A preparation method of a medical nanozyme, the medical nanozyme, application of the medical nanozyme and a medicine

Abstract

This invention relates to a method for preparing medical nanozymes, the medical nanozymes themselves, their applications, and pharmaceuticals, belonging to the field of biomedicine. It addresses at least one of the following problems in existing technologies for treating deep tissue infections caused by drug-resistant bacteria: poor antibacterial and anti-biofilm effects, insufficient deep tissue penetration, lack of inflammation regulation and tissue repair functions, biosafety risks, and high material preparation costs. The preparation method includes: mixing rhodium trichloride, urea, and polyvinylpyrrolidone to obtain a mixture; subjecting the mixture to staged pyrolysis under inert gas protection to obtain a nitrogen-doped carbon-supported single-atom rhodium catalyst; and modifying the nitrogen-doped carbon-supported single-atom rhodium catalyst with polyethylene glycol. The medical nanozymes prepared by this invention achieve excellent antibacterial and anti-biofilm effects both in vivo and in vitro, can regulate the inflammatory microenvironment, promote tissue repair, exhibit good biocompatibility, and have strong tissue penetration ability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing medical nanozymes, the medical nanozymes, their applications, and pharmaceuticals. Background Technology

[0002] Deep tissue infections, such as subcutaneous abscesses and bacterial pneumonia, especially those caused by multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA), have become a significant global public health challenge. Traditional antibiotic therapies are limited in effectiveness due to drug resistance and struggle to penetrate biofilms and deep tissues, resulting in high treatment failure rates.

[0003] In recent years, medical nanozyme catalysis therapy has attracted widespread attention as an emerging anti-infection strategy. It achieves chemokinetic therapy (CDT) by mimicking the activity of natural enzymes to catalyze the generation of highly reactive oxygen species (ROS) at the site of infection. However, existing medical nanozyme materials still have the following shortcomings: Low catalytic efficiency: Most medical nanozymes exhibit weak enzyme-mimicking activity, especially under complex infection microenvironments (such as acidic pH, hypoxia, and high concentrations of biofilm matrix), where activity significantly decreases. Poor tissue penetration: Nanomaterials have limited diffusion and retention capabilities in deep tissues, making it difficult to effectively reach the core infection area. Limitations of single treatment modality: Relying solely on CDT is insufficient to completely eliminate drug-resistant bacteria and stubborn biofilms, easily leading to infection recurrence. Material stability and biocompatibility issues: Some metal-based nanozymes (such as platinum-based catalysts) are costly and prone to inactivation or systemic toxicity in physiological environments.

[0004] Therefore, there is an urgent need to develop a novel medical nanozyme platform that is efficient, safe, and has a synergistic therapeutic mechanism to achieve complete eradication and tissue repair of deep drug-resistant bacterial infections. Summary of the Invention

[0005] In view of the above analysis, the present invention aims to provide a method for preparing medical nanozymes, medical nanozymes and their applications and drugs, in order to solve at least one of the following problems in the prior art when medical nanozymes are used to treat deep tissue drug-resistant bacterial infections: poor antibacterial and anti-biofilm effects, insufficient deep tissue penetration ability, lack of inflammation regulation and tissue repair functions, biosafety risks, and high material preparation costs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a method for preparing a medical nanozyme for treating drug-resistant bacterial infections in deep tissues, the method comprising the following steps: Rhodium trichloride, urea, and polyvinylpyrrolidone are mixed to obtain a mixture, wherein the mass ratio of rhodium trichloride, urea, and polyvinylpyrrolidone is (3.0~6.0) g : (80~120) g : (10~30) g; The mixture was subjected to staged pyrolysis under inert gas protection to obtain a nitrogen-doped carbon-supported single-atom rhodium catalyst. The nitrogen-doped carbon-supported single-atom rhodium catalyst was modified with polyethylene glycol to obtain the medical nanozyme.

[0008] Furthermore, the segmented pyrolysis includes: the temperature of the first pyrolysis stage is 400~600℃, and the temperature of the second pyrolysis stage is 900~1100℃.

[0009] Furthermore, the holding time of the first pyrolysis stage is 2 to 4 hours; and / or, the holding time of the second pyrolysis stage is 0.5 to 1.5 hours.

[0010] Further, the polyethylene glycol modification step includes: The nitrogen-doped carbon-supported single-atom rhodium catalyst was dispersed in a first solvent to obtain a catalyst dispersion. Polyethylene glycol is dissolved in a second solvent to obtain a polyethylene glycol solution; The catalyst dispersion was mixed with the polyethylene glycol solution and stirred at 20-30°C for 12-48 hours. The mixture after stirring reaction is subjected to solid-liquid separation and washing to obtain the medical nanozyme.

[0011] Further, in the polyethylene glycol modification, the mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to the polyethylene glycol is 1:(0.05~0.30); and / or, The molecular weight (Mn) of the polyethylene glycol is 2000-10000 Da.

[0012] Furthermore, the first solvent and the second solvent are each independently selected from water or a buffer solution with a pH value of 6.5-8.0.

[0013] This invention provides a medical nanozyme prepared according to the described method.

[0014] This invention provides the medical nanozyme obtained by the preparation method described above, or the application of the medical nanozyme described above in the preparation of drugs for the prevention and / or treatment of deep tissue infections caused by drug-resistant bacteria.

[0015] Furthermore, the drug is applied in conjunction with second near-infrared laser irradiation.

[0016] This invention provides a medicament for preventing and / or treating deep tissue infections caused by drug-resistant bacteria, wherein the medicament comprises a medical nanozyme obtained by the preparation method or the medical nanozyme described above.

[0017] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: This invention controls the feeding ratio of rhodium trichloride (rhodium precursor), urea (nitrogen source), and polyvinylpyrrolidone (carbon source), combined with staged pyrolysis, to form a nitrogen-doped nanosheet structure in a carbon framework under an inert atmosphere. The reaction atmosphere generated by urea decomposition promotes the dispersion and anchoring of rhodium species generated from the decomposition of rhodium trichloride within the carbon framework. A hydrophilic surface layer is then formed through polyethylene glycol modification. This process, based on the synergistic effect of precursor decomposition, framework growth, metal anchoring, and surface modification, produces a medical nanozyme with a specific core-shell structure: it consists of a core structural unit and a surface functional layer; the core structural unit comprises atomically dispersed rhodium monoatoms anchored within a nitrogen-doped carbon framework, forming a stable Rh-NC coordination structure; the nitrogen-doped carbon framework has a nanosheet structure; and the surface functional layer consists of polyethylene glycol modified on the surface of the core structural unit.

[0018] Based on the structural features obtained through the above-mentioned process control, the medical nanozyme of the present invention can achieve at least one of the following beneficial effects: (1) Achieve excellent antibacterial and anti-biofilm effects; This invention relates to a medical nanozyme that utilizes atomically dispersed rhodium single atoms anchored within a nitrogen-doped carbon framework to form a stable Rh-NC coordination structure, achieving highly efficient antibacterial activity. This structure makes each rhodium atom an exposed active center, significantly improving atom utilization. By modulating electronic states (such as the d-band center position), the energy barrier for hydrogen peroxide activation to generate hydroxyl radicals is significantly reduced, enhancing peroxidase-like and oxidase-like mimicry activities. In an acidic infection microenvironment, multiple nitrogen species and nitrogen vacancy defects in the nitrogen-doped carbon framework synergistically regulate the electronic structure of the carbon support, further optimizing the catalytic performance of the rhodium active sites and maintaining high ROS generation capacity. In the presence of H₂O₂, highly active •OH is generated through catalysis, directly oxidizing and destroying bacterial cell membrane lipids, proteins, and DNA, leading to bacterial death. The nitrogen-doped carbon framework has a nanosheet-like structure, and its high specific surface area and edge active sites can penetrate into the biomembrane matrix through physical adsorption and intercalation, disrupting its dense structure. Simultaneously, the ROS generated by rhodium single-atom catalysis can degrade extracellular polysaccharides (EPS) and protein components in the biomembrane, disintegrating the three-dimensional network of the biomembrane.

[0019] (2) Possesses the ability to penetrate deep tissues; Surface-modified polyethylene glycol forms a hydrophilic canopy, reducing non-specific protein adsorption and immune recognition, prolonging blood circulation time, and enhancing material accumulation and penetration at the infection site. The size effect of the nanosheet structure facilitates diffusion in the interstitial spaces, penetrating deep infection areas.

[0020] (3) Regulating the inflammatory microenvironment In the medical nanozymes (Rh SACs) of this invention, atomically dispersed rhodium single atoms are anchored in a nitrogen-doped carbon framework, forming a stable Rh-NC coordination structure with a unique electronic structure. This structure can influence the signaling pathways of immune cells at the site of infection by regulating the local redox microenvironment. Specifically, it regulates inflammatory signaling pathways such as NF-κB and MAPK by catalyzing the moderate production of reactive oxygen species, thereby reducing the release of pro-inflammatory cytokines such as TNF-α and IL-6. Simultaneously, it induces macrophages to transform from the pro-inflammatory M1 type to the anti-inflammatory / repair M2 type (decreased CD86 expression and increased CD206 expression). This process is related to the regulation of cell metabolism and signaling pathways by the Rh-NC site. PEG modification enhances the biocompatibility and stability of the material, reduces the non-specific recognition and clearance of the material by the immune system, and prolongs its residence time at the site of infection, thus continuously and gently regulating the local immune microenvironment.

[0021] (4) Promotes tissue repair The structure of the Rh SACs (Rhodium Nanozymes) of this invention can promote angiogenesis and fibroblast proliferation, and accelerate collagen synthesis and deposition (as shown by Masson staining) by regulating macrophage polarization to the M2 type and releasing repair factors such as VEGF and TGF-β. The Rh-NC structure can also promote epithelial cell migration and proliferation (e.g., increased positive expression of PCNA immunohistochemistry) by regulating repair-related signaling pathways such as PI3K / Akt and Wnt / β-catenin. The nitrogen-doped carbon backbone with a nanosheet structure has a high specific surface area, which is beneficial for interaction with the extracellular matrix, providing physical support, promoting cell adhesion and migration, and facilitating the loading and sustained release of repair-related factors. Polyethylene glycol (PEG) modification enhances the biocompatibility and stability of the material, reduces non-specific recognition and clearance of the material by the immune system, and prolongs its residence time at the infection site, thereby continuously and gently regulating the local immune microenvironment. The PEG layer can also reduce non-specific adsorption and inflammatory responses between the material and tissue, maintain the stability of the local microenvironment, and provide favorable conditions for tissue repair.

[0022] (5) Good biocompatibility This invention utilizes a polyethylene glycol (PEG)-modified surface to form a hydrophilic protective layer for medical nanoenzymes. The steric hindrance effect of the long PEG chains creates a hydration layer, effectively shielding the hydrophobicity and positive charge on the nanoparticle surface. This reduces non-specific adsorption to plasma proteins, decreasing the recognition and clearance of immune cells such as macrophages (i.e., reducing opsonization), thereby prolonging in vivo circulation time. The PEG layer also isolates the Rh single-atom active sites from direct contact with the cell membrane, reducing membrane damage and oxidative stress to normal cells and improving the material's biocompatibility. Atomically dispersed Rh single atoms are anchored within a nitrogen-doped carbon framework, forming a stable Rh-NC coordination structure, ensuring stable Rh anchorage in single-atom form without metal aggregation or crystalline phase formation. This stable Rh-NC coordination structure significantly inhibits the dissolution and release of Rh ions in the physiological environment, avoiding cytotoxicity, oxidative damage, and systemic toxicity (such as liver and kidney damage) caused by free metal ions. The atomically dispersed active centers can precisely catalyze the generation of ROS in the local microenvironment, preventing excessive ROS diffusion and systemic oxidative stress, thus improving the selectivity and safety of treatment. Nitrogen-doped carbon frameworks exhibit a nanosheet morphology, with smooth edges and uniform size, reducing mechanical stimulation and physical penetration damage to cell membranes. Appropriate nanoscale size can prevent excessive accumulation in vital organs (such as the liver and spleen), and some can be gradually cleared through the kidneys or hepatobiliary pathways, reducing the risk of long-term toxicity.

[0023] (6) In some preferred embodiments, the medical nanozyme provided by the present invention synergistically optimizes its structure, catalytic performance, and biocompatibility by controlling the raw material ratio and the segmented pyrolysis process. Specifically, by regulating the specific ratio between rhodium trichloride, urea, and polyvinylpyrrolidone, the stable anchoring of rhodium atoms in the nitrogen-doped carbon framework can be promoted, forming high-density atomically dispersed active centers and avoiding metal aggregation. This specific ratio, combined with the segmented pyrolysis process, helps to construct a nanosheet-like carrier structure with a high specific surface area, further improving the photothermal conversion capability and catalytic stability of the material, and maintaining high efficiency in acidic infection microenvironments. At the same time, the process control also provides a suitable surface structure for subsequent polyethylene glycol modification, optimizing the hydrophilicity and cycle stability of the material while maintaining the accessibility of catalytic sites, ultimately achieving a comprehensive improvement in antibacterial, anti-biofilm, tissue repair regulation, and biocompatibility.

[0024] (7) The medical nanozyme provided by this invention, when combined with second near-infrared laser irradiation, can significantly enhance its therapeutic effect through multiple synergistic mechanisms: First, the deep tissue penetration capability of second near-infrared light can precisely deliver photothermal energy and catalytic effects to deep infection sites that are difficult to reach with traditional therapies; Second, under laser excitation, the Rh single-atom active center exhibits highly efficient photothermal conversion performance, with a photothermal conversion efficiency of up to 50.8% or higher. Local heating can not only directly kill pathogens, but also accelerate catalytic reactions and enhance the generation and diffusion of reactive oxygen species, achieving synergistic destruction of drug-resistant bacteria and biofilms; At the same time, the photothermal effect can also soften the biofilm structure, promote the penetration of nanozymes and reactive oxygen species, and further improve the biofilm clearance efficiency. In addition, the moderate thermal effect can improve local microcirculation, synergistically regulate the polarization of macrophages by the Rh-NC structure, jointly optimize the repair microenvironment, and ultimately achieve a triple synergistic deep anti-infection and tissue repair therapeutic effect of "photothermal-catalysis-immunomodulation".

[0025] (8) Compared with platinum-based catalysts, Rh materials have a cost reduction of about 60–70%, and the synthesis method is simple and scalable, making them suitable for large-scale preparation. In addition, Rh SACs maintain high catalytic activity (>90% activity retention) in acidic microenvironments (such as pH 5.6), adapting to the complex physiological environment of infection sites.

[0026] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0027] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0028] Figure 1The design and structural characterization of nitrogen-doped carbon-supported single-atom rhodium catalysts (Rh SACs) modified with polyethylene glycol provided in this embodiment of the invention are as follows: a) Schematic diagram of the synthesis route of Rh SACs; b) Transmission electron microscope image of the prepared Rh SACs, where the inset is the corresponding selected area electron diffraction pattern; c) High-resolution transmission electron microscope image; d) Aberration-corrected scanning transmission electron microscope image, confirming the atomic-level dispersion state of rhodium species; e) X-ray diffraction pattern of Rh SACs; f) High-angle annular dark-field scanning transmission electron microscope image and corresponding elemental distribution map; g) Electron paramagnetic resonance spectrum, reflecting the electronic state of rhodium sites; h) Full X-ray photoelectron spectrum of Rh SACs; High-resolution i.N 1s spectrum and j.Rh 3p spectrum further confirm the chemical coordination environment and bonding properties of rhodium species. Figure 2 The photothermal properties and multi-enzyme simulated activity of the nitrogen-doped carbon-supported rhodium single-atom catalyst (Rh SACs) modified with polyethylene glycol provided in this embodiment of the invention are as follows: a) Photothermal heating curves of different concentrations of Rh single-atom catalyst under 1064 nm laser irradiation (1.0 W / cm²); b) Infrared thermographic images of PBS and Rh SACs solution (80 µg / mL) at different time points under laser irradiation (1064 nm, 1.0 W / cm²); c) Heating curves of Rh SACs aqueous solution (80 µg / mL) under irradiation with different laser power densities (1064 nm); d) Photothermal stability of Rh SACs in five laser switching cycles (1064 nm, 1.0 W / cm²); e) Photothermal conversion efficiency quantitatively calculated based on heating and cooling curves; fh) Rh The absorbance of TMB catalyzed by SACs over time is shown in the following curves: f represents the oxidase-simulated activity; g represents the peroxidase-simulated activity; h represents the peroxidase-simulated activity enhanced by near-infrared II light; ik represents the TMB absorbance as a function of Rh SACs concentration: i represents the oxidase-simulated reaction conditions; j represents the peroxidase-simulated reaction conditions; k represents the peroxidase-simulated reaction conditions enhanced by near-infrared II light; l represents the optimized atomic structure model of Rh SACs; m represents the charge density difference diagram of Rh SACs; n represents the optimized adsorption configuration of TMB molecules on the surface of Rh single-atom catalysts; o represents the electrostatic potential distribution of Rh SACs; p represents the free energy change diagram of key intermediates in the peroxidase-simulated catalysis process; and q represents the schematic diagram of the mechanism of action of peroxidase-simulated activity on the surface of Rh SACs. Figure 3The in vitro antibacterial and antibiofilm activities of the polyethylene glycol-modified nitrogen-doped carbon-supported rhodium single-atom catalysts (Rh SACs) provided in this embodiment of the invention are as follows: a) Representative images of bacterial colonies on agar plates after different treatments, and b) Corresponding quantitative colony counts (n=3); c) Biomass of MRSA biofilm assessed by crystal violet staining after different treatments; d) Quantitative analysis of biofilm clearance rate (n=3); e) Analysis of the control group by flow cytometry using the DCFH-DA fluorescent probe; f) Intracellular reactive oxygen species level in MRSA cells in the Rh SACs+NIR-II+H2O2 treatment group; Bacterial activity was assessed by live / damaged / dead cell staining by flow cytometry: g) Control group, h) Rh SACs alone group, and i) RhSACs+NIR-II+H2O2 group; j) Schematic diagram of conformational changes of bacterial outer membrane at 0 ns, 1 ns, 10 ns, 20 ns, 30 ns, and 40 ns after interaction with the Rh single-atom catalyst; k) Rh The change in the Z-axis position of SACs during the simulated cell membrane penetration process; l represents the evolution of cell membrane density; Figure 4 Transcriptome profiling and functional enrichment analysis of the MRSA-treated and control groups of the polyethylene glycol-modified nitrogen-doped carbon-supported rhodium single-atom catalysts (Rh SACs) provided in this embodiment of the invention; a) is a heatmap showing the expression levels of 2659 protein-coding RNAs, clustered using an unsupervised method; b) is a principal component analysis plot showing the sample distribution, with blue dots representing control samples (C) and red dots representing treated samples (T); c) is a volcano plot of differentially expressed genes, with blue dots indicating significantly downregulated genes (log2FC < -1, corrected p-value < 0.05) and red dots indicating significantly upregulated genes (log2FC > 1, corrected p-value < 0.05); p-values ​​are calculated using B... The enjamini–Hochberg method was used for correction; d is a heatmap showing the expression patterns of differentially expressed genes; e is the gene set enrichment analysis of Gene Ontology (GO) entries and f is the enrichment analysis of KEGG orthologs (KO) entries, showing the significantly enriched entries (FDR < 0.05, estimated using the Storey method); g is the GO functional enrichment analysis of upregulated differentially expressed genes and h is the GO functional enrichment analysis of downregulated differentially expressed genes, showing the entries with a corrected p-value < 0.05 (corrected using the Bonferroni stepwise descent method); Figure 5This invention provides an embodiment of a protein-protein interaction network construction and hub gene identification based on differentially expressed genes for a single-atom rhodium catalyst (Rh SACs) modified with polyethylene glycol and supported on nitrogen-doped carbon. a) The protein-protein interaction network constructed from differentially expressed genes; b) Key functional modules extracted from the PPI network, where node color represents MCODE clustering score (darker color indicates higher score); node size corresponds to degree centrality (larger node indicates higher connectivity), and isolated nodes have been removed; c) A heatmap showing the expression levels of identified hub genes in each sample group after z-score normalization; d) Gene Ontology (GO) functional enrichment analysis of hub genes, showing entries with corrected p-values ​​< 0.05 (Bonferroni stepwise descent correction); e) A visualization of the hub genes and their significantly enriched KEGG pathway network. Figure 6 This invention relates to the in vivo anti-biofilm efficacy of polyethylene glycol-modified nitrogen-doped carbon-supported rhodium single-atom catalysts (Rh SACs) in a mouse subcutaneous abscess model. The data are presented as follows: a) Schematic diagram of abscess healing after treatment with Rh SACs dressings; b) Representative images of wound healing processes in each treatment group; c) Quantitative tracking results of abscess area changes over time; d) Representative photographs of MRSA colonies cultured on agar plates in each treatment group; e) Statistical analysis of abscess area at different time points; f) Quantitative results of bacterial load in infected tissue on day 10 of treatment; gi) Histopathological examination of infected tissue collected on day 10: g is H&E staining, h is Masson's trichrome staining, and i is Gram staining; j is quantitative analysis of inflammation intensity and k is quantitative analysis of collagen deposition. Data are presented as mean ± standard deviation (n=3), and the results represent three independent experiments. Figure 7 Immunohistochemical and immunofluorescence analyses of wound healing in a subcutaneous abscess model treated with polyethylene glycol-modified nitrogen-doped carbon-supported rhodium single-atom catalysts (Rh SACs) provided in this embodiment of the invention are presented below. a) Representative images of PCNA immunohistochemical staining in wound sections; b) Quantitative analysis of PCNA protein levels in wound tissues of each group; c) Immunofluorescence staining images of CD86 in wound sections; d) Corresponding CD86 expression levels in wounds of each group; e) Immunofluorescence staining images of CD206 in wound sections; f) Corresponding CD206 expression levels in wounds of each group. Data are presented as mean ± standard deviation (n=3), and the results represent three independent experiments. Figure 8This invention relates to the therapeutic effect of polyethylene glycol-modified nitrogen-doped carbon-supported single-atom rhodium catalysts (Rh SACs) in an MRSA-induced mouse pneumonia model. a) is a schematic diagram of the experimental design for evaluating antibacterial activity in the MRSA pneumonia model; b) is a representative photograph of mouse lung tissue isolated after specified treatment; c) is an evaluation of the anti-biofilm effect in the MRSA pneumonia infection model; d) is a quantitative analysis of bacterial load in lung tissue (n=3); eg) is the staining of representative pathological sections of lung tissue: e) H&E staining, f) Masson's trichrome staining, g) Gram staining; h) is immunofluorescence staining of lung sections showing CD206 (M2 macrophages) and CD86 (M1 macrophages) markers; i, j are the concentrations of CD86(i) and CD206(j) in bronchoalveolar lavage fluid of different treatment groups; data are presented as mean ± standard deviation (n=3), and the results represent three independent experiments. Figure 9 Aberration-corrected high-angle annular dark-field scanning transmission electron microscope image of Rh SACs provided in this embodiment of the invention; Figure 10 X-ray energy dispersive spectroscopy analysis of Rh SACs provided in the embodiments of the present invention confirmed the coexistence of Rh, C, N and O elements; Figure 11 X-ray energy spectra of different elements of Rh SACs provided in this embodiment of the invention; Figure 12 The high-resolution X-ray photoelectron spectra of Rh SACs provided in this embodiment of the invention are as follows: a is O 1s and b is C 1s. Figure 13 The ultraviolet-visible-near-infrared spectrum of Rh SACs provided in the embodiments of the present invention; Figure 14 The figure shows the gene set enrichment analysis (GSEA) results of the Rh SACs gene ontology (GO) entries provided in the embodiments of the present invention. All entries that meet the significance threshold of nominal P value < 0.05 are shown in the figure. Entries that meet the significant enrichment condition (FDR estimated by Storey method < 0.05) are marked in bold. Figure 15 Gene set enrichment analysis (GSEA) results of KEGG orthologous (KO) entries of Rh SACs provided in this embodiment of the invention. The figure shows all entries that meet the significance threshold of nominal P value < 0.05, and entries that meet the significant enrichment condition (FDR estimated by Storey method < 0.05) are marked in bold. Figure 16The biosafety assessment of Rh SACs provided in this embodiment of the invention; ac refers to the blood biochemical indicators 24 hours after subcutaneous injection of 50 μL of the nanozyme (Rh SACs) suspension (80 μg / mL) or PBS into healthy mice, and di refers to the results of routine blood tests. Figure 17 H&E staining images of major organs (heart, liver, spleen, and kidney) in mice after receiving different treatments with Rh SACs according to the embodiments of the present invention; Figure 18 The diagram illustrates the preparation of Rh SACs provided in this invention and their application in the treatment of subcutaneous abscesses and bacterial pneumonia; a) shows the preparation of Rh SACs using PEG-modified Rh single-atom medical nanozymes; b) shows the antibacterial process of Rh SACs; c) shows Rh SACs acting on subcutaneous abscesses and bacterial pneumonia sites in mice via injection and atomization, respectively, killing bacteria, clearing biofilms, synergistically downregulating the expression of pro-inflammatory factors and upregulating the expression of anti-inflammatory factors, thereby promoting the healing of skin and lung tissue. Detailed Implementation

[0029] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0030] In clinical practice, the inventors found that the treatment of deep tissue multidrug-resistant (MDR) bacterial infections (such as subcutaneous abscesses and bacterial pneumonia) faces multiple challenges, mainly manifested in the following key issues: (1) Limited efficacy of traditional treatment methods: Conventional antibiotic therapy has declined in efficacy due to increasingly serious drug resistance problems, and existing nanomaterial therapies are also difficult to effectively eliminate deep drug-resistant bacteria and their biofilm structures due to insufficient catalytic efficiency and limited tissue penetration. (2) Low catalytic efficiency in complex infection microenvironments: Deep tissue infections are often accompanied by acidic pH environments, hypoxia, and dense biofilm barriers. These complex microenvironments significantly reduce the catalytic efficiency of existing therapeutic materials. (3) Limitations of single treatment modalities: Individual catalytic therapy or photothermal therapy often cannot simultaneously achieve deep tissue penetration, efficient sterilization, and complete elimination of biofilms, leading to repeated infections and hindering the tissue repair process. (4) Lack of immune regulation and tissue repair promotion functions: Most existing treatment plans only focus on sterilization, neglecting the regulation of excessive inflammation in the infected area and the promotion of the tissue repair process, affecting the overall treatment effect.

[0031] It is worth noting that current nanozyme technology primarily serves non-biological applications. In industrial catalysis, design goals are entirely focused on catalytic efficiency and thermal stability; in environmental remediation, material design only needs to consider chemical stability; and in analytical detection, optimization focuses solely on detection sensitivity. These applications do not require consideration of core biomedical requirements such as biocompatibility, tissue penetration, adaptability to complex physiological environments, and immunomodulatory functions. Biomedicine, particularly in the treatment of deep tissue drug-resistant bacterial infections, faces a substantial adaptation dilemma. Existing nanozymes exhibit significantly reduced catalytic activity in acidic, hypoxic infection microenvironments, lack tissue penetration, and do not possess immunomodulatory functions, resulting in poor biocompatibility and safety because these characteristics were not considered in the original structural design of existing traditional nanozymes.

[0032] Therefore, developing a single-atom catalyst specifically designed to treat drug-resistant bacterial infections in deep tissues requires a comprehensive overhaul from material design and preparation processes to application strategies. This invention addresses these needs by providing a nitrogen-doped rhodium single-atom medical nanozyme exhibiting highly efficient NIR-II photothermal conversion, multi-enzyme mimicry catalytic activity, excellent biocompatibility, and active immunomodulation capabilities. It also includes a complete preparation method, synergistic application scheme, and in vitro and in vivo validation data, as detailed below: In a first aspect, the present invention provides a method for preparing a medical nanozyme for treating drug-resistant bacterial infections in deep tissues, the preparation method comprising the following steps: Rhodium trichloride, urea, and polyvinylpyrrolidone are mixed to obtain a mixture, wherein the mass ratio of rhodium trichloride, urea, and polyvinylpyrrolidone is (3.0~6.0) g : (80~120) g : (10~30) g; The mixture was subjected to staged pyrolysis under inert gas protection to obtain a nitrogen-doped carbon-supported single-atom rhodium catalyst. The nitrogen-doped carbon-supported single-atom rhodium catalyst was modified with polyethylene glycol to obtain the medical nanozyme.

[0033] Compared with existing technologies, the preparation process provided by this invention achieves comprehensive optimization of structure and performance through the synergistic control of specific raw material ratios and polyethylene glycol modification. Using urea as the nitrogen source, polyvinylpyrrolidone as the carbon source and structure directing agent, and rhodium trichloride as the rhodium source, and controlling their mass ratio within a suitable range, a nitrogen-doped carbon framework with a nanosheet morphology, high specific surface area, and rich in various nitrogen species and nitrogen vacancy defects can be constructed through segmented pyrolysis. This framework not only serves as a structural support, but its multi-component nitrogen doping and defect structure synergistically optimize the electronic properties of the carrier, providing abundant sites for rhodium atom anchoring, promoting the formation of a stable Rh-NC coordination structure, and ensuring that rhodium exists in an atomically uniformly dispersed form. This greatly improves atom utilization and catalytic efficiency, while effectively inhibiting the dissolution and release of metal ions, thus avoiding the cytotoxicity and systemic toxicity of free metals at the source.

[0034] For example, the mass ratio of rhodium trichloride, urea and polyvinylpyrrolidone is (3.0, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.6, 4.8, 5.0, 5.4, 5.8, 6.0) g : (80, 85, 90, 94, 96, 98, 100, 102, 104, 106, 110, 115, 120) g : (10, 14, 16, 18, 19, 20, 21, 22, 24, 26, 30) g.

[0035] Preferably, the mass ratio of rhodium trichloride, urea and polyvinylpyrrolidone is (3.6~4.8) g: (90~110) g: (14~26) g.

[0036] More preferably, the mass ratio of rhodium trichloride, urea and polyvinylpyrrolidone is (3.8~4.6) g: (95~105) g: (16~24) g.

[0037] Specifically, the segmented pyrolysis includes: the temperature of the first pyrolysis stage is 400~600℃, and the temperature of the second pyrolysis stage is 900~1100℃.

[0038] It should be noted that the two-stage pyrolysis process used in the preparation of the medical nanozyme provided by this invention plays a crucial role in regulating the final structure of the product. In the first pyrolysis stage, the decomposition of urea and polyvinylpyrrolidone occurs simultaneously with preliminary carbonization, and the rhodium trichloride precursor is moderately decomposed, achieving the initial doping of nitrogen species into the carbon framework, the pre-dispersion of Rh atoms, and the formation of a preliminary nanosheet structure. In the second pyrolysis stage, the high temperature enables Rh atoms to form a stable Rh-NC strong coordination structure with nitrogen sites, while optimizing the distribution and doping state of nitrogen species and improving the graphitization degree of the carbon framework. This gradual heating strategy synergistically avoids the high-temperature aggregation of metal atoms, ensures the single-atom-level dispersion of Rh, and constructs an ideal structure with high catalytic activity sites, high specific surface area carrier, and excellent stability. This is a key process guarantee for obtaining medical nanozymes with high antibacterial properties and good biocompatibility.

[0039] For example, the temperature of the first pyrolysis stage is 400℃, 420℃, 440℃, 450℃, 460℃, 480℃, 500℃, 520℃, 540℃, 550℃, 560℃, 580℃, or 600℃; preferably, the temperature of the first pyrolysis stage is 450℃ to 550℃.

[0040] For example, the temperature of the second pyrolysis stage is 900℃, 920℃, 940℃, 950℃, 960℃, 980℃, 1000℃, 1020℃, 1040℃, 1050℃, 1060℃, 1080℃, or 1100℃; preferably, the temperature of the second pyrolysis stage is 950℃ to 1050℃.

[0041] Specifically, the holding time for the first pyrolysis stage is 2-4 hours. The holding time for the second pyrolysis stage is 0.5-1.5 hours.

[0042] It should be noted that this invention significantly influences the material structure formation by controlling the holding time of the two-step pyrolysis. The longer first holding time ensures sufficient decomposition of the precursor and stable formation of the carbon framework, providing adequate thermodynamic conditions for effective nitrogen doping and rhodium pre-dispersion. Conversely, the shorter second holding time avoids excessive migration and aggregation of rhodium atoms at high temperatures and excessive graphitization of the carbon framework. This synergistic control of the time gradient ensures that rhodium atoms in the product are stably anchored in an atomically dispersed form on a moderately graphitized nitrogen-doped carbon framework, thereby simultaneously achieving high catalytic activity, structural stability, and ideal morphological characteristics.

[0043] For example, the holding time of the first pyrolysis stage is 2.0h, 2.2h, 2.5h, 2.6h, 2.8h, 3.0h, 3.2h, 3.4h, 3.5h, 3.8h, or 4.0h; preferably, the holding time of the first pyrolysis stage is 2.5 to 3.5h.

[0044] For example, the holding time of the second pyrolysis stage is 0.5h, 0.6h, 0.8h, 1.0h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, or 2.0h; preferably, the holding time of the second pyrolysis stage is 0.6 to 1.5h.

[0045] Preferably, during the pyrolysis process, the temperature is increased to the temperature of the first pyrolysis stage at a first heating rate of 4~6℃ / min (e.g., 4.5℃ / min, 4.8℃ / min, 5.0℃ / min, 5.2℃ / min, 5.5℃ / min). The temperature is then increased to the temperature of the second pyrolysis stage at a second heating rate of 1~3℃ / min (e.g., 1.5℃ / min, 1.8℃ / min, 2.0℃ / min, 2.2℃ / min, 2.5℃ / min).

[0046] It should be noted that this heating rate design synergistically ensures that the material ultimately possesses ideal atomic dispersion, stable coordination structure, and complete support morphology. The first heating rate is controlled within a suitable range, allowing the precursors (urea, polyvinylpyrrolidone, and rhodium trichloride) to decompose smoothly and synchronously during the heating process. This avoids structural stress caused by excessively rapid heating leading to violent decomposition and disrupting the uniformity of the carbon-nitrogen framework, or asynchronous component decomposition due to excessively slow heating affecting the uniformity of elemental distribution. The second heating rate is controlled within a more moderate range, providing sufficient thermodynamic relaxation time in the higher temperature range for the ordered reconstruction of the carbon framework, stable doping of nitrogen species, and full bonding of Rh atoms to nitrogen sites. This promotes the formation of a high-strength Rh-NC coordination structure while preventing nitrogen loss or structural defects caused by excessively rapid heating.

[0047] The staged pyrolysis is carried out under an inert atmosphere, and the specific control parameters include: (1) Atmosphere type: High-purity argon or nitrogen with a purity of ≥99.999% is used, with argon preferred, to provide a chemically inert environment, avoid potential side reactions between oxygen and metal or carbon at high temperatures, and ensure that Rh species exist stably in the target metallic state; (2) Gas flow rate: Before staged pyrolysis, the reaction chamber is purged with inert gas. During the purging stage, the inert gas flow rate is 100-200 mL / min and lasts for 30-60 minutes. During the pyrolysis stage, the inert gas flow rate is 50-100 mL / min. The gas is thoroughly purified before heating (high flow rate during purging stage) and a stable environment is maintained during the reaction (moderate flow rate during pyrolysis stage) to prevent interference from oxygen or moisture. (3) Pressure control: During the pyrolysis process, the system is kept under a slightly positive pressure with a gauge pressure of 0.5-5 kPa (5-50 mbar), which effectively prevents the infiltration of outside air and avoids the premature volatilization of precursors due to negative pressure, thus ensuring the precise control of the carbon / nitrogen ratio. (4) Atmosphere purity monitoring: The residual oxygen content of the detection system is ≤10 ppm before the start of pyrolysis, and the atmosphere purity is continuously monitored during the pyrolysis process; to ensure that the chemical environment is consistent for each synthesis and to obtain reproducible high-quality single-atom structures; (5) After the segmented pyrolysis is completed, the sample is naturally cooled to room temperature. During the cooling process, an inert protective gas (e.g., inert gas flow rate of 30-80 mL / min) is continuously introduced and a slightly positive pressure (0.5-5 kPa) is maintained until the furnace temperature drops below 150°C or to room temperature. This is to prevent the high-temperature sample from being oxidized due to thermal expansion and contraction during the cooling process, and is the last barrier to maintain the chemical stability and structural integrity of the catalyst.

[0048] Specifically, the mixing includes: mixing rhodium trichloride, urea and polyvinylpyrrolidone by grinding to obtain a uniformly mixed powdery mixture.

[0049] For example, the grinding method is mortar and pestle grinding. For instance, manual grinding for 30 minutes or more to ensure uniform mixing of the materials.

[0050] For example, after grinding, the average particle size of the powdered mixture is 5~10μm. Preferably, the particle size distribution of the powdered mixture satisfies D90≤5μm.

[0051] Specifically, the polyethylene glycol modification involves mixing and reacting the nitrogen-doped carbon-supported single-atom rhodium catalyst with polyethylene glycol in a solvent, followed by solid-liquid separation and purification, and then redispersing the resulting product in a buffer medium.

[0052] In one embodiment, the polyethylene glycol modification step includes: The nitrogen-doped carbon-supported single-atom rhodium catalyst was dispersed in a first solvent to obtain a catalyst dispersion. Polyethylene glycol is dissolved in a second solvent to obtain a polyethylene glycol solution; The catalyst dispersion was mixed with the polyethylene glycol solution and stirred at 20-30°C for 12-48 hours. The mixture after stirring reaction is subjected to solid-liquid separation and washing to obtain the medical nanozyme.

[0053] Further, the medical nanozyme is redispersed in a buffer medium to obtain a suspension of the medical nanozyme.

[0054] Specifically, in the polyethylene glycol modification, the mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to the polyethylene glycol is 1:(0.05~0.30). Exemplarily, the mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to the polyethylene glycol is 1:0.05, 1:0.1, 1:0.15, 1:0.20, 1:0.25, or 1:30. Preferably, it is 1:(0.1~0.2).

[0055] Specifically, in the polyethylene glycol modification, the molecular weight Mn of the polyethylene glycol is 2000-10000 Da; preferably 4000-8000 Da, such as 6000 Da.

[0056] Specifically, the dispersion concentration of the nitrogen-doped carbon-supported single-atom rhodium catalyst in the first solvent is 0.5-2.0 mg / mL; preferably 0.8-1.2 mg / mL.

[0057] Specifically, the concentration of polyethylene glycol in the second solvent is 0.05-0.30 mg / mL; preferably 0.1-0.2 mg / mL.

[0058] Specifically, the first solvent and the second solvent are each independently selected from water or a buffer solution with a pH value of 6.5-8.0; preferably, both the first solvent and the second solvent are water.

[0059] Preferably, the catalyst dispersion is mixed with the polyethylene glycol solution and reacted at 20-30°C with a stirring speed of 200-500 rpm for 20-30 hours.

[0060] Specifically, the solid-liquid separation is performed by centrifugation, with a centrifugal force of 8000-12000g and a centrifugation time of 5-15 minutes. The unit of centrifugal force, g, is relative centrifugal force (RCF), and its conversion relationship with centrifuge speed (RPM, revolutions per minute) and rotor radius (r) is: RCF (g) = 1.118 × 10⁻⁶. -5 ×r (cm) × RPM 2 .

[0061] Specifically, the washing is performed 1-3 times, and the washing solvent is water or a buffer solution with a pH of 6.5-8.0.

[0062] Specifically, the buffer medium is a phosphate buffer solution with a pH of 7.2-7.6; the concentration of the redispersed medical nanozyme suspension is 0.5-2.0 mg / mL, more specifically 0.8-1.2 mg / mL.

[0063] In a second aspect, the present invention provides a medical nanozyme obtained according to the preparation method described in the first aspect.

[0064] Specifically, the medical nanozyme is a single-atom rhodium catalyst supported on nitrogen-doped carbon and modified with polyethylene glycol; The medical nanozyme consists of a core structural unit and a surface functional layer: The core structural unit consists of atomically dispersed rhodium single atoms anchored in a nitrogen-doped carbon framework, forming a stable Rh-NC coordination structure; the nitrogen-doped carbon framework has a nanosheet structure. The surface functional layer is composed of polyethylene glycol modified on the surface of the core structural unit.

[0065] Specifically, the average thickness of the nanosheet structure is ≤10 nm, and the average lateral dimension is ≤2000 nm. This size structure brings several synergistic advantages: its ultrathin shape and suitable lateral dimension together endow the material with a high specific surface area, fully exposing Rh-NC catalytic active sites and enhancing enzyme-like catalytic efficiency; the abundant active sites at the edges facilitate direct action on bacteria and biofilms; at the same time, this size range makes it easy to diffuse in the interstitial space, improving the physical penetration ability to deep infection areas; in addition, the appropriate nanoscale facilitates gradual clearance through metabolic pathways, reducing the risk of long-term accumulation in the body, thereby improving therapeutic efficacy while ensuring biosafety.

[0066] For example, the average thickness of the nanosheet structure is 1~10 nm, and the average lateral dimension is 100~2000 nm.

[0067] Specifically, the nitrogen-doped carbon framework has a specific surface area of ​​200–800 m². 2 / g. For example, the nitrogen-doped carbon framework has a specific surface area of ​​200 m². 2 / g、300m 2 / g、400m 2 / g、500m 2 / g、600m 2 / g、700m 2 / g、800m 2 / g. This structural feature brings the following important advantages: First, the high specific surface area provides ample dispersion and anchoring sites for Rh single-atom active centers, significantly improving atom utilization and catalytic efficiency; second, the abundant surface active regions enhance contact and interaction with hydrogen peroxide and the biofilm matrix, promoting the efficient generation of hydroxyl radicals and physical penetration and disruption of biofilms; furthermore, the high specific surface area structure facilitates the loading and slow release of repair-related factors, synergistically promoting tissue regeneration; this characteristic also optimizes the photothermal conversion performance of the material, enhancing energy absorption and thermal conversion efficiency for near-infrared lasers. In summary, the high specific surface area is one of the key structural foundations for achieving efficient catalysis, deep penetration, synergistic therapy, and good biocompatibility in this invention.

[0068] Specifically, the nitrogen species in the nitrogen-doped carbon framework include oxidized N, graphitic N, pyrrole N, and pyridine N. This composite nitrogen-doped structure has the following synergistic advantages: different nitrogen species, by regulating the electronic structure and charge distribution of the carbon framework, synergistically optimize the catalytic performance of the Rh single-atom active center, reduce the activation energy barrier of hydrogen peroxide, and enhance peroxidase-like and oxidase-like mimicry activities; conductive nitrogen species such as pyridine nitrogen and graphitic nitrogen enhance the electronic conductivity of the material and promote charge transfer during the catalytic process; polar groups such as oxidized nitrogen improve the hydrophilicity and biocompatibility of the material, which is beneficial for dispersion and stability in physiological environments; the coexistence of multiple nitrogen species also enhances the structural stability of the carbon framework, preventing the inactivation or shedding of active sites in acidic infection microenvironments. This multi-component nitrogen-doped system provides important electronic structure and chemical environment support for medical nanozymes to maintain efficient and stable catalytic activity under complex physiological environments.

[0069] Specifically, the nitrogen-doped carbon framework contains nitrogen vacancy defects. These nitrogen vacancies, as active defect sites, can modulate the local electronic structure and charge distribution of the carbon framework, enhancing the electronic interaction between the support and rhodium single atoms, thereby stabilizing the Rh-NC active center and optimizing its catalytic performance. This defect structure can also participate in the catalytic process as an additional active site, synergistically promoting the activation of hydrogen peroxide and the generation of hydroxyl radicals, thus improving enzyme-like catalytic efficiency. The presence of nitrogen vacancies can also improve the conductivity and charge transport capacity of the carbon framework, accelerating the electron transfer process in the catalytic reaction. Furthermore, the defect structure enhances the chemical reactivity of the material surface, facilitating interaction with biofilm matrix components and improving the physicochemical synergistic anti-biofilm effect. Nitrogen vacancy defects, together with multiple nitrogen species, constitute key microstructural features for optimizing electronic structure, enhancing catalytic activity, and improving material stability.

[0070] Specifically, in the core structural unit, rhodium single atoms are atomically uniformly dispersed within a nitrogen-doped carbon framework, and no rhodium crystalline phase diffraction peaks are observed in the X-ray diffraction pattern. This structural feature brings the following key advantages: atomically uniform dispersion makes each rhodium atom an independent catalytic active center, achieving near 100% atomic utilization and greatly improving catalytic efficiency; the complete absence of rhodium crystalline phase diffraction peaks indicates that rhodium atoms are completely captured by the carbon framework in an amorphous form, without the formation of metal nanoparticles or clusters, avoiding the reduction of active sites and catalytic performance caused by metal agglomeration; this highly dispersed structure enhances the stability of the Rh-NC coordination bond, effectively inhibiting the dissolution and release of rhodium ions in the physiological environment and significantly reducing the risk of metal toxicity; simultaneously, the atomically dispersed structure optimizes the electronic state distribution of the material, giving it stronger light absorption in the near-infrared region and improving photothermal conversion efficiency. This structural feature is one of the core guarantees for the efficient, safe, and stable catalytic therapy achieved by this invention.

[0071] For example, the polyethylene glycol is attached to the surface of the core structural unit by covalent bonds or physical adsorption.

[0072] Specifically, the nitrogen-doped carbon-supported single-atom rhodium catalyst is prepared by mixing urea as a nitrogen source, polyvinylpyrrolidone as a carbon source and structure directing agent, and rhodium trichloride as a rhodium source, and then undergoing segmental pyrolysis under an inert atmosphere. The prepared nitrogen-doped carbon-supported single-atom rhodium catalyst is then modified with polyethylene glycol. The mass ratio of rhodium trichloride, urea and polyvinylpyrrolidone is (3.0~6.0) g: (80~120) g: (10~30) g.

[0073] For example, the CAS number of the rhodium trichloride is 20765-98-4, the molecular formula is RhCl3·xH2O, and the molecular weight is 209~210, for example 209.26.

[0074] For example, the urea has a CAS number of 57-13-6, a molecular formula of H2NCONH2, and a molecular weight of 60~61, for example 60.06.

[0075] For example, the polyvinylpyrrolidone has the CAS number 9003-39-8, the molecular formula is (C6H9NO)n, and the molecular weight Mw is 1,000,000~1,500,000 Da (level K88~96).

[0076] Specifically, in the polyethylene glycol modification, the mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to the polyethylene glycol is 1:(0.05~0.30).

[0077] For example, the molecular weight Mn of the polyethylene glycol is 1000 Da to 20000 Da, preferably 4000 to 8000 Da, such as 6000 Da.

[0078] The medical nanozyme provided by this invention achieves synergistic optimization of structure, catalytic activity, and biocompatibility by controlling the mass ratio of polyethylene glycol (PEG) to the nitrogen-doped carbon-supported rhodium single-atom catalyst during its preparation. Within this feed ratio range, the PEG-modified layer can form a complete hydrophilic protective layer on the nanozyme surface, effectively shielding the hydrophobicity and charge characteristics of the nanoparticle surface through steric hindrance, significantly reducing non-specific protein adsorption and immune recognition clearance, thereby prolonging in vivo circulation time and enhancing enrichment at the infection site. Simultaneously, this feed ratio ensures sufficient accessibility to the Rh-NC active sites, avoiding a decrease in catalytic activity due to excessive PEG coating. Using PEG with a molecular weight of 1000 to 20000 Da in conjunction with the nanozyme allows for the formation of a hydration layer with suitable thickness and stability, maintaining good material dispersibility and tissue penetration while ensuring effective protection.

[0079] The raw material ratios, the process and parameters described in detail in the preparation method of the first aspect, such as the staged pyrolysis, mixing, and polyethylene glycol modification, are all applicable to the product of the second aspect. The raw material ratios, the process and parameters described in detail in the product of the second aspect, such as the staged pyrolysis, and polyethylene glycol modification, are all applicable to the preparation process of the first aspect.

[0080] Thirdly, the present invention provides the use of medical nanozymes obtained by the preparation method described in the first aspect or medical nanozymes described in the second aspect in the preparation of medicaments for the prevention and / or treatment of deep tissue infections caused by drug-resistant bacteria.

[0081] Specifically, the concentration of the medical nanozyme used in vitro is 10~100 μg / mL.

[0082] For example, the concentration of the medical nanozyme when used in vitro is 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, or 100 μg / mL.

[0083] Specifically, the concentration of the medical nanozyme when applied locally in vivo is 50~120 μg / mL.

[0084] For example, the concentration of the medical nanozyme when applied locally in vivo is 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, or 120 μg / mL, preferably 60~100 μg / mL.

[0085] Specifically, the drug is applied in conjunction with second near-infrared laser irradiation.

[0086] Specifically, when used in combination, the wavelength of the second near-infrared laser is 1000~1350nm, and the power density is 0.25~1.50 W / cm². 2 The irradiation time is 3~10 minutes.

[0087] For example, the wavelength of the second near-infrared laser is 1064nm, 1100nm, 1150nm, 1200nm, 1250nm, 1270nm, 1300nm, or 1340nm.

[0088] For example, the power density of the second near-infrared laser is 0.25 W / cm². 2 0.50W / cm 2 0.75W / cm 2 1.0W / cm 2 1.25W / cm 2 1.5W / cm 2 , .

[0089] For example, the irradiation time of the second near-infrared laser is 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, and 10 min.

[0090] Preferably, the wavelength of the second near-infrared laser is 1064 nm, and the power density is 0.75 W / cm². 2 ~1.25W / cm 2 The irradiation time is 5-8 minutes.

[0091] Specifically, the drug includes the medical nanozyme and hydrogen peroxide, wherein the concentration of hydrogen peroxide is 0.05~0.5mM.

[0092] The volume of hydrogen peroxide is determined based on the body weight and concentration of the drug-treated subject. Preferably, within the above concentration range, the volume of hydrogen peroxide is 0.01~0.1 μL per gram of mouse body weight. Further, the volume of hydrogen peroxide is 0.01~0.02 μL per gram of mouse body weight.

[0093] For example, the concentration of hydrogen peroxide is 0.05mM, 0.08mM, 0.1mM, 0.12mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.4mM, or 0.5mM; preferably 0.05~0.15mM.

[0094] Preferably, in the aforementioned application, the medical nanozymes (Rh SACs) are used in combination with hydrogen peroxide (H2O2) and second near-infrared (NIR-II) laser irradiation. This achieves a synergistic therapeutic effect against drug-resistant bacterial infections in deep tissues through a triple synergistic mechanism of "photothermal-catalytic-immunomodulatory," resulting in better antibacterial, anti-biofilm, and tissue repair-promoting effects. Using the above-mentioned preferred parameter combination (e.g., medical nanozyme 60~100 μg / mL, H2O2: 0.05~0.15 mM, 1064 nm laser 0.75 W / cm²),... 2 ~1.25W / cm 2 Irradiation for 5-8 minutes showed good synergistic effects and controllable biocompatibility in in vitro and in vivo experiments in terms of antibacterial, anti-biofilm, and tissue repair properties.

[0095] Fourthly, the present invention provides a medicament for preventing and / or treating deep tissue infections caused by drug-resistant bacteria, the medicament comprising a medical nanozyme obtained by the preparation method described in the first aspect or a medical nanozyme described in the second aspect. The medicament also comprises hydrogen peroxide.

[0096] In some embodiments, the drug is a composite suspension containing the medical nanozyme and hydrogen peroxide, wherein the concentration of the medical nanozyme is 60-100 μg / mL and the concentration of the hydrogen peroxide is 0.05-0.15 mM.

[0097] The polyethylene glycol-modified nitrogen-doped rhodium single-atom catalysts (Rh SACs) prepared by the method provided in this invention exhibit significant beneficial effects in near-infrared II (NIR-II) photothermal enhanced nanozyme catalysis therapy, particularly in combating multidrug-resistant bacterial infections, promoting wound healing, and treating bacterial pneumonia. Specifically, these effects are manifested in the following aspects: 1. Highly effective synergistic antibacterial and anti-biofilm effects Rh SACs combine NIR-II photothermal conversion with multi-enzyme mimicry activity to achieve a synergistic "photothermal-catalytic" therapy. In in vitro experiments, Rh SACs, in combination with low-concentration H2O2 and NIR-II laser, achieved a bactericidal efficiency of over 99.9% against methicillin-resistant Staphylococcus aureus (MRSA) and effectively disrupted mature biofilm structures. This synergistic mechanism not only disrupts biofilm integrity through localized heat generation but also catalyzes the generation of highly reactive hydroxyl radicals (•OH) at Rh single-atom sites, achieving dual damage to both bacterial membrane structures and biomolecules, thus overcoming the limitations of traditional antibiotics and single-atom therapies in deep tissue infections.

[0098] 2. Promotes tissue repair and immune regulation In mouse models of subcutaneous abscess and bacterial pneumonia, RhSACs combined with H2O2 and NIR-II irradiation significantly accelerated the repair of infected tissue. On day 10 post-treatment, the wound area decreased by 92.1%, and pulmonary inflammatory infiltration was significantly reduced. Histological analysis showed that collagen deposition was more compact and orderly in the treatment group, and epithelial regeneration was complete. Furthermore, immunofluorescence and ELISA analyses confirmed that RhSACs could induce macrophage polarization from pro-inflammatory M1 to reparative M2 (CD86↓, CD206↑), thereby regulating the inflammatory microenvironment and promoting tissue repair.

[0099] 3. Excellent biosafety and biocompatibility Systematic in vitro and in vivo safety evaluations showed that Rh SACs have good blood compatibility, with hemolysis rates below the safety threshold; no significant hematological abnormalities or liver and kidney function damage were observed in mouse models. Histopathological examination revealed no pathological changes in vital organs such as the heart, liver, spleen, lungs, and kidneys, demonstrating low systemic toxicity at therapeutic doses and good potential for clinical translation.

[0100] 4. High catalytic efficiency and stability DFT calculations showed that atomically dispersed Rh-NC sites significantly reduced the H2O2 activation barrier, optimized the adsorption of reaction intermediates, and thus improved the activities of peroxidase-like (POD) and oxidase-like (OXD) enzymes. Experiments revealed that the photothermal conversion efficiency of Rh SACs was 50.8%, and it remained stable after five cycles of heating-cooling, indicating good catalytic and photothermal stability, making it suitable for multiple or long-term treatments.

[0101] 5. Economy and scalability Compared to platinum-based catalysts, Rh materials reduce costs by approximately 60-70%, and their synthesis methods are simple, scalable, and suitable for large-scale preparation. Furthermore, Rh SACs maintain high catalytic activity (>90% activity retention) even in acidic microenvironments, adapting to the complex physiological environment of infection sites.

[0102] In summary, the medical nanozyme of this invention, through optimized structural features and process control, ensures good blood compatibility, low immunogenicity, and acceptable in vivo accumulation behavior at therapeutic concentrations, thereby achieving systemic biocompatibility. The medical nanozyme utilizes PEG surface modification to provide a hydrophilic protective layer, a stable Rh-NC single-atom structure to inhibit metal ion release, nanosheet morphology and appropriate size to reduce physical damage and facilitate metabolism, and an optimized synthesis process to control the surface properties of the material. All these factors collectively ensure its excellent biocompatibility in vitro and in vivo, making it suitable for safety assessment requirements before clinical translation.

[0103] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and comparative examples.

[0104] Example 1: This embodiment provides a medical nanozyme for treating drug-resistant bacterial infections in deep tissues, and its preparation method is as follows: Step 1: Mix the raw materials Rhodium trichloride (4.2 g), urea (100 g), and polyvinylpyrrolidone (20 g) were mixed by grinding to obtain a uniformly mixed powder.

[0105] Step 2: Segmented pyrolysis The mixture was transferred to a quartz boat and placed in a tube furnace for programmed heating and staged pyrolysis under an inert atmosphere (argon, purity ≥99.999%). Specific parameters were as follows: First pyrolysis stage: Heat to 500℃ at a heating rate of 5℃ / min and hold for 3 h; Second pyrolysis stage: Heat to 1000℃ at a heating rate of 2℃ / min and hold for 1 h; After pyrolysis, the catalyst was naturally cooled to room temperature to obtain the nitrogen-doped carbon-supported single-atom rhodium catalyst (black powder).

[0106] Step 3: Polyethylene glycol modification (1) Preparation of catalyst dispersion: 10 mg of the nitrogen-doped carbon-supported single-atom rhodium catalyst was dispersed in 10 ml of deionized water to obtain the catalyst dispersion; Preparation of polyethylene glycol solution: Dissolve 1.5 mg of polyethylene glycol in 10 ml of deionized water to obtain a polyethylene glycol solution; The mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to polyethylene glycol is 1:0.15.

[0107] (2) Mixing reaction: The catalyst dispersion was mixed with the polyethylene glycol solution and reacted at 25°C with a stirring speed of 300 rpm for 24 hours to obtain the reaction mixture; (3) Separation and purification: Centrifuge the mixture after reaction at 10000 g for 10 minutes, collect the precipitate, and wash it twice with deionized water; (4) Redispersion: The washed solid product is redispersed in a phosphate buffer solution at pH 7.4 to obtain a medical nanozyme suspension with a concentration of 1.0 mg / mL, which is a single-atom rhodium catalyst modified with polyethylene glycol and supported on nitrogen-doped carbon.

[0108] The parameters that changed in Examples 2-10 and Comparative Examples 1-2 compared to Example 1 are shown in Table 1. The other parameters are the same as in Example 1.

[0109] Table 1 Comparison of key process parameters between the examples and comparative examples

[0110] Table 2: Comparison of structural characterization and performance test results between the examples and comparative examples (test methods and conditions are the same as in Example 1)

[0111] The following describes the structural characterization and performance testing of the nitrogen-doped carbon-supported single-atom rhodium catalyst (Rh SACs) modified with polyethylene glycol, using Example 1 as an example.

[0112] (1) Structural characterization of Rh SACs; 1.1 Test Conditions: A series of characterization tests, such as transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and X-ray photoelectron spectroscopy (XPS), were performed on the prepared Rh SACs to study their morphology, composition, and electronic states. Ultraviolet-visible-near-infrared spectroscopy (Shimadzu UV3600i Plus) was used to determine optical properties. The atomic distribution of the Rh species was confirmed using an aberration-corrected TEM TitanCubed Themis G2 300.

[0113] 1.2 Results and Analysis: like Figure 1 As shown in Figure a, Rh SACs medical nanozymes were successfully synthesized using a controlled release strategy of metal atoms. Specifically, polyvinylpyrrolidone, urea, and rhodium trichloride were placed in a high-temperature quartz reactor for thermal decomposition. Through controlled release, Rh atoms can be efficiently captured by N-doped carbon, generating stable mononuclear Rh active sites, ultimately forming an Rh SACs medical nanozyme platform anchored in N-doped carbon. Figure 1 Images b and c in the figure show TEM and high-resolution TEM images of the synthesized Rh SACs, clearly demonstrating the nanosheet morphology with high specific surface area. Selected area electron diffraction patterns further reveal the exposed crystal planes. The direct state of Rh was observed at sub-angstrom resolution using aberration-corrected high-angle annular dark-field scanning transmission electron microscopy. Representative HAADF-STEM images ( Figure 1 d and Figure 9 The isolated bright spots were uniformly distributed on the carbon matrix, confirming the atomic-level isolation of the Rh species corresponding to the Rh-N / C structural motif. X-ray diffraction analysis ( Figure 1The peak at (e) in the image is near 25.2°, indicating the (002) crystal plane of the carbon support. No evidence of a Rh crystalline phase was found; the X-ray diffraction pattern shows no diffraction peaks for any rhodium crystalline phase, further demonstrating the strong atomic dispersion of Rh. This is compared with the diffraction card for standard hexagonal Rh (PDF#05-0685). (Elemental distribution map of energy-dispersive X-ray spectra...) Figure 1 f) shows the overlapping distribution of C, N, O, and Rh, indicating atomically uniform doping of Rh in the carbon matrix. The elemental composition of the prepared material was investigated by X-ray energy-dispersive spectroscopy. Figure 10 Spectroscopic analysis confirmed the presence of four elements: Rh, C, N, and O in the material. The results indicate that the target element Rh has been incorporated into the material system, which is primarily composed of a CNO substrate. Nitrogen vacancies in the sample were characterized using EPR spectroscopy. Figure 1 (g in the image). The results confirmed the presence of nitrogen vacancy defects in the N-doped carbon framework. The full spectrum of X-ray photoelectron spectroscopy (e.g.) Figure 1 h and Figure 11 The image shows clear signals for C, N, O, and Rh. This confirms the successful incorporation of all target elements. High-resolution N 1s spectra (shown in the image) Figure 1 In the i) group, the deconvolution can be divided into four nitrogen species: oxidized N, graphitic N, pyrrole N, and pyridine N. High-resolution Rh 3d spectra ( Figure 1 As shown in j), Rh mainly exists in the metallic state (Rh 0 ) exists, and at the same time, due to Rh Electron interaction between N / C ( Figure 12 ), contains a certain amount of oxidized species (Rh δ+ In summary, these detailed characterization results verify the formation of Rh single atoms stabilized by Rh-NC coordination and elucidate the chemical composition, microstructure, and elemental state of the prepared material.

[0114] The above structural characterization results are mainly illustrated using Example 1 as an example. This invention employs systematic structural characterization methods to conduct a comprehensive structural analysis of the medical nanozymes in each example, and all relevant characteristics have been fully verified. 1) Core Structure Verification: Multiple characterization methods confirmed that the core structural unit consists of atomically dispersed rhodium single atoms anchored in a nitrogen-doped carbon framework, forming a stable Rh-NC coordination structure. Specifically, the rhodium single atoms are atomically uniformly dispersed within the nitrogen-doped carbon framework, and no rhodium crystalline phase diffraction peaks are observed in the X-ray diffraction pattern.

[0115] 2) Verification of nitrogen doping characteristics: X-ray photoelectron spectroscopy analysis confirmed that the nitrogen species in the nitrogen-doped carbon framework include oxidized N, graphitic N, pyrrole N, and pyridine N. Electron paramagnetic resonance spectroscopy and other tests further confirmed the presence of nitrogen vacancy defects in the nitrogen-doped carbon framework.

[0116] 3) Confirmation of carrier morphology: Through joint observation by transmission electron microscopy (TEM), atomic force microscopy (AFM) and scanning electron microscopy (SEM), it was clearly confirmed that the nitrogen-doped carbon framework has a nanosheet structure, and the average thickness of the nanosheet structure is 1-10 nm, and the average lateral size is 100-2000 nm.

[0117] 4) Specific surface area determination: The specific surface area of ​​the nitrogen-doped carbon framework was accurately determined by nitrogen adsorption-desorption isotherm test (BET method), confirming that it was 200-800 m² / g.

[0118] 5) Surface functional layer confirmation: The analysis results confirmed that the surface functional layer is composed of polyethylene glycol modified on the surface of the core structural unit, forming a complete core-shell structure.

[0119] (2) Photothermal properties and multi-enzyme mimicry activity of Rh SACs; 2.1 Test conditions: Photothermal performance: Under NIR-II laser (1064 nm), at different power densities (0.25, 0.5, 0.75 and 1.0 W / cm²), 2 Each of the described medical nanozymes (Rh SACs) suspensions was irradiated for 6 minutes. Temperature changes were recorded every 20 seconds using an infrared thermal imaging camera.

[0120] To investigate the photothermal properties of the aforementioned medical nanozymes (Rh SACs), a power-tunable 1064 nm laser (power density 1.0 W / cm²) was used. 2 The medical nanozyme (RhSACs) suspension at different concentrations (0, 20, 40, 60, and 80 µg / mL) was irradiated with 100 µL of the solution, and the temperature change over 10 minutes was recorded using an infrared thermal imager (FLIR A350, USA). Infrared thermal imaging images were also acquired simultaneously. To investigate the photostability of the material, the Rh single-atom catalyst solution, i.e., the Rh SACs suspension (80 µg / mL, 100 μL), was first irradiated for 10 minutes, then the laser was turned off and allowed to cool naturally, and then the laser was turned back on. This cycle was repeated 5 times, and the temperature change was recorded. The photothermal conversion efficiency (η) was calculated using the classical formula of the heat balance model established by Roper et al. (Roper et al., J.Phys. Chem. C, 2007, 111, 3636-3641).

[0121] Multienzyme mimicry: The OXD mimicry activity of Rh SACs was assessed using TMB as a substrate in acetate buffer (pH 5.6). UV-Vis-NIR absorption spectra of different concentrations of Rh SACs (0–80 μg / mL) incubated with 1.0 mM TMB at room temperature for specific time intervals were recorded. POD mimicry activity was assessed using TMB and H2O2 under similar acidic conditions. Mixtures of Rh SACs (0–80 μg / mL) with 1.0 mM TMB and 1.0 mmol H2O2 were prepared. One group was incubated in the dark at 25°C, and the other group was treated with NIR-II (1.0 W / cm²). 2 Irradiation was performed at 1064 nm. Spectral changes were monitored over time. The effect of different concentrations of H2O2 was examined, with varying amounts of H2O2 added to a mixture of 1.0 mM TMB and 80 μg / mL Rh SACs. After 1 minute of reaction, samples were obtained from the light-shielded group and the NIR-II irradiated group (1.0 W / cm²). 2 The absorbance of the group at the characteristic wavelength (1064nm).

[0122] 2.2 Results and Analysis: Temperature variations and •OH formation can be used to investigate the photothermal conversion performance and biocatalytic activity of Rh SACs. Ultraviolet-Visible-Near Infrared Spectroscopy (UV-Vis-NIR spectroscopy) Figure 13 This study demonstrated its significant absorption capacity within the NIR-II biological window and confirmed its potential as a photothermal agent. The present invention investigated the temperature response under 1064 nm laser irradiation at different concentrations and power densities. Results showed that in Rh SACs solution (80 μg / mL) and at a power density of 1.0 W / cm², the temperature rose to 51.7°C within 6 minutes (e.g., [missing information]). Figure 2 As shown in ac), this demonstrates its excellent photothermal conversion effect. After five heating and cooling cycles, the material exhibits stable photothermal properties (as shown in ac). Figure 2 (d) The photothermal conversion efficiency was calculated to be 50.8% ( Figure 2 (e in the text).

[0123] The inventors further investigated the multi-enzyme mimicry capabilities of Rh SACs, utilizing the catalytic properties of Rh single-atom sites. Colorimetric analysis using TMB confirmed that the Rh SACs materials exhibited OXD and POD mimicry activities. OXD mimicry ( Figure 2 f and i in the POD simulation ( Figure 2 The catalytic activity of (g and j) in the catalyst increased with increasing reaction time and catalyst concentration. The simulated POD activity was observed to be enhanced under NIR-II laser irradiation. Figure 2(h and k in the original text). To elucidate the catalytic mechanism, DFT calculations were used in this study. Through structural optimization (… Figure 2 The l in the figure reveals that Rh atoms can be stably anchored on the support surface. Furthermore, charge density differential analysis (CDI) Figure 2 The m in the figure indicates a strong electronic interaction between the Rh site and the support. The TMB adsorption energy results ( Figure 2 The n and o values ​​in the figure show that Rh SACs exhibit a high adsorption energy (-1.91 eV) and favorable electron transfer capabilities, which is confirmed by work function calculations. The free energy calculations of the intermediates (e.g., ...) Figure 2 (As shown by p and q in the figure) indicates that the energy barriers of the rate-determining steps of H2O and H2O2 activation are significantly reduced on Rh SACs. In particular, OH... The near-thermal free energy of desorption indicates that the single-atom Rh site further optimizes the reaction pathway. The electron distribution of Rh was shown to enhance electron mobility. These findings confirm the experimental observations and collectively elucidate the structural origin of the superior dual-enzyme mimicry activity of Rh SACs.

[0124] (3) In vitro antibacterial and antibiofilm activities of Rh SACs; 3.1 Test Conditions: MRSA was selected as the model microorganism. The bacteria were cultured overnight in TSB at 37°C using logarithmic growth phase cultures. The antibacterial experiment consisted of six groups: control, NIR-II only, Rh SACs, Rh SACs+H2O2, RhSACs+NIR-II, and Rh SACs+H2O2+NIR-II. Final concentration: MRSA was 10... 7 CFU / mL, H2O2 concentration of 0.1 mM and volume of 1 μL (mouse weight 60-80g), Rh SACs of 80 μg / mL. After treatment at room temperature for 2 hours, groups (2), (5) and (6) were treated with NIR-II laser (1.0 W / cm²). 2 Irradiation was performed at 1064 nm for 6 minutes. Subsequently, the bacterial suspension was diluted 625-fold, spread on TSB agar plates, and incubated at 37°C for 16 hours to count colonies. For biofilm experiments, MRSA was cultured in 6-well plates (250 µL per well, 10⁻¹⁰ μL). 7 (CFU / mL bacteria + 5 mL TSB) was incubated at 37°C for 24 h. After biofilm formation, the supernatant was aspirated, and the plate was treated using a method similar to that used for antibacterial testing. The plate was dried and stained with 5% crystal violet for 20 minutes. It was washed and dried again. The biofilm was dissolved in 33.3% glacial acetic acid, and the absorbance was recorded at 590 nm.

[0125] 3.2 Results and Analysis: Excellent biocatalytic activity, NIR-II photothermal properties, and good biocompatibility make Rh SACs a highly efficient medical nanozyme platform for antibacterial and anti-biofilm applications. Due to its two-dimensional structure and atomically dispersed Rh active sites, Rh SACs can effectively disrupt bacterial membrane integrity and diffuse through dense biofilm matrices. The inventors believe that the strong antibacterial efficacy of their products stems from the synergistic effect of CDT (chemodynamic therapy) and PTT (photothermal therapy). Quantitative colony formation assay (…) Figure 3 Figures a and b in the table show that the combined use of Rh SACs with H2O2 and NIR-II laser irradiation resulted in a biomass eradication efficiency exceeding 99.9% against MRSA. To further evaluate the biofilm eradication capacity, crystal violet staining was used to assess biofilm biomass (…). Figure 3 (c and d in the original text). The combined treatment group showed a significant decrease in biofilm formation and bacterial activity, indicating that Rh SACs can disrupt biofilm structures and kill bacteria embedded within them. According to flow cytometry data (… Figure 3 The intracellular ROS levels of MRSA cells were measured using the DCFH-DA fluorescent probe (e and f in the figure), showing an increase in ROS fluorescence intensity from 42.57% in the control group to 65.06% after Rh SACs+H2O2+NIR-II treatment. This indicates that the treatment generated a large number of •OH free radicals on or inside the bacteria, demonstrating its high efficiency. Furthermore, the live / damaged / dead bacterial ratio was analyzed by flow cytometry. Figure 3 The presence of gi in the Rh SACs further confirmed these antibacterial effects. The damaged / dead bacteria ratios in the control group (7.71% / 2.81%) and the Rh SACs alone group (8.34% / 3.01%) were actually quite similar. These ratios increased more significantly when stimulated with NIR-II laser and H2O2 (9.67% / 3.56%), indicating a synergistic effect of the combined treatment. Based on the significant increase in the damaged / dead bacteria ratio compared to the control group, the Rh SACs+H2O2+NIR-II system can be confirmed to have higher antibacterial efficacy. The inventors performed molecular dynamics simulations to obtain mechanistic insights. The results showed that Rh SACs adsorbed within 40 nanoseconds and subsequently penetrated the bacterial outer membrane (OM). Density distribution curves showed initial contact at 0 nanoseconds, while complete transmembrane transport was observed at 40 nanoseconds. Figure 3 The results (jl) indicate the membrane permeation characteristics of Rh SACs materials. In summary, these results demonstrate that Rh SACs exhibit excellent antibacterial and anti-biofilm activity in vitro due to the synergistic effect of CDT and PTT. This will lay a solid foundation for the future development of anti-infective drugs based on single-atom catalysts.

[0126] (4) Transcriptional profiling analysis after MRSA treatment; 4.1 Test conditions: Principal component analysis was performed using the `prcomp` function in the `stats` package of R to assess overall sample similarity. Differential expression analysis was performed using the `limma` package. Functional enrichment analysis of biological processes, gene ontology terms, and KEGG pathways was performed using Metascape. Gene set enrichment analysis was performed using the `gseGO` function in the `clusterProfiler` package. The `Mm.eg.db` database was used. Visualization was performed using GraphPad Prism 8.0, R software, and Cytoscape 3.10.0.

[0127] 4.2 Results and Analysis: After treatment, the inventors found that the expression of 2659 mRNAs in MRSA changed. Unsupervised clustering and principal component analysis of the expression profiles showed good separation between the treatment group and the control group. Figure 4 (a and b in the original text). Differential expression analysis showed that 403 mRNAs were upregulated and 455 mRNAs were downregulated. Figure 4 c and d in the gene set enrichment analysis revealed many significantly altered GO terms and KEGG pathways (c and d). Figure 14 , Figure 15 According to the results () Figure 4 (e) Some upregulated biological processes were "molypterin cofactor biosynthesis", "histidine biosynthesis", and "protein hydrolysis", while "de novo IMP biosynthesis" and "ribosome structural components" were significantly downregulated. Based on KEGG pathway analysis results ( Figure 4 In the f group, "riboflavin metabolism" and "galactose metabolism" were upregulated. However, "one-carbon library via folic acid," "MRSA infection," "ribosomes," and "two-component system" were downregulated. GO and KEGG enrichment analyses of differentially expressed genes showed ( Figure 4 The gene (g) is enriched in biological processes, including histidine biosynthesis, lactose catabolism, urea metabolism, cofactor biosynthesis, and organic nitrogen compound metabolism; cellular components such as cytoplasm; molecular functions including serine peptidase activity, nickel cation binding, purine nucleotide binding, and unfolded protein binding; and pathways including histidine metabolism, biosynthesis of various other secondary metabolites, and nucleotide excision repair. Biological processes with significantly downregulated gene enrichment include purine ribonucleotide metabolism, nucleotide biosynthesis, ribonucleotide metabolism, pathogenesis, and tetraterpenoid metabolism. Figure 4(h in the original text). Furthermore, these genes are highly enriched in biological processes including intracellular, cytoplasmic, and cytoplasmic fractions; molecular functions such as cytochrome c oxidase activity, carbon-nitrogen bond ligase activity, and rRNA binding. Additionally, pathways including purine metabolism, carotenoid biosynthesis, and ribosomes are highly enriched in these genes. Based on the results, this treatment induced extensive metabolic reprogramming in MRSA, enhancing protein quality control and proteolytic activity while inhibiting growth-related processes such as ribosome biogenesis and nucleotide synthesis. Downregulation of pathways associated with infection and the two-component system may further contribute to the attenuation of treatment-encoded virulence mechanisms.

[0128] To detect key genes in MRSA, the inventors constructed a protein-protein interaction network of differentially expressed genes affected after treatment. Of the 858 differentially expressed genes, 778 were involved in PPI (protein-protein interaction). Figure 5 (a) Using the MCODE plugin, the inventors discovered 36 clusters, of which 3 clusters (MCODE score > 10) contained 68 genes. Figure 5 (b) After treatment, 15 genes were found to be upregulated, while 53 were downregulated ( Figure 5 c). Functional enrichment analysis showed that ( Figure 5 (d) The above gene sets are enriched in biological processes, such as "purine nucleobase metabolism," "translation," "ribonucleoside monophosphate biosynthesis," and organic nitrogen compound biosynthesis; cellular components such as "intracellular," "cytoplasmic," "ribosomes," and "ribosomal subunits"; molecular functions such as "translation factor activity, RNA binding," "ligase activity, carbon-nitrogen bond formation," and "rRNA binding"; KEGG pathways such as "purine metabolism," "alanine, aspartate, and glutamate metabolism," and ribosomes. Almost all genes enriched in these KEGG pathways were downregulated after treatment. Figure 5 (e). Based on these results, functions related to translation mechanisms and nucleotide biosynthesis were extensively suppressed. The small number of upregulated genes found in the enrichment analysis may suggest compensatory metabolic responses or cellular stress mechanisms. These may indicate survival strategies against antimicrobial agents. Transcriptional profiling analysis after MRSA treatment showed that the catalytic properties of the medical nanozymes (Rh SACs) described in the embodiments of the present invention include both oxidase and peroxidase properties.

[0129] (5) Evaluation of Rh SACs' promotion of wound healing in mice; 5.1 Test conditions: 50 μL of MRSA suspension (1×10⁻⁶) was injected into the dorsal region of female BALB / c mice (5-6 weeks old). 7A mouse subcutaneous abscess model was established using CFU / mL. After 24 hours, the mice were randomly divided into five groups (n=10): (1) PBS; (2) Rh SACs; (3) Rh SACs+NIR-II; (4) Rh SACs+H2O2; (5) Rh SACs+H2O2+NIR-II. 50 μL of Rh SACs (80 μg / mL) was injected locally into groups (2), (3), and (5). Groups (3) and (5) were irradiated with NIR-II laser (1.0 W / cm²). 2 Irradiation was performed at 1064 nm for 6 minutes, and temperature changes were monitored using an infrared camera. Groups (4) and (5) were injected with 0.1 mM H2O2 at a volume of 1 μL. Abscess formation was photographed on days 0, 1, 3, 5, 7, and 10, and mice were sacrificed on day 10. Skin samples were homogenized to obtain bacterial load, and major organs (heart, liver, spleen, lung, and kidney) were collected for histopathological studies, including H&E staining, Masson staining, Gram staining, and immunofluorescence.

[0130] 5.2 Results and Analysis: To systematically evaluate the therapeutic capabilities of Rh SACs, including photothermal, catalytic, and antibacterial properties, a subcutaneous abscess model was established in MRSA-infected mice. Figure 6 (a) Fifty mice were randomly divided into five groups: PBS, Rh SACs, Rh SACs+NIR-II, Rh SACs+H2O2, and Rh SACs+H2O2+NIR-II. During the 10-day observation period, the wound contraction was most significant in the Rh SACs+H2O2 and Rh SACs+H2O2+NIR-II groups. Figure 6 (b and c in the text). According to quantitative analysis, the wound area reduction rate was greatest in the Rh SACs+H2O2+NIR-II group (92.1%). Figure 6 (e). On the tenth day, the inventors homogenized the abscess tissue and counted the bacterial colonies (the bacterial colony count results are shown in [reference]). Figure 6 (d and f in the text). The results showed that the bacterial load in the RhSACs+H2O2+NIR-II group was significantly lower (p < 0.001). This is consistent with the inventors' in vitro studies. Histological analysis by H&E staining ( Figure 6 Masson's trichrome staining (g and j) showed that the PBS and Rh SACs groups resulted in severe crusting. The Rh SACs+H2O2 and Rh SACs+NIR-II groups showed partial healing. Ultimately, the Rh SACs+H2O2+NIR-II group resulted in complete epithelial regeneration and wound closure. Masson's trichrome staining of the latter group (g and j) showed... Figure 6 The h and k values ​​indicate that the collagen is more tightly packed and organized. Gram staining ( Figure 6 (i) demonstrated the presence of frequent bacteria in the PBS group, exhibiting scattered MRSA colonization, while only sparse bacteria were found in the Rh SACs+H2O2+NIR-II group. Using antibodies targeting macrophage polarization and cell proliferation, the molecular mechanisms of inflammation resolution and tissue regeneration during wound healing were determined by immunohistochemistry and immunofluorescence. Immunohistochemical staining for proliferating cell nuclear antigens (…) Figure 7 (a) and (b) show that PCNA expression was lower in the PBS group. These immunohistochemical results demonstrate suppressed treatment response and chronic inflammation. In contrast, PCNA expression was significantly upregulated in the Rh SACs+H2O2+NIR-II group, indicating enhanced proliferation of keratinocytes, skin appendage cells, and other types of regenerative cells. Immunofluorescence results of the combined treatment group ( Figure 7 The cf. (see figure) showed that the expression of the M1 marker CD86 (red) was significantly downregulated, while the expression of the M2 marker CD206 (green) was upregulated, indicating a shift in macrophage polarization towards the M2 type. This suggests that the inflammatory response was effectively regulated, while simultaneously creating a microenvironment conducive to tissue repair. In summary, Rh SACs, under the synergistic effect of H2O2 and NIR-II irradiation, can effectively clear deep tissue infections. Furthermore, this treatment can alleviate inflammation by promoting M2 macrophage polarization. This also helps to enhance collagen deposition and angiogenesis, thereby accelerating wound healing. This invention opens up new possibilities for Rh SACs as a universal and effective bactericide and wound healing agent.

[0131] (6) In vivo evaluation of Rh SACs in an MRSA-infected pneumonia model; 6.1 Test conditions: Male BALB / c mice (6-8 weeks old) were used. After exposing the trachea through a midline neck incision, 50 μL of a bacterial suspension was injected into the trachea. The bacteria in the suspension were MRSA, type ATCC 43300, at a concentration of 1×10⁻⁶. 7 CFU / mL was used to establish a pneumonia model, and the wound was then sutured. Five mice from each treatment group (n=10) were randomly assigned to (1) the control group; (2) the Rh SACs group; (3) the Rh SACs+NIR-II group; (4) the Rh SACs+H2O2 group; and (5) the Rh SACs+H2O2+NIR-II group. Mice were sacrificed 24 hours later. Lung tissue was collected for histopathological and immunofluorescence staining. The heart, liver, spleen, and kidneys were stained with H&E. The inventors collected bronchoalveolar lavage fluid and measured the CD86 and CD206 values ​​using a commercial ELISA kit. All procedures involving animals were approved by the Animal Ethics Committee of Wenzhou Medical University (Approval No.: SYXK-2021-0020).

[0132] 6.2 Results and Analysis: Based on the strong in vitro antibacterial activity and wound healing effects of Rh SACs, the inventors further evaluated their in vivo antibacterial and anti-biofilm properties using an MRSA-induced mouse pneumonia model to simulate deep tissue infections caused by bacteria. Figure 8 (a) In this context, mice were treated with MRSA via nebulization 12 hours after infection. Lung tissue was collected and examined grossly 48 hours after treatment. Figure 8 (b) According to the results, Rh SACs exhibited superior antibacterial and anti-biofilm effects through a synergistic mechanism involving CDT and PTT. Subsequently, lung tissue homogenates were used to estimate bacterial viability using the standard plate dilution method. Figure 8 (c and d in the text). The results showed that the Rh SACs+H2O2+NIR-II treatment group had the fewest bacterial colonies in the lung tissue. To further evaluate lung repair and bacterial clearance, the inventors performed histological analysis, including H&E staining (…). Figure 8 e), Masson staining ( Figure 8 f) and Gram staining ( Figure 8 The g in the text is missing. H&E and Masson staining showed significant neutrophil infiltration in the lungs of other treatment groups. Conversely, the Rh SACs+H2O2+NIR-II group showed significantly reduced inflammatory infiltration and improved tissue repair; Gram staining confirmed the absence of bacteria (purple areas) that should have been present in the plate count in this group's lung tissue. M1 macrophages primarily secrete pro-inflammatory cytokines, while M2 macrophages are involved in tissue repair and stimulate anti-inflammatory functions. CD86 is a surface marker of M1 macrophages, and CD206 is a surface marker of M2 macrophages. Immunofluorescence staining of lung sections from an MRSA-infected pneumonia model (…) Figure 8 The h-values ​​in the image show that Rh SACs combination therapy significantly upregulated CD206 expression (green) and downregulated CD86 expression (red), indicating a shift in macrophage polarization from pro-inflammatory and pro-repairing to anti-inflammatory and tissue-repairing. To further analyze inflammatory changes, the inventors used ELISA to quantitatively detect the levels of CD86 and CD206 in bronchoalveolar lavage fluid. Figure 8 (i and j in the text). The results showed that after Rh SACs+H2O2+NIR-II treatment, CD86 levels in BALF decreased while CD206 levels increased, effectively clearing bacteria from deep tissue infections, reducing pneumonia-related inflammation, and providing crucial protection against lung injury caused by an overactive inflammatory response.

[0133] (7) Safety assessment of Rh SACs; 7.1 Test Conditions: An in vitro hemolysis test was performed to assess the blood compatibility of Rh SACs. Mouse plasma was mixed with an equal volume of PBS (pH 7.4) and incubated with Rh SACs at final concentrations of 50 µg / mL, 100 µg / mL, 200 µg / mL, 400 µg / mL, and 80 µg / mL at 37°C for 2 hours. After centrifugation at 8000 rpm for 5 minutes, the absorbance of the supernatant was measured at 540 nm. The hemolysis rate was calculated using the formula: hemolysis rate (%) = (As - An) / (Ap - An) × 100%, where As, Ap, and An are the absorbances of the test sample, positive control (complete hemolysis), and negative control (PBS), respectively. For in vivo hematologic analysis, mice were divided into two groups (n=3): the PBS group and the Rh SACs group (80 µg / mL, 50 μL, intraperitoneal injection). Blood was drawn from the posterior orbital venous plexus 24 hours later for complete blood cell count and biochemical analysis.

[0134] 7.2 Results and Analysis: To assess the biosafety of Rh SACs, the inventors systematically conducted in vivo hematological studies. Complete blood count reports indicated that all parameters were within the normal range, including red blood cell count, mean corpuscular volume, platelet count, granulocyte percentage, lymphocyte percentage, and monocyte percentage. Furthermore, as... Figure 16 As shown in the ac data, there were no significant differences. Analysis of blood biochemical parameters indicating liver and kidney function, including blood urea nitrogen, alanine aminotransferase, and aspartate aminotransferase, showed no significant changes. Figure 16 The results (di) demonstrated that Rh SACs did not cause significant hematologic toxicity or liver and kidney damage. Furthermore, histopathological sections of vital organs (such as the liver, heart, lungs, kidneys, and spleen) were evaluated to examine for any organ toxicity that Rh SACs treatment of bacterial pneumonia might induce. Figure 17 Histological results showed that all organ structures remained intact without any pathological changes, including no congestion, edema, inflammatory cell infiltration, or necrosis, indicating that Rh SACs did not cause significant organ toxicity during treatment. In conclusion, hematological and histopathological results demonstrate that Rh SACs possess good biocompatibility and high in vivo safety, making them suitable for biomedical applications.

[0135] All chemicals and reagents used in the embodiments of this invention are of analytical grade. 3,3',5,5'-Tetramethylbenzidine (TMB), rhodium trichloride hydrate (catalog number R347826-1g), urea (catalog number U111902), hydrogen peroxide, polyvinylpyrrolidone (catalog number P110610-500g), and polyethylene glycol (catalog number P615504-250g, Mn 6000) were purchased from Aladdin. Tryptone soybean broth and agar were purchased from Solarbio. The MVL-210 laser light source was provided by CERMA PRECISION.

[0136] Theoretical calculations: Spin-polarized density functional theory calculations were performed using the CP2K program. A mixture of Gaussian functions and plane-wave basis sets was employed. Under the generalized gradient approximation, the Perdew-Burke-Ernzerhof functional was used to handle exchange-based interactions. The Goedecker-Teter-Hutter pseudopotential was used to simulate the core electrons. Simultaneously, the DZVP-MOLOPT-SR-GTH basis set was used to expand the valence electrons. A plane-wave energy cutoff of 600 Ry was applied. The maximum atomic force was calculated when it was below 4.5 × 10⁻⁶. -4 Hartley / Bohr and the energy change between successive steps is less than 1 × 10 -5 At electron volts, geometric optimization convergence is assumed.

[0137] Statistical analysis: All data were from three independent experiments and are expressed as mean ± standard deviation. Student's t-test was used to evaluate comparisons among multiple groups. GraphPad Prism 8.0 was used for all statistical analyses and graphing.

[0138] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a medical nanozyme for treating drug-resistant bacterial infections in deep tissues, characterized in that, The preparation method includes the following steps: Rhodium trichloride, urea, and polyvinylpyrrolidone are mixed to obtain a mixture, wherein the mass ratio of rhodium trichloride, urea, and polyvinylpyrrolidone is (3.0~6.0) g : (80~120) g : (10~30) g; The mixture was subjected to staged pyrolysis under inert gas protection to obtain a nitrogen-doped carbon-supported single-atom rhodium catalyst. The nitrogen-doped carbon-supported single-atom rhodium catalyst was modified with polyethylene glycol to obtain the medical nanozyme.

2. The preparation method according to claim 1, characterized in that, The segmented pyrolysis includes: the temperature of the first pyrolysis stage is 400~600℃, and the temperature of the second pyrolysis stage is 900~1100℃.

3. The preparation method according to claim 2, characterized in that, The holding time of the first pyrolysis stage is 2-4 hours; and / or, the holding time of the second pyrolysis stage is 0.5-1.5 hours.

4. The preparation method according to claim 1, characterized in that, The polyethylene glycol modification step includes: The nitrogen-doped carbon-supported single-atom rhodium catalyst was dispersed in a first solvent to obtain a catalyst dispersion. Polyethylene glycol is dissolved in a second solvent to obtain a polyethylene glycol solution; The catalyst dispersion was mixed with the polyethylene glycol solution and stirred at 20-30°C for 12-48 hours. The mixture after stirring reaction is subjected to solid-liquid separation and washing to obtain the medical nanozyme.

5. The preparation method according to claim 1, characterized in that, In the polyethylene glycol modification, the mass ratio of the nitrogen-doped carbon-supported single-atom rhodium catalyst to the polyethylene glycol is 1:(0.05~0.30); and / or, The molecular weight (Mn) of the polyethylene glycol is 2000-10000 Da.

6. The preparation method according to claim 4, characterized in that, The first solvent and the second solvent are each independently selected from water or a buffer solution with a pH of 6.5-8.

0.

7. A medical nanozyme obtained by the preparation method according to any one of claims 1-6.

8. The use of the medical nanozyme obtained by the preparation method according to any one of claims 1 to 6 or the medical nanozyme according to claim 7 in the preparation of medicaments for the prevention and / or treatment of deep tissue infections caused by drug-resistant bacteria.

9. The application according to claim 8, characterized in that, The drug is used in conjunction with second near-infrared laser irradiation.

10. A drug for the prevention and / or treatment of deep tissue infections caused by drug-resistant bacteria, characterized in that, The drug comprises a medical nanozyme obtained by any one of the preparation methods of claims 1 to 6 or a medical nanozyme as described in claim 7.

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