An injectable AuPd-Fe monatomic nanoszyme composite drug source antibacterial hydrogel, a preparation method and application thereof

Abstract

This invention discloses an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, its preparation method, and its applications, belonging to the field of biomedical nanomaterials technology. This invention uses an iron-based single-atom nanozyme with an irregular octahedral carbon framework structure as the core catalytic carrier, and in situ loads ultra-small AuPd alloy nanoparticles onto its surface to obtain the AuPd-Fe single-atom nanozyme. Then, using the AuPd-Fe single-atom nanozyme as the active center and oleanolic acid drug-derived hydrogel as the delivery carrier, the AuPd-Fe single-atom nanozyme is uniformly dispersed in the three-dimensional network of the OA hydrogel through physical embedding and hydrogen bonding, resulting in the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel. The provided antibacterial hydrogel exhibits excellent ocular surface biocompatibility, no significant corneal toxicity, and no systemic organ damage. It also shows significant clinical application potential for blinding ocular infections such as drug-resistant bacterial keratitis and corneal ulcers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to an injectable AuPd-Fe single-atom nanoenzyme composite drug-derived antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogel dressings, with their unique three-dimensional network structure, possess excellent exudate absorption, water retention, and gas exchange capabilities, providing an ideal moist and breathable microenvironment for wound healing and reducing wound complications. Their application in clinical wound care and tissue repair is increasingly widespread. Common antibacterial hydrogels are categorized into topical, dressing, and injectable types. Among these, injectable hydrogels require no surgery, allowing for precise injection and flexible adaptation to wound shapes, solving the adaptation problems of traditional dressings and becoming a research hotspot in the medical field.

[0003] Existing injectable antibacterial hydrogels mostly use high-surface-area, high-porosity hydrogels as carriers to load antibiotics for antibacterial effects. However, in clinical practice, wounds often have high bacterial colonization, which can easily lead to cross-infection and bacterial quorum effects, resulting in antibiotic resistance, reduced treatment efficacy, and even worsening of infection, thus limiting their clinical application.

[0004] To address this challenge, this field proposes using photothermal nanomaterials to mimic nanobioenzymes as an alternative to antibiotics. These nanobioenzymes leverage their enzymatic catalytic activity for highly efficient antibacterial activity, fundamentally circumventing drug resistance. Nanoenzymes combine the properties of nanomaterials with the catalytic function of natural enzymes, achieving multi-target antibacterial activity through the generation of reactive oxygen species. Among these, alloy nanoenzymes, with their synergistic effect of multiple metals and the high atomic utilization rate of single-atom nanoenzymes, are considered the preferred choice.

[0005] However, existing antibacterial hydrogels suffer from several drawbacks in their preparation and application, including limited antibacterial effects, susceptibility to bacterial resistance, difficulty in adapting to the ocular environment, and insufficient wound inflammation relief. These limitations hinder their ability to heal wounds infected with drug-resistant bacteria. For example, patent CN118403005A reports a hydrogel formulation that combines antibacterial, anti-inflammatory, and injectable properties with synergistic effects. However, this type of antibacterial hydrogel lacks the ability to combat multidrug-resistant bacteria and has insufficient anti-inflammatory capacity, limiting its clinical translation and application. Therefore, developing an injectable alloy / single-atom nanoenzyme composite drug-derived antibacterial hydrogel that combines multi-enzyme activity with synergistic antibacterial, injectability, and anti-inflammatory properties, while also being convenient to use and adaptable to the complex ocular environment, has become a pressing challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, its preparation method, and its application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a method for preparing an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, comprising the following steps: Tetraethyl silicate and a surfactant were dissolved in an alkaline solution, and ZIF-8 was added. After stirring, SiO2-coated ZIF-8 was obtained. The SiO2-coated ZIF-8 was pyrolyzed under an inert atmosphere, and the pyrolysis product was etched with an alkaline solution to obtain Zn single-atom nanozymes. The Zn single-atom nanozymes were dispersed in a solvent, and then Fe was added. 2+ The solution was stirred to separate the solid product, which was then reacted in a sulfuric acid solution to obtain Fe single-atom nanozymes; a reducing agent and Au were then added. 3+ Source and Pb 2+ The Fe single-atom nanozyme was dissolved in an alkaline solution, and then methoxy polyethylene glycol thiol (SH-PEG-OMe) was added. After the reaction, ultrafiltration was performed to obtain AuPd ultra-small alloy particles with a particle size of 5-10 nm. The Fe single-atom nanozyme and the AuPd ultra-small alloy particles were dispersed together in water, and AuPd-Fe composite nanoparticles were obtained after the reaction. The AuPd-Fe composite nanoparticles were dispersed in water, and choline-oleanolic acid hydrogel lyophilized powder prepared from choline and oleanolic acid was added. After gelation, the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel was obtained.

[0008] This invention considers AuPd ultra-small alloy nanoparticles as a nanocatalytic material with excellent electron transfer capabilities and synergistic catalytic properties. It possesses superior properties such as synergistic enhancement of multi-enzyme activity, high stability, low susceptibility to induced drug resistance, and good biocompatibility. However, recent studies have focused primarily on its single-component enzyme-mimicking activity, failing to fully utilize its coupling with single-atom nanozymes to achieve d-band electron regulation and synergistic amplification of multi-enzyme activity. Furthermore, Fe-based single-atom nanozymes possess highly dispersed Fe-N4 active sites on their surface. 2+ and Fe 3+ It can undergo reversible valence state transitions, thus possessing excellent peroxidase-like, superoxide dismutase-like, and myeloperoxidase-like catalytic activities. Therefore, this invention employs a d-band regulation and spatial confinement strategy, using a gold-palladium ultra-small alloy as the electronic regulation unit and an Fe single-atom nanozyme as the core catalytic site, loaded into an OA drug-derived hydrogel to form a long-lasting ocular surface delivery system. Through the synergistic electronic effect between AuPd and Fe single atoms, the d-band center is upregulated, significantly enhancing the cascade nanozyme effect and fully utilizing its triple enzyme-like catalytic activity. Under H2O2 enrichment conditions, it rapidly catalyzes the production of reactive oxygen species (·OH) and reactive chlorine (HClO), which are then released into the tear fluid. - with in-situ O2 -Under the condition of continuous supply of reaction substrate, strong oxidizing bactericidal substances are continuously accumulated, and the bactericidal and anti-drug resistance effects are enhanced through the dual effects of multi-enzyme cascade and molecular transcriptional regulation.

[0009] This invention employs a redox method to prepare gold-palladium ultra-small alloy nanozymes, ensuring that the obtained gold-palladium ultra-small alloy nanozymes have an ultra-small size of 5-10 nm and uniform size. Furthermore, after preparing the gold-palladium ultra-small alloy nanozymes by the redox method, defective iron single-atom nanozymes are introduced as spatial confinement carriers, so that the gold-palladium ultra-small alloy nanozymes prepared by the redox method can be uniformly distributed and loaded on the surface of divalent iron single-atom nanozymes to regulate the d-band centers of iron single atoms.

[0010] Preferably, the ZIF-8 is prepared using zinc salt and 2-methylimidazole, wherein the molar ratio of zinc salt to 2-methylimidazole is 1:4.

[0011] Preferably, the surfactant is hexadecyltrimethylammonium bromide, the alkaline solution is NaOH solution, the molar ratio of hexadecyltrimethylammonium bromide to NaOH is 1:15, the tetraethyl silicate is dissolved in methanol before addition, and the ratio of hexadecyltrimethylammonium bromide, tetraethyl silicate and ZIF-8 is 150mg:1.2mL:100mg.

[0012] Preferably, the pyrolysis temperature is 800℃, the time is 2h, and the heating rate is 10℃ / min.

[0013] Preferably, the alkaline solution used for etching the pyrolysis products is a 3-4 mol / L NaOH solution.

[0014] Preferably, the Zn single-atom nanozyme and the Fe-containing 2+ Fe in solution 2+ The mass ratio is 30:24~30.

[0015] Preferably, the concentration of the sulfuric acid solution is 4 mol / L.

[0016] Optionally, the reducing agent is tetrahydroxymethylphosphoric acid, and the Au 3+ The source is HAuCl4, and the Pb 2+ The source is Na2PdCl4.

[0017] When tetrahydroxymethylphosphoric acid is used as a reducing agent, the reduction temperature is 30℃ and the pH value of the system is controlled at 12.1.

[0018] Preferably, the Au 3+ Source Au 3+ With the Pb 2+ Pb in source 2+ The molar ratio is 1:1~2; the molar amount of SH-PEG-OMe and Au3+ Pb 2+ The ratio of total molar amounts is 1:5~10.

[0019] To obtain pure AuPd ultrafine alloy particles for further application or composite processing, the reaction solution obtained after the reaction was subjected to ultrafiltration. An ultrafiltration tube with a molecular weight cutoff of 100kDa to 1000kDa was used, and the centrifugation rate was 4000 r / min for 10 min. Subsequently, to avoid residual solvent on the surface of the AuPd ultrafine alloy particles potentially affecting subsequent processing or application performance, they were dried to remove excess solvent. Considering the impact of solvent residue and nanoparticle agglomeration on product performance, this invention preferably uses freeze-drying as the drying method, with a drying temperature of -50℃ to -55℃ and a drying time of 12 h.

[0020] Preferably, the mass ratio of the Fe single-atom nanozyme to the AuPd ultra-small alloy particles is 3:1.

[0021] Preferably, the mass ratio of choline to oleanolic acid is 0.145:0.456.

[0022] Preferably, the mass ratio of the AuPd-Fe composite nanoparticles to the choline-oleanolic acid hydrogel lyophilized powder is 1:100.

[0023] The second technical solution of the present invention provides an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel prepared according to the above-mentioned preparation method of injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel.

[0024] The third technical solution of the present invention provides an application of the above-mentioned injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel in the preparation of antibacterial hydrogels.

[0025] The antibacterial hydrogel can resist one or more of the following pathogenic bacteria: Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa.

[0026] The beneficial technical effects of the present invention are as follows: This invention uses iron-based single-atom nanozymes with an irregular octahedral carbon framework as the core catalytic carrier. These nanozymes are introduced during the room-temperature ion exchange process to prepare single-atom nanozymes. Through room-temperature electron coupling, ultra-small AuPd alloy nanoparticles are in situ loaded onto the surface of the iron-based single-atom nanozymes, resulting in AuPd-Fe single-atom nanozymes. Then, using the composite nanozyme particles as the active center and oleanolic acid-derived hydrogel as the delivery carrier, the AuPd-Fe single-atom nanozymes are uniformly dispersed within the three-dimensional network of the OA hydrogel through physical encapsulation and hydrogen bonding, yielding an injectable AuPd-Fe single-atom nanozyme composite antibacterial hydrogel.

[0027] This invention prepares nitrogen-doped carbon-supported Fe single-atom nanozymes via a room-temperature exchange method; subsequently, ultra-small and uniform AuPd bimetallic alloy nanozymes are prepared using a low-temperature liquid-phase reduction method. AuPd alloy sites are successfully combined with Fe single-atom active centers through electronic coupling and in-situ loading. Finally, the prepared AuPd-Fe composite nanozymes are composited with oleanolic acid-choline hydrogel using a room-temperature dissolution-cooling molding method, resulting in a locally administered nanozyme composite hydrogel with good ocular surface compatibility, long-lasting retention, and high-efficiency antibacterial activity. The preparation method of this invention is mild and controllable, requires no high temperature or high pressure, and has good reproducibility, making it very suitable for further translation and clinical application.

[0028] The injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel provided by this invention fully utilizes the triple enzyme activity (SOD-like, POD-like, MPO-like) and electronic synergistic regulation effect of AuPd-Fe single-atom nanozymes. Through cascade catalytic reactions, it generates reactive oxygen species and reactive chlorine in the infection microenvironment for efficient sterilization. At the same time, the OA drug-derived hydrogel prolongs the residence time on the ocular surface, downregulates inflammatory pathways, and accelerates corneal epithelial repair. The local ocular delivery system reduces the ocular irritation of the nanozyme and improves drug bioavailability.

[0029] This invention relates to an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, which simultaneously combines the electronic regulation effect of AuPd alloy, the high catalytic activity of Fe single atoms, and the long-term delivery and anti-inflammatory repair functions of OA hydrogel. Through multi-enzyme synergistic chemokinetics, it continuously generates strong oxidizing bactericidal substances, significantly improving the antibacterial efficiency of a single nanozyme. Furthermore, employing a targeted infection microenvironment activation strategy, it triggers catalytic reactions in situ at acidic, H2O2-rich corneal infection foci without external light / heat / electric stimulation, achieving precise antibacterial action. An injectable / applied oleanolic acid drug-derived hydrogel reduces ocular toxicity and prolongs ocular surface retention time to over 2 hours, significantly improving the treatment effect of deep corneal infections. A multidrug-resistant bacterial keratitis model demonstrates that this hydrogel system has extremely strong antibacterial and corneal repair efficacy against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa, especially exhibiting rapid and thorough killing effects against clinically highly drug-resistant ocular pathogens.

[0030] The injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel prepared by this invention has excellent ocular surface biocompatibility, no obvious corneal toxicity, and no systemic organ damage. Compared with traditional antibiotic gels such as levofloxacin commonly used in clinical practice, it has higher antibacterial effect and lower risk of drug resistance induction. At the same time, it has significant clinical application prospects for blinding ocular infections such as drug-resistant bacterial keratitis and corneal ulcers. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The flowchart shows the preparation process of the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel of Example 1.

[0033] Figure 2 The images show transmission electron microscope (TEM) images of Fe-SACs in Comparative Example 3 and AuPd-Fe SACs in Comparative Example 5; the left image is a TEM image of Fe-SACs in Comparative Example 3, and the right image is a TEM image of AuPd-Fe SACs in Comparative Example 5.

[0034] Figure 3 Micrographs and elemental distribution diagrams of AuPd-Fe SACs in Comparative Example 5.

[0035] Figure 4The images show photographs of the injectable AuPd-Fe single-atom nanozyme composite antibacterial hydrogel of Example 1 and the OA drug-derived hydrogel of Comparative Example 1; wherein, the left image is a photograph of the OA drug-derived hydrogel of Comparative Example 1, and the right image is a photograph of the injectable AuPd-Fe single-atom nanozyme composite antibacterial hydrogel of Example 1.

[0036] Figure 5 X-ray photoelectron spectroscopy (XPS) spectra of the materials prepared in Comparative Examples 2, 3, and 5.

[0037] Figure 6 The X-ray photoelectron spectroscopy (XPS) spectra of the materials prepared in Comparative Examples 3 and 5 are shown in detail.

[0038] Figure 7 The images show the K-edge XANES and EXAFS spectra of Fe in AuPd-Fe SACs, Fe Foil, and Fe2O3; the left image shows the K-edge XANES spectra of Fe in AuPd-Fe SACs, Fe Foil, and Fe2O3, and the right image shows the K-edge EXAFS spectra of Fe in AuPd-Fe SACs, Fe Foil, and Fe2O3.

[0039] Figure 8 The images show the Fe element K-space and R-space spectra of AuPd-Fe SACs; the left image shows the Fe element K-space spectra of AuPd-Fe SACs, and the right image shows the Fe element R-space spectra of AuPd-Fe SACs.

[0040] Figure 9 The wavelet transform spectra of Fe element in AuPd-Fe SACs, Fe Foil, and Fe2O3 are shown below; where A is the wavelet transform spectra of Fe element in AuPd-FeSACs, B is the wavelet transform spectra of Fe element in Fe Foil, and C is the wavelet transform spectra of Fe element in Fe2O3.

[0041] Figure 10 The images show the antibacterial activity of MRSA and MDR-PA plates on samples prepared in Example 1, Comparative Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5.

[0042] Figure 11 The figures show the bacterial counts of MRSA and MDR-PA in the samples prepared in Example 1, Comparative Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5; the left figure shows the bacterial count of MRSA and the right figure shows the bacterial count of MDR-PA.

[0043] Figure 12The images show pathological sections of corneal tissue from the samples prepared in Example 1, Comparative Example 1, and Comparative Example 5, as well as corneal tissue from SD rats treated with levofloxacin gel.

[0044] Figure 13 Histopathological sections of the brain, heart, liver, spleen, lung, and kidney of the samples prepared in Example 1, Comparative Example 1, and Comparative Example 5, as well as SD rats treated with levofloxacin gel.

[0045] Figure 14 Representative slit-lamp micrographs of the samples prepared in Example 1, Comparative Example 1, and Comparative Example 5, and SD rats with bacterial corneal ulcers treated with levofloxacin gel, at different observation time points during the treatment period. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0047] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0048] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0050] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0051] In the following embodiments of the present invention, the ultrasonic power of the ultrasonic treatment is 300 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 5 min, which mainly promotes dispersion and dissolution.

[0052] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0053] The weight-average molecular weight (Mw) of SH-PEG-OMe used in the embodiments and comparative examples of this invention is 5000.

[0054] Example 1 This embodiment provides an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, which is obtained through the following preparation steps: Step 1: Preparation of Fe single-atom nanozymes 1.1 Preparation of ZIF-8 vector Accurately weigh 2.38 g Zn(NO3)2·6H2O and 2.77 g 2-methylimidazole, dissolve them separately in 100 mL methanol, mix and stir at room temperature for 2 h. After the reaction is complete, centrifuge at 6000 rpm for 5 min, wash three times with methanol to obtain ZIF-8.

[0055] 1.2 Preparation of SiO2-coated ZIF-8 (ZIF-8@SiO2) 100 mg ZIF-8 was dispersed in 20 mL of deionized water, and 150 mg of cetyltrimethylammonium bromide and 57.6 mg of NaOH were added sequentially. The mixture was stirred until homogeneous to obtain a homogeneous solution. 1.2 mL of tetraethyl silicate (dissolved in 6 mL of methanol) was slowly added dropwise to the homogeneous solution, and the mixture was stirred for 1 h to obtain ZIF-8@SiO2. After centrifugation and washing, the solution was dried at 60 °C for 12 h.

[0056] 1.3 Preparation of Zn single-atom nanozymes 5.0 g of ZIF-8@SiO2 was pyrolyzed in a nitrogen atmosphere at a temperature increased to 800 °C at a rate of 10 °C / min for 2 h. The resulting black powder was etched with 60 mL of 4 mol / L NaOH solution for 12 h, centrifuged at 6000 rpm for 5 min, washed three times with ultrapure water, and dried at 60 °C for 12 h to obtain Zn single-atom nanozymes.

[0057] 1.4 Preparation of Fe single-atom nanozymes by ion exchange FeCl2·4H2O was dissolved in anhydrous ethanol to prepare Fe 2+ A 10 mg / mL solution was prepared. 30 mg of Zn single-atom nanozyme was dispersed in 15 mL of anhydrous ethanol, and 3 mL of FeCl2 solution was added. The mixture was stirred at room temperature for 12 h. After centrifugation and washing with anhydrous ethanol, the mixture was placed in 30 mL of 4 mol / L H2SO4 solution and reacted for 6 h. The mixture was then centrifuged at 6000 rpm for 5 min and washed three times with ultrapure water to obtain the Fe single-atom nanozyme.

[0058] Step 2: Preparation of AuPd ultra-small alloy nanoenzymes 4 μL of tetrahydroxymethylphosphoric acid and 667 μL of 2 mol / L NaOH solution were added dropwise to 100 mL of ultrapure water, and the mixture was stirred in an ice bath for 5 min. Then, 4.84 mg of HAuCl4 and 12.58 mg of Na2PdCl4 were added, and the mixture was stirred thoroughly for 10 min until the pH of the system reached 12.1. Next, 11 mg of SH-PEG-OMe was added, and the reaction was carried out at 30 °C for 3 h. The mixture was then centrifuged at 4000 rpm for 10 min using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. Finally, the mixture was freeze-dried at -50 °C to -55 °C for 12 h to obtain 5 nm AuPd ultraalloy nanoparticles.

[0059] Step 3: Preparation of AuPd-Fe single-atom nanozymes 30 mg of Fe single-atom nanozyme was dissolved in 10 mL of ultrapure water and thoroughly dispersed by stirring. Then, 10 mg of AuPd ultra-small alloy nanozyme was added, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 6000 rpm for 5 min, and the precipitate was freeze-dried to obtain AuPd-Fe single-atom nanozyme.

[0060] Step 4: Preparation of OA drug-derived hydrogel lyophilized powder Weigh 0.33 g of choline aqueous solution (44wt% in water), add 20 mL of ultrapure water, mix well, add 0.456 g of oleanolic acid, react at 60℃ for 24 h, and freeze dry to obtain OA gel lyophilized powder.

[0061] Step 5: Crosslinking to construct AuPd-Fe@OA nanozyme composite antibacterial hydrogel 5.1 Preparation of composite dispersion Weigh 2 mg of AuPd-Fe single-atom nanozyme, dissolve it in 3.8 mL of ultrapure water, and disperse it evenly by ultrasonication to obtain AuPd-Fe single-atom nanozyme dispersion.

[0062] 5.2 Preparation of hydrogel precursor solution Add 200 mg of OA gel lyophilized powder to AuPd-Fe single-atom nanozyme dispersion, and sonicate in a 50°C water bath until completely dissolved to obtain hydrogel precursor solution.

[0063] 5.3 Preparation of injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel The precursor solution was removed and placed at room temperature to cool and solidify naturally, resulting in an injectable AuPd-Fe single-atom nanoenzyme composite drug-derived antibacterial hydrogel, denoted as AuPd-Fe@OA Gel.

[0064] Example 2 This embodiment provides an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, which is obtained through the following preparation steps: Step 1: Preparation of Fe single-atom nanozymes 1.1 Preparation of ZIF-8 vector Accurately weigh 2.38 g Zn(NO3)2·6H2O and 2.77 g 2-methylimidazole, dissolve them separately in 100 mL methanol, mix and stir at room temperature for 2 h. After the reaction is complete, centrifuge at 6000 rpm for 5 min, wash three times with methanol to obtain ZIF-8.

[0065] 1.2 Preparation of SiO2-coated ZIF-8 (ZIF-8@SiO2) ZIF-8 was dispersed in 20 mL of deionized water, and 150 mg of cetyltrimethylammonium bromide and 57.6 mg of NaOH were added sequentially. The mixture was stirred until homogeneous to obtain a homogeneous solution. 1.0 mL of tetraethyl silicate (dissolved in 6 mL of methanol) was slowly added dropwise to the mixture, and the mixture was stirred for 1 h to obtain ZIF-8@SiO2. After centrifugation and washing, the solution was dried at 60 °C for 12 h.

[0066] 1.3 Preparation of Zn single-atom nanozymes 2.0 g of ZIF-8@SiO2 was pyrolyzed in a nitrogen atmosphere at a temperature increased to 800 °C at a rate of 10 °C / min for 2 h. The resulting black powder was etched with 60 mL of 3 mol / L NaOH solution for 12 h, centrifuged at 6000 rpm for 5 min, washed three times with ultrapure water, and dried at 60 °C for 12 h to obtain Zn single-atom nanozymes.

[0067] 1.4 Preparation of Fe single-atom nanozymes by ion exchange FeCl2·4H2O was dissolved in anhydrous ethanol to prepare Fe 2+ A solution with a concentration of 8 mg / mL was prepared. 30 mg of Zn single-atom nanozyme was dispersed in 15 mL of anhydrous ethanol, and 3 mL of FeCl2 solution was added. The mixture was stirred at room temperature for 12 h. After centrifugation and washing with anhydrous ethanol, the mixture was placed in 30 mL of 4 mol / L H2SO4 solution and reacted for 6 h. The mixture was then centrifuged at 6000 rpm for 5 min and washed three times with ultrapure water to obtain Fe single-atom nanozyme.

[0068] Step 2: Preparation of AuPd ultra-small alloy nanoenzymes Same as Example 1.

[0069] Step 3: Preparation of AuPd-Fe single-atom nanozymes 30 mg of Fe single-atom nanozyme was dissolved in 10 mL of ultrapure water and thoroughly dispersed by stirring. Then, 10 mg of AuPd ultra-small alloy nanozyme was added, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 6000 rpm for 5 min, and the precipitate was freeze-dried to obtain AuPd-Fe single-atom nanozyme.

[0070] Step 4: Preparation of OA drug-derived lyophilized powder Weigh 0.33 g of choline aqueous solution (44wt% in water), add 20 mL of ultrapure water, mix well, add 0.456 g of oleanolic acid, react at 60℃ for 24 h, and freeze dry to obtain OA gel lyophilized powder.

[0071] Step 5: Crosslinking to construct AuPd-Fe@OA nanozyme composite antibacterial hydrogel 5.1 Preparation of composite dispersion Weigh 2 mg of AuPd-Fe single-atom nanozyme, dissolve it in 3.8 mL of ultrapure water, and disperse it evenly by ultrasonication to obtain AuPd-Fe single-atom nanozyme dispersion.

[0072] 5.2 Preparation of hydrogel precursor solution Add 200 mg of OA gel lyophilized powder to AuPd-Fe single-atom nanozyme dispersion, and sonicate in a 50°C water bath until completely dissolved to obtain hydrogel precursor solution.

[0073] 5.3 Preparation of injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel The precursor solution was removed and placed at room temperature to cool and solidify naturally, resulting in an injectable AuPd-Fe single-atom nanoenzyme composite drug-derived antibacterial hydrogel.

[0074] Example 3 This embodiment provides an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, which is obtained through the following preparation steps: Step 1: Preparation of Fe single-atom nanozymes 1.1 Preparation of ZIF-8 vector Accurately weigh 2.38 g Zn(NO3)2·6H2O and 2.77 g 2-methylimidazole, dissolve them separately in 100 mL methanol, mix and stir at room temperature for 2 h. After the reaction is complete, centrifuge at 6000 rpm for 5 min, wash three times with methanol to obtain ZIF-8.

[0075] 1.2 Preparation of SiO2-coated ZIF-8 (ZIF-8@SiO2) ZIF-8 was dispersed in 20 mL of deionized water, and 150 mg of cetyltrimethylammonium bromide and 57.6 mg of NaOH were added sequentially. The mixture was stirred until homogeneous to obtain a homogeneous solution. 1.0 mL of tetraethyl silicate (dissolved in 6 mL of methanol) was slowly added dropwise to the mixture, and the mixture was stirred for 1 h to obtain ZIF-8@SiO2. After centrifugation and washing, the solution was dried at 60 °C for 12 h.

[0076] 1.3 Preparation of Zn single-atom nanozymes 2.0 g of ZIF-8@SiO2 was pyrolyzed in a nitrogen atmosphere at a temperature increased to 800 °C at a rate of 10 °C / min for 2 h. The resulting black powder was etched with 60 mL of 3 mol / L NaOH solution for 12 h, centrifuged at 6000 rpm for 5 min, washed three times with ultrapure water, and dried at 60 °C for 12 h to obtain Zn single-atom nanozymes.

[0077] 1.4 Preparation of Fe single-atom nanozymes by ion exchange FeCl2·4H2O was dissolved in anhydrous ethanol to prepare Fe 2+ A solution with a concentration of 8 mg / mL was prepared. 30 mg of Zn single-atom nanozyme was dispersed in 15 mL of anhydrous ethanol, and 3 mL of FeCl2 solution was added. The mixture was stirred at room temperature for 12 h. After centrifugation and washing with anhydrous ethanol, the mixture was placed in 30 mL of 4 mol / L H2SO4 solution and reacted for 6 h. The mixture was then centrifuged at 6000 rpm for 5 min and washed three times with ultrapure water to obtain Fe single-atom nanozyme.

[0078] Step 2: Preparation of AuPd ultra-small alloy nanoenzymes Same as Example 1.

[0079] Step 3: Preparation of AuPd-Fe single-atom nanozymes 30 mg of Fe single-atom nanozyme was dissolved in 10 mL of ultrapure water and thoroughly dispersed by stirring. Then, 10 mg of AuPd ultra-small alloy nanozyme was added, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 6000 rpm for 5 min, and the precipitate was freeze-dried to obtain AuPd-Fe single-atom nanozyme.

[0080] Step 4: Preparation of OA drug-derived lyophilized powder Weigh 0.33 g of choline aqueous solution (44wt% in water), add 20 mL of ultrapure water, mix well, add 0.456 g of oleanolic acid, react at 60℃ for 24 h, and freeze dry to obtain OA gel lyophilized powder.

[0081] Step 5: Crosslinking to construct AuPd-Fe@OA nanozyme composite antibacterial hydrogel 5.1 Preparation of composite dispersion Weigh 2 mg of AuPd-Fe single-atom nanozyme, dissolve it in 3.8 mL of ultrapure water, and disperse it evenly by ultrasonication to obtain AuPd-Fe single-atom nanozyme dispersion.

[0082] 5.2 Preparation of hydrogel precursor solution Add 200 mg of OA gel lyophilized powder to AuPd-Fe single-atom nanozyme dispersion, and sonicate in a 50°C water bath until completely dissolved to obtain hydrogel precursor solution.

[0083] 5.3 Preparation of injectable AuPd-Fe single-atom nanozyme drug-derived antibacterial hydrogel The precursor solution was removed and placed at room temperature to cool and solidify naturally, resulting in an injectable AuPd-Fe single-atom nanoenzyme composite drug-derived antibacterial hydrogel.

[0084] Comparative Example 1 The only difference between this comparative example and Example 1 is that: This comparative example does not support AuPd-Fe single-atom nanozyme material. That is, the hydrogel material provided in this comparative example is OA drug-derived hydrogel. The specific preparation steps are as follows: weigh 200 mg of OA gel lyophilized powder, dissolve it in 3.8 mL of ultrapure water, ultrasonically stir in a 50℃ water bath until completely dissolved, and let it stand at room temperature to obtain OA drug-derived hydrogel, denoted as OA Gel.

[0085] Comparative Example 2 The only difference between this comparative example and Example 1 is that: In this comparative example, the Zn single-atom nanozymes do not undergo Fe exchange; that is, the material provided in this comparative example is Zn single-atom nanozyme particles, which are denoted as Zn-SACs.

[0086] Comparative Example 3 The only difference between this comparative example and Example 1 is that: In this comparative example, AuPd ultra-small alloy particles and hydrogel materials are not combined; that is, the material provided in this comparative example is a single Fe single-atom nanoenzyme particle, denoted as Fe-SACs.

[0087] Comparative Example 4 The only difference between this comparative example and Example 1 is that: In this comparative example, neither Fe single-atom nanozymes nor hydrogel materials are composited; that is, the material provided in this comparative example is a nanocomposite particle, which is denoted as AuPd NPs.

[0088] Comparative Example 5 The only difference between this comparative example and Example 1 is that: In this comparative example, no composite hydrogel material is used; that is, the material provided in this comparative example is a composite alloy / single-atom nanoparticle, denoted as AuPd-Fe SACs.

[0089] Through analysis, this invention has found that Examples 1 to 3 have similar structural features. Those skilled in the art should know that structure determines properties. Similar structures indicate that Examples 1 to 3 have similar performance characteristics. Therefore, to avoid redundancy, this invention will take Example 1 as an example to analyze and explain its structure and performance.

[0090] I. Preparation Process Example 1 of this invention describes the preparation of an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel. Fe-SACs single-atom nanozymes were prepared using a room-temperature exchange method. First, ZIF-8 was synthesized at room temperature, then protected with SiO2, followed by high-temperature calcination to obtain Zn-SACs. Fe... 2+ Zn ions in Zn-SACs were exchanged to obtain Fe-SACs. Then, the synthesized AuPd ultra-small alloy nanozyme was loaded onto the surface of Fe-SACs to obtain AuPd-Fe SACs composite nanozyme. Choline aqueous solution and oleanolic acid were reacted under heating conditions to prepare OA drug-derived hydrogel lyophilized powder, which was dissolved in water to obtain OA hydrogel base solution. The AuPd-Fe SACs composite nanozyme was dispersed in the OA hydrogel base solution, uniformly mixed, and self-crosslinked at room temperature to obtain the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel. A schematic diagram of the preparation process is shown below. Figure 1 As shown.

[0091] II. Transmission Electron Microscopy Testing The present invention also conducted transmission electron microscopy tests on the Fe-SACs of Comparative Example 3 and the AuPd-Fe SACs of Comparative Example 5, and the test results are as follows: Figure 2 As shown.

[0092] Depend on Figure 2 The test results clearly show that Fe-SACs exhibit a typical irregular octahedral carbon support morphology with a relatively uniform size distribution. No obvious nanoparticles or aggregates were observed on the carbon support surface, indicating that Fe elements are anchored on the carbon substrate in a highly dispersed single-atom form, consistent with the design expectation of single-atom catalysts. Compared with Fe-SACs, the overall morphology of AuPd-Fe SACs did not change significantly after the introduction of AuPd ultra-small alloy nanozymes, maintaining the irregular octahedral structure of the carbon support. Simultaneously, the support surface remained highly uniform, without obvious metal agglomeration, indicating that both AuPd and Fe single-atom sites can achieve good dispersion on the carbon support.

[0093] III. Element Distribution Analysis The present invention performed elemental distribution analysis on the material provided in Comparative Example 5, and the test analysis results are as follows: Figure 3 As shown, AuPd NPs are uniformly modified on Fe-SACs single-atom nanozymes. The corresponding elemental distribution results show that C, N, and O elements exhibit a uniform and continuous distribution throughout the region, consistent with the elemental composition of the support; while Fe, Au, and Pd elements appear as discrete point-like signals, and their distribution areas highly overlap. This clearly confirms the successful synthesis of Fe single-atom nanozymes and the successful synthesis and uniform loading of AuPd NPs, without obvious elemental segregation or aggregation. This demonstrates the successful preparation of AuPd-Fe SACs with a well-defined structure.

[0094] IV. Morphological Test In this invention, the macroscopic morphology of the injectable AuPd-Fe single-atom nanozyme drug-derived antibacterial hydrogel of Example 1 and the OA drug-derived hydrogel of Comparative Example 1 were photographed and recorded, and the recording results are as follows: Figure 4 As shown in the figure, the right image is a photograph of the injectable AuPd-Fe single-atom nanozyme drug-derived antibacterial hydrogel of Example 1, and the left image is a photograph of the OA drug-derived hydrogel of Comparative Example 1.

[0095] Depend on Figure 4 The test results show that the hydrogel materials obtained in Example 1 and Comparative Example 1 have similar macroscopic morphologies.

[0096] V. XPS Analysis The present invention performed X-ray photoelectron spectroscopy analysis on the materials provided in Comparative Examples 2, 3, and 5, respectively, and the test and analysis results are as follows: Figure 5As shown, the results indicate that Zn-SACs, Fe-SACs, and AuPd-Fe SACs all exhibited characteristic peaks for C 1s, N 1s, and O 1s at 284.8 eV, 400 eV, and 532 eV, respectively. This is completely consistent with the elemental composition of the N-doped carbon material, indicating that the support maintained a stable chemical state in each catalyst. Zn-SACs detected characteristic peaks for Zn 2p at 1021.7 eV and 1044.7 eV, confirming the successful introduction of Zn and its dispersion in single-atom form within the support. Fe-SACs, after room-temperature exchange with divalent iron, showed characteristic signals for Fe 2p at 711.6 eV and 724.6 eV, indicating that Fe was uniformly loaded onto the support in single-atom form without forming obvious metal agglomerates or crystalline phases. In addition to the characteristic peaks of Fe 2p, C 1s, N 1s and O 1s, AuPd-Fe SACs also detected characteristic signals of Au 4f and Pd 3d at approximately 83.9 eV and 332.3 eV, respectively, directly proving that AuPd bimetal and Fe single atoms were successfully co-loaded on the same support.

[0097] The present invention performed fine X-ray photoelectron spectroscopy analysis on the materials provided in Comparative Example 3 and Comparative Example 5, respectively, and the test and analysis results are as follows: Figure 6 As shown, based on the results, the Fe 2p XPS peak division and d-band center theory indicate that AuPd has a regulatory effect on the d-band of the Fe single-atom electronic structure. In Fe-SACs, the valence state distribution of Fe is as follows: Fe 3+ Accounting for 61.6%, Fe 2+ It accounts for 38.4%. After introducing AuPd NPs, the Fe in AuPd-Fe SACs... 3+ The proportion increased significantly to 78.59%, with a higher average valence state of Fe ions and a decrease in the number of d-orbital electrons. This indicates that AuPd shifts the d-band center of Fe sites upwards through electron transfer. According to the D-band center theory, a moderate upward shift of the d-band center can enhance the adsorption of reaction intermediates and lower the energy barrier. In this system, the electronic coupling effect of AuPd shifts the d-band center of Fe sites upwards, optimizing the adsorption and catalytic ability for H2O2. High-valence Fe... 3+ The increased proportion is beneficial for the efficient catalysis of H2O2 decomposition as an electron acceptor, and enhances the activity of peroxidase-like enzymes.

[0098] VI. Synchrotron Radiation Absorption Analysis To verify the valence state and local coordination structure of the Fe single atom in Comparative Example 5, this invention employs X-ray absorption near-edge structure (XANES), X-ray absorption near-edge structure (EXAFS), and wavelet transform (WT) for analysis, and the analysis results are as follows: Figure 7 , Figure 8 and Figure 9As shown in the Fe K-edge XANES spectrum, the absorption edge of AuPd-Fe SACs is significantly higher than that of Fe foil, and close to but slightly lower than that of Fe2O3, indicating that the average valence state of Fe species in the sample is between that of Fe. 0 with Fe 3+ The presence of positive valence states indicates that Fe atoms undergo electron transfer within a support or coordination environment, forming partially oxidized Fe^δ+ species. Furthermore, the lower intensity of the white line compared to Fe2O3 further suggests that Fe is not in a completely oxidized state. 3+ It is not in a highly symmetric oxygen coordination environment, but rather in a dispersed state with low coordination and regulated electronic structure.

[0099] like Figure 7 As shown, Fe Foil exhibits a distinct Fe–Fe metallic bond characteristic peak at approximately 2.2 Å, while Fe₂O₃ shows a characteristic peak of the Fe–O bond at approximately 1.5 Å. AuPd-Fe SACs, however, does not show distinct characteristic peaks at either of these positions; instead, a new main peak appears at approximately 1.6 Å, which closely matches the bond length range of the Fe–N bond (1.6–2.0 Å). Combined with the k-space fitting results... Figure 8 As shown, the EXAFS curves of AuPd-Fe SACs highly overlap with the fitted curves, further verifying that Fe atoms are mainly coordinated with N atoms, forming Fe–N. x Single-atom sites, rather than Fe–Fe metallic bonds or Fe–O bonds.

[0100] Wavelet transform (WT) results are as follows Figure 9 As shown, the differences in local structure among the three samples are clearly illustrated. The WT contour plot of Fe Foil shows a strong Fe–Fe signal at approximately 2.2 Å, while Fe2O3 shows a Fe–O signal at approximately 1.5 Å. In contrast, AuPd-Fe SACs only exhibit a characteristic Fe–N signal at approximately 1.6 Å, with no Fe–Fe or Fe–O signals detected. This result directly demonstrates that Fe atoms are highly dispersed in AuPd-Fe SACs in single-atom form and are mainly coordinated with N atoms, forming a stable Fe–N group. x Single-atom active center.

[0101] The comprehensive analysis of the results indicates that the Fe atoms in Comparative Example 5 exist in monatomic form and mainly coordinate with N atoms to form Fe–N. x The site has a valence state between 0 and +3 and has not formed an Fe–Fe metallic bond or an Fe–O bond.

[0102] VII. Antibacterial Performance Test The antibacterial properties of the samples prepared in Example 1, Comparative Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 were tested according to the present invention.

[0103] 1) Preparation of primary seed culture: 1 mL of fresh LB nutrient broth (Shanghai Sangon Biotech) was added to the lyophilized powders of methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) and multidrug-resistant Pseudomonas aeruginosa (MDR-PA, D25207) purchased from the American Type Culture Collection (ATCC) to dissolve the bacterial strains. The entire bacterial solution was transferred to an Erlenmeyer flask containing 20 mL of fresh LB broth and placed in a constant temperature shaker at 37°C for overnight incubation to activate the bacteria and obtain the primary seed culture.

[0104] 2) Preparation of secondary seed culture: Take 10 μL from each of the primary seed cultures and transfer them to 10 mL of fresh LB broth. Incubate in a constant temperature shaker at 37℃ for 8 h. Measure the absorbance (OD) of the bacterial solution at 600 nm. 600 ) Determine bacterial concentration, OD 600 The range is between 0.6 and 0.8, resulting in a secondary seed solution in the logarithmic growth stage.

[0105] 3) Determination of plate counting experiment: Add 10 μL of 10 to each experimental group. 6 CFU / mL bacterial culture; place in a shaker at 37 ℃ and incubate for 3 h; then take the reaction solution, dilute it appropriately, spread it on a solid plate, and incubate in an incubator for 18 h.

[0106] The antibacterial activity of AuPd-Fe@OA Gel against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDR-PA) was evaluated using a plate count method. Results are as follows: Figure 10 The results showed that both the control group and the OA hydrogel group exhibited a large number of bacterial colonies, indicating weak antibacterial effects. Fe-SACs, AuPd NPs, and AuPd-Fe SACs all demonstrated some antibacterial activity, with AuPd-Fe SACs showing significantly better antibacterial effects than the single-component materials. The AuPd-Fe@OA Gel group showed almost no visible colonies on its plates, exhibiting near-complete inhibition against both MRSA and MDR-PA.

[0107] Quantitative statistical results as follows Figure 11As shown, in the MRSA model, the colony count in the control group was approximately 1000 CFU, the OA hydrogel group was approximately 750 CFU, while the AuPd-Fe@OA Gel group had a colony count reduced to 0, with an antibacterial rate greater than 99%. In the MDR-PA model, the colony count in the control group was approximately 750 CFU, the OA hydrogel group was approximately 500 CFU, and the AuPd-Fe@OA Gel group also achieved a near-complete antibacterial effect.

[0108] The above antibacterial experiments effectively demonstrate the high antibacterial activity of the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel obtained in Example 1, making it a candidate for antibacterial materials.

[0109] VIII. Corneal Toxicity Evaluation and Analysis To verify the biocompatibility of the alloy / single-atom nanoenzyme composite material of the present invention, the AuPd-Fe@OA Gel sample prepared in Example 1 was used as an example, and the following tests were performed on it: First, Sprague-Dawley (SD) rats were acclimatized for one week. They were then divided into six groups of three rats each: a control group, a normal saline (NS) group, an OA gel group (comparative example 1), an AuPd-Fe SACs group (comparative example 5), a levofloxacin gel (LVFX Gel) group, and an AuPd-Fe@OA Gel group. Normal corneal drug administration was performed for 18 days, twice daily at 0.2 mL each time. After the experiment, corneal, brain, heart, liver, spleen, lung, and kidney tissues from the SD rats were harvested for H&E staining and sectioning to observe the morphology of each tissue.

[0110] Figure 12 H&E stained sections of the cornea of ​​SD rats after drug administration in each group and Figure 13 Images of H&E-stained sections of brain, heart, liver, spleen, lung, and kidney tissues from SD rats in each group after drug administration. Figure 12 It was found that the corneal structure in the Control group was intact, with tightly and orderly arranged epithelial cells and dense collagen fibers in the stromal layer, and no obvious inflammatory infiltration or tissue structure destruction was observed. The corneal morphology of the AuPd-Fe@OAGel group was closest to that of the Control group, with neatly arranged epithelial cells, clear stromal structure, and no obvious toxicity to normal corneal tissue. Figure 13 It was found that the tissue structure of the brain, heart, liver, spleen, lungs, kidneys, and other major organs of rats in each treatment group was not significantly different from that in the normal group. No obvious pathological changes such as cell degeneration, necrosis, inflammatory infiltration, or tissue structure destruction were observed, further confirming that each treatment had no significant toxic effects on the major organs of rats at the experimental doses. This indicates that the alloy / single-atom nanoenzyme composite material prepared in this invention has good biosafety.

[0111] IX. Analysis of In vivo therapeutic effects To verify the good in vivo therapeutic effect of the alloy / single-atom nanoenzyme composite material of the present invention, the present invention still uses the AuPd-Fe@OA Gel sample prepared in Example 1 as an example to conduct a bacterial corneal ulcer test: Establishment of a rat model for evaluating the efficacy of antibacterial treatment in patients with bacterial corneal ulcers: First, promecaine hydrochloride eye drops were instilled onto the surface of the eyeballs of anesthetized SD rats to anesthetize the eyeballs. Then, the corneal stroma of the SD rats was scraped off. Next, the suspension of multidrug-resistant Pseudomonas aeruginosa was diluted to a concentration of 1×10⁻⁶. 6 Following CFU, 100 μL of bacterial solution was instilled onto the ocular surface to induce corneal ulceration. Thirty successfully modeled infected rats were selected and randomly divided into five groups (n=3 per group) according to their body weight, corneal opacity, and average ulcer area. Each group received a different treatment: Normal group, normal saline (NS) group, OA Gel group (Comparative Example 1), AuPd-Fe SACs group (Comparative Example 5), levofloxacin gel (LVFX Gel) group, and AuPd-Fe@OA Gel group. The treatment was administered to the affected ocular area twice daily (normal saline or different groups of hydrogel composite nanomaterials). The treatment lasted for 18 consecutive days, with 0.2 mL administered each time. Every two days, the corneal surface condition was observed and recorded using a slit-lamp microscope. The extent of corneal damage was assessed by sodium fluorescein staining, and the changes in corneal damage in each group were recorded and compiled as follows: Figure 14 The image shows the corneal damage changes in rats with bacterial corneal ulcers after injection of AuPd-Fe@OA Gel, in response to multidrug-resistant Pseudomonas aeruginosa infection.

[0112] Depend on Figure 14 The test results showed that, compared with the drug-treated group, the rats in the blank group had obvious grayish-white opacity, ulcers and purulent secretions in their corneas, and the corneal structure was severely damaged, and it had not fully recovered until the 18th day. In contrast, the rats in the drug-treated group treated with the alloy / single-atom nanoenzyme composite material had significantly reduced corneal opacity after the 6th day, and the cornea was basically restored to transparency by the 12th day of treatment, with only slight epithelial defects remaining. By the 18th day, it was close to the normal state of the normal group, which shows that the alloy / single-atom nanoenzyme composite material has excellent in vivo antibacterial activity and the ability to promote corneal repair.

[0113] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel, characterized in that, Includes the following steps: Tetraethyl silicate and a surfactant were dissolved in an alkaline solution, and ZIF-8 was added. After stirring, SiO2-coated ZIF-8 was obtained. The SiO2-coated ZIF-8 was pyrolyzed under an inert atmosphere, and the pyrolysis product was etched with an alkaline solution to obtain Zn single-atom nanozymes. The Zn single-atom nanozymes were dispersed in a solvent, and then Fe was added. 2+ The solution was stirred to separate the solid product, which was then reacted in a sulfuric acid solution to obtain Fe single-atom nanozymes; a reducing agent and Au were then added. 3+ Source and Pb 2+ The Fe single-atom nanozyme was dissolved in an alkaline solution, and then methoxy polyethylene glycol thiol was added. After the reaction, ultrafiltration was performed to obtain AuPd ultra-small alloy particles with a particle size of 5-10 nm. The Fe single-atom nanozyme and the AuPd ultra-small alloy particles were dispersed together in water, and AuPd-Fe composite nanoparticles were obtained after the reaction. The AuPd-Fe composite nanoparticles were dispersed in water, and choline-oleanolic acid hydrogel lyophilized powder prepared from choline and oleanolic acid was added. After gelation, the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel was obtained.

2. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The surfactant is hexadecyltrimethylammonium bromide, the alkaline solution is NaOH solution, the molar ratio of hexadecyltrimethylammonium bromide to NaOH is 1:15, the tetraethyl silicate is dissolved in methanol before being added, and the ratio of hexadecyltrimethylammonium bromide, tetraethyl silicate and ZIF-8 is 150mg:1.2mL:100mg.

3. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The pyrolysis temperature is 800℃, the time is 2h, and the heating rate is 10℃ / min; and / or, the alkaline solution used to etch the pyrolysis products is a 3~4mol / L NaOH solution.

4. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The Zn single-atom nanozyme and the Fe-containing 2+ Fe in solution 2+ The mass ratio is 30:24~30; and / or the concentration of the sulfuric acid solution is 4 mol / L.

5. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, Au 3+ Source Au 3+ With the Pb 2+ Pb in source 2+ The molar ratio is 1:1~2; the molar amount of SH-PEG-OMe and Au 3+ Pb 2+ The ratio of total molar amounts is 1:5~10.

6. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The mass ratio of the Fe single-atom nanozyme to the AuPd ultra-small alloy particles is 3:

1.

7. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The mass ratio of choline to oleanolic acid is 0.145:0.

456.

8. The method for preparing the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel according to claim 1, characterized in that, The mass ratio of the AuPd-Fe composite nanoparticles to the choline-oleanolic acid hydrogel lyophilized powder is 1:

100.

9. An injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel prepared by the method of any one of claims 1 to 8.

10. The application of the injectable AuPd-Fe single-atom nanozyme composite drug-derived antibacterial hydrogel of claim 9 in the preparation of antibacterial hydrogels.

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