Bimetal alloy nano-enzyme for treating multidrug-resistant bacterial infection as well as preparation method and application of bimetal alloy nano-enzyme

Abstract

The invention relates to a bimetallic alloy nano-enzyme for treating multi-drug-resistant bacterial infection as well as a preparation method and application thereof, and belongs to the field of biomedical nano-materials. The problems that in the prior art, the catalytic activity of metal and metal-based nano enzyme is limited, the bactericidal effect is poor due to poor targeting performance, the bactericidal effect in vivo (especially deep drug-resistant bacteria) is poor, and biocompatibility is poor are solved. The invention relates to a bimetallic alloy nano-enzyme for treating multidrug-resistant bacterial infection, which is mainly composed of two metal elements of Pt and Ru, and the atomic ratio of Pt / Ru is 90 / 10-70 / 30. The bimetallic alloy nano-enzyme disclosed by the invention has efficient biological catalytic activity, has a very good treatment effect on deep drug-resistant bacterial infection under ultrasonic activation, shows remarkable antibacterial, anti-biofilm and anti-inflammatory multiple effects, and is good in biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections, its preparation method, and its application. Background Technology

[0002] Multidrug-resistant (MDR) bacteria have become significant hospital-acquired pathogens, posing a major threat to global public health. These bacteria can cause a variety of infections, particularly in immunocompromised patients, including pneumonia, abscesses, keratitis, and sepsis. Antibiotic treatment is a common clinical practice for bacterial infections; however, prolonged antibiotic use has led to increased resistance in MDR bacteria and the emergence of superbugs, seriously threatening human health. Therefore, there is an urgent need to find new bactericides to combat drug-resistant bacterial infections.

[0003] Nanozymes exert their bactericidal effect by catalyzing the generation of reactive oxygen species (ROS), and their catalytic mechanism makes them less prone to drug resistance. Furthermore, as nanomaterials, nanozymes possess unique physicochemical properties, and when combined with photothermal, magnetothermal, and sonodynamic therapies, their ability to kill pathogenic microorganisms can be enhanced. Nanozymes developed include those based on metal oxides, metals, metal sulfides, and carbon-based nanostructures.

[0004] However, existing nanozyme antibacterial systems are still hampered by three major limitations: low catalytic efficiency, insufficient biocompatibility, and inherent defects of single-therapy approaches. Phototherapy is limited by its limited tissue penetration depth; chemokinetic therapy (CDT) experiences a sharp decline in activity in hypoxic microenvironments, making it difficult to simultaneously eradicate planktonic bacteria and biofilms; while ultrasound (US) has the advantage of deep penetration, existing sonosensitive agents (ROS) have low yields and lack targeting, making it difficult to eradicate deep drug-resistant bacterial infections (such as pneumonia and abscesses) due to biofilm barriers and hypoxic microenvironments. Therefore, there is an urgent need to develop a novel nanozyme with high catalytic activity, precise targeting, biocompatibility, and multi-mechanism synergy to overcome the three major bottlenecks of hypoxia, deep penetration, and biofilms, and achieve efficient, safe, and thorough treatment of multidrug-resistant (MDR) bacterial infections. Summary of the Invention

[0005] In view of the above analysis, the present invention aims to provide a bimetallic alloy nanozyme for treating multidrug-resistant bacterial infections, its preparation method and application, in order to solve at least one of the following problems in the prior art: limited catalytic activity of metal and metal-based nanozymes, poor bactericidal efficacy due to poor targeting, poor bactericidal effect in vivo (especially for deep-seated drug-resistant bacteria), and poor biocompatibility.

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

[0007] This invention provides a bimetallic alloy nanozyme for treating multidrug-resistant bacterial infections. The bimetallic alloy nanozyme is mainly composed of two metal elements, Pt and Ru, with an atomic ratio of Pt / Ru of 90 / 10 to 70 / 30.

[0008] Furthermore, the bimetallic alloy nanozyme is activated by ultrasound.

[0009] Furthermore, the bimetallic alloy nanozyme has a spherical profile and an average particle size of 1-10 nm; and / or,

[0010] The specific surface area of ​​the bimetallic alloy nanozyme is 500-2000 m². 2 / g.

[0011] Furthermore, the bimetallic alloy nanozyme has a face-centered cubic single-phase solid solution structure; and / or, the interplanar spacing of the bimetallic alloy nanozyme is 0.223 nm, corresponding to the 111 crystal plane of platinum crystal.

[0012] Furthermore, regarding the peroxidase-like POD activity, under conditions without ultrasonic activation, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanozyme include: K m [TMB-POD] = 0.2-0.8 mM, V max [TMB-POD] = 2 × 10 -7 -8×10 -7 M / s.

[0013] Furthermore, regarding the peroxidase-like POD activity, under ultrasonic activation conditions, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanozyme include: K m [TMB-POD] = 0.24-0.96 mM, V max [TMB-POD] = 2.4 × 10 -7 -9.6×10 -7 M / s.

[0014] Furthermore, the bimetallic alloy nanozyme was prepared using a glycine-mediated co-reduction method.

[0015] This invention provides a method for preparing the bimetallic alloy nanozyme for treating multidrug-resistant bacterial infections, the method comprising the following steps:

[0016] (1) Mix the soluble platinum source and ruthenium source with solvent, glycine and stabilizer in the required molar ratio for the first stage of mixing until all materials are completely dissolved to form a homogeneous precursor solution.

[0017] (2) Under controlled temperature conditions, the precursor solution and the reducing agent are mixed together for a second-stage mixing process until the reaction is complete, and the dispersion of the bimetallic alloy nanoenzyme is obtained.

[0018] The present invention provides the use of the bimetallic alloy nanozyme as described above or the bimetallic alloy nanozyme prepared by the method described above in the application as a drug and / or bactericide for treating diseases caused by bacterial infections.

[0019] Furthermore, in the aforementioned application, the dispersion of the bimetallic alloy nanozyme and hydrogen peroxide are applied to the infection site, and the infection site is activated by ultrasonic irradiation. The mass concentration of the bimetallic alloy nanozyme dispersion is 10-200 μg / ml, the molar concentration of the hydrogen peroxide is 0.05-1.0 mM, and the ultrasonic irradiation frequency is 0.1-5 MHz with a power density of 0.1-5 W / cm². 2 The irradiation time is 1-10 minutes.

[0020] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0021] (1) This invention provides a bimetallic PtRu alloy nanozyme (PR) for treating multidrug-resistant bacterial infections. By optimizing the atomic ratio of platinum (Pt, catalytic site) and ruthenium (Ru, adsorption site), an ultrafine bimetallic alloy nanoplatform with highly efficient biocatalytic activity was obtained. Characterization of the examples confirmed that the PR of this invention exhibits excellent oxidase-like and peroxidase-like activities and has a significant bactericidal effect.

[0022] The highly efficient biocatalytic mechanism of PtRu alloy nanozymes mainly benefits from their optimized electronic structure, surface charge distribution, synergistic optimization of adsorption and activation energies, and enhanced adsorption strength of O atoms. These factors work together to enable PtRu alloy nanozymes to exhibit excellent performance in peroxidase-like activity (POD-like).

[0023] (2) In some preferred embodiments, the bimetallic PtRu alloy nanozyme (PR) is activated by ultrasound (US), which improves the penetration depth of the nanozyme PR. Addressing the problems of low ROS yield and targeting in existing US + sonosensitive agent bactericidal methods, this invention utilizes the synergistic effect of Pt and Ru to significantly improve ROS yield and targeting, thereby achieving better bactericidal effects, especially for deep-seated drug-resistant bacterial infections, exhibiting significant antibacterial, anti-biofilm, and anti-inflammatory multiple effects.

[0024] In this process, Pt acts as a catalytic site, efficiently catalyzing the formation of ROS from reactants; Ru acts as an adsorption site, adsorbing reactants and stabilizing their structure, making the reactants more readily catalyzed by Pt. This synergistic effect is similar to a relay race, with Ru responsible for adsorbing and stabilizing reactants, and Pt responsible for catalyzing the formation of ROS. Through this synergistic effect, PtRu nanoparticles can significantly improve the ROS formation efficiency.

[0025] PtRu nanoparticles possess a suitable small size (e.g., 1-10 nm) and a surface positive charge (e.g., PtRu). 2+ These properties enable PtRu nanoparticles to exhibit more significant targeting than traditional sonosensitive agents in bactericidal applications. Specifically, the positive charge on the surface of PtRu nanoparticles can electrostatically attract the negative charge on the surface of bacteria, enhancing their adhesion to bacterial surfaces. Simultaneously, the small size of PtRu nanoparticles allows for rapid absorption by bacteria and easy penetration of bacterial biofilms, directly acting on the bacterial interior and thus enhancing the bactericidal effect. Furthermore, PtRu nanoparticles, through their physicochemical properties (such as size, shape, and surface charge), readily accumulate at sites of inflammation. For example, the increased intercellular spaces in vascular endothelial cells at sites of inflammation allow PtRu nanoparticles to enter the inflamed tissue through these spaces, further increasing their accumulation in the bacterial infection and inflammation area. These combined properties enable PtRu nanoparticles to achieve highly efficient targeted bactericidal and anti-inflammatory effects even without surface modification.

[0026] (3) PtRu nanoparticles not only possess highly efficient bactericidal capabilities but also exhibit superior biocompatibility compared to other metal nanozymes (such as gold, palladium, platinum, silver, or their alloys). Specifically, the hemolysis rate of PR is <5% (0-200 μg / mL), and it is non-toxic to various organs (such as the heart, liver, spleen, lungs, and kidneys). Furthermore, blood biochemical indicators after using PtRu nanoparticles show no significant difference compared to the control group, making it suitable for in vitro and in vivo bactericidal therapy. Compared to traditional nanozyme bactericides, PtRu alloy nanozymes achieve superior bactericidal effects, particularly in deep sterilization, by reducing hydrogen peroxide dosage by 50% through the synergistic effect of ultrasonic activation's acoustic and chemodynamic forces, as well as optimized alloy ratios and other characteristic parameters. Through appropriate alloying ratios, PtRu alloy nanozyme particles optimize the electronic structure of the metal, enhancing not only catalytic activity but also stability, making them more stable in the biological environment and reducing potential toxicity. This allows for a reduction in the amount of hydrogen peroxide and PtRu nanoparticles used to achieve the same bactericidal effect, while leveraging their stability to further reduce potential toxicity to normal tissues, improve biocompatibility and patient compliance.

[0027] (4) In some embodiments, in vitro antibacterial and antibiofilm evaluation experiments demonstrated that the PtRu alloy nanozyme of the present invention, in the presence of H2O2 without US irradiation, achieved an inhibition rate of over 76% against multidrug-resistant bacteria such as Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), indicating that potent chemokinetic therapy (CDT) triggered by the PtRu alloy nanozyme can effectively combat bacterial infections. Under the synergistic effect of ultrasound (US) and CDT, the PtRu alloy nanozyme of the embodiments achieved a bacterial mortality rate of over 99% and a biofilm clearance rate of over 96%. The K of the PtRu alloy nanozyme of the embodiments of the present invention... m [TMB-POD]≤0.55mM.

[0028] (5) In some embodiments, the in vivo deep antibacterial effect of the PtRu alloy nanozyme of the present invention is as follows: In a subcutaneous abscess model infected with multidrug-resistant bacteria, the bacterial colony count in the infected tissue on day 10 showed that, under the synergistic effect of ultrasound (US) and CDT, the bactericidal rate of the PtRu alloy nanozyme of the embodiment exceeded 95%, and the wound healing rate reached over 95% on day 10. The PtRu alloy nanozyme of the present invention, through combined treatment with CDT and US, showed significant efficacy in clearing bacteria and biofilms in lung tissue and effectively reducing inflammation, with a 98% reduction in lung tissue bacterial load and a 70% downregulation of the inflammatory factor IL-6 (immunofluorescence).

[0029] 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

[0030] 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.

[0031] Figure 1 For the rational design and biological application of PR alloy nanozymes, (a) a schematic diagram of PR alloy nanozyme synthesis, (b) the SDT and CDT performance of PR and the synergistic effect of US-enhanced CDT, and (c) PR alloy nanozymes for dual-mode anti-infective therapy using US-enhanced CDT strategy for MDRPAO-induced pneumonia and MRSA-induced subcutaneous abscess models.

[0032] Figure 2 Pt in the embodiments of the present invention 80 Ru20 Synthesis and surface morphology characterization of PR alloy nanozymes; (a) TEM image of PR alloy nanozymes; (be) elemental distribution map of PR alloy nanozymes by scanning transmission electron microscopy energy-dispersive X-ray spectroscopy (STEM-EDS), scale bar 10 nm; (f) the position of the EDS line scan is indicated by the yellow dashed arrow; (g) selected area electron diffraction (SAED) pattern of PR alloy nanozymes; (h) high-resolution TEM (HRTEM) image of PR alloy nanozymes; (i) X-ray diffraction (XRD) pattern of PR alloy nanozymes; (j) electron paramagnetic resonance (EPR) spectrum of PR alloy nanozymes; (k) XPS measurement spectrum of PR alloy nanozymes, with 4 / 1 Pt 2+ / Ru 3+ High-resolution XPS spectra of (l)Pt4f and (m)Ru3p of PR alloy nanozymes prepared by atomic ratio; X-ray absorption near-edge structure (XANES) spectrum of (n)Pt L3 edge; Fourier transform extended X-ray absorption fine structure (FT-EXAFS) spectrum of (o)PR alloy nanozymes.

[0033] Figure 3 This study aims to provide insights into the biocatalytic performance and mechanism of PR alloy nanozymes. The findings include: (a) UV absorption changes of TMB under various conditions; (b) absorbance curves of TMB over time in the presence of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM), with inset photographs showing the color change of the corresponding samples over time; and (c) different ultrasonic (US) irradiation times (1.0 MHz, 0.5 W cm⁻¹) in the presence of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM). -2 (d) Absorbance changes of TMB during ultrasound treatment, with inset photographs showing the corresponding sample color changes over time; (d) Normalized absorbance changes of DPBF (in water) in the presence of PR under different environmental conditions, data expressed as mean ± standard deviation (n = 3); (e) and (f) Absorbance changes of PR in H2O2 (0.1 mM) and ultrasound irradiation (1.0 MHz, 0.5 W cm⁻¹), respectively. -2 ESR spectra detected in water under the following conditions;

[0034] Figure 4To assess the in vitro antibacterial and antimicrobial membrane activities of PR alloy nanozymes, the antibacterial activity of the alloy nanozymes against MDRPAO and MRSA was determined using the plate counting method. Photographs and corresponding survival rates of (a, b) MDRPAO and (c, d) MRSA colonies after different treatments were presented. Live / dead fluorescent staining images and corresponding survival rates of (e, f) MDRPAO and (g, h) MRSA after different treatments were also presented, scale bar, 50 μm. Representative scanning electron microscopy (SEM) images of (i) MDRPAO and (j) MRSA collected before and after incubation with PR alloy nanozymes were presented, scale bar, 1 μm. Optical micrographs and quantitative analysis of (k, l) MDRPAO and (m, n) MRSA biofilms collected from different groups and stained with crystal violet at specified treatment times were presented. 3D images of (o) MDRPAO and (p) MRSA biofilms collected from different groups at specified treatment times were also presented. CLSM images stained with a live / dead double staining scheme, scale bar, 100 μm; data are expressed as mean ± standard deviation of three independent experiments (n=3); *p<0.05, **p<0.01 and ***p<0.001 were considered statistically significant by a two-tailed Student's t-test.

[0035] Figure 5 The therapeutic performance of PR alloy nanozymes in MDRPAO-induced pneumonia in vivo; (a) digital images of lung tissue collected from mice after treatment; (b) in vivo antimicrobial smear results of MDRPAO-induced pneumonia infection under different treatment conditions; (c) representative histological images of H&E staining at different time points during different treatment processes, scale bar = 2 cm (for image (c) upward) and 200 μm (for image (b) downward);

[0036] Figure 6 Immunofluorescence analysis of lung tissue in vivo for PR alloy nanozymes induced by MDRPAO pneumonia; representative immunofluorescence staining images of (a) IL-6, (b) IL-10, (c) CD86 and (d) CD86 in lung sections from different treatment groups;

[0037] Figure 7(a) Schematic diagram of the experimental procedure for treating subcutaneous MRSA infection in mice with PR alloy nanozymes; (b) Representative photographs of infected wounds at different time points during treatment; (c) Relative wound areas of mice on day 0 (blue), day 5 (orange), and day 10 (dark red) after different treatments; (d) Photographs of MRSA colonies in infected tissue on day 10 after treatment; (e) Changes in mouse body weight during treatment; (f) Infected areas of mice after different treatments (n = 5 independent biological samples; mean ± standard deviation); (g) Quantification of viable bacteria in biofilm-infected tissue on day 10 after treatment (n = 5 independent biological samples; mean ± standard deviation); (h) h&E staining; (i) Masson staining; and (j) Gram-stained tissue sections of infected tissue from mice on day 10 after treatment, scale bar = 500 μm.

[0038] Figure 8 Immunofluorescence analysis of PR alloy nanozymes on MRSA-induced subcutaneous abscess skin tissue; representative immunofluorescence staining images and corresponding quantitative analyses of abscess tissue on day 10 after treatment with TNF-α (a, b), TGF-β (c, d), and CD31 (e, f); data are expressed as mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001;

[0039] Figure 9 The image shows a scanning electron microscope (SEM) image of the PR alloy nanozyme according to an embodiment of the present invention.

[0040] Figure 10 This is a transmission electron microscope (TEM) image of the PR alloy nanozyme according to an embodiment of the present invention;

[0041] Figure 11 This is an extended X-ray absorption fine structure (EXAFS) spectrum of the PR alloy nanozyme according to an embodiment of the present invention;

[0042] Figure 12 Schematic diagram of the color development principle and molecular structure changes of (a) TMB, (b) OPD and (c) DPBF after reacting with the corresponding reactive oxygen species;

[0043] Figure 13 (a) Changes in UV absorption of OPD under different conditions, inset photo: corresponding color change of the sample; (b) Changes in absorbance of OPD over time in the presence of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM), inset photo: corresponding color change of the sample; (c) Enhancement effect of PR producing ·OH under H₂O₂ and ultrasonic (US) irradiation with OPD as a trapping agent, inset photo: corresponding color change of the sample;

[0044] Figure 14 (a) Michaelis-Menten fitting of PR with different concentrations of H2O2 as substrates and (b) Lineweaver-Burk fitting;

[0045] Figure 15 Lung tissues from medullomyeloid mice were treated with PBS and PR (50 μg / mL, 100 μL) and then analyzed in vitro.

[0046] Figure 16 Quantitative analysis of (a) IL-6, (b) IL-10, (c) CD86 and (d) CD206 in each treatment group. Data were obtained from three independent experiments (mean ± standard deviation, n = 3).

[0047] Figure 17 Representative images of CD31 immunofluorescence staining in lung tissue sections from mice in different treatment groups;

[0048] Figure 18 Representative histological images of heart, liver, spleen, lung and kidney with H&E staining from different treatment groups;

[0049] Figure 19 Images of H&E staining of major organs (heart, liver, spleen, lung, and kidney) in mice after receiving different treatments;

[0050] Figure 20 To assess the biosafety of PR, healthy mice were injected intravenously with 50 μL of PR suspension (50 μg / mL) or PBS, and the above tests were performed 24 hours later. Among them, (a–c) blood biochemical indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN); (d–f) blood routine indicators: white blood cell count (WBC), mean corpuscular volume (MCV) and platelet count (PLT). Detailed Implementation

[0051] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which constitute a part of the present invention 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.

[0052] Compared to natural enzymes, nanozymes have attracted considerable attention in interdisciplinary research due to their stability and ease of synthesis. More than 50 types of nanomaterials have been developed as enzyme mimics, including metal oxides, metals, metal sulfides, and carbon-based nanostructures, showing broad potential in clinical diagnosis and disease treatment. Among them, metal and metal-based nanostructures are particularly noteworthy because they exhibit a variety of enzymatic activities. For example, platinum (Pt)-based nanoparticles (NPs) can mimic the activities of at least four redox enzymes, including peroxidase, oxidase, catalase, and superoxide dismutase. However, the catalytic effects of these NPs are contradictory: while peroxidase and oxidase activities promote substrate oxidation and produce pro-oxidative effects, catalase-like and superoxide dismutase-like activities enhance the reduction of hydrogen peroxide and superoxide, exhibiting antioxidant effects.

[0053] To address the bottleneck of pro-antioxidant reactions in single-metal nanozymes, this invention proposes designing bimetallic nanostructures (M1M2-NPs) as a synergistic platform with dual functions of "sound sensitizer + nanozyme"; further, it preferably uses ultrasonic activation to generate reactive oxygen species (sound dynamics) through acoustic kinetics, and simultaneously catalyzes the production of ·OH from H2O2. 1 O2 (chemical kinetics) enables synergistic effects of acoustic and chemical kinetics, thus providing a new solution to effectively address the problems of poor bactericidal effect, difficulty in eradicating pathogens, and easy recurrence of existing nanozymes in clinical applications of bacterial infections.

[0054] In a first aspect, the present invention provides a bimetallic alloy nanozyme for treating multidrug-resistant bacterial infections. The bimetallic alloy nanozyme is mainly composed of two metal elements, Pt and Ru, with an atomic ratio of Pt / Ru ranging from 90 / 10 to 70 / 30. Exemplary examples include atomic ratios of Pt / Ru of 87 / 13, 85 / 15, 82 / 18, 80 / 20, 78 / 22, 75 / 25, 73 / 27, and 70 / 30.

[0055] The chemical formula of the bimetallic alloy nanoenzyme of the present invention is Pt. x Ru 1-x The value of x is set between 70 and 90 at.% (atomic percentage).

[0056] In a preferred embodiment, the bimetallic alloy nanozyme is activated by ultrasound.

[0057] In some embodiments, the bimetallic alloy nanozymes have a spherical profile and an average particle size of 1-10 nm. Exemplarily, the average particle size is 1 nm, 2 nm, 4 nm, 5 nm, 6 nm, 8 nm, or 10 nm.

[0058] In some embodiments, the specific surface area of ​​the bimetallic alloy nanozyme is 500-2000 m². 2 / g. For example, the specific surface area is 600m². 2 / g、800m 2 / g, 1000m 2 / g、1200m 2 / g, 1400m 2 / g, 1600m 2 / g、1800m 2 / g、2000m 2 / g.

[0059] In some embodiments, the bimetallic alloy nanozyme has a face-centered cubic single-phase solid solution structure.

[0060] For example, the interplanar spacing of the bimetallic alloy nanozyme is 0.223 nm, corresponding to the 111 crystal plane of platinum crystal.

[0061] In some embodiments, the bimetallic alloy nanozyme has peroxidase-like POD activity and oxidase-like OXD activity.

[0062] In some embodiments, for peroxidase-like POD activity, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanozyme under conditions without ultrasonic activation include: K m [TMB-POD] = 0.2-0.8 mM, V max [TMB-POD] = 2 × 10 -7 -8×10 -7 M / s. For example, K m [TMB-POD] = 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM; preferably 0.2-0.5mM. V max [TMB-POD] = 2 × 10 -7 M / s, 3×10 -7 M / s, 4×10 -7 M / s, 5×10 -7 M / s, 6×10 -7 M / s, 7×10 -7 M / s, 8×10 -7 M / s; preferably 4×10 -7 M / s~8×10 -7 M / s.

[0063] In some embodiments, for peroxidase-like POD activity, under ultrasonic activation conditions, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanozyme include: K m[TMB-POD] = 0.24-0.96 mM, V max [TMB-POD] = 2.4 × 10 -7 -9.6×10 -7 M / s. For example, K m [TMB-POD] = 0.24mM, 0.36mM, 0.48mM, 0.60mM, 0.72mM, 0.84mM, 0.96mM; preferably 0.24-0.6mM. V max [TMB-POD] = 2.4 × 10 -7 M / s, 3.6×10 -7 M / s, 4.8×10 -7 M / s, 6.0×10 -7 M / s, 7.2×10 -7 M / s, 8.4×10 -7 M / s, 9.6×10 -7 M / s; preferably 4.8 × 10 -7 M / s~9.6×10 -7 M / s.

[0064] It is understandable that the Michaelis constant K m and maximum reaction rate V max These are two key indicators for evaluating the catalytic activity of nanozymes. They are obtained through steady-state kinetic experiments combined with Michaelis-Menten equation fitting and linearization methods (such as Lineweaver-Burk plots) in existing technologies.

[0065] Wherein, the Michaelis constant K m [TMB-POD] indicates that in peroxidase (POD) type reactions, the bimetallic alloy nanozyme catalyzes the oxidation of the substrate TMB (3,3',5,5'-tetramethylbenzidine) to half of the maximum reaction rate (1 / 2 V). max ) Required TMB concentration; K m [TMB-POD] reflects the affinity of nanozymes for TMB. The smaller the Km value, the more efficient the enzyme can catalyze at low TMB concentrations, indicating high affinity; conversely, the larger the Km value, the lower the affinity.

[0066] Maximum reaction rate V max [TMB-POD] indicates that in peroxidase (POD) type reactions, when the TMB substrate concentration is much greater than K... m (i.e., [TMB] >> K) m The maximum initial reaction rate achievable by bimetallic alloy nanozyme catalysis of TMB oxidation ([TMB-POD]). V max A higher value indicates a greater number of active sites or stronger intrinsic catalytic activity in the bimetallic alloy nanozyme.

[0067] It should be noted that the bimetallic PtRu alloy nanozyme of this invention exhibits highly efficient biocatalytic activity. Through in-depth research, the inventors believe that this is mainly due to the unique structure-activity relationship formed by the two metal elements (Pt and Ru) in an appropriate ratio, which facilitates the realization of a synergistic catalytic mechanism.

[0068] (1) Electronic structure optimization

[0069] Under appropriate alloy ratios, Ru incorporation induces the formation of Pt vacancies, and the electronic structure of PtRu alloy nanozymes is more conducive to electron-hole separation, thereby improving catalytic efficiency. This structure allows electrons and holes to be separated more effectively, reducing recombination and thus improving the efficiency of biocatalytic reactions.

[0070] (2) Surface charge distribution and catalytic sites

[0071] The charge distribution of Pt and Ru atoms on the surface of the alloy nanozyme is uneven, with negative charges mainly concentrated at the top of the nanozyme and at the bonding sites between components. This charge distribution allows both Pt and Ru atoms to contribute to the catalytic activity, especially Pt. x Ru 1-x When x is 70–90 at.%, this synergistic effect is more pronounced.

[0072] (3) Adsorption energy and activation energy

[0073] The synergistic optimization of the adsorption and activation energies of H2O2 at Pt and Ru sites makes biocatalytic reactions more efficient. Especially in Pt... x Ru 1-x At a ratio of 70–90 at.%, the adsorption rate of H2O2 on Ru is higher than that on Pt, but Pt has a higher ability to generate OH*. This synergistic effect makes the adsorption and activation of H2O2 more efficient.

[0074] (4) Adsorption and activation of H2O2

[0075] The adsorbed H₂O₂ is first activated by Pt and Ru atoms to H₂O₂*, where the O=O bonds are strongly bonded to the metal atoms, forming an unstable intermediate (TS). This intermediate rapidly breaks the O=O bonds, generating OH*. This synergistic effect makes the adsorption and activation of H₂O₂ more efficient, especially in Pt. x Ru 1-x This synergistic effect is more pronounced when x is 70–90 at.%.

[0076] (5) Differences in activation energy:

[0077] The activation energy from H₂O₂ to OH indicates that Pt has a higher ability to generate OH than Ru. This difference makes the synergistic effect of Pt and Ru more efficient, especially in Pt. x Ru 1-x This synergistic effect is more pronounced when x is 70–90 at.%.

[0078] (6) Adsorption strength of O atoms:

[0079] The addition of Ru enhances the adsorption capacity of O atoms, thereby improving the overall biocatalytic activity. This enhanced adsorption capacity makes the catalytic reaction more efficient, especially in Pt. x Ru 1-x This synergistic effect is more pronounced when x is 70–90 at.%.

[0080] In summary, the highly efficient biocatalytic mechanism of PtRu alloy nanozymes mainly benefits from their optimized electronic structure, surface charge distribution, synergistic optimization of adsorption and activation energies, and enhanced adsorption strength of O atoms. These factors work together to enable PtRu alloy nanozymes to exhibit superior performance in peroxidase-like activity (POD-like).

[0081] In a preferred embodiment, the bimetallic alloy nanozyme is prepared by a glycine-mediated co-reduction method, which involves dissolving a soluble platinum source and a ruthenium source in a solvent at the desired molar ratio, adding glycine and a stabilizer until fully dissolved, and then adding a reducing agent and stirring under controlled temperature conditions to obtain the bimetallic alloy nanozyme.

[0082] In some embodiments, the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 10-100 μmol, and the volume of the solvent is 1-20 ml. Preferably, the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, and the volume of the solvent is 3-10 ml.

[0083] In some embodiments, the total molar amount of Pt and Ru in the soluble platinum source and ruthenium source is 10-100 μmol, and the mass of glycine is 100-400 mg. Preferably, the total molar amount of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, and the mass of glycine is 150-200 mg.

[0084] In one embodiment, in step (1), the stabilizer is polyvinylpyrrolidone (PVP), and when the total molar concentration of the soluble platinum source and ruthenium source is 10-100 μmol, the amount of PPVP added is 300-500 mg. Preferably, when the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, the amount of PPVP added is 400-480 mg.

[0085] In one embodiment, the reducing agent is ascorbic acid, and the ratio of the molar amount of ascorbic acid to the total molar amount of the soluble platinum source and ruthenium source is 10-40; preferably 20-30.

[0086] In one embodiment, the temperature control condition is to control the temperature of the reaction system at a specific temperature point within the range of 40 to 80°C (preferably 55°C to 65°C), and to add the reducing agent after reaching thermal equilibrium at this temperature.

[0087] In one embodiment, the bimetallic alloy nanozyme further includes glycine and a stabilizer, which are additives added during the preparation of bimetallic nanozymes, i.e., PtRu nanoparticles.

[0088] In one embodiment, in the bimetallic alloy nanozyme, Ru is replaced with the metallic element iridium (Ir) or rhodium (Rh).

[0089] It is understood that the "bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections" described in this invention can be replaced with other similar expressions, such as: PtRu alloy nanoenzyme bactericide.

[0090] In a second aspect, the present invention provides a method for preparing the bimetallic alloy nanozyme for treating multidrug-resistant bacterial infections as described in the first aspect, the method comprising the following steps:

[0091] (1) Mix the soluble platinum source and ruthenium source with solvent, glycine and stabilizer in the required molar ratio for the first stage of mixing until all materials are completely dissolved to form a homogeneous precursor solution.

[0092] (2) Under controlled temperature conditions, the precursor solution and the reducing agent are mixed together for a second-stage mixing process until the reaction is complete, and the dispersion of the bimetallic alloy nanoenzyme is obtained.

[0093] In some embodiments, the concentration of the dispersion of the bimetallic alloy nanozyme is 10-100 μg / ml. Exemplarily, the concentration of the dispersion of the bimetallic alloy nanozyme is 10 μg / ml, 25 μg / ml, 45 μg / ml, 48 μg / ml, 50 μg / ml, 52 μg / ml, 55 μg / ml, 60 μg / ml, 75 μg / ml, 90 μg / ml, or 100 μg / ml; preferably 45-55 μg / ml.

[0094] It should be noted that the glycine-mediated co-reduction method of this invention has the following advantages: This method is mild and controllable. Glycine, as a reducing agent and ligand, promotes the uniform nucleation and growth of metal ions, forming uniformly sized and distributed nanoparticles, thus optimizing the microstructure of the nanozyme. In principle, glycine interacts with metal ions during synthesis, regulating particle morphology and increasing specific surface area, thereby improving catalytic activity. Simultaneously, the protective layer formed by glycine enhances the stability of the nanozyme, making it less prone to aggregation or inactivation during storage and reaction. In terms of macroscopic performance, this preparation method enables the nanozyme to exhibit higher catalytic efficiency and better stability, allowing for more efficient catalytic reactions. Regarding bactericidal effects, the uniformly distributed active sites and optimized structure allow the nanozyme to efficiently generate reactive oxygen species (ROS) at lower concentrations, improving bactericidal efficiency while reducing potential toxicity to normal tissues and enhancing biocompatibility.

[0095] For example, the soluble platinum source is selected from K2PtCl4, PtCl2, H6Cl2N2Pt, and PtN2O6.

[0096] At least one of the following. Preferably, the soluble platinum source is K2PtCl4.

[0097] For example, the soluble ruthenium source is selected from RuCl3, (NH4)2RuCl6, C 15 H 21 At least one of O6Ru. Preferably, the soluble ruthenium source is RuCl3.

[0098] It is understandable that in step (1), the required molar ratio can be calculated based on the atomic ratio of Pt and Ru in the bimetallic alloy nanoenzyme.

[0099] Specifically, the chemical formula of the bimetallic alloy nanozyme of the present invention is Pt. x Ru 1-x The value of x is set between 70–90 at.% (atomic percentage). Since all the metals are derived from the precursors during synthesis, and the two metals are ultimately reduced to an alloy almost quantitatively, the atomic ratio can be directly used as the molar ratio of the feed materials. The molar ratio n of the two metals... Pt :n Ru The ratio is 7:3 to 9:1. (According to n) Pt and n Ru Multiply by the molecular weight of their respective precursors (i.e., soluble platinum source and ruthenium source) to obtain the actual weighing mass, and complete the feeding process.

[0100] For example, in step (1), the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 10-100 μmol, and the volume of the solvent is 1-20 ml. Preferably, the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, and the volume of the solvent is 3-10 ml.

[0101] For example, in step (1), the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 10-100 μmol, and the mass of glycine is 100-400 mg. Preferably, the total molar number of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, and the mass of glycine is 150-200 mg.

[0102] For example, the total molar amounts of Pt and Ru in the soluble platinum and ruthenium sources are 10 μmol, 20 μmol, 40 μmol, 60 μmol, 80 μmol, and 100 μmol. The volumes of the solvents are 1 ml, 3 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, and 20 ml. The mass of the glycine is 100 mg, 150 mg, 180 mg, 200 mg, 250 mg, 300 mg, 350 mg, and 400 mg.

[0103] For example, in step (1), the solvent is selected from at least one of water and ethanol.

[0104] For example, in step (1), the stabilizer is selected from at least one of polyvinylpyrrolidone (PVP) and acetone. Preferably, the stabilizer is polyvinylpyrrolidone (PVP).

[0105] For example, in step (1), the stabilizer is polyvinylpyrrolidone (PVP), and when the total molar amount of the soluble platinum source and ruthenium source is 10-100 μmol, the amount of PPVP added is 300-500 mg. For example, the amount of PPVP added is 300 mg, 350 mg, 400 mg, 420 mg, 450 mg, 470 mg, 480 mg, or 500 mg.

[0106] Preferably, when the total molar amount of Pt and Ru in the soluble platinum source and ruthenium source is 40-60 μmol, the amount of polyvinylpyrrolidone added is 400-480 mg.

[0107] For example, in step (2), the reducing agent is selected from at least one of ascorbic acid, sodium citrate, and sodium borohydride. Preferably, the reducing agent is ascorbic acid.

[0108] For example, in step (2), the reducing agent is ascorbic acid, and the ratio of the number of moles of ascorbic acid to the total number of moles of the soluble platinum source and ruthenium source is 10-40; for example, the ratio of the number of moles of ascorbic acid to the total number of moles of the soluble platinum source and ruthenium source is 10, 15, 20, 23, 25, 28, 30, 35, or 40; preferably 20-30.

[0109] For example, in step (1), the first-stage mixing process includes stirring or sonicating until all added materials are completely dissolved and form a homogeneous, clear solution. For instance, the first-stage mixing process may employ ultrasonic mixing at a frequency of 0.1-0.5 MHz and a power density of 0.5-2 W / cm². 2 And a duty cycle of 30%-50%.

[0110] For example, in step (2), the second-stage mixing process includes stirring or sonicating until the solution turns into a uniform dark gray color, indicating the formation of bimetallic alloy nanozymes, and then ending the reaction. For example, the second-stage mixing process is carried out by stirring at a speed of 100-200 rpm for 2-4 hours.

[0111] For example, in step (2), the temperature control condition refers to placing the precursor solution obtained in step (1) at 40-80℃, such as 40℃, 50℃, 60℃, 70℃, or 80℃, preferably 55℃ to 65℃, and stirring until the solution temperature reaches thermal equilibrium before adding the reducing agent. For example, thermal equilibrium means that the temperature reading fluctuation is ≤0.5℃ within 30 seconds.

[0112] It should be noted that the preparation method of this invention optimizes the electronic structure and surface properties of the nanozyme by adjusting the amounts of soluble platinum and ruthenium sources, solvent, glycine, stabilizer, and reducing agent, as well as the temperature control conditions, thereby achieving a uniform morphology and size distribution and improving biocatalytic activity and stability. Furthermore, appropriate temperature control ensures reaction uniformity, avoids side reactions, and further enhances the purity and performance of the nanozyme, enabling PtRu alloy nanozymes to exhibit excellent performance in applications such as biocatalysis and bactericides.

[0113] Thirdly, the present invention provides the use of the bimetallic alloy nanozyme according to the first aspect or the bimetallic alloy nanozyme obtained according to the preparation method of the second aspect in the use as or in the preparation of a medicament and / or bactericide for treating diseases caused by bacterial infections.

[0114] In some embodiments, the drugs and / or bactericides used to treat diseases caused by bacterial infections are applied for in vitro and in vivo sterilization and exhibit good biocompatibility. Furthermore, when the bimetallic alloy nanozyme treats drug-resistant bacterial infections, it can exert a highly efficient bactericidal effect using ultrasound (US) irradiation without the addition of hydrogen peroxide, or it can be supplemented with hydrogen peroxide to further enhance the bactericidal effect.

[0115] In some embodiments, the bactericide can kill Gram-positive and Gram-negative bacteria, such as methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, but is not limited to these two types of pathogens. The infections include pulmonary pneumonia and subcutaneous abscesses.

[0116] In some embodiments, in the application, the bimetallic alloy nanoenzyme dispersion and hydrogen peroxide are applied to the infection site, followed by activation by ultrasonic irradiation of the infection site. The mass concentration of the bimetallic alloy nanoenzyme dispersion is 10-200 μg / ml, the molar concentration of the hydrogen peroxide is 0.05-1.0 mM, and the ultrasonic irradiation frequency is 0.1-5 MHz with a power density of 0.1-5 W / cm². 2 The irradiation time is 1-15 minutes.

[0117] For example, in the aforementioned application, the mass concentration of the bimetallic alloy nanoenzyme dispersion is 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 120 μg / ml, 140 μg / ml, 160 μg / ml, 180 μg / ml, and 200 μg / ml. The molar concentration of hydrogen peroxide is 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, and 1.0 mM; the frequency of the ultrasonic irradiation is 0.1 MHz, 0.5 MHz, 1.0 MHz, 1.5 MHz, 2.0 MHz, 3.0 MHz, 4.0 MHz, and 5.0 MHz, and the power density is 0.1 W / cm³. 2 0.5W / cm 2 1.0W / cm 2 1.5W / cm 2 2.0W / cm 2 3.0W / cm 2 4.0W / cm 2 5.0W / cm 2The irradiation times were 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 12 min, and 15 min.

[0118] For example, in the application, the duty cycle of the ultrasound irradiation is 30%-50%.

[0119] For example, the method of application to the infection site may be adapted to different infection sites, such as local injection, nebulized inhalation, intravenous injection, local application or spraying, or crossing the blood-brain barrier.

[0120] In some embodiments, in the said application, the bimetallic alloy nanozyme is used to kill surface and / or deep-seated bacterial species in vivo, including various drug-resistant bacteria. Specifically, the drug-resistant bacteria are genera / species listed in the latest WHO Bacterial Priority Pathogens List (WHO-BPPL). For example, these include multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), which are included in the WHO-BPPL High-priority level and pose a significant threat to human health. The embodiments of this invention fully demonstrate that the bimetallic alloy nanozyme Pt ​​provided by this invention... x Ru 1-x Where x is 70–90 at.% (atomic percentage), it has a good therapeutic effect on infections caused by multidrug-resistant MDRPA and / or MRSA, especially deep bacterial infections in the body. Clinically, it has shown significant efficacy in clearing bacteria and biofilms in lung tissue and subcutaneous abscesses and effectively reducing inflammation. This invention provides a promising strategy for antibacterial and anti-inflammatory treatment.

[0121] In some embodiments, the present invention provides the bimetallic alloy nanoenzyme Pt ​​suitable for infections caused by two typical multidrug-resistant bacteria. x Ru 1-x The preferred combination of amounts of hydrogen peroxide.

[0122] Preferably, for diseases caused by multidrug-resistant bacteria MDRPA and / or MRSA infection, in the application, the bimetallic alloy nanoenzyme dispersion and hydrogen peroxide are applied to the infection site, and the infection site is activated by ultrasonic irradiation; wherein, the mass concentration of the bimetallic nanoenzyme dispersion is 20-60 μg / ml, and the molar concentration of the hydrogen peroxide is 0.1-0.5 mM; the frequency of the ultrasonic irradiation is 0.5-2 MHz, and the power density is 0.5-2 W / cm². 2 The irradiation time is 2-6 minutes.

[0123] It should be noted that the present invention can achieve deep sterilization with low doses of hydrogen peroxide. Specifically, compared with the prior art, the present invention can achieve a good deep sterilization effect by reducing the amount of hydrogen peroxide by 50% and by using US activation and the synergistic effect of acoustic and chemical kinetics of PtRu bimetal.

[0124] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and comparative examples.

[0125] The materials and instruments used in the embodiments of this invention are from the following sources:

[0126] Potassium chloroplatinate (II) (K₂PtCl₄), ruthenium chloride hydrate (III) (RuCl₃), polyvinylpyrrolidone K₃O (PVP), 3,3,5,5-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 1,3-diphenylisobenzofuran (DPBF), dimethyl sulfoxide (DMSO), L-ascorbic acid (AA), and acetate were purchased from Aladdin. Trypsin-treated soybean broth (TSB), Luria-Bertani broth (LB), and agar powder were purchased from Solarbio. BacLight TM Bacterial viability kits were purchased from Thermo Fisher Scientific. Phosphate-buffered saline (PBS) was also used. The morphology and size of PR were analyzed using transmission electron microscopy (TEM) (Thermo Fisher Scientific, Talos F200S). The valence states of various elements were determined using Thermo Fisher K-Alpha X-ray photoelectron spectroscopy (XPS). The UV-Vis-NIR spectra of the solutions were recorded using a CARY 5000 spectrophotometer (Agilent Technologies, USA). Fluorescence images were captured using a confocal laser scanning microscope (CLSM, Nikon A1, Japan).

[0127] Drug-resistant strains (MDRPA and MRSA) were obtained from Wenzhou Medical University and handled strictly in accordance with the guidelines (Wenzhou Medical University Biosafety Operating Procedures). Additionally, mice (Balb / c and C57BL / 6J) were purchased from Wenzhou Medical University. All animal procedures were performed in accordance with the Wenzhou Medical University Guidelines for Laboratory Animal Care and Use and were approved by the Wenzhou Medical University Animal Ethics Committee (SYXK-2021-0020).

[0128] Example 1:

[0129] This embodiment prepared a bimetallic PtRu alloy nanozyme (PR for short), and the specific preparation process is as follows:

[0130] Step 1: Massage the area with a small amount of water (n). Pt :n Ru The ratio was 4:1. 2.08 ml of 20 mM K2PtCl4 solution and 0.52 ml of 20 mM RuCl3 (total moles of Pt and Ru were 52 μmol) were added to a 50 ml round-bottom flask, along with 6.7 ml of ultrapure water, 180 mg of glycine, and 440 mg of polyvinylpyrrolidone (PVP). The mixture was sonicated at room temperature until all added materials were completely dissolved and a homogeneous, clear solution was formed, thus creating a homogeneous precursor solution.

[0131] Step 2: Place the round-bottom flask containing the precursor solution in a 60°C water bath and stir continuously (120 rpm). Once the system reaches thermal equilibrium (i.e., the temperature fluctuation is ≤0.5°C for 30 seconds), under vigorous stirring (120 rpm), rapidly inject 1.3 ml of 1M ascorbic acid solution AA (the molar number of AA is related to the Ru...) into the precursor solution. 3+ and Pt 2+ The total molar ratio was 25), and the mixture was stirred at 120 rpm for 2 hours until the solution turned into a uniform dark gray color, indicating that the reaction was complete and a dispersion of bimetallic PtRu alloy nanozymes was obtained. Polyvinylpyrrolidone (PVP) ligand modification was used to prevent the nanozyme particles from agglomerating and to play a stabilizing role.

[0132] Examples 1-2

[0133] This embodiment is the same as Embodiment 1-1, except that the molar ratio n Pt :n Ru The ratio is 9:1. Take 2.34 ml of 20 mM K2PtCl4 solution and 0.26 ml of 20 mM RuCl3 solution.

[0134] Examples 1-3

[0135] This embodiment is the same as Embodiment 1-1, except that the molar ratio n Pt :n Ru The ratio is 7:3. Take 1.82 ml of 20 mM K2PtCl4 solution and 0.78 ml of 20 mM RuCl3 solution.

[0136] Examples 1-4

[0137] This embodiment is the same as Embodiment 1-1, except that 100 mg of glycine is added.

[0138] Examples 1-5

[0139] This embodiment is the same as Embodiment 1-1, except that 300mg of PVP is added.

[0140] Examples 1-6

[0141] This embodiment is the same as Embodiment 1-1, except that 0.78 ml of 1 M ascorbic acid solution AA (the molar number of AA is the same as that of Ru) is rapidly injected into the precursor solution. 3+ and Pt 2+ The ratio of total moles is 15).

[0142] Examples 1-7

[0143] This embodiment is the same as Embodiment 1-1, except that it is placed in an 80°C water bath.

[0144] Examples 1-8

[0145] This embodiment is the same as Embodiment 1-1, except that the molar ratio n Pt :n Ru The ratio is 4:1. Take 1.2 ml of 20 mM K2PtCl4 solution and 0.3 ml of 20 mM RuCl3 solution (the total number of moles of Pt and Ru is 30 μmol).

[0146] Examples 1-9

[0147] This embodiment is the same as Embodiment 1-1, except that the molar ratio n Pt :n Ru The ratio is 4:1. Take 2.8 ml of 20 mM K2PtCl4 solution and 0.7 ml of 20 mM RuCl3 solution (the total moles of Pt and Ru are 70 μmol).

[0148] Comparative Example 1

[0149] This comparative example is the same as Example 1-1, except that the molar ratio n is... Pt :n Ru The ratio was 9.5:0.5. 2.47 ml of 20 mM K2PtCl4 solution and 0.13 ml of 20 mM RuCl3 solution were taken (the total moles of Pt and Ru were 52 μmol).

[0150] Comparative Example 2

[0151] This comparative example is the same as Example 1-1, except that the molar ratio n is... Pt :n Ru The ratio was 6.5:3.5. 1.69 ml of 20 mM K2PtCl4 solution and 0.91 ml of 20 mM RuCl3 solution were taken (the total number of moles of Pt and Ru was 52 μmol).

[0152] Comparative Example 3

[0153] This comparative example is the same as Example 1-1, except that glycine is not added.

[0154] Table 1 Performance data of the embodiments and comparative examples

[0155]

[0156]

[0157] The data for the above examples and comparative examples were obtained under the same test conditions. As can be seen from Table 1, among all examples and comparative examples, Example 1-1 exhibited the best catalytic activity (expressed as K0). m Smaller, V max The bacterial mortality rate was higher (larger) and the sterilization effect was the best (manifested as a higher bacterial mortality rate and biofilm removal rate); in contrast, the sterilization effect of Comparative Examples 1-3 was not as good as the above-mentioned examples (manifested as a decrease in bacterial mortality rate and biofilm removal rate).

[0158] Example 2:

[0159] This embodiment investigates the structural characterization, oxidase-like activity, and peroxidase-like activity of the bimetallic PtRu alloy nanozyme prepared in Example 1-1. The specific implementation process is as follows:

[0160] (1) Structural characterization of bimetallic PtRu alloy nanozymes

[0161] The above-mentioned synthesis using glycine-mediated co-reduction method, in Pt 2+ / Ru 3+ The structure of the bimetallic PtRu alloy nanozyme dispersion obtained at a molar ratio of 4:1 was characterized.

[0162] TEM and scanning electron microscopy (SEM) analyses revealed uniformly distributed ultrafine PtRu nanodots (indicated by red dashed circles). Figure 2 a, Figure 9 and Figure 10 ).

[0163] Energy dispersive spectroscopy (EDS) confirmed the presence of Pt and Ru, and that they were uniformly distributed throughout the sample. Figure 2 b-2e).

[0164] Line scan analysis of the sample ( Figure 2 f (yellow dashed arrow) indicates that the Pt content is significantly higher than that of Ru (Pt:Ru = 4:1), and the different distribution indicates that most of the Pt is exposed on the alloy surface.

[0165] Selected area electron diffraction (SAED) images show the lattice structure of PR. Figure 2 g).

[0166] High-resolution TEM (HR-TEM) analysis showed that PR exhibited an ultrafine quasi-spherical morphology. Figure 2 h). Both samples showed clear lattice fringes with an adjacent fringe spacing of 0.223 nm, corresponding to the (111) crystal plane of PR. In addition, PR showed certain defect structures (green dashed circles), which can be attributed to Pt vacancies induced by Ru occupying Pt sites.

[0167] X-ray diffraction (XRD) patterns indicate that PtRu has a face-centered cubic (fcc) phase structure. Figure 2 i) Typical diffraction peaks are observed at (111), (200), (220), and (311), consistent with the fcc structure of Pt. No additional peaks associated with hexagonal close-packed (hcp)Ru were detected due to the formation of the PtRu alloy.

[0168] The electron paramagnetic resonance (EPR) spectrum of PtRu is as follows: Figure 2 As shown in j, a pair of peaks (g = 2.003) are displayed, indicating the presence of high-density defects in the PR nanozyme.

[0169] XPS measurements showed signals corresponding to Pt 4f, Pt 4d, Ru 3p, C 1s, and O1s. Figure 2 The presence of Pt and Ru in the PtRu alloy nanozyme was confirmed. The C1s and O1s peaks at 284.8 eV and 532 eV, respectively, mainly originated from additives used in the preparation of nanoparticles, such as glycine and PVP. High-resolution XPS spectra of Pt 4f and Ru 3p were fitted. Figure 2 l and 2m). Pt 4f 7 / 2 At 75.41 eV and Pt 4f 5 / 2 The binding energy of 71.63 eV indicates that Pt exists in its elemental form (Pt 0 The presence of one peak and the presence of another set of bimodal peaks (76.02 eV and 72.18 eV) can be attributed to Pt. 2+ Chemical state. The Ru 3p peaks at 461.93 eV (Ru 3p3 / 2) and 484.09 eV (Ru 3p5 / 2) correspond to metallic Ru. 0 The peaks at 463.69 eV and 487.96 eV indicate that RuO2 is in a high valence state.

[0170] The oxidation state and electronic structure of the metal elements were further characterized using X-ray absorption spectroscopy (XAS). The white line intensity, edge energy, and shape of the X-ray absorption near-edge structure (XANES) region are similar to those of the Pt foil, indicating that the oxidation state of Pt in PtRu is approximately zero. The Pt L3- edge position in PtRu shifts negatively, becoming almost identical to that of the Pt and Ru foils. Figure 2 n).

[0171] Furthermore, the corresponding Fourier transform extended X-ray absorption fine structure (EXAFS) did not show any scattering peaks associated with PtO2 bonds. Figure 2 o and Figure 11 This indicates that H2 annealing will oxidize PtRuO. x It is reduced to a metallic PtRu alloy.

[0172] These results indicate that the target PtRu alloy nanozyme was successfully prepared, and its structure was confirmed by various characterization techniques.

[0173] (2) Determination of peroxidase and oxidase-like activities of bimetallic PtRu alloy nanozymes

[0174] 2.1 Type Peroxidase Activity

[0175]

[0176] 2.2 Oxidase Activity

[0177]

[0178] 2.3 Ultrasound (US) Enhancement Effect

[0179]

[0180] 2.4 Analysis of Activity Assay Results

[0181] 2.4.1 Catalytic activity of PR under H2O2 and US stimulation

[0182] TMB is used to monitor the generation of ·OH, because it reacts with ·OH to form oxidized TMB, which produces a characteristic absorption peak at 652 nm. Figure 12 ).like Figure 3 As shown in figure a, the combination of PR and TMB resulted in slight changes, indicating the presence of some oxidase activity. A significant absorption peak at 652 nm was observed under H2O2 and US stimulation, demonstrating that PR can generate a large amount of ·OH through its peroxidase activity and sonodynamic properties. The generation of ·OH was most significant when CDT and SDT acted synergistically. Subsequently, the time-dependent changes of PR in the presence of H2O2 were examined. Figure 3As shown in b, the characteristic peaks increased significantly with time, indicating that PR exhibits strong CDT performance. Furthermore, the effects of different US conditions on the binding of PR to H2O2 were explored. Figure 3 As shown in c, under US irradiation, the combination of PR and H2O2 produced more ·OH, indicating that SDT effectively enhanced CDT to produce more ROS.

[0183] To further explore the generation of 1O2, DPBF was used as an 1O2 scavenger because it can specifically degrade 1O2 in its presence. In the presence of PR, the typical absorption peak of DPBF at 419 nm gradually decreased with increasing US irradiation time, indicating that 1O2 generation is time-dependent. Figure 3 d). Compared to the DPBF+US group (5.7%), the PR+US group showed a 68.0% reduction in DPBF absorption after 4 minutes, indicating optimal acoustic dynamic performance under these conditions. Furthermore, the PR+H2O2+US group showed a 77.3% reduction in DPBF absorption, suggesting that the generation of 1O2 by PR is primarily due to acoustic dynamic effects, and the presence of H2O2 promotes 1O2 generation.

[0184] 2.4.2PR's CDT and SDT performance

[0185] To further investigate the CDT and SDT performance of PR, it was found that the control group and free PR were insufficient in terms of energy required to generate ·OH. Therefore, 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) was used as a ·OH scavenger. To verify the contribution of PR to CDT and SDT, the generation of ·OH was tested under PR+SDT, PR+CDT, and PR+CDT+SDT conditions. Figure 3 As shown in Figure e, significant ·OH signal peaks were generated under both CDT and SDT conditions, with the most satisfactory ·OH signal observed under the synergistic condition of CDT and SDT, indicating that CDT and SDT can effectively generate ·OH. Furthermore, ·OH can also be detected using an OPD scavenger. Figure 13 Furthermore, electron spin resonance (ESR) spectroscopy confirmed that significant 1O2 production was observed under photodynamic conditions (PR+US) when 2,2,6,6-tetramethylpiperidine (TEMP) was used as the 1O2 scavenger. In contrast, no significant signal peaks were detected in the control group, PR group, and PR+CDT group, indicating no 1O2 production. Moreover, 1O2 production was enhanced during the synergistic process of CDT and SDT. Figure 3 f). According to the Lineweaver-Burk plot, when H2O2 is used as the substrate at 25°C, PR shows V. max It is 5.8 × 10 -7 M / s, K m 0.55mM ( Figure 14 ).

[0186] Example 3:

[0187] This embodiment evaluates the in vitro antibacterial and anti-biofilm effects of the bimetallic PtRu alloy nanozyme prepared in Example 1-1. The specific implementation process is as follows:

[0188] 1. Experimental Methods

[0189] (1) Evaluation of in vitro antibacterial activity

[0190] During the antimicrobial study, multidrug-resistant Pseudomonas aeruginosa (MDRPA) was used as a Gram-negative bacterium, and methicillin-resistant Staphylococcus aureus (MRSA) as a Gram-positive strain. The strains were revived from frozen glycerol stock solutions and inoculated overnight in a humidified incubator at 37°C. The bacterial cell population density was determined by measuring absorbance at 600 nm. In a typical antimicrobial assay, MDRPA and MRSA were divided into six groups: (1) bacteria; (2) bacteria + US; (3) bacteria + PR; (4) bacteria + PR + US; (5) bacteria + PR + H2O2; and (6) bacteria + PR + H2O2 + US. Groups (2), (4), and (6) were further exposed to US irradiation for 5 minutes (1.0 MHz, 0.5 W / cm²). -2 Following US irradiation, the treatment procedures were the same as those for groups (1), (3), and (5). The final concentrations of PR, H2O2, and bacteria were 50 μg / mL, 100 μM, and 1 × 10⁻⁶, respectively. 6 Colony forming units (CFU / mL). The total volume of solution in each well is 0.1 mL. After incubation for 2 hours, 20 μL of diluted bacterial culture (10⁻⁶ dilution) is added. 3 The bacterial colonies were spread (multiplied by 1) onto agar plates and incubated in a shaker at 37°C for 15 hours. The colony counts were then recorded. All experiments were repeated three times. Bacterial survival rate was calculated using the following formula: Survival rate (%) = Nt / Nc × 100%, where Nt represents the number of colonies formed in the experimental group, and Nc refers to the number of colonies formed in the control group (PBS treatment).

[0191] (2)Live / Dead staining

[0192] Bacterial viability was imaged using a dual-fluorescent dye method. All samples were centrifuged at 8000 rpm for 5 minutes and washed twice with double-distilled water (DDW). After different treatments, each group was co-stained with green fluorescent dye (Calcein-AM) and red fluorescent dye (PI) for 20 minutes. MDRPA and MRSA samples with different treatments were imaged using a laser scanning confocal microscope (NIKON).

[0193] (3) Bacterial morphology studies

[0194] SEM was used to further examine the damage to bacteria caused by PR. Bacterial solutions treated with different methods were fixed in 4% paraformaldehyde for 4 hours. Subsequently, all samples were washed five times with deionized distilled water (DDW) and treated for 10 minutes each with ethanol of increasing concentrations (30%, 40%, 50%, 65%, 75%, 85%, 95%, and 100% v / v). Afterward, a gradient dehydration process was performed at 8000 rpm for 5 minutes. Finally, the dried dead bacteria were adhered to conductive tape and sputter-plated with gold for observation.

[0195] (4) In vitro biomembrane inhibition test

[0196] 20 μL of MDRPA or MRSA (OD) cultured overnight 600 =0.05) was added to 24-well plates and incubated at 37°C to establish a biofilm. After 24 hours, the top layer of LB or TSB medium after the formation of MDRPA or MRSA biofilms was removed. Simultaneously, 200 μL of plain PBS, H2O2 (0.1 mM), and PR (50 μg / mL) were added to each well. The plain PBS treatment group served as a negative control. All groups were incubated at 37°C for 2 hours. For the US irradiation group (1.0 MHz, 0.5 W / cm²), 2 The same parameters were used for 5 minutes. Then, the supernatant in each well was removed and the membrane was dried to fix MDRPA or MRSA. After fixation, 200 μL of 5% crystal violet dye was added to stain the MDRPA or MRSA biofilm. After staining for 30 minutes, the MDRPA or MRSA biofilm was rinsed twice with PBS to remove unbound crystal violet. Each well was then allowed to dry. Finally, 200 μL of acetic acid was added to each well to dissolve the dye bound to the MDRPA or MRSA biofilm, and the absorbance of all samples was measured at 590 nm using a microplate reader.

[0197] 2. Analysis of in vitro antibacterial and anti-biofilm effects

[0198] Alloy nanozymes exhibit significant biocatalytic activity, acoustic-dynamic properties, and biocompatibility, prompting exploration of their antibacterial effects. Due to their unique properties and tiny size, alloy nanozymes can be rapidly absorbed by bacteria and easily penetrate bacterial biofilms. Therefore, it is hypothesized that they can rapidly accumulate within bacteria, exhibiting potent bactericidal effects and biofilm scavenging capabilities through the synergistic action of CDT and SDT.

[0199] The antibacterial activity of PR against multidrug-resistant Pseudomonas aeruginosa (MDRPAO) and methicillin-resistant Staphylococcus aureus (MRSA) was quantitatively evaluated using the standard colony counting method. Figure 4(a-4d). Compared with the control group cultured in PBS, free PR showed no significant bactericidal effect against MDRPAO and MRSA. Under US conditions, PR exhibited some antibacterial activity against MDRPAO and MRSA. Furthermore, in the presence of H2O2, PR effectively inhibited P. aeruginosa PAO and MRSA by 76.3% and 79.5%, respectively, confirming that the potent CDT triggered by PR can combat bacterial infection. As expected, the synergistic effect of US and CDT (CDT / SDT group) resulted in the most significant bacterial death in MDRPAO and MRSA.

[0200] Quantitative analysis showed that PR had an antibacterial activity exceeding 99%, attributed to the effective enhancement of catalytic activity and efficient bactericidal effect under US irradiation. The antibacterial effects of PR against MDRPAO and MRSA were visually demonstrated through live / dead staining and quantitative analysis, as shown in the CLSM images. Figure 4 (e-4h) Viable bacteria stained with SYTO-9 showed green fluorescence, while dead bacteria were labeled with red propidium iodide (PI). As expected, the PBS-treated group and the free PR group showed predominantly green fluorescence in MDRPAO and MRSA, indicating viable bacteria. Simultaneously, significant bacterial death (red fluorescence) was observed in the PR+CDT and PR+US treatment groups. The synergistic effect of US and CDT significantly improved the bactericidal effect, achieving a bacterial mortality rate of over 99%. Under US, almost all bacteria in the PR+CDT treatment group showed red fluorescence, confirming the excellent antibacterial effect of the synergistic CDT / SDT combination.

[0201] To further verify the disruption of MDRPAO and MRSA bacterial membranes, scanning electron microscopy (SEM) was used to observe the morphological changes of bacteria within the bacterial membranes. Figure 4 As shown in i and 4j, minimal membrane disruption was observed in PBS-treated bacterial membranes, with MDRPAO and MRSA exhibiting densely packed, intact rod-like and spherical shapes, respectively. In contrast, PR disrupted bacterial membrane integrity after US or CDT treatment, confirming that ROS generated by PR via US and CDT can partially disrupt bacterial membranes. As expected, the most severe membrane damage occurred after synergistic CDT / SDT treatment, with most bacteria exhibiting shrunken and twisted shapes, indicating severe cell membrane damage (as shown by the red and yellow arrows in the figure). Studies have shown that over 80% of chronic infections are associated with biofilms, posing a significant challenge to clinical treatment. Biofilms protect bacteria from host immune attack and prevent the penetration of antimicrobial agents. Inspired by the excellent antimicrobial activity of PR, its antimicrobial capacity against MDRPAO and MRSA biofilms was further investigated using crystal violet staining and fluorescent staining methods. Figure 4As shown in k-4n, biofilm biomass or bacterial activity were not significantly reduced in the PBS group and the free PR group. In contrast, when PR was combined with H2O2 (0.1 mM) or US radiation (0.5 W), it significantly reduced the biofilm of MDRPAO and MRSA. Notably, compared with the PR treatment group, the PR+US+CDT group showed a significant reduction in biofilm biomass and bacterial activity, indicating that PR has excellent anti-biofilm effects through synergistic eradication of biofilms via CDT / SDT. These results suggest that PR has the potential to eliminate biofilms and exhibits excellent bactericidal properties due to its effective internalization ability. Furthermore, the anti-biofilm effect of PR was observed using live / dead staining. Figure 4 As shown in o and 4p, 3D CLSM images reveal the corresponding fluorescence patterns of biofilms after different treatments. After PBS and PR treatment, intact and dense green fluorescent biofilms appeared. Compared to biofilms treated with US or CDT alone (showing abundant yellow fluorescence), biofilms treated with PR-mediated synergistic CDT / SDT primarily showed red fluorescent spots, accompanied by biofilm loss. This indicates significant biofilm diffusion and bacterial death. Therefore, this further validates the strong biofilm diffusion ability and bactericidal effect of PR under external US and CDT stimulation.

[0202] In summary, PR can effectively eliminate biofilms and has great potential in combating biofilm-related infections through the synergistic effect of CDT and SDT.

[0203] Example 4:

[0204] This embodiment evaluates the antibacterial efficacy of the bimetallic PtRu alloy nanozyme prepared in Example 1-1 in pneumonia infection. The specific implementation process is as follows:

[0205] 1. Experimental Methods

[0206] An acute MDRPA-induced pneumonia model was established based on previous research. Specifically, mice were anesthetized, and the trachea was fully exposed by blunt dissection. A total of 50 μL of MDRPA (5 × 10⁻⁶) suspended in PBS was injected using a syringe. 7 The bacteria (CFU / mL) were slowly injected into the mouse trachea, followed by gentle agitation to ensure even distribution in both lungs. Two hours after infection, 100 μL of PR (50 μg / mL) and H2O2 (0.1 mM) suspended in PBS were slowly injected into the trachea using a syringe. Lung tissue samples were then examined using H&E staining and immunofluorescence analysis. Additionally, major organs, including the heart, spleen, kidneys, and liver, were collected for H&E staining.

[0207] 2. Analysis of the antibacterial results of PR in pneumonia infection

[0208] Based on its strong in vitro antibacterial activity, this invention uses an MDRPAO-induced pneumonia mouse model to simulate deep tissue bacterial infection and evaluates the in vivo antibacterial and antibiofilm properties of PR alloy nanozymes. Twelve hours after MDRPAO infection, infected mice were administered various treatments via nebulization. Forty-eight hours after treatment, the infection and inflammation status in the lung tissue were assessed. Figure 5 a).

[0209] The results showed that PR exhibited significant antibacterial and antibiofilm effects through the combined mechanism of CDT and SDT, while effectively reducing pulmonary edema and ignoring the potential toxicity of PR to lung tissue. Figure 5 b and Figure 15 These findings further confirm the potential of PR in in vivo antimicrobial therapy.

[0210] Subsequently, lung tissue was removed and homogenized using the standard plate dilution method to assess its bactericidal effect. Figure 5 c). CDT+US group (US irradiation 1.0MHz, 0.5W / cm²) 2 The lowest number of bacterial colonies was observed in lung tissue after 5 minutes of treatment with infected tissue, indicating that the PR alloy nanozyme has significant antibacterial activity in a mouse lung infection model.

[0211] To further assess the histological changes in lung tissue, H&E staining was performed on lung tissue from different treatment groups. Figure 5 As shown in Figure d, the PBS group exhibited a significant increase in neutrophil infiltration and marked edema, which did not improve significantly over time. In contrast, the CDT treatment group effectively alleviated edema, but the number of inflammatory cells remained high. However, in the CDT+US treatment group, pulmonary edema was significantly reduced, and the number of inflammatory cells returned to near-normal levels, indicating that combined CDT and US treatment significantly promoted lung tissue repair and recovery.

[0212] In addition, immunofluorescence analysis was performed to examine inflammatory changes in lung tissue under various treatment conditions. Lung inflammatory changes were assessed by evaluating the expression levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and CD86 / CD206 after treatment. Figure 6 a-6b and Figure 16 As shown in a-16b, compared with the PBS group, the CDT+US group significantly downregulated IL-6 expression (weakened green fluorescence) and upregulated IL-10 expression (enhanced red fluorescence), indicating effective inhibition of the inflammatory response. Furthermore, the expression patterns of CD86 and CD206 confirmed these findings. The CDT+US group exhibited downregulated CD86 expression (weakened red fluorescence) and upregulated CD206 expression (enhanced green fluorescence), further confirming its ability to inhibit pro-inflammatory responses and promote the transition to anti-inflammatory responses. Figure 6c-6d and Figure 16 b- Figure 16 d). Detection of CD31 expression showed that the CDT+US group exhibited significant CD31 overexpression ( Figure 17 ).

[0213] Meanwhile, H&E sections were performed on the heart, liver, spleen, and kidneys of different treatment groups under different conditions, and the results showed no toxicity to other organs. Figure 18 ).

[0214] In summary, PR alloy nanozymes, through combined CDT and US therapy, demonstrated significant efficacy in clearing bacteria and biofilms from lung tissue and effectively reducing inflammation, with a 98% reduction in lung tissue bacterial load and a 70% downregulation of the inflammatory factor IL-6 (immunofluorescence). This provides a promising strategy for antibacterial and anti-inflammatory therapy.

[0215] Example 5:

[0216] This embodiment evaluates the in vivo simulated deep antibacterial effect of the bimetallic PtRu alloy nanozyme prepared in Example 1-1. The specific implementation process is as follows:

[0217] 1. Experimental Methods

[0218] To establish a bacterial infection model, all mice were subcutaneously inoculated with MRSA in the dorsal region. One day after infection, mice were randomly assigned to three groups (n=5): a PBS group, a CDT group, and a CDT+US group. In the CDT group, PR solution (50 μg / mL) and H2O2 (0.1 mM) were injected in situ into the infection site. In the CDT+US treatment group, after the in situ injection of PR (50 μg / mL) and H2O2 (0.1 mM), mice were irradiated with US (1.0 MHz, 0.5 W / cm²). 2 Infected tissue was treated for 5 minutes. Wound photographs were taken every other day after different treatments. Ten days after treatment, infected skin was collected and homogenized in PBS for colony counting to assess anti-infective efficacy. Infected skin samples were also used for hematoxylin-eosin (H&E), Masson, Gram staining, and immunofluorescence analysis. Major organs (including heart, spleen, lung, kidney, and liver) were obtained for H&E staining. Sections were then observed under a fluorescence microscope. Blood samples were obtained from treated mice after in vivo wound healing experiments and used for biochemical analysis. Finally, mouse weight was recorded, and mouse survival was assessed every 24 hours.

[0219] 2. Analysis of the results of PR simulating deep antibacterial effects in vivo

[0220] To further evaluate the generalizability of PR's antibacterial and antibiofilm effects in vivo, a subcutaneous abscess model of MRSA infection was established. Figure 7 a). Subcutaneous injection of MRSA (50 μL, 10) into mice. 7 (CFU / mL) and randomly assigned to three treatment groups: (i) PBS group, (ii) CDT group, and (iii) CDT+US group. After treatment under various conditions, we monitored the resolution of infected tissue abscesses and wound area to assess wound healing. Figure 7 As shown in b, after 10 days of treatment, the abscesses in the infected tissue of the US+CDT group had dissipated, and the wounds were almost completely healed compared to the PBS and CDT groups. Furthermore, in situ superimposed images depicting changes in wound area on days 0, 5, and 10 in different groups more visually demonstrate the role of US+CDT in promoting wound healing. Figure 7 c) The wound healing rate reached over 95% in 10 days. Furthermore, bacterial colony counts in the infected tissue on day 10 showed that the US+CDT group had a sterilization rate exceeding 95%, significantly higher than other groups. Figure 7 (d and 7g). This indicates that US-mediated enhancement significantly improved the bactericidal efficiency of CDT. For example... Figure 7 As shown in Figure e, the mice's body weight increased slowly but steadily during treatment, indicating no toxic side effects. Based on wound healing statistics at different stages, the PBS group showed the slowest wound healing rate, with more noticeable scarring and the largest relative wound area by day 10. Figure 7 f).

[0221] In contrast, the CDT+US group showed the fastest wound healing rate, with almost complete healing by day 10, indicating that US-enhanced CDT accelerated abscess resolution and promoted wound healing by generating more ROS in vivo. Pathological sections were examined to assess the debridement effect and wound healing promotion capacity of PR treatment on deeply infected tissues. Figure 7 As shown in h, almost no granulation tissue was observed in the PBS group, a thin layer of granulation tissue appeared in the CDT group, and the thickest granulation tissue was found in the CDT+US group.

[0222] Furthermore, local high-magnification H&E images showed that the CDT and CDT+US groups had significantly fewer inflammatory cells than the PBS group, indicating effective reduction of tissue inflammation. In Masson staining ( Figure 7 i) The blue collagen fibers in the CDT+US group were significantly higher than those in the PBS group and the CDT group, indicating that CDT+US can promote collagen deposition and accelerate wound healing, with collagen deposition increasing by at least 50%.

[0223] Similarly, Gram staining was performed to assess residual bacteria and biofilm in the repaired infected tissue on day 10. Figure 7As shown in Figure j, the number of residual bacteria in the CDT group and the US+CDT group was significantly reduced, indicating that PR can effectively clear residual bacteria in deep tissues through the combined action of US and CDT. Inflammation is inevitable during bacterial infection, and the ratio of pro-inflammatory macrophages (M1) to anti-inflammatory macrophages (M2) is a key indicator of the inflammatory state. TNF-α and TGF-β are typical biomarkers of M1 and M2 macrophages, respectively.

[0224] In addition, immunofluorescence analysis was performed to assess the inflammatory characteristics in the infected tissue. Figure 8 As shown in 8a and 8b, when inflammatory cytokines were detected, the CDT+US group significantly downregulated the expression of TNF-α (red fluorescence) and upregulated the expression of TGF-β (green fluorescence) in infected tissues. Figure 8 (c and 8d). These results indicate that macrophages gradually polarize from the M1 phenotype to the M2 phenotype, thereby promoting the healing process of infected tissue. Furthermore, the pro-angiogenic marker CD31 was used to assess and regulate angiogenesis. Correspondingly, due to reduced infection severity, the CD31 fluorescence intensity was significantly increased in the CDT+US group compared to the PBS and CDT groups, reflecting a rapid repair process in the infected area (c and 8d). Figure 8 e and 8f).

[0225] Furthermore, H&E sections of the heart, liver, spleen, lungs, and kidneys from different treatment groups under different conditions were analyzed, and the results showed no toxicity to other organs. Figure 19 ).

[0226] In addition, the potential toxicity of PR will be further explored through complete blood count and blood biochemistry tests. Figure 20 As shown, there were no significant differences in ALT, AST, BUN, WBC, MCV, and PLT levels between the PR group and the PBS group, and all were within the normal range, indicating that the potential toxicity of PR is negligible. This suggests that the bimetallic PtRu nanozyme has good biocompatibility, superior to other metal nanozymes (such as gold, palladium, platinum, silver, or their alloys). Experiments showed that the hemolysis rate of PR was <5% (0-200 μg / mL). These data indicate that the ultrasound-enhanced CDT antibacterial strategy can more effectively promote the healing of infected wounds.

[0227] The results of the above embodiments were obtained using statistical analysis methods: all experiments involved at least three independent measurements. The obtained data are expressed as mean ± standard deviation. Statistical significance among multiple groups was analyzed using a two-tailed Student's t-test, with significance determined using GraphPad Prism 8.0 software. Quantitative statistics were based on mean ± standard deviation (SD), with *p < 0.05, **p < 0.01, and ***p < 0.001.

[0228] 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 bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections, characterized in that, The bimetallic alloy nanozyme is mainly composed of two metal elements, Pt and Ru, with an atomic ratio of Pt / Ru of 90 / 10 to 70 / 30.

2. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to claim 1, characterized in that, The bimetallic alloy nanozyme was activated by ultrasound.

3. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to claim 1, characterized in that, The bimetallic alloy nanozymes are spherical in shape with an average particle size of 1-10 nm; and / or, The specific surface area of the bimetallic alloy nanoszyme is 500-2000 m 2 / g.

4. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to claim 1, characterized in that, The bimetallic alloy nanozyme has a face-centered cubic single-phase solid solution structure; and / or, The interplanar spacing of the bimetallic alloy nanozyme is 0.223 nm, corresponding to the 111 crystal plane of platinum crystal.

5. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to claim 1, characterized in that, For peroxidase-like POD activity, under the condition without ultrasonic activation, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanoszyme include: K m [TMB-POD] = 0.2-0.8mM, V max [TMB-POD] = 2×10 -7 -8×10 -7 M / s.

6. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to claim 1, characterized in that, For peroxidase-like POD activity, the enzyme catalytic reaction kinetic parameters of the bimetallic alloy nanoszyme under the condition of ultrasonic activation include: K m [TMB-POD] = 0.24-0.96 mM, V max [TMB-POD] = 2.4 x 10 -7 -9.6 x 10 -7 M / s.

7. The bimetallic alloy nanoenzyme for treating multidrug-resistant bacterial infections according to any one of claims 1-6, characterized in that, The bimetallic alloy nanozyme was prepared using a glycine-mediated co-reduction method.

8. The method for preparing bimetallic alloy nanozymes for treating multidrug-resistant bacterial infections according to any one of claims 1-7, characterized in that, The preparation method includes the following steps: (1) Mix the soluble platinum source and ruthenium source with solvent, glycine and stabilizer in the required molar ratio for the first stage of mixing until all materials are completely dissolved to form a homogeneous precursor solution. (2) Under controlled temperature conditions, the precursor solution and the reducing agent are mixed together for a second-stage mixing process until the reaction is complete, and the dispersion of the bimetallic alloy nanoenzyme is obtained.

9. The use of the bimetallic alloy nanozyme according to any one of claims 1-7 or the bimetallic alloy nanozyme obtained by the preparation method according to claim 8 in the preparation of a medicament and / or bactericide for treating diseases caused by bacterial infections.

10. The application according to claim 9, characterized in that, In this application, the bimetallic alloy nanoenzyme dispersion and hydrogen peroxide are applied to the infected site, and the site is activated by ultrasonic irradiation. The mass concentration of the bimetallic alloy nanoenzyme dispersion is 10-200 μg / ml, the molar concentration of the hydrogen peroxide is 0.05-1.0 mM, and the ultrasonic irradiation frequency is 0.1-5 MHz with a power density of 0.1-5 W / cm². 2 The irradiation time is 1-10 minutes.

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