A nano-enzyme for photocatalytic cascade enhancement of peroxidase reaction and a preparation method and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a kind of nano-enzyme for photocatalytic cascade enhancement of peroxidase reaction and a preparation method and purposes thereof.The nano-enzyme is copper-doped nano-enzyme CuCo(OH)2, wherein the doping amount of Cu in CuCo(OH)2 is 3-5%.The nano-enzyme is synthesized by a simple one-step hydrothermal method.The application constructs photocatalytic cascade enhancement of peroxidase (POD) reaction nano-enzyme by doping Cu in Co(OH)2, and the light-enhanced POD-like enzyme activity enables CuCo(OH)2 to have efficient reactive oxygen species (ROS) generation and excellent antibacterial performance, and its excellent in-vivo antibacterial activity and biocompatibility indicate its potential in the treatment of drug-resistant bacterial infection, and it can be used for treating drug-resistant bacterial infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nanozyme that enhances peroxidase reaction through photocatalytic cascade, its preparation method, and its uses. Background Technology

[0002] Inappropriate use of antibiotics can lead to the development of drug-resistant bacteria. Drug-resistant bacterial infections have become a common and complex clinical problem, posing a serious threat to human health. Reactive oxygen species (ROS), as promising antibacterial agents, can effectively eliminate bacteria by inducing oxidative stress, while reducing the likelihood of bacterial resistance. Since the discovery of ferrite (Fe3O4) nanoparticles with peroxidase (POD)-like activity, nanozymes have emerged as an emerging field. Among them, POD-like nanozymes, which catalyze the generation of hydroxyl radicals (•OH) from H2O2, have become promising candidates for eliminating drug-resistant bacteria. Compared with biological enzymes, nanozymes are reusable, low-cost, stable, and easily controllable in terms of morphology and composition. However, the single functionality and limited catalytic efficiency of nanozymes still fall short of biological enzymes, restricting their development and application.

[0003] The key steps in the POD reaction to generate •OH include the adsorption of H2O2 at the active site, electron transfer, and desorption of •OH. Enhancing the adsorption of H2O2 at the active site is crucial. Currently, the regulation of nanozyme catalysts mainly revolves around strategies such as constructing diverse nanostructures, introducing vacancies or defects, doping, and crystal engineering. Among these, oxygen vacancies (Ov), as a common defect type in nanomaterials, play a vital role in heterogeneous catalysis, and nanomaterials rich in oxygen vacancies exhibit excellent adsorption of H2O2 and O2. For example, Yu et al. significantly enhanced the number of oxygen vacancies on the surface of molybdenum oxide (Fe-MoOV) through iron doping. This enhancement significantly improved the adsorption and dissociation efficiency of H2O2, thereby improving its oxidase and peroxidase-like activities.

[0004] Typically, POD-like nanozymes generate •OH through OO cleavage via a Fenton-type mechanism. Photoexcitation of electron transitions promotes the directional migration of excited electrons from organic ligands to metal atoms, thereby accelerating Fenton or Fenton-like reactions. In recent years, the unique optical properties of nanomaterials have become a research hotspot. Combining the optical properties of nanozyme materials with enzyme-like activity to form photo-enhanced catalytic systems has attracted widespread attention due to its controllability and high efficiency. Ultraviolet (UV) nanozymes exhibit excellent catalytic performance due to their high photon energy; however, severe DNA damage limits their application. Therefore, visible light nanozymes, with their cost-effectiveness and excellent biocompatibility, have become a more ideal alternative. Short ROS lifetimes and limited diffusion distances also weaken the antibacterial properties of nanozymes. Due to electrostatic interactions, positively charged nanomaterials tend to adsorb onto negatively charged bacterial membranes, as confirmed in earlier studies, such as positively charged gold nanorods and gold nanorods modified with hexadecyltrimethylammonium bromide (CTAB). Therefore, developing a positively charged, photo-enhanced nanozyme system capable of efficiently generating ROS is expected to become an effective strategy for inactivating drug-resistant bacteria. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a photocatalytic cascade-enhanced peroxidase reaction nanozyme, its preparation method, and its applications. A three-dimensional (3D) flower-like nanozyme, CuCo(OH)₂, was synthesized via a simple one-step hydrothermal method. 2+ and Cu 2+ Ions form hydroxide compounds through hydrolysis, which further grow to form a three-dimensional layered flower-like structure with extremely thin, corrugated nanosheets. Copper-doped Co(OH)₂ exhibits efficient reactive oxygen species (ROS) generation and excellent antibacterial properties.

[0006] The technical solution provided by this invention is: a photocatalytic cascade enhanced peroxidase reaction nanozyme, wherein the nanozyme is a copper-doped nanozyme CuCo(OH)2; wherein the doping amount of Cu in CuCo(OH)2 is 3-5%.

[0007] Preferably, the Cu doping amount in CuCo(OH)2 is 3.96%.

[0008] The present invention further provides a method for preparing the copper-doped nanozyme, comprising:

[0009] Co(NO3)2·6H2O and Cu(NO3)2·3H2O were dissolved in methanol, and 2-methylimidazole was also dissolved in methanol; subsequently, the 2-methylimidazole solution was quickly poured into a container containing Co. 2+ and Cu 2+The solution was stirred magnetically until homogeneous. The final concentrations of Co(NO3)2·6H2O, Cu(NO3)2·3H2O, and 2-methylimidazole were 50-100 mmol / L, 20-50 mmol / L, and 150-200 mmol / L, respectively. The mixture was then transferred to a PTFE-lined stainless steel autoclave and heated at 120°C for 2 hours. After cooling to room temperature, the precipitate was washed several times with methanol by centrifugation and then dried at 60°C to collect the yellow product.

[0010] The present invention also provides the use of the nanozyme in the preparation of antibacterial compositions.

[0011] Preferably, the nanozyme is used in the preparation of compositions against drug-resistant bacteria.

[0012] The present invention further provides an antibacterial composition comprising an effective dose of the nanozyme, wherein the nanozyme promotes the generation of ROS in a photocatalytic cascade enhanced POD reaction.

[0013] The copper-doped nanoenzyme Co(OH)₂ provided by this invention forms abundant oxygen vacancies, promoting the adsorption of H₂O₂. Subsequently, the CuCo(OH)₂ system enhances photogenerated electron transfer, generating •OH to promote the POD reaction, and further enhances O₂• under visible light irradiation. - and 1 O2 generation. Photoenhanced POD-like enzyme activity endows CuCo(OH)2 with highly efficient reactive oxygen species (ROS) generation and excellent antibacterial properties. Furthermore, this invention demonstrates its superior in vivo antibacterial activity and biocompatibility, indicating its potential in the treatment of drug-resistant bacterial infections and promising broad clinical application prospects. Attached Figure Description

[0014] Figure 1 Schematic diagram of photo / enzyme synergistic catalysis mechanism and wound healing / antibacterial mechanism; wherein: (a) photo / enzyme synergistic catalysis mechanism; (b) wound healing and antibacterial mechanism;

[0015] Figure 2Characterization spectra of CuCo(OH)2; including: (a) Scanning electron microscope (SEM) image of CuCo(OH)2 nanoflowers; (b) Transmission electron microscope (TEM) image of CuCo(OH)2 nanoflowers; (c) High-resolution transmission electron microscope (HRTEM) image of CuCo(OH)2 nanoflowers; (d) Elemental distribution map of CuCo(OH)2 nanoflowers; (e) X-ray diffraction (XRD) spectra of Co(OH)2 and CuCo(OH)2; (f) High-resolution copper (Cu) 2p X-ray photoelectron spectrum of CuCo(OH)2; (g) High-resolution cobalt (Co) 2p X-ray photoelectron spectrum of Co(OH)2 and CuCo(OH)2.

[0016] Figure 3 Energy dispersive spectroscopy (EDS) for CuCo(OH)2.

[0017] Figure 4 Full scan X-ray photoelectron spectroscopy (XPS) spectra of Co(OH)2 and CuCo(OH)2;

[0018] Figure 5 High-resolution O1s X-ray photoelectron spectra of Co(OH)2 and CuCo(OH)2;

[0019] Figure 6 The POD-like activity and optical properties of Co(OH)2 were determined; including: (a) the POD activity of CuCo(OH)2 / H / L, Co(OH)2 / H / L, CuCo(OH)2 / H and Co(OH)2 / H was characterized using a tetramethylbenzidine (TMB) method; (b) the electron paramagnetic resonance (EPR) spectra of Co(OH)2 and CuCo(OH)2; (c) the UV-Vis reflectance spectra of Co(OH)2 and CuCo(OH)2; (d) the EPR spectra of Co(OH)2 and CuCo(OH)2 for the detection of •OH in the presence of 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO); and (e) the detection of singlet oxygen (•OH) in the presence of 2,2,6,6-tetramethylpiperidine (TEMP). 1 (f) Electron paramagnetic resonance (EPR) spectrum of O2; (f) Detection of superoxide anion radicals (O2•) in the presence of 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO). - The electron paramagnetic resonance (EPR) spectrum of H₂O₂ is shown, where H represents H₂O₂ and L represents visible light; the illumination time is 10 minutes.

[0020] Figure 7 The study included the following: (a) bacterial culture results of ESBL-producing *E. coli* treated with different methods, H2O2, and visible light; (c) staining of ESBL-producing *E. coli* with SYTO 9 and propidium iodide (PI); (d) scanning electron microscope (SEM) images of the morphology of ESBL-producing *E. coli*; (e) protein leakage of ESBL-producing *E. coli* incubated with different methods, A: Co(OH)2 / H, B: CuCo(OH)2 / H, C: Co(OH)2 / H / L, D: CuCo(OH)2 / H / L; (f) DNA agarose gel electrophoresis results of ESBL-producing *E. coli* treated with different methods; (g) in vitro cytotoxicity characteristics of L929 cells incubated with different concentrations of CuCo(OH)2 / H / L; and (g) the effect of different treatments on cell migration (scale bar, 200). (μm); (h) Statistical data on scratch width for different treatment groups, with error bars representing standard deviation (n = 3); *** indicates p < 0.001; H represents H2O2; L represents visible light; illumination time was 10 minutes;

[0021] Figure 8 Survival of ESBL-producing Escherichia coli under different treatments;

[0022] Figure 9 The effect of CuCo(OH)2 on wound healing was tested; (a) images of rat back wounds after different treatments, A: Co(OH)2 / H, B: CuCo(OH)2 / H, C: Co(OH)2 / H / L, D: CuCo(OH)2 / H / L; (b) bacterial samples collected from the treated wounds; (c) relative wound area of ​​different groups on day 10 and (d) bacterial count at the wound site; error bars represent standard deviation (n = 3); *** indicates p < 0.001; (e) H&E staining data of corresponding skin wounds after various treatments; (f) immunohistochemical images of rat wounds; (g) H&E sections of major organs (heart, liver, spleen, lungs, and kidneys) of mice in different groups on day 10; H represents H2O2; L represents visible light; illumination time was 10 minutes. Detailed Implementation

[0023] To facilitate understanding of this research, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. However, this research can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of this research.

[0024] This invention proposes a photocatalytic cascade-enhanced peroxidase (POD) nanozyme and a photocatalytic cascade-enhanced POD reaction strategy. It utilizes copper-doped nanozyme CuCo(OH)₂, rich in oxygen vacancies and positively charged, to promote ROS generation for antibacterial therapy. Studies show that copper-doped Co(OH)₂ forms abundant oxygen vacancies, promoting H₂O₂ adsorption. Subsequently, the CuCo(OH)₂ system enhances photogenerated electron transfer, generating •OH to promote the POD reaction, and further enhances O₂•OH adsorption under visible light irradiation. - and 1 The generation of O2, such as Figure 1 As shown in (a), CuCo(OH)₂ exhibits photoenhanced POD-like enzyme activity, resulting in highly efficient ROS generation and excellent antibacterial properties. It achieves an elimination rate exceeding 99.999% against drug-resistant ESBL-producing Gram-negative Escherichia coli (E. coli), and its superior antibacterial properties accelerate wound healing. Further investigation into the antibacterial mechanism reveals that ROS can lead to cell membrane damage and DNA degradation, thereby causing irreversible cell death, such as… Figure 1 As shown in (b). Furthermore, the excellent antibacterial activity and biocompatibility of CuCo(OH)2 in vivo suggest its potential in the treatment of drug-resistant bacterial infections.

[0025] I. Reagents and Materials

[0026] Cobalt nitrate hexahydrate (Co(NO3)2·6H2O), copper nitrate hydrate (Cu(NO3)2·3H2O), 2-methylimidazole (2-MIM), and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. Hydrogen peroxide (H2O2) and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). The live / dead BacLight bacterial viability and counting kit was purchased from Thermo Fisher Scientific, USA. The BCA protein concentration assay kit was purchased from Elabscience Biotechnology Co., Ltd. The TIANamp bacterial DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. All experiments used ultrapure water with a resistivity of 18.2 MΩ·cm. All chemical reagents were commercially available and used directly without further purification.

[0027] II. Sample Synthesis

[0028] (1) Synthesis of Co(OH)2

[0029] A simple one-step solvothermal method was used to synthesize three-dimensional flower-like Co(OH)₂. Specifically, 3 mmol of Co(NO₃)₂·6H₂O and 4 mmol of 2-MIM were dissolved in 25 mL of methanol, respectively. Then, the 2-MIM solution was quickly poured into the Co… 2+The solution was stirred magnetically for 15 minutes. The mixture was then transferred to a 100 mL PTFE-lined stainless steel autoclave and heated at 120°C for 2 hours. After cooling to room temperature, the precipitate was washed several times with methanol by centrifugation, and then dried at 60°C to collect the yellow product.

[0030] (2) Synthesis of CuCo(OH)2

[0031] A simple one-step solvothermal method was used to synthesize three-dimensional flower-like CuCo(OH)₂. Specifically, 2 mmol Co(NO₃)₂·6H₂O and 1 mmol Cu(NO₃)₂·3H₂O were dissolved in 25 mL of methanol, and 4 mmol 2-MIM was dissolved in 25 mL of methanol. Then, the 2-MIM solution was quickly poured into a solution containing Co. 2+ and Cu 2+ The solution was stirred magnetically for 15 minutes. The mixture was then transferred to a 100 mL PTFE-lined stainless steel autoclave and heated at 120°C for 2 hours. After cooling to room temperature, the precipitate was washed several times with methanol by centrifugation, and then dried at 60°C to collect the yellow product.

[0032] III. Characterization

[0033] To investigate the microstructure of the samples, scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy (EDS) elemental mapping / line scanning were obtained on a Sigma 500 (200 kV). Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images were acquired using a JEM-2100F. The crystal structure of the samples was measured by X-ray diffraction (XRD, SmartLab 3KW) using Cu Ka radiation at a scan rate of 10° / min. X-ray photoelectron spectroscopy (XPS) data were obtained using a Thermo VG Scientific ESCALAB 250 spectrometer. UV-Vis absorption spectra were detected using a Mapada UV-6300 spectrophotometer (Shanghai, China). Electron spin resonance (ESR) spectra were evaluated at 77 K using a Bruker A300-10. Microplate readers were purchased from Thermo Fisher Scientific. Zeta potentials were measured using a Malvern nanoparticle potentiometer. Bacterial flow cytometry data were measured using a FACS Celesta flow cytometer, and live / dead bacteria assays were performed using a fluorescence microscope (Nikon ECLIPSE Ni-U, Japan).

[0034] IV. Electrochemical Measurement

[0035] (1) Characterization of POD-like activity

[0036] The POD-like catalytic activity of the samples was evaluated in an acetate buffer system containing 50 μL TMB (20 mM) and 10 μL H2O2 (100 mM). The absorbance of the reaction system was measured at 652 nm in the presence of 50 μL nanozyme (250 μg / mL). Absorbance was determined using a microplate reader.

[0037] (2) Free radical scavenging test

[0038] Free radical species were detected by electron spin resonance (EPR) using 5,5-dimethyl-1-pyrrolidine (DMPO) and 2,2,6,6-tetramethyl-4-piperidinol (TEMP) as capture reagents. Detailed parameters were as follows: 100 μL of the sample aqueous suspension was mixed with 1.0 mL of TEMP (50.0 mM) solution. •OH capture-EPR assays were also performed as described above, except that DMPO (50.0 mM) was used as the capture reagent. For O2• - Mix 1 mL of methanol and water (volume ratio 20:1) with a solution containing DMPO (50.0 mM).

[0039] V. Bacterial Culture

[0040] This study used broad-spectrum β-lactamase-producing *Escherichia coli* (ESBL-producing *E. coli*) as the model organism. Hospital wastewater was collected from a hospital in Qingdao, and airborne bacteria were collected in the microbiology laboratory. Monoclonal bacteria were cultured on Luria-Bertani (LB) solid medium and then transferred to 50 mL of LB liquid medium, incubated at 37°C and 150 rpm for 12 hours until reaching the logarithmic growth phase.

[0041] VI. In vitro antibacterial test of CuCo(OH)2

[0042] More than three bacterial colonies were randomly selected from LB solid medium and inoculated into 50 mL of sterile LB liquid medium, and incubated at 37°C and 150 rpm for 12 hours. The suspension was then diluted with sterile physiological saline to 0.5 MCF (McFarland) using a McFarland turbidimeter. 0.5 MCF corresponds to 1 × 10⁻⁶ mcg / mL. 8 CFU / mL. The bacterial suspension was then diluted to 1×10⁻⁶. 6CFU / mL. Bacterial suspensions (500 μL) containing different concentrations of nanozymes were incubated at 37°C for 30 minutes, followed by the addition of 10 μL of H2O2 (100 mM) and irradiation under visible light for 10 minutes. After incubation, the bacterial suspension was removed from the water bath, and 100 μL of the bacterial suspension was evenly spread onto solid LB medium using a spreader. The culture plates were placed upright for 30 minutes, then inverted and incubated at 37°C for 24 hours. Each experiment was repeated three times.

[0043] VII. Detection of live / dead bacteria

[0044] To further observe the effect of ROS on ESBL-producing *Escherichia coli*, the bacteria were labeled using a Syto 9 / PI fluorescent probe. After culturing the bacteria for 12 hours, 500 μL of bacterial suspension was taken and washed twice with PBS (pH 7.4). The bacterial suspension was then treated with different concentrations of CuCo(OH)₂ for 30 minutes, centrifuged at 37°C at 3000 rpm for 5 minutes, and the precipitate was retained and washed twice. Finally, 25 μL of Syto 9 / PI dye mixture was added, incubated for 15 minutes, and observed using a standard vertical fluorescence microscope.

[0045] 8. Bacterial Protein Leakage Test

[0046] The bacteria were cultured in liquid medium for 12 hours. 500 μL of the bacterial suspension was then washed twice with PBS (pH 7.4). The bacterial suspension was then treated with different concentrations of CuCo(OH)₂ for 30 minutes, centrifuged at 37°C at 8000 rpm for 5 minutes, and the cell-free supernatant was collected. The protein concentration in the supernatant was determined using a BCA protein assay kit according to the manufacturer's instructions.

[0047] IX. DNA Degradation Experiment

[0048] ESBL-producing E. coli were treated with CuCo(OH)2 for 30 minutes and then washed twice with PBS (pH 7.4). Bacterial DNA was extracted using a DNA extraction kit, the DNA concentration was measured, and DNA samples of the same concentration were loaded onto the substrate. Finally, identification was performed using agarose gel electrophoresis.

[0049] 10. Bacterial morphology detection

[0050] To observe the changes in bacterial morphology after CuCo(OH)2 treatment, treated ESBL-producing Escherichia coli strains were collected and centrifuged at 3000 rpm for 15 minutes. Single colonies were cultured on LB agar to OD200. 600 The OD value reached 1.0. This was achieved by centrifugation of 1.5 mL OD. 600Bacterial cultures reaching pH 1.0 were collected, and the enriched bacteria were washed three times with PBS (pH 7.4). Two bacterial treatment groups were then established: a) bacteria only, and b) bacteria + CuCo(OH)₂ / H₂O₂ / light (10 min light exposure). Both treatments were incubated at 37°C for 5 h, followed by washing three times with PBS (pH 7.4), collecting the precipitate, fixing it with 2.5% glutaraldehyde for 1 h, and storing at 4°C. Subsequently, the fixative was washed with PBS (pH 7.4), and dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol for 15 min each time, followed by treatment with 50% ethyl acetate. Finally, the bacterial cells were characterized using an S-4700 scanning electron microscope (Hitachi, Japan).

[0051] XI. In vitro toxicity tests

[0052] To evaluate the cytotoxicity of the prepared CuCo(OH)2, mouse L929 fibroblasts were used as a cell model. Cell viability was determined using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were cultured in 96-well plates for 24 hours, followed by treatment with different concentrations of CuCo(OH)2 / H2O2 / light. After 24 hours of incubation, 10 μL of MTT solution was added to each well, and incubation continued for another 4 hours. Finally, 10 μL of DMSO was added to each well, and incubation continued for another 4 hours. The absorbance was measured at 490 nm using a microplate reader.

[0053] 12. In vitro scratch healing experiment

[0054] Cell migration ability was assessed using a scratch assay. L929 cells (4 × 10⁶) were used to assess cell migration. 5 Cells were seeded in 6-well plates. When the cells reached 80% confluence, three uniform scratches were made using a 200 μL pipette tip. The cells were then washed twice with PBS (pH 7.4), and 2 mL of serum-free medium was added. Co(OH)₂ / H₂O₂ / light and CuCo(OH)₂ / H₂O₂ / light solutions at concentrations of 12.5 mg / mL were added. Photographs were taken at the same locations every 12, 24, and 48 hours to assess cell migration. Cell migration rate × (%) = (0-hour scratch width - post-culture scratch width) / 0-hour scratch width × 100%.

[0055] XIII. Animal Experiments

[0056] Sprague Dawley (SD) rats and Kunming mice (KM) (6 weeks old) were purchased from Beijing Sipufu Biotechnology Co., Ltd. All experiments involving animals were approved by the Ethics Committee of Qingdao University Medical College. After anesthetizing the rats with 20% urea, a wound model was created on the rats' backs using a 1 cm diameter circular punch, and then 20 μL of 1×10⁻⁶ urea solution was injected. 6 CFU / mL of ESBL-producing *E. coli* was added to the wound. The wound was then treated with 10 μL of 12.5 mg / mL Co(OH)₂ / CuCo(OH)₂ and 10 μL of 100 μM H₂O₂ under visible light for 10 minutes. SD rats were divided into 5 groups of 3 rats each. Wound condition was recorded daily. Rats were sacrificed on day 10, and wound tissue was collected, stored in 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and subjected to immunohistochemical treatment. After 14 days, the tissue was observed using a standard upright fluorescence microscope.

[0057] XIV. Data Analysis

[0058] Wound area, colony count, and scratch width in rats were quantitatively analyzed using Image J 1.46. Data were from three independent experiments, and error bars represent mean ± standard deviation. Data were analyzed using Origin 2023 or GraphPad Prism 8. Statistical significance was determined using a two-tailed Student's t-test and one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant between groups.

[0059] XV. Experimental Results

[0060] (1) This invention synthesizes three-dimensional (3D) flower-like nanozymes Co(OH)2 and CuCo(OH)2 using a simple one-step hydrothermal method. 2+ (and Cu) 2+ The ions undergo hydrolysis to form hydroxide compounds, which then grow further, ultimately forming a three-dimensional, layered, flower-like structure with extremely thin, corrugated nanosheets. Scanning electron microscopy (SEM) images of Co(OH)₂ show this blooming flower-like morphology. Figure 2 The SEM image shown in (a) indicates that the introduction of copper ions did not alter the overall morphology, but led to the formation of a bimetallic layered flower-like structure. Transmission electron microscopy (TEM) images of CuCo(OH)₂ reveal that this three-dimensional layered flower-like structure is composed of ultrathin and smooth-surfaced nanosheets, such as... Figure 2 (b) Figure 2The high-resolution TEM (HRTEM) image shown in (c) further demonstrates the well-defined crystal structure of CuCo(OH)₂. Through elemental mapping and energy dispersive spectroscopy (EDS), this invention confirmed the uniform distribution of oxygen, cobalt, and copper elements in CuCo(OH)₂, with a copper content of 3.96%, proving successful copper incorporation. Figure 2 (d) and Figure 3 As shown.

[0061] The crystal structures of Co(OH)₂ and CuCo(OH)₂ were further analyzed by X-ray diffraction (XRD). Figure 2 As shown in (e), the physical phases of Co(OH)₂ and CuCo(OH)₂ conform to the cubic spinel structure of Co(OH)₂ (PDF#: 46-0605). Due to the similar atomic radii of copper and cobalt atoms, the incorporation of copper has a relatively small impact on the crystal phase of Co(OH)₂. X-ray photoelectron spectroscopy (XPS) further reveals the effect of copper incorporation on the electronic structure of Co(OH)₂. Figure 4 The XPS spectrum of CuCo(OH)₂ shows distinct characteristic peaks for copper, cobalt, and oxygen, while Co(OH)₂ exhibits only peaks for cobalt and oxygen. The peaks for copper are 932.6 eV, 935.7 eV, and 943.4 eV, corresponding to Cu... + Cu 2+ And the corresponding satellite peaks, such as Figure 2 (f) Figure 2 The high-resolution Co 2p spectrum shown in (g) indicates that Co in Co(OH)2 and CuCo(OH)2... 2+ (784.4 eV and 784.1 eV) and Co 3+ Satellite peaks corresponding to the peak values ​​of 781.3 eV and 781.1 eV. For example... Figure 5 The O 1s spectrum shown indicates that all samples contain lattice oxygen (531.4 eV) and oxygen vacancy (Ov) (532.0 eV) species, and the Ov region in CuCo(OH)2 is significantly larger than that in Co(OH)2, indicating that a large number of oxygen vacancies are generated after copper doping.

[0062] (2) To verify the enhancing effect of Cu doping with Co(OH)₂ on its POD-like activity and optical properties, this invention tested the POD-like activity of Co(OH)₂ and CuCo(OH)₂ under visible light irradiation. The absorbance at 652 nm was measured by catalyzing the oxidation reaction of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the presence of H₂O₂. For example... Figure 6The results shown in (a) indicate that, compared to Co(OH)₂, the POD-like activity of CuCo(OH)₂ is significantly enhanced, with a marked increase in POD-like activity under visible light irradiation, while the change in Co(OH)₂ is relatively small. This suggests that CuCo(OH)₂ possesses superior visible light photocatalytic performance. Further analysis using electron spin resonance (EPR) and ultraviolet-visible reflectance spectroscopy (UV-Vis) revealed that CuCo(OH)₂ has a higher oxygen vacancy content and stronger absorption capacity for visible light, such as... Figure 6 As shown in (b) and (c). These results indicate that after Cu is incorporated into Co(OH)2, the formation of oxygen vacancies promotes the adsorption of H2O2, which in turn enhances the transfer of photogenerated electrons in the CuCo(OH)2 system and promotes the generation of •OH.

[0063] (3) To further reveal the types and rates of ROS formation catalyzed by CuCo(OH)2 and Co(OH)2, a series of EPR tests were conducted in this invention. •OH and •O2 were captured by using the trapping agents 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and 2,2,6,6-tetramethyl-4-piperidinol (TEMP), respectively. - and 1 The generation of O2. For example... Figure 6 The test results shown in (d)–(f) indicate that CuCo(OH)₂ generates significantly more •OH groups under visible light irradiation than Co(OH)₂, demonstrating that CuCo(OH)₂ exhibits stronger POD-like activity. Furthermore, CuCo(OH)₂ generates O₂• more efficiently than Co(OH)₂. - and 1 O2 indicates that CuCo(OH)2 has a stronger ability for photoelectron transfer and free radical generation.

[0064] (4) To verify the antibacterial effect of CuCo(OH)2 against common drug-resistant bacteria (such as ESBL-producing Escherichia coli) in vitro, the present invention conducted antibacterial performance tests under different experimental conditions. The results showed that under visible light irradiation, CuCo(OH)2 could effectively eliminate ESBL-producing Escherichia coli at a concentration of 12.5 μg / mL, with a bactericidal rate exceeding 99.9999%. Figure 7 (a) and Figure 8 As shown, this demonstrates its excellent antibacterial properties. Laser confocal microscopy (LCSM) revealed that CuCo(OH)₂ can disrupt bacterial membranes, and the charge interaction between CuCo(OH)₂ and the bacterial membrane promotes bacterial aggregation on the nanozyme surface, such as... Figure 7 As shown in (b) and (c), DNA agarose gel electrophoresis and protein leakage experiments demonstrate that ROS causes severe damage to bacterial DNA, ultimately leading to cell death. Figure 7As shown in (d) and (e).

[0065] (5) Further cytotoxicity experiments showed that CuCo(OH)2 had no significant toxic effect on fibroblasts (L929), such as Figure 7 As shown in (f).

[0066] (6) To evaluate the role of nanozymes in the actual healing process of bacterial-infected wounds, an ESBL-producing Escherichia coli-infected wound model was established in 6-week-old Sprague-Dawley (SD) rats. Representative images of the treated wounds and surrounding areas are shown on days 0, 2, 4, 6, 8, and 10. The CuCo(OH)2 / H2O2 / light-treated group showed the highest degree of wound healing and the highest wound repair rate, as shown in Figures 9(a) and (c). On day 10, bacterial counts showed that the CuCo(OH)2 / H2O2 / light-treated group almost completely eliminated all bacteria around the wound, while viable bacteria remained in the wounds of other groups, such as... Figure 9 (b) and (d). Since re-epithelialization is an important process in wound healing, hematoxylin-eosin (H&E) staining was performed on the wound tissue of rats on day 10 to further evaluate the wound healing process. In the CuCo(OH)2 / H2O2 / light-treated group, inflammatory cell infiltration was reduced, granulation tissue was formed, and the skin layer remained intact, indicating that wound re-epithelialization was enhanced and the treatment effect was significant, as shown in Figure 9(e).

[0067] The expression of relevant cytokines during wound healing was further investigated using immunohistochemistry, such as... Figure 9As shown in (f). Tumor necrosis factor-α (TNF-α) was used as a biomarker to study collagen formation and inflammation at the wound site. TNF-α expression was lowest in the CuCo(OH)2 / H2O2 / light-treated group, indicating almost complete resolution of inflammation, consistent with hematoxylin-eosin (H&E) staining results. Vascular endothelial growth factor-α (VEGF-α) promotes angiogenesis, which is particularly important in wound healing, while CD31, also known as platelet endothelial cell adhesion molecule 1, is a biomarker of angiogenic capacity. Compared with other control groups, the expression levels of VEGF-α and CD31 were significantly increased in the CuCo(OH)2 / H2O2 / light-treated group. These results suggest that CuCo(OH)2 / H2O2 / light irradiation has the potential to promote angiogenesis in fibrotic tissue and thus promote wound healing. Furthermore, the histomorphology of several important organs, including the heart, liver, spleen, lungs, and kidneys, was studied using hematoxylin and eosin (H&E) staining, as shown in Figure 9(g). No organ damage or histological lesions were observed in mice in any group after 10 days. This indicates that CuCo(OH)2 is non-toxic and harmless, and its excellent biocompatibility suggests broad application prospects in the biomedical field.

Claims

1. The application of nanozymes with photocatalytic cascade-enhanced peroxidase reactions in the preparation of antibacterial compositions, characterized in that: The nanozyme is a copper-doped nanozyme CuCo(OH)2, wherein the Cu doping amount in CuCo(OH)2 is 3-5%. The copper-doped Co(OH)2 forms abundant oxygen vacancies, which promotes the adsorption of H2O2. The CuCo(OH)2 system enhances photogenerated electron transfer, generates •OH to promote the POD reaction, and enhances the generation of O2•- and ¹O2 under visible light irradiation. The bacteria are ESBL-producing Escherichia coli.

2. The application according to claim 1, characterized in that: The Cu doping content in CuCo(OH)2 is 3.96%.

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