Ultrasound-activated bimetallic PtRu alloy nanozymes for synergistic sonodynamic and chemodynamic therapy of multidrug-resistant bacterial infection

Abstract

Deep-seated infections caused by multidrug-resistant (MDR) bacteria, such as pneumonia and abscesses, present significant therapeutic challenges due to their complex pathological microenvironments, which often limit the efficacy of conventional antibiotic treatments. The increasing emergence of MDR bacteria, along with their ability to rapidly acquire resistance, has intensified the need for novel therapeutic strategies. The advancement of nanotechnology has facilitated the development of non-antibiotic-dependent treatment modalities, which are increasingly preferred due to their high efficiency, non-invasiveness, and resistance-free properties. In this study, guided by density functional theory (DFT) predictions, we designed an ultrasound (US)-activated bimetallic Pt_xRu_y alloy nanozyme (PR) that synergistically combines US-activated sonodynamic therapy (SDT) with chemodynamic therapy (CDT) for precise control of reactive oxygen species (ROS) generation. By carefully optimizing the atomic ratio of platinum (Pt, catalytic sites) to ruthenium (Ru, adsorption sites), we synthesized ultrafine bimetallic alloy nanoplatforms with enhanced functional performance. Both theoretical simulations and experimental characterizations confirmed that PR exhibits exceptional oxidase-like and peroxidase-like activity, facilitating enhanced US-triggered ROS production through amplified sonodynamic effects. The PR demonstrated significant in vitro antibacterial activity, effectively disrupted biofilms, and showed excellent biocompatibility. In mouse models of pneumonia and subcutaneous abscesses, PR facilitated rapid bacterial clearance and modulation of the inflammatory microenvironment. This study presents a novel, non-antibiotic biocatalytic platform that provides a rational design strategy for bimetallic alloy nanozymes, offering a promising therapeutic approach for the synergistic treatment of MDR bacterial infections. These findings underscore the translational potential of multifunctional nanoplatforms in addressing the growing challenge of antibiotic resistance.

Graphical abstract

Highlights

• •DFT-guided design optimizes Pt/Ru alloy nanozyme for catalytic ROS generation.
• •PR nanozyme enables synergistic CDT/SDT therapy against MDR bacterial infection.
• •US activation boosts .•OH and ¹O₂ production for potent antibacterial effect.
• •PR shows high antibiofilm efficiency in MDR-induced pneumonia and abscess models.
• •Exhibits excellent biocompatibility with no observable systemic toxicity in vivo.

1. Introduction

Multidrug-resistant (MDR) bacteria have emerged as significant hospital pathogens, posing a considerable threat to global public health. These bacteria can lead to various infections, particularly in immunocompromised patients, including pneumonia, abscesses, keratitis, and septicemia [1]. Furthermore, the ability of MDR bacteria to acquire resistance easily and their rapid transmission exacerbate the challenges they pose to human health. In recent years, there has been a marked increase in severe antibiotic resistance, resulting in limited treatment options and drawing significant attention to the difficulties these infections present in clinical practice. Among these infections, bacterial pneumonia (BP) is one of the leading causes of morbidity and mortality worldwide, and the rise of MDR BP has made its treatment increasingly challenging. With advancements in nanotechnology various non-antibiotic-dependent treatment strategies have emerged, including photodynamic therapy (PDT) [2], photothermal therapy (PTT) [[3], [4], [5]], chemodynamic therapy (CDT) [[6], [7], [8], [9]], and sonodynamic therapy (SDT) [10,11]. These strategies are particularly promising as they can eliminate bacteria and disrupt biofilms without easily developing resistance, unlike traditional antibiotics. While phototherapy has been widely utilized for treating skin infections, its effectiveness is limited in deep tissue infections due to the shallow tissue penetration depth of lasers. In contrast, SDT offers significant advantages for treating deep tissue diseases, as ultrasound (US) waves possess superior tissue penetration capabilities. SDT operates by activating sound sensitizers with US waves to generate ROS, such as singlet oxygen (¹O₂) and hydroxyl radicals (•OH) [[12], [13], [14]], which are expected to exhibit potent antibacterial effects.

CDT represents an emerging anti-infective strategy that leverages nanozyme activity with high bactericidal efficacy. Nanozymes have garnered considerable attention in interdisciplinary research due to their stability and ease of synthesis compared to natural enzymes. Over 50 types of nanomaterials, including metal oxides, metals, metal sulfides, and carbon-based nanostructures, have been developed as enzyme mimics, demonstrating promising potential in clinical diagnosis and disease treatment [[15], [16], [17]]. Among these, metal and metal-based nanostructures are particularly noteworthy due to their advanced synthesis methods, well-defined and controllable structures, and ease of surface modification. Some of these materials exhibit multiple enzyme activities. For instance, platinum (Pt)-based nanoparticles (NPs) can mimic the activity of at least four oxidoreductases, including peroxidase, oxidase, catalase, and superoxide dismutase [[18], [19], [20]]. Interestingly, the catalytic effects of these NPs appear contradictory: while peroxidase and oxidase activities promote substrate oxidation and generate a prooxidant effect, catalase and superoxide dismutase-like activities enhance the reduction of hydrogen peroxide (H₂O₂) and superoxide, exhibiting antioxidant effects. Regulating both prooxidant and antioxidant activities simultaneously, along with understanding the structure-activity relationship of nanozymes, is essential for their development and specific applications [[21], [22], [23], [24]]. To achieve sustainable artificial catalysis, it is critical to explore and develop efficient biocatalysts and sonosensitizer. Bimetallic alloy systems, which can adjust both electronic and geometric structures, offer distinct advantages over their monometallic counterparts. These systems, particularly those with solid solution structures, exhibit enhanced physical and chemical properties. Notably, recent advancements in bimetallic platinum-ruthenium (PtRu) systems have showcased their high catalytic efficiency, positioning them as promising candidates for biocatalytic applications [25]. The incorporation of ruthenium (Ru) into platinum (Pt) nanoparticles enhances not only charge separation and transfer efficiency but also the adsorption and decomposition of H₂O₂

In this study, we developed a novel bimetallic alloy nanozyme (PR) to serve as a sonosensitizer and biocatalyst. We administered PR to the lungs of mice through tracheal delivery, achieving a synergistic strategy of SDT and CDT for the treatment of MDR BP (Scheme 1). By integrating Ru into Pt nanoparticles, we engineered stable PtRu alloys characterized by uniform size and a high specific surface area. The optimal Pt-to-Ru ratio was determined through calculations, resulting in narrower band gaps and lower activation energy for the H₂O₂ reaction. Under low-dose H₂O₂ and ultrasound irradiation, PR generated substantial ¹O₂ and •OH, effectively eradicating MDR bacteria, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). In a mouse lung infection model, the synergistic effects of CDT and SDT using PR facilitated efficient bacterial clearance and promoted lung tissue healing. Furthermore, PR demonstrated safety and no significant toxicity at both the cellular and animal levels in subcutaneous abscess models. As a safe and effective sonosensitizer and biocatalyst, the metal alloy nanozyme offers a promising solution for the treatment of MDR bacterial infections.

Scheme 1.

The rational design and bioapplications of PR alloy nanozymes. (a) Schematic illustration of the synthesis of the PR alloy nanozymes. (b) The SDT and CDT performance of PR and the synergistic effect of US enhancing CDT. (c) PR alloy nanozymes for dual-modal anti-infective therapy with MDRPAO-induced pneumonia and MRSA-induced subcutaneous abscess models using US Enhanced CDT strategy.

2. Experimental section

2.1. Materials and instruments

Potassium tetrachloro platinate(II) (K₂PtCl₄), Ruthenium (III) chloride hydrate (RuCl₃), polyvinylpyrrolidone K30 (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. Tryptic soy broth (TSB) medium, Luria-Bertani broth (LB), and agar powder were obtained from Solarbio. The LIVE/DEAD® BacLight™ bacterial viability kit was purchased from Thermo Fisher Scientific. Phosphate-buffered saline (PBS) was also utilized. 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 via X-ray photoelectron spectroscopy (XPS) with the Thermo Scientific K-Alpha. UV–vis–NIR spectroscopy of the solution was recorded using a CARY 5000 spectrophotometer (USA, Agilent Technologies). Fluorescent photographs were captured with a confocal laser scanning microscope (CLSM, Nikon Japan A1).

2.2. Synthesis of PR

PR alloy nanozymes were synthesized following a modified version of a previously established method [26]. In brief, 20 mM Ru³⁺ and 20 mM PtCl₄²⁻ were combined in appropriate volumes with 6.7 mL of water in a 50 mL round-bottom flask. Subsequently, 180 mg of glycine and 440 mg of PVP were added to this solution and mixed ultrasonically until clarity was achieved. The flask was then placed in a water bath at 60 °C, and stirring was maintained. After reaching thermal equilibrium, 1.3 mL of a 1M AA solution (with a molar ratio of AA to (Ru³⁺ and Pt²⁺) of 25) was introduced into the homogeneous solution while stirring vigorously for 2 h, resulting in a color change to dark grey, indicative of bimetallic PR formation.

2.3. Peroxidase-like and oxidase-like activities assays of PR

The generation of •OH was analyzed based on its ability to oxidize TMB or OPD probes, thereby enhancing the absorbance of TMB at 652 nm or OPD at 420 nm. TMB and OPD served as indicators to visualize and monitor •OH generation [27,28]. Specifically, 1.0 mL of PBS buffer containing PR (10 μg mL⁻¹), TMB (0.1 mM), and H₂O₂ (1.0 mM) was incubated at room temperature (25 °C) for varying time intervals. Alternatively, PR was incubated for different durations (0, 1, 2, 3, and 4 min) under the same conditions with consistent concentrations. The color of the solution was captured, and the UV–vis–NIR spectra were recorded. The catalytic performance of PR was further assessed using an OPD probe. Solutions of PR (10 μg mL⁻¹), OPD (0.1 mM), and H₂O₂ (1.0 mM) were mixed with or without ultrasound (US) irradiation. After various incubation times, the solution's color was imaged, and the UV–vis–NIR spectra were measured. These reactions were also conducted in the absence of H₂O₂ to evaluate oxidase activity.

US enhances nanozymes activity: PR (50 μg mL⁻¹) was mixed with DPBF (60 μL, 20 μg mL⁻¹) in 1.0 mL phosphate buffer saline (PBS, pH 7.4) with 5 % dimethyl sulfoxide (v/v). The mixture was exposed to US irradiation for 5 min (1.0 MHz, 0.5 W cm⁻²). After high-speed centrifugation, the supernatant was passed through UV–vis–NIR spectroscopy to detect the attenuation trend at 420 nm (DPBF) with different time points to test whether ¹O₂ was produced.

2.4. Antibacterial activity evaluation in vitro

During antibacterial investigations, multidrug-resistant Pseudomonas aeruginosa (MDRPA) was used as the Gram-negative bacteria and MRSA as the Gram-positive bacterial strain. The strains were revived from frozen glycerol stocks by inoculation overnight at 37^oC in a humidified incubator. The population density of the bacterial cells was determined by measuring the absorbance at 600 nm. In a typical antibacterial assay, MDRPA and MRSA were divided into the following six groups: (1) bacteria; (2) bacteria + US; (3) bacteria + PR; (4) bacteria + PR + US; (5) bacteria + PR + H₂O₂; (6) bacteria + PR + H₂O₂ + US. The groups (2), (4), and (6) were further exposed to US irradiation for 5 min (1.0 MHz, 0.5 W cm⁻²). After the US irradiation, the procedure was the same as groups (1), (3), and (5). The final concentrations of PR, H₂O₂, and bacteria were 50 μg mL⁻¹, 100 μM, and 1 × 10⁶ colony-forming units (CFU/mL), respectively. The total volume of solution in each well was 0.1 mL. After incubation for 2 h, 20 μL of the diluted bacterial culture (10³ times) was spread onto the agar plate and incubated in a shaker at 37 °C for 15 h, and the number of colonies was counted. All experiments were repeated three times. The bacteria survival rate was calculated using the following equation:

$$\text{Survival}\text{viability}(\%)=\text{Nt}/\text{Nc}\times 100\%$$

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 treated).

2.5. Live/dead staining

Bacterial viability was imaged using the dual fluorescent dye method [29]. All samples were centrifuged at 8000 rpm for 5 min and washed 2 times with double distilled water (DDW). After each group was treated differently, it was then co-stained with a green fluorescent dye (Calcein-AM) and a red fluorescent dye (PI) for 20 min. Differently treated MDRPA and MRSA were imaged under a laser scanning confocal microscope (NIKON).

2.6. Bacterial morphology study

The damage inflicted by PR on bacteria was further examined using SEM. Bacterial solutions subjected to various treatments were fixed in 4 % paraformaldehyde for 4 h. Subsequently, all samples were washed five times with deionized distilled water (DDW) and treated with increasing concentrations of ethanol (30 %, 40 %, 50 %, 65 %, 75 %, 85 %, 95 %, and 100 % v/v) for 10 min each. Following this, gradient dehydration was performed at 8000 rpm for 5 min. Finally, the dried, dead bacteria were adhered to conductive tape and sputter-coated with gold for observation.

2.7. Biofilm inhibition tests in vitro

20 μL of MDRPA or MRSA (OD₆₀₀ = 0.05) cultured overnight was added into a 24-well plate and incubated at 37 °C to establish biofilm [30]. After 24 h, the upper LB or TSB medium after the formation of MDRPA or MRSA biofilms was removed. Simultaneously, 200 μL of normal PBS, H₂O₂ (0.1 mM), and PR (50 μg mL⁻¹) was added into each well. A normal PBS-treated group was used as a negative control. All groups were cultured at 37 °C for 2 h. For US irradiation groups, the same parameters were adopted as described above. Then, the supernatant in each well was removed and dried to fix MDRPA or MRSA. After fixation, 200 μL of 5 % crystal violet dye was added to stain MDRPA or MRSA biofilms. MDRPA or MRSA biofilms were rinsed twice with PBS to remove unbound crystal violet after staining for 30 min. Then allow each well to dry. At last, 200 μL of acetic acid was added into each well to dissolve the bound dye of MDRPA or MRSA biofilms followed by the measurement of the absorbance of all samples at 590 nm using a microplate reader.

2.8. Hemolysis analysis of PR

The hemolytic properties were evaluated using erythrocytes from fresh white mice. In brief, whole blood was centrifuged at 3000 rpm for 10 min to isolate the erythrocytes. Following three washes with PBS, 1 mL of erythrocytes was combined with 19 mL of PBS and stored in a refrigerator for subsequent use. Subsequently, 500 μL of the erythrocyte suspension was mixed with 500 μL of PR dispersions at concentrations of 0, 25, 50, 100, and 200 μg mL⁻¹. DDW and PBS served as the positive and negative control groups, respectively. The mixed solutions were then incubated at 37 °C for 6 h. After incubation, the absorbance at 540 nm was measured following centrifugation at 12000 rpm for 10 min. Finally, the hemolysis ratio was calculated using the following formula:

$$\text{Hemolysis}\text{ratio}(\%)=({\mathrm{A}}_{\text{sample}}-{\mathrm{A}}_{\text{NC}})/({\mathrm{A}}_{\text{PC}}-{\mathrm{A}}_{\text{NC}})\times 100\%\text{.}$$

2.9. Statements

The drug-resistant bacterial strains (MDRPA and MRSA) used in this experiment were obtained from Wenzhou Medical University and strictly operated and executed according to the guidelines (Wenzhou Medical University Biosafety Operating Regulations). Additionally, mice (Balb/c and C57BL/6J) were purchased from Wenzhou Medical University. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Wenzhou Medical University and approved by the Animal Ethics Committee of SYXK-2021-0020.

2.10. Subcutaneous bacterial infection model

All mice were subcutaneously inoculated with MRSA in the dorsal region to establish a bacterial infection model [31,32]. After one day of infection, the mice were randomly divided into 3 groups (n = 5) including PBS, CDT, and CDT + US groups. For the group of CDT, PR solution (50 μg mL⁻¹) and H₂O₂ (0.1 mM) were in situ injected into the infected site. For the treatment group of CDT + US, the infected tissue was treated with US irradiation for 5 min (1.0 MHz, 0.5 W cm⁻²) after in situ injection of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM). After different treatments, photographs of the wounds in each group were taken every other day. After 10 days of therapy, the infectious skin was collected and homogenized in PBS for colony counting to evaluate the anti-infective effect. The infected skin samples were also prepared for hematoxylin and eosin (H&E), Masson, Gram staining, and immunofluorescence assay. And main organs (including the heart, spleen, lung, kidney, and liver) were acquired for H&E staining. Then the slices were observed with a fluorescence microscope. Blood samples were obtained from the treated mice after an in vivo wound healing experiment and then used to carry out the biochemical analyses. Finally, the body weight of mice was recorded and mice survival was evaluated per 24 h.

2.11. Acute MDRPA-infected pneumonia model

The acute MDRPA-infected pneumonia model was established based on previous studies [33]. Specifically, mice were anesthetized, and the trachea was fully exposed through blunt dissection. A total of 50 μL of MDRPAO (5 × 10⁷ CFU/mL), resuspended in PBS, was slowly injected into the trachea of the mice using a syringe, followed by gentle shaking to ensure even distribution of the bacteria in both lungs. After 2 h, 100 μL of PR (50 μg/mL) and H₂O₂ (0.1 mM), also resuspended in PBS, was slowly administered into the trachea using a syringe. Lung tissue samples were subsequently examined using H&E staining and immunofluorescence assays. Additionally, major organs, including the heart, spleen, kidney, and liver, were collected for H&E staining.

2.12. Statistical analysis

All the experiments were performed with at least three independent measurements. The obtained data were expressed as the mean value standard deviation, and the statistical significance for multiple groups was analyzed by the two-tailed Student t-test, and significance was achieved using GraphPad Prism 8.0. Quantitative statistics according to mean ± standard deviation (SD), and ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

3. Results and discussion

3.1. Rational design and theoretical calculation for biocatalytic activity

To investigate the efficient biocatalytic activity (peroxidase-like activity, POD-like) and the structure-activity relationship of Pt_xRu_y alloy nanozymes, as well as their catalytic mechanism, first-principles calculations were conducted using density functional theory (DFT) based on the Vienna Ab initio Simulation Package (VASP). The optimized full geometric structure of Pt_xRu_y alloy nanozymes with varying Pt and Ru ratios (i.e., Pt₈₀Ru₂₀, Pt₅₀Ru₅₀, and Pt₂₀Ru₈₀) were modeled using a face-centered cubic (fcc) lattice (Fig. 1a). Before calculating the catalytic reaction pathway, the overall catalytic activity and catalytic sites of the alloy nanozymes were assessed through electrostatic potential (ESP) simulation calculations [34]. The blue area represents positive charges, while the red area indicates negative charges. The blue region is conducive to the adsorption of H₂O₂. As shown in Fig. 1b, the negative ESP is mainly distributed at the top of the alloy nanozymes and the connection sites between the components. This observation suggests that both metals in the alloy nanozymes contribute to the enzymatic catalytic activity. As illustrated in Fig. 1c, the adsorbed H₂O₂ is first activated into H₂O₂∗ by Pt and Ru atoms in the alloy nanozymes. The O=O bond in the activated H₂O₂∗ strongly binds to the metal atom, resulting in an unstable intermediate (TS) that quickly breaks the O=O bond, producing OH∗. DFT calculations further confirm this result, with the adsorption energies of H₂O₂ at Pt and Ru sites in the alloy nanozymes being −0.56 and −1.16 eV (Pt₈₀Ru₂₀), −0.46 and −0.9 eV (Pt₅₀Ru₅₀), and −0.58 and −1.22 eV (Pt₂₀Ru₈₀), respectively (Fig. 1d and Table S1). These results indicate that the adsorption rate of H₂O₂ on Ru is higher than on Pt in the alloy nanozymes. However, the actual catalytic activity depends on the activation energy during the catalytic reaction. Therefore, the catalytic activity of the alloy nanozymes is further characterized by calculating the activation energy of the reaction. To achieve this, the free energy variation of each atom along the optimal reaction path in the alloy nanozymes was calculated. As shown in Fig. 1e and Table S2, the activation energies for Pt and Ru atoms from H₂O₂ to OH∗ are 0.28 and 0.5 eV (Pt₈₀Ru₂₀), 0.37 and 0.63 eV (Pt₅₀Ru₅₀), and 0.61 and 0.8 eV (Pt₂₀Ru₈₀), respectively. These results suggest that the ability to produce OH∗ is higher for Pt than for Ru in the alloy nanozymes. After obtaining sufficient desorption energy, OH∗ ultimately generates •OH. Furthermore, the DFT calculation results confirm that the designed alloy nanozymes exhibit efficient POD-like activity, with both metal elements in the alloy nanozymes contributing to enhancing their overall activity. DFT modeling was also used to examine how the adsorption strength of O atoms (derived from O atoms in H₂O₂) depends on the surface composition of the alloy nanozymes. Understanding the adsorption trend of O atoms is crucial for determining whether the binding energy of O atoms serves as a descriptor for the activity trend of the alloy nanozymes. As the Ru content of the alloy catalyst increases, the binding strength of O atoms becomes stronger (Fig. 1f), which is consistent with the above calculation results. The band structures of Pt₈₀Ru₂₀, Pt₅₀Ru₅₀, and Pt₂₀Ru₈₀, obtained based on the HSE06 functional, are shown in Fig. 1g-i. Research has found that the minimum conduction band (CBM) and maximum valence band (VBM) of Pt₈₀Ru₂₀ almost overlap, while the CBM and VBM of Pt₅₀Ru₅₀ and Pt₂₀Ru₈₀ are partially overlapping, but a clear separation trend can be observed. This indicates that alloy nanozymes do not have significant CBM and VBM separation. Notably, the energy band gap between the CBM and VBM of Pt₈₀Ru₂₀ is smaller, suggesting that this structure is more conducive to electron-hole separation, which can improve acoustic power efficiency. In summary, based on theoretical calculations, the structure of bimetallic Pt_xRu_y alloy nanozymes (PR), with a ratio around x/y = 80/20, appears to be the most favorable for optimal catalytic performance.

Fig. 1.

Design, screening, and chemodynamic mechanism of Pt_xRu_y alloy nanozymes. (a) Optimized structures of Pt_xRu_y alloy nanozymes (Pt₈₀Ru₂₀, Pt₅₀Ru₅₀, Pt₂₀Ru₈₀). (b) Electrostatic potential (ESP) simulation calculations for the Pt_xRu_y alloy nanozymes. (c) Optimized adsorption configurations of H₂O₂ and •OH on Pt and Ru sites within the alloy nanozymes. (d) Calculated Gibbs free energy changes for the conversion of H₂O₂ to •OH on the alloy nanozymes. (e) The energy barrier for H₂O₂ decomposition on the alloy nanozymes. (f) Top view of binding energies and geometries of the strongest-binding H₂O₂ positions on the (111) alloy nanozyme surface slab models at the Ru site. The solid black line represents the supercell. (g–i) Band structures for the valence band maximum (VBM) and conduction band minimum (CBM) of (g) Pt₈₀Ru₂₀, (h) Pt₅₀Ru₅₀, and (i) Pt₂₀Ru₈₀. Atom color legend: Maroon = Pt, Teal = Ru. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.2. Preparation and characterization of PR alloy nanozymes

The PR was synthesized using a glycine-mediated co-reduction method. The PR alloy nanozyme dispersion, obtained at a Pt²⁺/Ru³⁺ molar ratio of 4:1, was modified with polyvinylpyrrolidone (PVP) ligands to prevent aggregation. TEM and scanning electron microscope (SEM) analysis revealed that ultrafine nanodots of PR (indicated by red dashed circles) were obtained with a uniform distribution (Fig. 2a–S1, and S2). Energy dispersive spectroscopy (EDS) confirmed the presence of both Pt and Ru, which were uniformly distributed throughout the sample (Fig. 2b-e). Line scan analysis of the sample (Fig. 2f, yellow dashed arrow) demonstrated that the Pt content was significantly higher than that of Ru (Pt: Ru = 4:1), with differing distributions indicating that most of the Pt was exposed on the alloy surface. The selected area electron diffraction (SAED) image showed the lattice structure of PR (Fig. 2g). High-resolution TEM (HR-TEM) analysis indicated that PR exhibited an ultrafine quasi-spherical morphology (Fig. 2h). Both samples exhibited clear lattice stripes with adjacent stripe spacing of 0.223 nm, corresponding to the (111) plane of PR. In addition, PR displayed certain defect structures (green dashed circles), which could be attributed to Pt vacancies induced by Ru occupying Pt sites. The X-ray diffraction (XRD) pattern demonstrated that PR has a face-centered cubic (fcc) phase structure (Fig. 2i), with typical diffraction peaks at (111), (200), (220), and (311), consistent with the fcc structure of Pt [35]. Due to the formation of the Pt-Ru alloy, no additional peaks associated with hexagonal close-packed (hcp) Ru were detected. The electron paramagnetic resonance (EPR) spectrum of PR, shown in Fig. 2j, exhibited a pair of peaks (g = 2.003), indicating the presence of high-density defects in the PR nanozymes. XPS measurements showed signals corresponding to Pt 4f, Pt 4d, Ru 3p, C 1s, and O 1s (Fig. 2k), confirming the presence of Pt and Ru in PR. The C 1s and O 1s peaks at 284.8 eV and 532 eV, respectively, primarily 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 separately fitted (Fig. 2l and m). The binding energies of Pt 4f_7/2 at 75.41 eV and Pt 4f_5/2 at 71.63 eV indicated that Pt existed in its elemental form (Pt⁰), while another set of bimodal peaks (76.02 eV and 72.18 eV) could be attributed to the Pt²⁺ chemical state. The Ru 3p peaks at 461.93 eV (Ru 3p₃/₂) and 484.09 eV (Ru 3p₅/₂) corresponded to metallic Ru⁰, while the peaks at 463.69 eV and 487.96 eV indicated the high valence state of RuO₂. Subsequently, 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 in the X-ray absorption near-edge structure (XANES) region were similar to those of Pt foil, suggesting that the oxidation state of Pt in PR was approximately zero. The Pt L₃-edge positions of PR shifted in a negative direction and became almost identical to the positions of Pt and Ru foils (Fig. 2n). Furthermore, the corresponding Fourier transform extended X-ray absorption fine structure (EXAFS) did not show any scattering peaks related to PtO₂ bonds (Fig. 2o and S3), indicating that H₂ annealing reduced the oxidized PtRuO_x to a metallic PtRu alloy [36]. Based on these results, we successfully prepared the target PR alloy nanozymes and confirmed their structure through various characterization techniques.

Fig. 2.

Synthesis and surface morphology characterization of Pt₈₀Ru₂₀ alloy nanozymes (PR). (a) TEM image of PR alloy nanozymes. (b–e) Scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) elemental distribution maps for PR alloy nanozymes. Scale bar, 10 nm. (f) The location of the EDS line scan is indicated by the dotted yellow 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) patterns of PR alloy nanozymes. (j) Electron paramagnetic resonance (EPR) spectra of PR alloy nanozymes. (k) XPS survey spectra of PR alloy nanozymes. High-resolution XPS spectra of (l) Pt 4f and (m) Ru 3p from PR alloy nanozymes prepared with a Pt²⁺/Ru³⁺ atomic ratio of 3/1. (n) Pt L₃-edge X-ray absorption near-edge structure (XANES) spectra. (o) Corresponding Fourier-transformed extended X-ray absorption fine structure (FT-EXAFS) profiles of PR alloy nanozymes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. US-activated enhanced CDT in vitro

Based on the theoretical calculations and structural optimization screening, the PR was selected for its exceptional biocatalytic performance, attributed to its efficient H₂O₂ capture and catalytic capabilities. Furthermore, the surface piezoelectric potential induced by ultrasound (US) irradiation can enhance the interaction among polarized charges, charge carriers, and surrounding molecules (H₂O, O₂), thereby facilitating the generation of ROS through redox reactions [37]. Initially, the catalytic activity of PR under H₂O₂ and US stimulation was investigated. TMB was employed to monitor the production of •OH, as it reacts with •OH to yield oxidized TMB, producing a characteristic absorption peak at 652 nm (Fig. S4). As illustrated in Fig. 3a, the combination of PR and TMB resulted in minor changes, indicating some oxidase activity. Under both H₂O₂ and US stimulation, a significant absorption peak emerged at 625 nm, demonstrating that PR can generate substantial amounts of •OH through its peroxidase activity and sonodynamic characteristics. The most pronounced production of •OH occurred when CDT and SDT acted synergistically. Subsequently, the temporal variation of PR in the presence of H₂O₂ was examined. As depicted in Fig. 3b, the characteristic peaks increased significantly over time, indicating that PR exhibits robust CDT performance. Additionally, the effects of varying US conditions on PR in conjunction with H₂O₂ were explored. As shown in Fig. 3c, with US irradiation, the combination of PR and H₂O₂ produced an increased amount of •OH, suggesting that SDT effectively enhances CDT to generate more ROS. To further explore ¹O₂ production, DPBF was used as a ¹O₂ scavenger, as it can specifically degrade in the presence of ¹O₂. In the presence of PR, the typical absorption peak of DPBF at 419 nm gradually decreased with the extension of US irradiation time, indicating that ¹O₂ production is time-dependent (Fig. 3d). Compared to the DPBF + US group (5.7 %), the PR + US group exhibited a 68.0 % decrease in DPBF absorption after 4 min, indicating optimal acoustic dynamic performance under these conditions. Furthermore, the PR + H₂O₂+US group showed a 77.3 % decrease in DPBF absorption, indicating that the ¹O₂ generation by PR was mainly due to the acoustic dynamic effect and that the presence of H₂O₂ promotes the generation of ¹O₂. To further investigate the CDT and SDT performance of PR, the control group and free PR were found to be insufficient in terms of energy required to produce •OH, when using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the •OH capture agent. To verify the contributions of PR to CDT and SDT, the production of •OH was tested under PR + SDT, PR + CDT, and PR + CDT + SDT conditions. As shown in Fig. 3e, significant •OH signal peaks were generated under both CDT and SDT conditions, with the most satisfactory •OH signals generated under the synergistic conditions of CDT and SDT, indicating that CDT and SDT can effectively produce •OH. Moreover, the detection of •OH can also be achieved using an OPD capture agent (Fig. S5). Additionally, electron spin-resonance (ESR) spectroscopy confirmed that when 2,2,6,6-tetramethylpiperidine (TEMP) was utilized as a ¹O₂ capture agent, significant production of ¹O₂ was observed under photodynamic conditions (PR + US). In contrast, no notable signal peaks were detected in the control group, PR group, and PR + CDT group, indicating the absence of ¹O₂ production. Furthermore, in the cooperative process of CDT and SDT, the production of ¹O₂ was enhanced (Fig. 3f). According to the Lineweaver-Burk plot, the PR displayed a V_max of 5.8 × 10⁻⁷ M s⁻¹ and a K_m of 0.55 mM when H₂O₂ was applied as the substrate at 25^oC (Fig. S6). Moreover, the catalytic mechanism of Pt_xRu_y alloy nanozymes was deeply explored through theoretical calculations [[38], [39], [40]]. As shown in Fig. 3g and h, the results indicate that when there are a certain number of Pt vacancies in the alloy, the PDOS peak significantly decreases. This suggests that the interaction between Pt and Ru reduces the electron density of the Pt element, which in turn affects the catalytic activity. This further highlights that Pt significantly contributes to the electronic states of the alloy and may play a dominant role in catalytic reactions. Meanwhile, the calculation results for the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) demonstrate that the electron cloud density is predominantly localized around Pt, further substantiating the significant contribution of Pt to the alloy's electronic states (Fig. 3i and S7). As shown in Fig. 3j-l, the distribution of electrons and holes in alloys with varying Pt/Ru ratios (Pt₈₀Ru₂₀, Pt₅₀Ru₅₀, and Pt₂₀Ru₈₀) was analyzed. It was found that as the Pt ratio increased, the electron cloud gradually shifted toward Pt, indicating that Pt can effectively capture electrons and improve the catalytic performance of the alloy.

Fig. 3.

Biocatalytic performance and mechanistic insights of PR alloy nanozymes. (a) UV absorption changes of TMB under various conditions. (b) Time-dependent absorption changes of TMB in the presence of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM). Inset digital photos show the color change of the corresponding samples. (c) Absorption changes of TM in the presence of PR (50 μg mL⁻¹) and H₂O₂ (0.1 mM) under different ultrasound (US) irradiation times (1.0 MHz, 0.5 W cm⁻²). Inset digital photos show the color change of the corresponding samples. (d) Normalized absorbance of DPBF (in H₂O) in the presence of PR under different environmental conditions. Data are presented as mean ± S.D. (n = 3). ESR spectra of (e) DMPO/•OH and (f) TEMP/¹O₂ (in H₂O) for PR under H₂O₂ (0.1 mM) and US irradiation (1.0 MHz, 0.5 W cm⁻²). (g) Density of states (DOS) for pristine PR and (h) PR with platinum vacancies. (i) Highest occupied molecular orbital (HOMO) - lowest unoccupied molecular orbital (LUMO) energy levels and interfacial plots of the molecular orbitals for PR. Realspace representation of hole and electron distributions for Pt_xRu_y alloy nanozymes: (j) Pt₈₀Ru₂₀, (k) Pt₅₀Ru₅₀, and (l) Pt₂₀Ru₈₀, calculated using DFT with the range-separated hybrid functional CAM-B3LYP and the basis set 6-31G(d). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Antibacterial and antibiofilm effect of PR in vitro

Alloy nanozymes demonstrate remarkable biocatalytic activity, sonodynamic performance, and biocompatibility, which has prompted an exploration of their antibacterial effects [41]. Due to their unique characteristics and diminutive size, alloy nanozymes can be rapidly absorbed by bacteria and easily penetrate bacterial biofilms. Consequently, it is hypothesized that they can accumulate swiftly within bacteria, exhibiting potent bactericidal effects and biofilm clearance capabilities through the synergy of CDT and SDT. The antibacterial performance of PR against multidrug-resistant Pseudomonas aeruginosa (MDRPAO) and MRSA was quantitatively evaluated using the standard colony counting method (Fig. 4a-d). Compared with the control group cultured with PBS, free PR showed no significant bacterial death on MDRPAO and MRSA. Under US conditions, PR exhibited certain antibacterial activity against both MDRPAO and MRSA. Additionally, PR effectively inhibited PAO and MRSA by 76.3 % and 79.5 %, respectively, in the presence of H₂O₂, confirming that strong CDT triggered by PR can combat bacterial infections. As expected, the synergistic effect of US and CDT (CDT/SDT group) caused the most significant bacterial death in both MDRPAO and MRSA. Quantitative analysis showed that the antibacterial activity of PR exceeded 99 %, attributable to the effective enhancement of catalytic activity and efficient bactericidal action under US irradiation. The antibacterial effect of PR on MDRPAO and MRSA was visually demonstrated through live/dead staining and quantitative analysis. As shown in the CLSM images (Fig. 4e-h), live bacteria stained with SYTO-9 exhibited green fluorescence, while dead bacteria were labeled with red propidium iodide (PI). As expected, the PBS-treated group and free PR mainly showed green fluorescence in MDRPAO and MRSA, indicating live bacteria. Meanwhile, significant bacterial death (red fluorescence) was observed in the PR + CDT group and PR + US treatment group. The bactericidal effect was greatly improved with the synergistic effect of US and CDT, achieving over 99 % bacterial death. Under US, almost all bacteria in the PR + CDT treatment group showed red fluorescence, confirming that the synergistic CDT/SDT exhibited excellent antibacterial effects. To further verify the destruction of MDRPAO and MRSA bacterial membranes, the morphological changes of bacteria in the bacterial membranes were observed using scanning electron microscopy (SEM). As shown in Fig. 4i and j, minimal membrane destruction was observed in the bacterial membrane treated with PBS, with MDRPAO and MRSA exhibiting dense stacking with intact rod-shaped and spherical shapes, respectively. In contrast, after US or CDT treatment, the integrity of the bacterial membranes was disrupted by PR, verifying that the ROS produced by PR through US and CDT can partially destroy the bacterial membrane. As expected, the most severe membrane damage occurred after the synergistic CDT/SDT treatment, with most bacteria exhibiting wrinkled and twisted shapes, and the cell membranes were severely damaged (as shown by the red and yellow arrows in the figure). Research has shown that over 80 % of chronic infections are related to biofilms, which pose a serious challenge in clinical treatment. Biofilms protect bacteria from host immune attacks and prevent the penetration of antibacterial agents. Motivated by the excellent antibacterial activity of PR, we further investigated its antibacterial ability against MDRPAO and MRSA biofilms using crystal violet staining and fluorescence staining methods. As shown in Fig. 4k-n, there was no significant reduction in biofilm biomass or bacterial activity in the PBS and free PR groups. In contrast, PR significantly reduced the biofilm of MDRPAO and MRSA when combined with H₂O₂ (0.1 mM) or US radiation (0.5 W). Notably, compared to the PR treatment group, the biofilm biomass and bacterial activity of the PR + US + CDT group were significantly reduced, indicating that PR has excellent anti-biofilm effects through the synergistic eradication of biofilms via CDT/SDT. These results suggest that PR has the potential to eliminate biofilms and exhibits excellent bactericidal performance due to its effective internalization ability. Additionally, the anti-biofilm effect of PR was observed using live/dead staining. As shown in Fig. 4o and p, the 3D CLSM images display corresponding fluorescence images of biofilms with different treatments. After PBS and PR treatment, a complete and dense biofilm with green fluorescence appeared. In comparison to biofilms treated with US or CDT alone, which exhibited a large amount of yellow fluorescence, biofilms treated with PR-mediated synergistic CDT/SDT displayed predominantly red fluorescence spots, accompanied by biofilm loss. This indicates significant biofilm diffusion and bacterial death. Therefore, the strong biofilm diffusion ability and bactericidal effect of PR under external US and CDT stimulation were further verified. In conclusion, PR can effectively eliminate biofilms and has great potential in combating biofilm-related infections through the synergistic effects of CDT and SDT.

Fig. 4.

In vitro antibacterial and antibiofilm activity of PR alloy nanozymes. The antibacterial activity of the alloy nanozymes against MDRPAO and MRSA was determined by the plate counting method. Photographs and corresponding viability of (a, b) MDRPAO and (c, d) MRSA colonies after different treatments. Live/dead fluorescence staining images and corresponding viability of (e, f) MDRPAO and (g, h) MRSA after different treatments. Scale bar, 50 μm. Representative scanning electron microscopy (SEM) images of (i) MDRPAO and (j) MRSA were collected before and after incubation with PR alloy nanozymes. Scale bar, 1 μm. Optical micrographs and quantitative analysis of biofilms of (k, l) MDRPAO and (m, n) MRSA collected from different groups at specified treatment times and stained with crystal violet. 3D CLSM images of biofilms of (o) MDRPAO and (p) MRSA collected from different groups at specified treatment times and stained with the live/dead double-stain protocol. Scale bar, 100 μm. The data are presented as mean ± SD from three independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, calculated by two-tailed Student's t-test, considered statistically significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. Evaluation of antibacterial efficacy of PR in pneumonia infection

Building on the strong in vitro antibacterial activity, this study employed an MDRPAO-induced pneumonia mouse model to simulate deep-tissue bacterial infections and evaluate the antibacterial and antibiofilm properties of PR alloy nanozymes in vivo [42]. Twelve hours post-MDRPAO infection, various treatments were administered to the infected mice via nebulization [43]. Following 48 h of treatment, infection and inflammation in the lung tissue were assessed (Fig. 5a). The results demonstrated that PR exhibited significant antibacterial and antibiofilm effects through the combined mechanisms of CDT and SDT, while effectively reducing pulmonary edema and ignore the potential toxicity that the PR may have on lung tissue (Fig. 5b and S8). These findings further confirm the potential of PR in in vivo antibacterial therapy. Subsequently, lung tissues were excised and homogenized using the standard plate dilution method to evaluate their bactericidal effects (Fig. 5c). The CDT + US group exhibited the lowest number of bacterial colonies in the lung tissues, indicating that PR alloy nanozymes possess significant antibacterial effects in the mouse lung infection model. To further evaluate histological changes in lung tissue, H&E staining was performed on the lung tissues from different treatment groups. As shown in Fig. 5d, the PBS group displayed significantly increased neutrophil infiltration and noticeable edema, with no significant improvement over time. In contrast, the CDT treatment group effectively alleviated edema, but the number of inflammatory cells remained elevated. 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 the combined CDT and US treatment significantly promoted lung tissue repair and recovery. Additionally, immunofluorescence analysis was conducted to examine the inflammatory changes in lung tissues under various treatment conditions. By assessing the expression levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and CD86/CD206 post-treatment, we evaluated the inflammatory alterations in the lungs. As illustrated in Fig. 6a and b and S9a-S9b, the CDT + US group significantly downregulated IL-6 expression (indicated by reduced green fluorescence) and upregulated IL-10 expression (indicated by enhanced red fluorescence) compared to the PBS group, indicating effective suppression of the inflammatory response. Furthermore, the expression patterns of CD86 and CD206 corroborated these findings. The CDT + US group exhibited downregulation of CD86 expression (shown by reduced red fluorescence) and upregulation of CD206 expression (indicated by enhanced green fluorescence), further confirming its ability to suppress the pro-inflammatory response and promote the shift toward an anti-inflammatory response (Fig. 6c and d and S9b-S9d). The detection of CD31 expression showed that the CDT + US group exhibited significant overexpression of CD31 (Fig. S10). 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 (Fig. S11). In conclusion, PR alloy nanozymes, through the combined treatment of CDT and US, demonstrate significant efficacy in clearing bacteria and biofilms in lung tissue while effectively reducing inflammation. This offers a promising strategy for antibacterial and anti-inflammatory treatments.

Fig. 5.

Therapeutic performance of PR alloy nanozymes in MDRPAO-induced pneumonia in vivo. (a) Digital images of lung tissues collected from mice post-treatment. (b) In vivo antibiofilm results for the MDRPAO pneumonia infection under different treatment conditions. (c) Representative histological images of H&E staining at different time points for different treatment processes. Scale bar = 2 cm (for images in (c) up) and 200 μm (for images in (c) down).

Fig. 6.

Immunofluorescence analysis of lung tissue with PR alloy nanozymes in MDRPAO-induced pneumonia in vivo. Representative images of immunofluorescence staining for (a) IL-6, (b) IL-10, (c) CD86, and (d) CD86 in lung sections from different treatment groups.

3.6. Simulated deep-seated antibacterial effect of PR in vivo

To further evaluate the universality of the antibacterial and antibiofilm effects of PR in vivo, an MRSA-infected subcutaneous abscess model was established (Fig. 7a) [44]. Mice were subcutaneously injected with MRSA (50 μL, 10⁷ CFU/mL) and randomly divided into three treatment groups: (i) PBS group, (ii) CDT group, and (iii) CDT + US group. After treatment under various conditions, we monitored the dissipation of the infected tissue abscess and the wound area to evaluate wound healing. As illustrated in Fig. 7b, after 10 days of treatment, the abscess in the infected tissue had dissipated in the US + CDT group, and the wound was nearly completely healed compared to the PBS and CDT groups. Furthermore, the in situ overlay images depicting changes in the wound area at days 0, 5, and 10 across different groups provide a more intuitive view of the role of US + CDT in promoting wound healing (Fig. 7c). Additionally, bacterial colony counts in the infected tissues on day 10 showed that the bactericidal rate in the US + CDT group exceeded 95 %, significantly surpassing that of the other groups (Fig. 7d and g). This indicates that US-mediated enhancement significantly improved the bactericidal efficiency of CDT. As shown in Fig. 7e, the mice's weight showed a slow but steady increase during the treatment process, suggesting that there were no toxic side effects. According to the wound healing statistics at different stages, the PBS group had the slowest wound healing rate by day 10, with prominent scars and the largest relative wound area (Fig. 7f). In contrast, the CDT + US group showed the fastest wound healing rate, with nearly complete healing by day 10, indicating that US-enhanced CDT, through the production of more ROS in the body, accelerated abscess dissipation and promoted wound healing. To evaluate the debridement efficacy and promotion of wound healing in PR-treated deep-seated infection tissues, pathological tissue sections were examined. As shown in Fig. 7h, almost no granulation tissue was observed in the PBS group, while thin granulation tissue appeared in the CDT group, and the thickest granulation tissue was found in the CDT + US group. Additionally, localized high-magnification H&E images revealed that inflammatory cells in the CDT and CDT + US groups were significantly fewer than those in the PBS group, indicating the effective reduction of tissue inflammation. In Masson's staining (Fig. 7i), the blue collagen fibers in the CDT + US group were significantly higher than those in the PBS and CDT groups, indicating that CDT + US can promote collagen deposition and accelerate wound healing. Similarly, Gram staining was performed to assess the residual bacteria and biofilm in the repaired infected tissue on day 10. As shown in Fig. 7j, the number of residual bacteria in the CDT and US + CDT groups significantly decreased, indicating that PR can effectively remove residual bacteria in deep tissues through the combined action of US and CDT. During bacterial infection, inflammation is inevitable, and the ratio of pro-inflammatory macrophages (M1) to anti-inflammatory macrophages (M2) is a key indicator of the inflammatory status. TNF-α and TGF-β are typical biomarkers for M1 and M2 macrophages, respectively. Immunofluorescence analysis was performed to evaluate the inflammatory characteristics in the infected tissues. As shown in Fig. 8a and b, when inflammatory cytokines were detected, the CDT + US group significantly downregulated the expression of TNF-α (red fluorescence) in the infected tissues and upregulated the expression of TGF-β (green fluorescence) (Fig. 8c and d). These results suggest that macrophages gradually polarize from the M1 to the M2 phenotype, thereby promoting the healing process of infected tissues. Additionally, the pro-angiogenic marker CD31 was employed to evaluate and regulate vascular growth. Correspondingly, due to the reduced severity of infection, the CDT + US group exhibited a significant increase in CD31 fluorescence intensity compared to the PBS and CDT groups, which reflects the rapid repair process in the infected area (Fig. 8e and f). Furthermore, H&E sections were analyzed from the heart, liver, spleen, lung, and kidneys across different treatment groups under varying conditions, and the results indicated no toxicity to other organs (Fig. S12). In addition, through blood routine and blood biochemistry experiments, we can further explore the potential toxicity of PR. As shown in Fig. S13, there were no significant differences in ALT, AST, BUN, WBC, MCV, and PLT indicators between the PR group and the PBS group, and they were all within the normal range, indicating that the potential toxicity of PR can be ignored. These data suggest that the ultrasound-enhanced CDT antibacterial strategy can effectively promote the healing of infected wounds.

Fig. 7.

Treatment of subcutaneous MRSA-infected mice with PR alloy nanozymes. (a) Schematic illustration of the experimental procedure for treating MRSA-infected subcutaneous abscess model. (b) Representative photographs of the infected wounds at different time points during therapy. (c) Relative wound area of mice on day 0 (blue), day 5 (orange), and day 10 (crimson) after different treatments. (d) Photographs of MRSA colonies from infected tissues on day 10 post-treatment. (e) Changes in mice body weight during therapy. (f) Infected area of mice after various treatments (n = 5 biologically independent samples; mean ± SD). (g) Quantification of viable bacteria inside biofilm-infected tissues on day 10 post-treatment (n = 5 biologically independent samples; mean ± SD). (h) H&E staining, (i) Masson staining, and (j) Gram staining tissue sections of infected tissues from mice on day 10 post-treatment. Scale bar = 500 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8.

Immunofluorescence analysis of skin tissue with PR alloy nanozymes in MRSA-induced subcutaneous abscess in vivo. Representative immunofluorescence staining images and corresponding quantitative analysis of abscess tissue on the 10th day post-treatment for (a, b) TNF-α, (c, d) TGF-β, and (e, f) CD31. Data are presented as mean ± S.D. (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

4. Conclusion

In summary, the development of bimetallic Pt_xRu_y alloy biocatalysts with enhanced activity and stability has been systematically investigated. Theoretical simulations have identified the optimal Pt/Ru molar ratio (x/y = 80/20, denoted as PR) for maximizing catalytic performance. Through the controlled co-reduction of Pt²⁺ and Ru³⁺ precursors, PR alloy nanozymes have been successfully synthesized with precisely tuned composition. The strategic alloying of Pt and Ru at this optimal ratio significantly enhances the catalytic efficiency of Pt across multiple reaction systems. Notably, the incorporation of Ru induces the formation of Pt vacancies, which synergistically improve both enzymatic activity and sonodynamic performance. The engineered PR nanoplatforms offer four key advantages for biomedical applications: Exceptional biocatalytic activity combined with superior ultrasound-responsive capabilities; US-activated activation that enhances nanozyme performance, enabling robust generation of various ROS, particularly ¹O₂ and •OH; Ultracompact nanoparticle dimensions that facilitate efficient alveolar penetration, which is crucial for the effective treatment of BP and proven efficacy in subcutaneous abscess therapy, coupled with excellent biocompatibility. This study not only establishes a promising paradigm for alloy nanozyme applications in stimulus-responsive infection therapy but also advances a fundamental understanding of the composition-structure-performance relationships in bimetallic catalytic systems. The findings provide valuable insights for the rational design of next-generation nanozymes, paving the way for precision nanomedicine.

CRediT authorship contribution statement

Xiang Zheng: Writing – original draft, Conceptualization. Lingxia Pang: Investigation, Formal analysis, Data curation, Conceptualization. Youpei Wang: Methodology, Formal analysis. Qianlei Zhao: Supervision, Methodology. Guoquan Pan: Supervision, Methodology. Xiaojun He: Supervision, Resources, Project administration, Funding acquisition. Yafeng Liang: Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.

Data availability

Data will be made available on request.