the healing time of diabetic wounds. Furthermore, the moist environment provided by the hydrogel and the beneficial activities within the QPCCAP hydrogel both promote wound healing. As a dressing, the hydrogel can integrate multiple active molecules with different functions to promote the healing of diabetic wounds.

[0018] This invention also provides an application of a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery in the preparation of wound dressings for treating diabetes. The hydrogel, used as a wound dressing, slowly releases the CeO2 / Cu@Au-Pt nanozyme. The nanozyme encapsulated within the hydrogel and the positively charged quaternary ammonium groups in the QCSG of the gel itself enhance the antibacterial effect of the hydrogel. The multiple functions of the QPCCAP hydrogel enhance ROS clearance and promote wound angiogenesis. Through tissue adhesion, hemostasis, bacterial elimination, ROS clearance, and significant promotion of human umbilical vein endothelial cell (HUVEC) migration, proliferation, and angiogenesis, the QPCCAP hydrogel's synergistic effect creates a protective environment for diabetic wound healing and avoids the high-cost and time-consuming work required for growth factor or cell / cell-derived therapies by promoting angiogenesis, anti-inflammation, and increasing the number of healing-promoting M2 macrophages.

[0019] During application, in the early stages of wound healing, the composite hydrogel uses endogenous high glucose as "fuel," combining photothermal therapy and ROS catalytic therapy to achieve highly efficient sterilization. Simultaneously, it accelerates the reduction of local blood glucose concentration and pH levels in the wound, effectively inhibiting early infection and the aggravation of inflammatory responses. As glucose concentration decreases and nanozymes are further released, the SOD / GOx-CAT self-cascade reaction program is initiated, clearing excess ROS and releasing large amounts of oxygen to alleviate tissue hypoxia, reshaping the microenvironment of diabetic wounds, and promoting wound angiogenesis. Attached Figure Description

[0020] Figure 1 Macroscopic photographs of the hydrogel precursor solution and hydrogel of the present invention; Figure 2 Photographs showing the adhesion properties of the hydrogel of the present invention after complete gelation on bent and stretched fingers (A) and pigskin (B); Figure 3 Photographs showing the injectability of the hydrogel of the present invention; Figure 4 A photograph illustrating the self-healing properties of the hydrogel of this invention; Figure 5 The statistical results of the shear adhesion strength test of the QP(I), QPC(II), QPCC(III) and QPCCAP(IV) hydrogels of the present invention are shown in the figure, expressed as mean ± SD (n=5); Figure 6 The FT-IR spectra of the QCSG, PEG-CHO, QP, and QPCCAP hydrogels of this invention are shown below. Figure 7 A schematic diagram of the rheological properties of the QP(I), QPC(II), QPCC(III) and QPCCAP(IV) hydrogels of the present invention; Figure 8 Figure 1 shows the swelling and degradation curves of the QP(I), QPC(II), QPCC(III), and QPCCAP(IV) hydrogel samples of the present invention in the physiological microenvironment characterizing their drug and ion release kinetics. Figure 2 shows the swelling ratio of the QP(I), QPC(II), QPCC(III), and QPCCAP(IV) hydrogels, and Figure 3 shows the in vitro degradation of the QP(I), QPC(II), QPCC(III), and QPCCAP(IV) hydrogels. Figure 9 This is a representative schematic diagram of live and dead cell staining after NFB and HUVECs were co-cultured with QP, QPC, QPCC and QPCCAP hydrogels for 24 h. Figure 10 The graph shows the reactive oxygen species (ROS) scavenging capacity test results of the Control (I), QP (II), QPC (III), QPCC (IV), and QPCCAP (V) hydrogels of the present invention. Figure 11 The diagram shows the antibacterial activity test results of the Control (I), QP (II), QPC (III), QPCC (IV), and QPCCAP (V) hydrogels of the present invention. Figure 12 Figure 1 shows the hemostatic performance test of the QP hydrogel of the present invention in rat (Rat) and mouse (Mouse) liver hemorrhage models; wherein, Figure (A) is a representative bleeding image of the rat liver incision; Figure (B) is a statistical chart of bleeding volume; Figure 13 The diagram shows the in vivo diabetic wound healing performance test results of the Control (I), QP (II), QPC (III), QPCC (IV), and QPCCAP (V) hydrogels of the present invention. Figure 14 The wound size statistics of the Control (I), QP (II), QPC (III), QPCC (IV) and QPCCAP (V) hydrogels of the present invention and the control group are shown in the figure. Figure 15 This is a statistical chart showing the number of CD31-positive neovascularizations in the Control (I), QP (II), QPC (III), QPCC (IV), and QPCCAP (V) hydrogels of the present invention and the control group. Figure 16 This is a schematic diagram of the synthesis route of QCSG and PEG-CHO used in this invention. Detailed Implementation

[0021] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0022] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0023] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0024] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0025] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0026] This invention provides a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, its preparation method, and its application.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0029] Hydrogels, as network structures formed by the cross-linking of hydrophilic polymer chains in a water-rich environment, have shown unique advantages in tissue repair and regeneration, attracting increasing attention. However, traditional hydrogels have limited functionality and cannot effectively address the complex and variable microenvironment of diabetic wounds. In recent years, hydrogel-based composite systems have emerged as promising alternatives, especially hydrogels with immunomodulatory functions, which have been widely proposed for the management of chronic wounds. Simultaneously, many efforts are being made to develop functional hydrogel dressings for diabetic wound healing, capable of loading bioactive molecules and delivering therapeutic agents to the wound site through responsive release therapy protocols. Therefore, developing artificial nanoenzyme hydrogel platforms with multi-enzyme cascade reaction activity will help address the complex microenvironment of diabetic wounds, characterized by a complex interplay of hyperglycemia, high ROS, and severe hypoxia, and improve wound angiogenesis and revascularization in diabetic patients. This may be an ideal strategy for managing chronic diabetic wounds and accelerating wound healing.

[0030] In this study, we developed a composite hydrogel platform with multiple enzyme-like activities and photothermal-enhanced antibacterial properties. The aim was to achieve programmed management of chronic diabetic wound healing through dynamic, phase-specific adaptive reprogramming of the wound microenvironment. We proposed a fully customized programmed treatment strategy: In the early stages of wound healing, the composite hydrogel uses endogenous high glucose as "fuel," combining photothermal therapy and ROS catalytic therapy to achieve highly efficient sterilization while accelerating the reduction of local blood glucose concentration and pH levels, effectively inhibiting early infection and the exacerbation of inflammatory responses. As glucose concentration decreases and nanozymes are further released, a SOD / GOx-CAT self-cascade reaction program is initiated, clearing excess ROS and releasing large amounts of oxygen to alleviate tissue hypoxia and remodel the diabetic wound microenvironment. Furthermore, through the synergistic effect of multiple signaling pathway molecular mechanisms, oxidative stress damage is reduced, the inflammatory response is balanced, and a smooth transition from the inflammatory phase to the proliferative phase is promoted. Once healing successfully enters the proliferative and remodeling phases, it can also promote angiogenesis, granulation tissue formation, and timely collagen deposition at the wound site. In summary, unlike existing strategies that primarily regulate wound healing at a single stage, this platform focuses on tracking the continuity of different healing stages and has dynamic adaptive adjustment capabilities, making it a promising new direction for future diabetes wound management.

[0031] In summary, the artificial nanoenzyme hydrogel platform developed in this invention with multi-enzyme cascade reaction activity will help solve the complex microenvironment of diabetic wounds, which is characterized by a complex interplay of hyperglycemia, high ROS, and severe hypoxia. It can also improve wound angiogenesis and revascularization in diabetic patients, which may be an ideal strategy for treating chronic diabetic wounds and accelerating wound healing.

[0032] Hydrogels are polymer materials with a three-dimensional network structure, exhibiting excellent biocompatibility, effective adhesion, good antibacterial properties, and high water content (70-95%). They can absorb wound exudate, provide oxygen permeability and hemostasis. Furthermore, the three-dimensional network structure of hydrogels facilitates the loading of active agents and drugs, enabling slow release, thus showing broad application prospects in wound dressings. Hydrogels overcome the shortcomings of conventional dressings by providing not only a physical barrier against secondary infection but also a compatible physiological environment—a suitable biocompatibility, moisture, and antibacterial interface for the wound. Hydrogel dressings are widely used in the treatment of diabetic wounds due to their extracellular matrix (ECM)-like softness, ability to fill wounds of any irregular shape, excellent biocompatibility, effective drug encapsulation, and sustained drug delivery. In particular, viscous hydrogels can adhere to and bind damaged tissue, acting as hemostatic agents or sealants to stop bleeding, blocking the flow of fluid or gas in the wound, and serving as a barrier to protect against microorganisms.

[0033] The precursor solution of the hydrogel comprises two components, A and B. Component A is gallic acid-modified quaternized chitosan (QCSG), which exerts cationic antibacterial effects, seals wounds, stops bleeding (the cations react with the negative charges on platelets to aggregate platelets), and promotes wound repair (QCSG promotes macrophage secretion of PDGF and IL-1, which in turn promotes angiogenesis and fibroblast proliferation). Component B is 4-arm polyvinyl alcohol benzaldehyde (4-arm PEG-BA or PEG-CHO). PEG-CHO exhibits excellent biocompatibility. Furthermore, linear / multi-arm PEG can indeed combine with various materials to improve the mechanical properties of the hydrogel. The multi-arm structure of PEG-CHO provides abundant aldehyde groups, which crosslink with the amino groups of QCSG through dynamic Schiff base reactions to form imine bonds (C=N bonds), a key driving force for the formation of the three-dimensional network structure of the QP hydrogel. Simultaneously, the phenolic hydroxyl groups of gallic acid can further enhance the mechanical stability of the hydrogel through hydrogen bonding and π-π stacking synergistic mechanisms with the aldehyde groups of PEG-CHO. Furthermore, the quaternary ammonium salt of QCSG carries a positive charge, which can form an electrostatic interaction with the composite nanozyme with a negative charge on the surface. Combined with the metal coordination between the phenolic groups provided by GA and the nanozyme, a unique CeO2 / Cu@Au-Pt nanozyme delivery hydrogel carrier is constructed.

[0034] The antioxidant activity of CeO2 / Cu@Au-Pt nanozymes is attributed to the coexistence of Ce on the particle surface. 3+ / Ce 4 The autonomous switching between two valence states. Simultaneously, by doping CeO2 nanoparticles with Cu ions, oxygen in the crystal lattice can be better released to promote the formation of oxygen vacancies, allowing Ce... 3+ / Ce 4+ The redox transitions between CeO2 and CeO2 are more readily realized, further enhancing the superoxide dismutase (SOD) and catalase (CAT) activities of CeO2. Au-Pt is then deposited on its surface, successfully creating a composite nanozyme with multifunctional cascade catalytic properties exhibiting SOD, CAT, and glucose oxidase (GOx) activities. This composite nanozyme can regulate local blood glucose levels in diabetic wounds, reduce glucose in the wound microenvironment, and scavenge reactive oxygen species (ROS) through its multi-enzyme cascade activity, mitigating oxidative stress damage and promoting rapid wound healing.

[0035] The preparation of PEG-CHO used in this invention is based on the method described in Wang L, Yang K, Li X, Zhang X, Zhang D, Wang LN, Lee CS. A double-crosslinked self-healing antibacterial hydrogel with enhanced mechanical performance for wound treatment. Acta Biomater. 2021Apr 1;124:139-152. doi: 10.1016 / j.actbio.2021.01.038. Epub 2021 Jan 30. PMID:33524557. In summary, 4-carboxybenzaldehyde (150 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 191.7 mg), and 4-dimethylaminopyridine (DMAP, 61.08 mg) were dissolved in dry dichloromethane (CH2Cl2, 40 mL), and then tetraarm polyethylene glycol (4-arm-PEG-OH, 1 g) was added under a nitrogen atmosphere. The system was stirred at 37 °C for 24 hours, and then the mixture was washed with 1 M HCl (3 × 40 mL), saturated NaHCO3 (3 × 40 mL), and brine (3 × 40 mL). The organic layer was then partially dried under reduced pressure, and the polymer was obtained as a white solid after drying under vacuum.

[0036] The preparation of QCS used in the present invention refers to X. Zhao, B. Guo, H. Wu, Y. Liang, PX Ma, Injectable antibacterial conductive nanocomposite cryogels with rapid shaperecovery for noncompressible hemorrhage and wound healing, Nat Commun 9(1)(2018) 2784. and J. Qu, X. Zhao, Y. Liang, T. Zhang, PX Ma, B. Guo, Antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing, Biomaterials 183 (2018) 185-199. In short, chitosan (1g, Sigma-Aldrich) was reacted with GTMAC (glycidyltrimethylammonium chloride) at 55°C for 18 hours to generate quaternary ammonium chitosan (QCS), which was then separated, dialyzed (or precipitated), and freeze-dried. Specific steps are as follows: Figure 16 The synthetic route for QCS is shown below. 0.5 g of chitosan was suspended in 18 mL of glacial acetic acid solution (0.5% v / v). Then, 773 μL of GTMAC was added dropwise, and the reaction was stirred at 55 °C for 18 hours. Finally, the solution was centrifuged (6500 rpm, 8 min). The supernatant was precipitated with 5–8 volumes of acetone. The entire purification process was repeated three times, and the final product was dried in a vacuum oven.

[0037] The preparation of the QCSG used in this invention is based on the work of Shan Y, Cao F, Zhao X, Luo J, Mei H, Zhang L, Huang Y, Yang Y, Yan L, Huang Y, Han Y, Guo B. Procoagulant, antibacterial and antioxidant high-strength porous hydrogel adhesives in situformed via self-gelling hemostatic microsheets for emergency hemostasis and wound repair. Biomaterials. 2025 Apr;315:122936. doi: 10.1016 / j.biomaterials.2024.122936. Epub 2024 Oct 30. In short, gallic acid was grafted onto QCS to obtain quaternized chitosan-g-gallic acid (QCS-GA). Specifically, 3.03 g of QCS was dissolved in 300 mL of deionized water, and then 2.82 g of 1-hydroxybenzotriazole (HOBt) was added, with stirring at room temperature for 12 h. Next, 2.33 g of gallic acid was weighed and added to the above solution, and stirring continued until completely dissolved. 2.61 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and dissolved in 10 mL of anhydrous ethanol, and then added dropwise to the above solution, reacting at room temperature for 24 h. The purified QCS-GA (QCSG) was obtained by lyophilization.

[0038] The preparation method of the CeO2 / Cu@Au-Pt nanozyme used in this invention is as follows: Ce(NO3)3•6H2O aqueous solution (0.5 M, 2 mL) was added to 60 mL of ethylene glycol and stirred vigorously for 40 min. The mixture was then transferred to a high-pressure reactor and maintained at 180 °C for 12 h to obtain CeO2 nanoparticles. Subsequently, 57.85 mg of Cu(NO3)2•4H2O was added to the obtained CeO2 product, and the mixture was stirred for 1 h and maintained at 180 °C in the reactor for 4.5 h. The precipitate was centrifuged, washed alternately with anhydrous ethanol and deionized water, and freeze-dried to obtain copper-doped cerium oxide nanoparticles with a hollow structure (CeO2 / Cu NPs). For the synthesis of CeO2 / Cu@Au-Pt, 36 mg of CeO2 / Cu nanoparticles were dissolved in 36 mL of deionized water, and the reaction flask was wrapped with tin foil to protect it from light. Then, 36 μL of HAuCl4 solution (20 mM) and 36 μL of H2PtCl6 solution (20 mM) were added, and the pH of the reaction solution was quickly adjusted to 10 with 0.1 M NaOH. After mixing, 30 μL of NaBH4 solution (0.1 M) containing 1% sodium citrate and 0.5% citric acid was added, and the mixture was stirred at 25 °C for 3.5 h. After centrifugation, washing, and freeze-drying, CeO2 / Cu@Au-Pt NPs were obtained. The obtained product was stored at 4 °C for future use.

[0039] 1. Hydrogel preparation method 1.1 The preparation process of QPCCAP hydrogel is as follows Figure 16 As shown, the specific steps are as follows: Step 1: Gallic acid-modified quaternized chitosan (QCSG) and 4-arm polyethylene glycol benzaldehyde (PEG-CHO) are added sequentially to deionized water under water bath heating conditions to obtain a hydrogel precursor solution; wherein, the temperature of the water bath heating conditions is 50~80℃; the mass ratio of QCSG to PEG-CHO is (18~20):(80~100).

[0040] Specifically, 18-20 mg of lyophilized gallic acid-modified quaternary ammonium chitosan (QCSG) was weighed and placed in a 5 mL centrifuge tube. 2 mL of deionized water was added, and the mixture was dissolved at 50-60 °C for 4-6 h. After complete dissolution, 80-100 mg of PEG-CHO was added, and the mixture was heated at 50-60 °C for 5-10 min. After complete dissolution, a hydrogel precursor solution was obtained. The optimal water bath heating temperature was 60 °C.

[0041] Step 2: Add the uniformly dispersed CeO2 / Cu@Au-Pt nanozyme to the hydrogel precursor solution and mix thoroughly until homogeneous.

[0042] Specifically, CeO2 / Cu@Au-Pt nanozyme (0.2g~0.8g) was added to 2ml of hydrogel precursor solution to obtain a precursor hydrogel solution containing nanozyme (the concentration of nanozyme in the precursor hydrogel solution was 100~400μg / mL).

[0043] Step 3: The precursor hydrogel solution containing nanozymes is subjected to thorough vortexing to allow it to undergo Schiff base crosslinking to form a QPCCAP multifunctional hydrogel network, thereby obtaining a multifunctional hydrogel.

[0044] Specifically, the precursor hydrogel solution containing nanozymes is subjected to thorough vortexing to induce Schiff base crosslinking, forming a QPCCAP multifunctional hydrogel network, thus obtaining a multifunctional hydrogel. PEG-CHO was selected as the main component of the multifunctional hydrogel because its phenolic groups possess significant antioxidant and adhesive properties; QCSG is a component with good solubility and antibacterial activity.

[0045] This invention provides a novel QPCCAP multifunctional hydrogel with biocompatibility, adhesion, antibacterial properties, sustained-release CeO2 / Cu@Au-Pt nanozyme, ROS scavenging, and angiogenesis promotion, offering new therapeutic targets and approaches for the treatment of diabetic wounds and laying the foundation for the clinical application of novel hydrogels. The aforementioned QPCCAP multifunctional hydrogel based on precise delivery of CeO2 / Cu@Au-Pt nanozyme can be used in the preparation of wound dressings for the treatment of diabetes.

[0046] 1.2 The preparation process of QP hydrogel is as follows: To synthesize the QP hydrogel, 500 μL of QCSG solution (20 mg / mL) was incorporated into 500 μL of PEG-CHO solution (80 mg / mL). The suspension was then mixed in a vortex mixer and left at room temperature to allow the hydrogel to form.

[0047] 1.3 The preparation process of QPC hydrogel is as follows: To synthesize the QPC hydrogel, 500 μL of QCSG solution (20 mg / mL) was incorporated into 500 μL of PEG-CHO solution (80 mg / mL) containing 200 μg / mL CeO2 nanoparticles. The suspension was then mixed in a vortex mixer and left at room temperature to allow the hydrogel to form.

[0048] The preparation process of 1.4QPCC hydrogel is as follows: To synthesize the QPC hydrogel, 500 μL of QCSG solution (20 mg / mL) was incorporated into 500 μL of PEG-CHO solution (80 mg / mL) containing 200 μg / mL CeO2 / Cu nanoparticles. The suspension was then mixed in a vortex mixer and left at room temperature to allow the hydrogel to form.

[0049] 1.5 Examples Example 1 Step 1: Weigh 20 mg of lyophilized gallic acid-modified quaternary ammonium chitosan (QCSG) and place it in a 5 mL centrifuge tube. Add 2 mL of deionized water and dissolve at 60 °C for 4 h. After complete dissolution, add 80 mg of PEG-CHO and heat at 60 °C for 2 min. After complete dissolution, obtain the hydrogel precursor solution.

[0050] Step 2: Mix the uniformly dispersed CeO2 / Cu@Au-Pt nanozyme (concentration of 100 μg / mL) with the hydrogel precursor solution to obtain a precursor hydrogel solution containing nanozyme.

[0051] Step 3: The precursor hydrogel solution containing nanozymes is subjected to thorough vortexing to allow it to undergo Schiff base cross-linking to form a QPCCAP multifunctional hydrogel network, thereby obtaining the QPCCAP multifunctional hydrogel.

[0052] Example 2 Step 1: Weigh 18 mg of lyophilized gallic acid-modified quaternary ammonium chitosan (QCSG) and place it in a 5 mL centrifuge tube. Add 2 mL of deionized water and dissolve at 60 °C for 4 h. After complete dissolution, add 90 mg of PEG-CHO and heat at 60 °C for 2 min. After complete dissolution, obtain the hydrogel precursor solution.

[0053] Step 2: Mix the uniformly dispersed CeO2 / Cu@Au-Pt nanozyme (concentration of 100 μg / mL) with the hydrogel precursor solution to obtain a precursor hydrogel solution containing nanozyme.

[0054] Step 3: The precursor hydrogel solution containing nanozymes is subjected to thorough vortexing to allow it to undergo Schiff base cross-linking to form a QPCCAP multifunctional hydrogel network, thereby obtaining the QPCCAP multifunctional hydrogel.

[0055] Example 3 Step 1: Weigh 19 mg of lyophilized gallic acid-modified quaternary ammonium chitosan (QCSG) and place it in a 5 mL centrifuge tube. Add 2 mL of deionized water and dissolve at 60 °C for 4 h. After complete dissolution, add 100 mg of PEG-CHO and heat at 60 °C for 2 min. After complete dissolution, obtain the hydrogel precursor solution.

[0056] Step 2: Mix the uniformly dispersed CeO2 / Cu@Au-Pt nanozyme (concentration of 100 μg / mL) with the hydrogel precursor solution to obtain a precursor hydrogel solution containing nanozyme.

[0057] Step 3: The precursor hydrogel solution containing nanozymes is subjected to thorough vortexing to allow it to undergo Schiff base cross-linking to form a QPCCAP multifunctional hydrogel network, thereby obtaining the QPCCAP multifunctional hydrogel.

[0058] Example 4 Unlike Example 1, the CeO2 / Cu@Au-Pt nanozyme (concentration of 100 μg / mL) was replaced with CeO2 / Cu@Au-Pt nanozyme (concentration of 200 μg / mL) to obtain QPCCAP hydrogel.

[0059] Example 5 Unlike Example 2, the 18 mg gallic acid-modified quaternized chitosan (QCSG) in step one was replaced with 20 mg gallic acid-modified quaternized chitosan (QCSG) to obtain QPCCAP hydrogel.

[0060] Example 6 Unlike Example 3, the CeO2 / Cu@Au-Pt nanozyme (concentration of 100 μg / mL) in step 2 was replaced with CeO2 / Cu@Au-Pt nanozyme (concentration of 400 μg / mL) to obtain QPCCAP hydrogel.

[0061] 2. Performance Testing and Results The properties of QPCCAP multifunctional hydrogels were explored using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), energy dispersive spectroscopy (EDS), nuclear magnetic resonance (1H NMR), degradation analysis, adhesive strength, and rheological testing.

[0062] 2.1 Viscoelasticity Test: like Figure 1 As shown in the macroscopic photographs, QCSG precursor solution, PEG-CHO precursor solution, and QP multifunctional hydrogel exhibit good tissue adhesion and can be used as wound dressings.

[0063] like Figure 2 As shown in the photograph, the hydrogel adheres to the bent and extended fingers after complete gelation. The hydrogel exhibits good viscoelasticity after cross-linking for 2 minutes and can be stretched well between the two fingers. The hydrogel can adhere firmly to the bent fingers without shrinkage or breakage, indicating that the hydrogel can establish a stable and close contact with the dynamic wound and has good viscoelasticity.

[0064] 2.2 Injectability and Adaptability Testing like Figure 3 and Figure 4 As shown, the modular in-situ gelation behavior gives QP hydrogel excellent injectability and allows it to be easily extruded from the needle of a disposable syringe into the shape of the letters "XXH". QP multifunctional hydrogel has excellent shape adaptability and can be repeatedly reconfigured into various shapes, including crescent, cross, triangle and star. This demonstrates the good ability of our synthesized hydrogel to adapt to irregular wound shapes, which is crucial for biomedical applications on uneven wound sites. It can bind tightly to the wound surface to form a protective environment, which helps prevent infection and keep the wound moist, further highlighting its clinical applicability.

[0065] 2.3 Adhesion strength test: The adhesion properties of the QP(I), QPC(II), QPCC(III), and QPCCAP(IV) hydrogels were determined using fresh pigskin. Pigskin was selected to simulate human tissue and cut into rectangular strips (10 mm × 30 mm), which were then washed with PBS prior to the adhesion test. 200 μL of the test hydrogel was then applied and sandwiched between two pieces of pigskin, with an adhesion area of ​​10 × 10 mm. The adhesion properties were then measured via an overlap shear test at a rate of 5 mm / min⁻¹ on an Instron material testing system (MTS Standard 43) equipped with a 50 N weighing sensor. Furthermore, the in vitro adhesion of the QPCCAP hydrogel was tested on eight organs (heart, liver, lung, spleen, kidney, bladder, colon, and stomach) removed from C57 mice. Specifically, 200 μL of the QPCCAP hydrogel was attached to stainless steel tweezers before being applied to the moist surfaces of these organs, and all tests were performed at least three times.

[0066] The results are as follows Figure 5 As shown, according to the pigskin overlap shear test, the adhesion strength can reach 11.4 kPa, and the adhesion strength is not affected after loading nanozymes, which is much higher than that of commercial dressings (about 5 kPa), and it is an effective value that keeps the skin in place on diabetic wounds under natural skin movement.

[0067] The excellent adhesion behavior exhibited by the QPCCAP multifunctional hydrogel demonstrates its great potential in wound repair. The tissue adhesion mechanism of the hydrogel is attributed to the following: the main contributor is the dynamic covalent cross-linking of Schiff base structures between the abundant aldehyde groups of the multi-armed PEG-CHO and the amino groups on the tissue surface. This interaction promotes excellent wet tissue adhesion, independent of the liquid environment. Furthermore, the catechol groups of GA have a high affinity for various nucleophiles of peptides and proteins present on the tissue surface, enabling the formation of hydrogen bonds, cation-π interactions, π-π interactions, and Michael addition reactions. Moreover, the positively charged quaternary ammonium groups from QCS also promote electrostatic interactions between the hydrogel and the tissue surface, collectively conferring excellent wet tissue adhesion properties. This broad adhesion to target tissues provides the hydrogel with the potential for extensive practical medical applications in wound healing.

[0068] 2.4 Fourier Transform Infrared Spectroscopy (FT-IR) Test: FT-IR spectra of QCSG, PEG-CHO, QP, and QPCCAP lyophilized hydrogels were obtained on an FT-IR spectrometer (Nicolet 6700, Thermo Scientific) in the range of 550-4000 cm⁻¹. -1 The study was conducted to investigate the chemical composition of the hydrogel.

[0069] Test results are as follows Figure 6 As shown, in the FT-IR spectrum, QCSG is present in the range of 1600–1450 cm⁻¹. -1 An absorption band appears at (the C=C stretching vibration of benzene), relative to quaternized chitosan, demonstrating the successful grafting of gallic acid. At 1723 cm⁻¹ -1 and 1113cm -1 The absorption peaks near the aldehyde group (–C=O–) and ether group (–OCO–) correspond to the stretching vibrations of the aldehyde group and the ether group (–OCO–), respectively, confirming the successful synthesis of PEG-CHO. The FTIR spectra of QP and QPCCAP show a peak at 1643 cm⁻¹. -1 The appearance of the new peak confirmed that a Schiff base reaction occurred between the phenolic groups of PEG-CHO and the amino groups in QCSG to form an imine bond.

[0070] 2.5 Rheological property test: The rheological properties of QP(I), QPC(II), QPCC(III), and QPCCAP(IV) hydrogels were evaluated using a rheometer (DHR-2). The test temperature was 37 °C. The storage modulus (G′) and loss modulus (G′′) of the different hydrogels were evaluated by placing the hydrogel solutions on 20 mm parallel plates with a 1 mm gap. Time-scan tests were performed at a strain amplitude of 1% and a frequency of 10 rad / s. The rheological behavior of the hydrogels was analyzed, and the storage modulus (G′′) and loss modulus (G′′) were determined using the rheometer in time-scan mode.

[0071] Rheological test results as follows Figure 7 As shown, QP(I), QPC(II), QPCC(III), and QPCCAP(IV) + The G′ value of the hydrogels is consistently about 5 times that of the loss modulus (G′′), indicating elastic properties. The storage modulus (G′) of the four hydrogels exceeds the loss modulus (G′′) across the entire angular frequency range, demonstrating the good elasticity, high mechanical strength, and structural stability of the hydrogels.

[0072] 2.6 In vitro swelling and degradation test of hydrogels: like Figure 8 As shown, the equilibrium swelling ratio (ESR) of four hydrogels—QP(I), QPC(II), QPCC(III), and QPCCAP(IV)—after immersion in PBS (pH 7.4) at 37°C for 24 hours is illustrated. Since water absorption capacity significantly affects the mechanical properties of hydrogel materials, the ESR decreased from 50% to 38% with increasing nanozyme loading concentration, indicating a decrease in the cross-linking density of the hydrogel network. The degradation capacity of hydrogels is an important characteristic for use as wound dressings. Loading nanozymes accelerates the degradation rate of hydrogels in PBS, increases the contact area with water, and improves material exchange capacity, making it more desirable for in vivo applications. Furthermore, all four hydrogels can maintain their colloidal state for more than 12 days, meeting the long-term needs of wound healing.

[0073] 2.7 In vitro cell compatibility and proliferation assays: Human fibroblasts (NFB) were incubated in DMEM (Gibco) medium supplemented with 10% FBS and 1% penicillin and streptomycin (Hyclone) at 37°C in an incubator with 5% CO2. (Using LIVE / DEAD...) ®The cytotoxicity of the hydrogel was studied using an activity / cytotoxicity kit (YESAN, Shanghai, China). NFB cells were seeded at a density of 10,000 cells / well in 96-well plates, and hydrogel sections (5 mm in diameter, 0.5 mm thick) were prepared and co-cultured with HFB for 24 h. The LIVE / DEAD® kit was used to assess the viability of cells co-cultured with the hydrogel plates. After 24 hours of co-culture, the hydrogel plates and culture medium were removed, and LIVE / DEAD® reagent was added according to the instructions. After a further 45 minutes of incubation, images were taken using a fluorescence microscope (Nikon, DS-Ri2, Japan).

[0074] NFB (human fibroblasts) and HUVEC (human vascular endothelial cells) were co-cultured with hydrogels, and the results of using a live / dead assay to visualize cell morphology were as follows: Figure 9 As shown, most NFB cells and HUVEC cells were stained green (live cells), and only a small number of cells were stained red (dead cells), demonstrating the high cell compatibility of the QP, QPC, QPCC, and QPCCAP hydrogels of the present invention.

[0075] 2.8 In vitro ROS scavenging ability test: The 2',7'-dichlorofluorescein diacetate (DCFH-DA, Maokang, Shanghai, China) probe was used to evaluate the intracellular ROS scavenging capacity of the hydrogel. In short, HUVECs were seeded in 6-well plates, and ROS was induced using ROS-up (MKbio, Shanghai, China), followed by co-culturing with the hydrogel samples for 12 hours. A negative control (PBS) was provided. Afterward, the hydrogel was removed, and DCFH-DA was added for staining at 37°C for 20 minutes. The ROS scavenging capacity of each group was measured using flow cytometry (BD Accuri® C6, USA), with each group repeated three times.

[0076] The antioxidant properties of the hydrogels were determined using ROS-specific probes, with 1,1-diphenyl-2-picrylhydrazine (DPPH∙) as the mode radical for detection. The ability of QP, QPC, QPCC, and QPCCAP hydrogels to reduce active ROS in HUVECs under intracellular oxidative stress stimulated by ROS-up reagents (ROS inducers) was tested.

[0077] The results are as follows Figure 10As shown, the hydrogel alone effectively alleviated ROS generation, while the addition of nanozymes further reduced the ROS fluorescence intensity in HUVECs. Among them, the QPCC and QPCCAP groups exhibited the most significant protective effect against oxidative damage, which may be attributed to the excellent multiple cascade enzyme activity of the CeO2 / Cu and CeO2 / Cu@Au-Pt nanozymes. During the hydrogel preparation process, the CeO2 / Cu@Au-Pt nanozyme was introduced into the physically cross-linked quaternary ammonium of QCSG, and promoted potent ROS scavenging through cascade multi-enzyme activity. This endowed the hydrogel with excellent ROS scavenging ability, contributing to wound healing. In summary, these findings highlight the potential of the QPCCAP multifunctional hydrogel to reduce excessive oxidative stress and protect cells in the microenvironment of diabetic wounds.

[0078] 2.9 In vitro antibacterial performance test: The hydrogel exhibited glucose-activated antibacterial activity against *E. coli* and MRSA. Briefly, 200 μL of hydrogel was prepared into 48-well microplates (Costar). After complete gelation, 10 μL of bacterial suspension (10... 7 CFU mL -1 Sterile saline solution was added to the surface of each hydrogel in a 48-well culture plate. The inoculated hydrogel was incubated at 37°C for 2 hours with a relative humidity of at least 90% inside the microplate. At the end of this time, 1 mL of sterile saline solution was added to each well to resuspend any bacterial survivors, and finally 10 μL of the suspension was added to the agar plate. Using 10 μL of bacterial suspension suspended in 1 mL of physiological saline (10... 7 CFU mL -1 As a negative control, colony-forming units were counted on agar plates (petition dishes) after incubation at 37°C for 18 hours. Each group was tested three times. The antibacterial ratio (%) was calculated as: (control bacterial count - hydrogel survivor count) / control bacterial count) × 100%.

[0079] Figure 11 Images show the contact antimicrobial activity of hydrogels treated with PBS (I), QP (II), QPC (III), QPCC (IV), and QPCCAP (V) against *Escherichia coli* and *Staphylococcus aureus*, respectively. Antimicrobial dressings can minimize infection by harmful microorganisms and improve delayed wound healing, a key challenge in the healing process of diabetic wounds. The intrinsic antimicrobial activity of QCSG has been extensively demonstrated previously, such as... Figure 11 As shown, the antibacterial activity of the hydrogel against *Escherichia coli* and *Staphylococcus aureus* was tested using a surface antibacterial activity assay. The pure QP hydrogel exhibited a certain degree of intrinsic antibacterial properties, which may be attributed to the positive charge of chitosan itself and the quaternary ammonium groups and NH3 after quaternization. +Both metal nanoparticles and gallic acid (GA) interact electrostatically with the carbonyl and phosphate groups on the bacterial phospholipid layer, causing bacterial cell membrane rupture and leakage of contents. Simultaneously, GA releases H⁺, disrupting the bacterial membrane potential. Under NIR light exposure, the QPC and QPCC groups showed antibacterial rates of 72.4% and 88.6% against *E. coli*, respectively, and clearance rates of 64.3% and 82.2% against MRSA, respectively, indicating that the loading of metal nanoparticles enhanced the antibacterial ability of the hydrogel. The QPCCAP group had the fewest colony-forming units, further significantly improving the inhibition rates against these two bacteria, reaching approximately 97.4% and 95.5%, respectively, essentially achieving complete bacterial removal. Notably, without NIR light exposure, the inhibitory effects of both the QPCC and QPCCAP groups on both bacteria were less than under NIR light, with MRSA being more sensitive to the photothermal effect, further demonstrating the photodynamic antibacterial properties of the composite gel system. The combination of all these properties significantly improved the antibacterial activity of the hydrogel, indicating its great potential in protecting diabetic wounds from contamination.

[0080] 2.10 In vivo hemostasis test: In short, mice / rats were anesthetized and then fixed to a surgical cork board. The liver of the mice / rats was exposed through an abdominal incision. Filter paper was weighted and placed under the liver. A 5 mm deep bleeding wound was then introduced into the liver of a Kunming mouse or SD rat with a blade, and 200–250 μL of hydrogel was immediately applied to the bleeding site. The control group was set as untreated. The filter paper was then weighed and the blood loss was calculated.

[0081] The first step in wound healing is hemostasis. It is hypothesized that viscous hydrogels can adhere to surrounding tissues and, due to their excellent tissue adhesion, act as a physical barrier to facilitate the hemostasis process. We evaluated the hemostatic performance of QP composite hydrogels using a mouse / rat liver hemorrhage model. Figure 12As shown, in all bleeding scenarios, the hydrogel-treated group exhibited better hemostasis, with almost no visible bloodstains, while the untreated group showed significant bleeding. Quantitative analysis of blood loss volume showed that the QP composite hydrogel group significantly reduced blood loss compared to the control group, with a reduction of approximately 35 mg in mice and approximately 70 mg in rats. This is mainly due to its rapid wound-closing ability. Furthermore, the electrostatic effect of QCS and the high affinity of GA catechol groups for erythrocytes also contribute to blood coagulation, playing an important procoagulant role. The strong adhesion between the hydrogel and the wound tissue surrounding the bleeding site is the reason for its excellent hemostatic ability; the hydrogel can act as a physical barrier for hemostasis. Besides superficial bleeding, intraoperative or unintentional deep vascular bleeding is also a significant problem. QPCCAP hydrogel adhesive is a promising wound-closing hemostatic barrier because it possesses the natural hemostatic properties of chitosan, positively charged quaternary ammonium groups, and excellent adhesion, thus synergistically improving hemostatic activity. These findings provide evidence for the excellent hemostatic properties of hydrogels in vivo and point to their potential applicability in repairing infected and / or hemorrhagic diabetic wounds.

[0082] 2.11 Diabetic Wound Healing Test: Diabetes was induced in male Kunming mice (35 g) using streptozotocin (STZ). One week after administration, blood glucose levels were measured using a tail vein glucometer (Yuyue Company). After another week, mice were considered to have diabetes when their fasting blood glucose level exceeded 16.7 mmol / L. All mice were randomly divided into 5 groups: Tegaderm... TM Membranes, QP gels, QPCC gels (loaded with CeO2 / Cu NPs), QPCCAP gels (loaded with CeO2 / Cu@Au-Pt NPs), and QPCCAP gels + NIR (under near-infrared irradiation) were used. All procedures were performed under sterile conditions. After standard anesthesia, the dorsal region of mice above the tail but below the back was shaved in preparation for surgery. A 10 mm diameter full-thickness circular wound was created by puncture biopsy. The hydrogel groups received appropriate treatment. All tissues were collected on days 3, 7, and 14 and then stored at -80°C before analysis. For wound contraction monitoring, mice in each group were under standard anesthesia, and wound contraction was measured using ImageJ software. A 10 mm diameter wound was created on the dorsal side of the mice using a skin punch, followed by treatment with QP(II), QPC(III), QPCC(IV), and QPCCAP(V) hydrogels, with Tegaderm dressing (I) serving as a control.

[0083] Figure 13Macroscopic photographs of the wounds at different time points and wound closure marks in the wound area are shown. A preliminary observation of the wound healing process shows that the wound size decreased significantly in all groups. The QPCCAP+NIR laser irradiation group showed the fastest wound healing speed and the largest reduction in wound area, while the control group showed the slowest wound healing speed. Figure 14 Quantitative analysis of wound closure rates showed that after 3 days of treatment, the wound contraction in the QPCCAP+NIR laser irradiation group was significantly better than that in other treatment groups. This was attributed to the QPCCAP composite gel unlocking a cascade reaction in response to the high glucose environment during the early stages of wound healing, significantly reducing local blood glucose concentration. Simultaneously, it synergistically inhibited bacterial growth and colonization in the microenvironment with PTT, further suppressing inflammation. On day 7, the wound healing area in both the QPCCAP and QPCCAP+NIR groups was significantly reduced, with wound healing rates as high as 68.6% and 78.2%, respectively. In contrast, the wound areas in the control group, QP group, and QPCC group remained relatively large, with wound healing rates of only 24.4%, 42.8%, and 46.1%, respectively. All wounds treated with different hydrogels showed stronger wound contraction than those treated with Tegaderm dressing (control group). After 14 days of healing, although skin reconstruction was not yet complete in the QPCCAP+NIR group, the closure rate reached approximately 92%, indicating that their diabetic wounds were almost completely healed. In contrast, approximately 56.6% of the wound area in the control group remained unhealed. These results indicate that the combined hydrogel and PTT treatment approach has strong potential in promoting the healing of diabetic wounds. Furthermore, the moist environment provided by the hydrogel and the beneficial activity of QPCCAP both promote wound healing.

[0084] Neovascularization is a crucial marker of the transition from the inflammatory to the proliferative phase of wound healing and is essential for normal wound healing. Blood flow delivers growth factors, nutrients, and oxygen to the wound site; therefore, the amount of neovascularization is key to the effectiveness of the healing process. Furthermore, we used immunofluorescence to measure CD31 expression at the wound site to assess the number of blood vessels in the newly formed skin tissue, confirming the ability of the composite hydrogel to reverse the lack of angiogenesis in diabetic wounds. Figure 15As shown, after 7 days of healing, the CD31 fluorescence intensity in the QP hydrogel group was not significantly different from that in the control group. This indicates that the QP hydrogel has a lower pro-angiogenic potential under high glucose conditions, a trend consistent with in vitro studies. Notably, the CD31 expression level was most significantly upregulated in the QPCCAP+NIR group, exhibiting the strongest CD31 fluorescence staining in the neovascularization area, indicating richer angiogenesis in its granulation tissue. This is attributed to the combined effect of QPCCAP and PTT, preventing vascular endothelial cells from being subjected to excessive oxidative stress and inflammation. Interestingly, the formation of a large number of new blood vessels promotes blood flow and accelerates oxygen transport to the wound site. This amplifies the oxygen-producing capacity of the composite nanozyme cascade to some extent, completely resolving the local hypoxia at the wound site. All results indicate that QPCCAP hydrogel directly promotes the formation of more blood vessels and prevents excessive oxidative stress and inflammation of vascular endothelial cells through the combined action of the hydrogel. Therefore, QPCCAP hydrogel can accelerate the healing of diabetic wounds by scavenging ROS, reducing inflammation, and promoting angiogenesis.

[0085] Unlike most current hydrogel dressings that target only one stage of diabetic wound healing, the QPCCAP developed in this invention has been shown to promote healing during the hemostasis, inflammation, and proliferation stages of wound repair. Thanks to the CeO2 / Cu@Au-Pt nanozyme, the hydrogel can significantly reduce ROS to achieve anti-inflammation. Furthermore, the inherent antibacterial properties of QCSG synergistically enhance the antibacterial efficacy of the hydrogel. This composite hydrogel can significantly promote fibroblast survival and greatly accelerate the healing of diabetic wounds by clearing ROS, enhancing angiogenesis, and reducing inflammation. These results indicate that the QPCCAP hydrogel is easy to prepare and apply, showing great promise for future clinical applications as a diabetic wound dressing.

[0086] In summary, this invention provides a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery. This hydrogel has good tissue adhesion, excellent biocompatibility, hemostasis, ROS scavenging, angiogenesis promotion and antibacterial ability. It can effectively accelerate the healing of diabetic wounds by continuously delivering drugs to the wound area.

[0087] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery, characterized in that, Includes the following steps: Under water bath heating conditions, gallic acid-modified quaternized chitosan and 4-arm polyvinyl alcohol benzaldehyde were added sequentially to deionized water to obtain a hydrogel precursor solution. A precursor hydrogel solution containing nanozyme was obtained by mixing CeO2 / Cu@Au-Pt nanozyme and hydrogel precursor solution. A precursor hydrogel solution containing nanozymes was subjected to vortex treatment to crosslink with Schiff bases to form a QPCCAP multifunctional hydrogel network, thus obtaining a multifunctional hydrogel based on the delivery of CeO2 / Cu@Au-Pt nanozymes.

2. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 1, characterized in that, The temperature of the water bath heating condition is 50~80℃.

3. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 1, characterized in that, The mass ratio of gallic acid-modified quaternized chitosan to 4-arm polyvinyl alcohol benzaldehyde is (18~20):(80~100).

4. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 1, characterized in that, The concentration of CeO2 / Cu@Au-Pt nanozyme in the precursor hydrogel solution containing nanozyme is 100~400 μg / mL.

5. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 1, characterized in that, The ratio of CeO2 / Cu@Au-Pt nanozyme to hydrogel precursor solution is 0.2g~0.8g:2mL.

6. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 1, characterized in that, The preparation method of the CeO2 / Cu@Au-Pt nanozyme is as follows: An aqueous solution of Ce(NO3)3•6H2O was added to ethylene glycol and stirred to carry out the first reaction, yielding CeO2 nanoparticles; Cu(NO3)2•4H2O was added to CeO2 nanoparticles and stirred to carry out the second reaction. Then, precipitation, centrifugation, washing and freeze-drying were performed sequentially to obtain CeO2 / Cu nanoparticles with hollow structure. CeO2 / Cu nanoparticles were dissolved in deionized water and wrapped in tin foil to protect them from light. Then, HAuCl4 solution and H2PtCl6 solution were added. The pH of the reaction solution was adjusted to the set value with NaOH, and then NaBH4 solution was added. After stirring at room temperature, the mixture was centrifuged, washed and freeze-dried to obtain CeO2 / Cu@Au-Pt nanozyme.

7. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 6, characterized in that, The concentration of the Ce(NO3)3•6H2O aqueous solution is 0.5 M, and the volume ratio of the Ce(NO3)3•6H2O aqueous solution to ethylene glycol is 1:

30. The mass ratio of Cu(NO3)2•4H2O and CeO2 nanoparticles is 57.85 mg: 172.11 mg; The ratio of CeO2 / Cu nanoparticles to deionized water is 1 mg: 1 mL; The concentrations of the HAuCl4 solution and the H2PtCl6 solution are 20 mM, and the volume ratio of the HAuCl4 solution to the H2PtCl6 solution is 1:

1. The set pH value is 10; the concentration of the NaBH4 solution is 0.1 M, and the NaBH4 solution includes 1% sodium citrate and 0.5% citric acid.

8. The method for preparing a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to claim 6, characterized in that, The temperature of the first reaction is 180℃ and the reaction time is 12h; the temperature of the second reaction is 180℃ and the reaction time is 4.5h.

9. A multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme prepared by the preparation method of the multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme according to any one of claims 1 to 8.

10. The application of a multifunctional hydrogel based on CeO2 / Cu@Au-Pt nanozyme delivery according to claim 9 in the preparation of wound dressings for treating diabetes.

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