High-entropy alloy nanoparticles for use as biocatalysts and applications thereof

Abstract

The application belongs to the technical field of catalytic materials, and particularly relates to high-entropy alloy nanoparticles used as biological catalysts and application thereof. The pH adaptive PtFeCuCoNi high-entropy alloy nanoparticles are developed by a one-step solvothermal synthesis method, and it is found that under acidic conditions, the PtFeCuCoNi high-entropy alloy nanoparticles can be used as a strong oxidase to generate reactive oxygen species (ROS) and effectively kill bacteria such as MRSA, and under neutral conditions, the PtFeCuCoNi high-entropy alloy nanoparticles can play the functions of superoxide dismutase and catalase, effectively remove intracellular reactive oxygen species, relieve oxidative stress and play a cell protection role.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of catalytic materials, and particularly relates to a high-entropy alloy nanoparticle used as a biological catalyst and application thereof. BACKGROUND

[0002] Antibiotic-resistant bacterial infections have a wide range of clinical manifestations, especially in chronic non-healing skin wounds, which have become a major burden on global health. The wound microenvironment undergoes dynamic changes in pH: during the acute phase, bacterial overgrowth and biofilm formation acidify the tissue by producing organic acids, both hindering host defense mechanisms and reducing antibiotic efficacy. When the infection enters the chronic inflammation stage, persistent M1 macrophage infiltration and pro-inflammatory cytokine release will cause the local pH to re-trend to neutral, while excessive reactive oxygen species damage endothelial cells and fibroblasts, delaying the repair process. This pH-driven paradox - the need to exert potent bactericidal effects in an acidic microenvironment, followed by the ability to scavenge reactive oxygen species and regulate immune function under neutral pH conditions - exceeds the capacity of existing antibiotics, especially for multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). Therefore, there is an urgent need to develop interventions that do not require antibiotics and can adapt to changes in pH, allowing them to gradually eliminate pathogens under acidic conditions and then switch to antioxidant and immune regulation functions to accelerate tissue regeneration.

[0003] Peroxisome-mediated reactive oxygen species (ROS) regulation is a core mechanism of innate immunity, which clears pathogens while minimizing collateral damage by driving an oxidative cascade. Specifically, oxidases (OXD) and peroxidases (POD) generate ROS to disrupt bacterial membrane structures, oxidize proteins and nucleic acids, and disintegrate biofilms. When pathogens are cleared, catalase (CAT) decomposes residual H2O2 into water and oxygen, maintaining redox balance and protecting host cells. However, sustained or excessive ROS production - especially during chronic inflammation - can overwhelm these defense mechanisms, exacerbate inflammatory signaling, and cause oxidative damage to surrounding tissues, ultimately delaying the repair process. Although peroxisomal enzymes can skillfully coordinate ROS metabolism, their direct therapeutic applications are limited by short circulating half-lives, poor stability, and potential immunogenicity. In this context, engineering ROS regulation has become a non-antibiotic strategy to achieve potent bactericidal and controlled inflammation resolution. In recent years, advances in nanotechnology have given rise to a variety of enzyme-mimicking materials, including inorganic nanoparticles, carbon-based nanostructures, nanoliposomes, and metal-organic framework materials, which combine ROS generation and scavenging functions. However, many of these platforms still have limited catalytic turnover rates and insufficient control over ROS release, leading to incomplete eradication of pathogens or unintended oxidative damage to host tissues.

[0004] High-entropy alloys (HEAs) provide an unprecedented platform for mimicking enzyme catalysis by virtue of their synergistic effects of multiple elements. By combining five or more transition metals at near-equiatomic ratios (5%-35%), HEAs form stable single-phase solid solutions with high configurational entropy, lattice distortion, and sluggish diffusion. This "cocktail effect" creates a dense array of chemically active sites, enabling precise tuning of the d-band center and oxygen-binding properties of the alloy, which outperform single-metal alloys in both catalytic performance and stability. Therefore, HEAs have become a cornerstone in the field of energy conversion and environmental catalysis. However, their application in the regulation of biomedically active oxygen is still in its infancy, limited by harsh activation conditions, limited aqueous stability, and biocompatibility issues. To bridge this gap, rational design of HEA nanoszymes with controllable size, surface chemical properties, and pH-responsive redox behavior is needed to achieve safe and effective regulation of active oxygen in vivo. SUMMARY

[0005] Inspired by the adaptability of natural peroxidase, the present application develops a pH-adaptive PtFeCuCoNi high-entropy alloy nanoparticle by one-step solvothermal synthesis, and finds that it can generate reactive oxygen species (ROS) as a powerful oxidase under acidic conditions, effectively killing bacteria such as MRSA, while under neutral conditions it can exhibit superoxide dismutase and catalase-like functions, effectively scavenging intracellular reactive oxygen species, relieving oxidative stress, and exerting cell protection.

[0006] Specifically, in one aspect, the present application provides a high-entropy alloy nanoparticle, the composition of which includes Pt, Fe, Cu, Co, and Ni.

[0007] Further, the atomic ratio of each of Pt, Fe, Cu, Co, and Ni in the high-entropy alloy nanoparticle is 5%-35%.

[0008] Further, the atomic ratio of Pt, Fe, Cu, Co, and Ni in the high-entropy alloy nanoparticle is 19.1:14.7:20.9:22.3:23.0.

[0009] Further, the crystal phase structure of the high-entropy alloy nanoparticle is a single-phase solid solution structure.

[0010] Further, the high-entropy alloy nanoparticle has the ability to promote the generation and clearance of reactive oxygen species with pH adaptability.

[0011] Further, the high-entropy alloy nanoparticle has pH-adaptive POD-like, SOD-like, and / or CAT-like enzyme activity.

[0012] As used herein, pH adaptability means that the high-entropy alloy nanoparticles exhibit different properties at different pH levels, such as the ability to promote the generation or scavenging of reactive oxygen species, or different enzyme catalytic activities.

[0013] Furthermore, the high-entropy alloy nanoparticles have the ability to promote the generation of reactive oxygen species under acidic conditions and the ability to promote the scavenging of reactive oxygen species under neutral conditions.

[0014] Furthermore, the high-entropy alloy nanoparticles exhibit POD-like enzyme activity under acidic conditions and SOD-like and / or CAT-like enzyme activity under neutral conditions.

[0015] The high-entropy alloy nanoparticles described herein can be prepared using methods known in the art for preparing high-entropy alloy materials.

[0016] As an example, the present invention provides a method for preparing high-entropy alloy nanoparticles as described herein, comprising the following steps: performing a solvothermal reaction of Pt salt, Fe salt, Cu salt, Co salt and Ni salt in a solvent to obtain high-entropy alloy nanoparticles.

[0017] Furthermore, as used herein, the salt of a metal is at least one of a metal acetate, acetylacetone salt, chloride salt, nitrate, and their hydrates.

[0018] Further, the solvent is one or a mixture of several of N,N-dimethylformamide (DMF), ethylene glycol (EG), methanol, ethanol and acetone.

[0019] Preferably, the solvent is a mixture of DMF and EG.

[0020] Furthermore, the solvothermal reaction includes reacting at 150-240°C for 1-10 hours.

[0021] Furthermore, the preparation method includes dissolving Ga salt, Pd salt, Ru salt, Ir salt and Mn salt in a solvent, ultrasonically mixing, carrying out a solvothermal reaction, collecting the product by centrifugation, washing and drying to obtain PtFeCuCoNi high-entropy alloy nanoparticles.

[0022] In another aspect, the present invention provides the application of high-entropy alloy nanoparticles as described herein as biocatalysts.

[0023] Furthermore, the application is not intended for diagnosis or treatment.

[0024] Furthermore, the biocatalyst has the ability to promote the generation and removal of reactive oxygen species in a pH-adaptive manner.

[0025] Further, the biological catalyst has pH-adaptable POD-like, SOD-like and / or CAT-like enzyme activity.

[0026] Further, the biological catalyst has the ability to promote active oxygen generation under acidic conditions and the ability to promote active oxygen scavenging under neutral conditions.

[0027] Further, the biological catalyst has POD-like enzyme activity under acidic conditions and SOD-like and / or CAT-like enzyme activity under neutral conditions.

[0028] In other aspects, the present application provides the use of the high-entropy alloy nanoparticles as described herein in the preparation of a material for scavenging bacteria and destroying biofilms under acidic conditions and / or scavenging active oxygen, protecting cells to alleviate damage caused by oxidative stress and anti-inflammation under neutral conditions.

[0029] Further, the bacteria are drug-resistant bacteria.

[0030] Further, the bacteria include, but are not limited to, drug-resistant Staphylococcus aureus.

[0031] Further, the bacteria include, but are not limited to, methicillin-resistant Staphylococcus aureus.

[0032] Further, the minimum inhibitory concentration of the high-entropy alloy nanoparticles is 8 μg / mL.

[0033] In other aspects, the present application also provides the use of the high-entropy alloy nanoparticles as described herein in the preparation of a product for treating wound infection and accelerating wound healing.

[0034] Further, the wound infection includes bacterial infection.

[0035] Further, the bacteria are drug-resistant bacteria.

[0036] Further, the bacteria include, but are not limited to, drug-resistant Staphylococcus aureus.

[0037] Further, the bacteria include, but are not limited to, methicillin-resistant Staphylococcus aureus.

[0038] Advantages of the present application

[0039] The PtFeCuCoNi high-entropy alloy nanoparticles of the application are composed of five transition metals with similar atomic radii, which can not only achieve a more ideal antibacterial effect, but also reduce the amount of noble metal. The strong interaction between d electrons in the nanoparticles optimizes the electron distribution around the electric field of the bulk material, thereby enhancing the adsorption and desorption capacity of the surface active sites to intermediates, significantly improving the performance of the nano-catalyst. When exposed to the acidic environment of infected tissue, its oxidase and peroxidase-like functions can release a ROS burst, effectively killing bacteria such as MRSA and destroying biofilms; as the local pH value returns to the physiological level during the healing process, the superoxide dismutase and catalase-like functions can efficiently scavenge excess ROS, relieving oxidative stress. Both theoretical modeling and experimental verification confirm its excellent catalytic performance: the peroxidase-like activity is 10.23 μM s⁻¹, and the catalase-like activity reaches 74.69 μM s⁻¹, surpassing mainstream nano-enzyme products, achieving ultra-low dose and high efficiency in killing bacteria with extremely low cytotoxicity. What is particularly key is that these high-entropy alloy nanoparticles can also regulate macrophage polarization in stages - first enhancing M1-type mediated bacterial clearance, and then inducing M2-type driven regeneration and repair reactions. Comprehensive in vitro and in vivo studies confirm that this technology can quickly clear bacteria, efficiently scavenge reactive oxygen species (ROS), and protect endothelial cells, fibroblasts, and macrophages, ultimately significantly accelerating wound healing. This pH-adaptive HEA nano-enzyme platform provides a time-programmable, antibiotic-free strategy that can overcome drug resistance and chronic inflammation, and promote tissue regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Synthesis and characterization of PtFeCuCoNi high-entropy alloy nanoparticles are shown. (a) Schematic diagram of synthesis and structure display of PtFeCuCoNi high-entropy alloy (NPs) nanoparticles. (b) Scanning electron microscope images of platinum nanoparticles and (c) PtFeCuCoNi high-entropy alloy nanoparticles. (d) Transmission electron microscope image of PtFeCuCoNi high-entropy alloy nanoparticles. (e) High-resolution transmission electron microscope image of PtFeCuCoNi high-entropy alloy nanoparticles, and (e1, e2) corresponding fast Fourier transform (FFT) images in the dashed box. (f) Inverse fast Fourier transform (FTT) images corresponding to e1, e2. (g) High-angle angular momentum scattering (HAADF)-STEM-EDS element distribution map of PtFeCuCoNi high-entropy alloy nanoparticles.

[0041] Figure 2(a) X-ray diffraction pattern of PtFeCuCoNi high-entropy alloy nanoparticles and its comparison with the standard PDF card matching results. (b) STEM-EDS energy spectrum of PtFeCuCoNi high-entropy alloy nanoparticles (inset is the corresponding ICP-OES detection results). XPS spectra of PtFeCuCoNi high-entropy alloy nanoparticles for Pt 4f (c), Fe 2p (d), copper 2p (e), cobalt 2p (f), and nickel 2p (g). (h) Pt L3 edge XANES spectra of PtFeCuCoNi high-entropy alloy nanoparticles and reference samples (PtO2 and platinum foil). (i) Fourier-transform k2-weighted EXAFS spectra. (j) In-situ probe spectroscopy analysis of different samples at Pt L3 edge.

[0042] Figure 3 (a) Schematic diagram of ROS generation activity of PtFeCuCoNi high-entropy alloy nanoparticles in acidic environment, and comparison of scavenging activity in neutral environment. (b) Quantitative analysis of peroxidase-like activity of platinum nanoparticles and PtFeCuCoNi high-entropy alloy nanoparticles and UV-Vis spectral data. Data are expressed as mean ± SD, n = 3 independent experiments. (c) PtFeCuCoNi nanoparticles exhibit pH-adaptive POD mimetic activity (n = 3 independent experiments, data are expressed as mean ± SD). a.u represents arbitrary units. (d) TMB-based UV-Vis spectra simulate the activity of POD in the presence of quaternary alloy nanoparticles and PtFeCuCoNi nanoparticles.

[0043] Figure 4 (a) Michaelis-Menten kinetic analysis of peroxidase-like activity of platinum nanoparticles and PtFeCuCoNi high-entropy alloy nanoparticles with hydrogen peroxide as substrate. (b) Comparison analysis of TON value and Vmax value compared with the reported advanced peroxidase catalysts. (c) Experimental results of tert-butyl alcohol (TBA) quenching •OH radicals, benzoquinone (BQ) quenching O2- radicals, and by hydroxyl radical (sodium azide) quenching singlet oxygen (O2) in the process of platinum iron copper cobalt nickel high-entropy alloy nanoparticle system catalyzing trimethyl bromide (TMB) oxidation. Data are expressed as mean ± SD, n = 3 independent experiments. (d) EPR spectra for recording •O2- and 1 O2-. 1O2signal. (e) SOD-like activity of Pt nanoparticles and PtFeCuCoNi high-entropy alloy nanoparticles (n = 3 independent experiments, data presented as mean ± SD). (f) CAT-like performance of the biocatalyst in the presence of hydrogen peroxide was detected based on the TiSO4method over time. (g) Oxygen production concentration was measured using a dissolved oxygen meter containing the biocatalyst and hydrogen peroxide. (h) Statistical analysis of oxygen production kinetic parameters of Pt nanoparticles and PtFeCuCoNi high-entropy alloy nanoparticles. (i) Comparison analysis of TON values and Vmaxvalues with reported advanced CAT-like catalysts.

[0044] Figure 5 MRSA in vitro bactericidal effect evaluation of PtFeCuCoNi high-entropy alloy nanoparticles is shown. (a) Survival rate of MRSA captured in different concentrations of platinum and platinum iron copper cobalt nickel alloy medium (n = 3 independent repeats). (b) Bacterial viability of MRSA after H2O2(0.2 mM) treatment, PtFeCuCoNi was 0, 2, 4, 6, 8, 16, 32, 64, 96, 128 pg mL-1, respectively. PtFeCuCoNi and H2O2(0.2 mM) were incubated with 1 mL of bacterial suspension (10 6 CFU mL-1) at 37°C for 12 h. The culture suspension was diluted 10 5 times, and agar plates were counted. The agar plates were incubated at 37°C for another 12 h, and then counted to determine the final colony count. Bacterial viability was calculated as the ratio of the number of colonies in the treatment group to the number of colonies in the control group (n = 3 independent repeats). Data are mean ± SD, * p < 0.05, *** p < 0.001; one-way ANOVA plus multiple comparison test, all tests were two-sided. (c) Comparison with the effective antibacterial concentration of the materials reported in the literature. (d) Bacterial protein exudation photos (n = 3 biological independent repeats). (e) MRSA agar plate colony formation statistics. (h) Comparison of agar plate antibacterial effect (n = 3 biological independent repeats), p value < 0.001 compared with PBS / H2O2 / Pt group; p value = 0.1059 compared with PBS group. Crystal violet staining shows biofilm inhibition effect (f) and destruction effect (g). Biofilm bacterial amount determination results of different sample treatments (i) (n = 3 biological independent repeats), p value < 0.001 compared with PBS / H2O2 / Pt group; p value = 0.0144 compared with PBS group.

[0045] Figure 6The in vitro ROS scavenging and cytoprotection of PtFeCuCoNi were shown. (a) Quantitative analysis of DCFH-DA fluorescence of different types of cells under different treatment conditions; left panel: compared with H2O2 / Pt group, p < 0.001 for PtFeCuCoNi group; compared with control group, p (hydrogen peroxide) < 0.001, p (PtFeCuCoNi) = 0.0886; middle panel: compared with H2O2 / Pt group, p < 0.001 for PtFeCuCoNi group; compared with control group, p (hydrogen peroxide) < 0.001, p (PtFeCuCoNi) = 0.0545; right panel: compared with hydrogen peroxide group, p < 0.001 for PtFeCuCoNi group; compared with Pt group, p < 0.001 for PtFeCuCoNi group; compared with control group, p (hydrogen peroxide) < 0.001, p (PtFeCuCoNi) = 0.3634; compared with Pt group, p = 0.0095 for PtFeCuCoNi group; (b) Quantitative analysis of cell viability under different treatment conditions; left panel: compared with hydrogen peroxide / Pt nanoparticle group, p (PtFeCuCoNi) < 0.001; p (PtFeCuCoNi) = 0.0505, p (hydrogen peroxide) < 0.001, compared with control group (n = 4 independent repeats); middle panel: compared with hydrogen peroxide / Pt nanoparticle group, p (PtFeCuCoNi) < 0.001; p (PtFeCuCoNi) = 0.5933, p (hydrogen peroxide) < 0.001, compared with control group (n = 4 independent repeats); right panel: compared with hydrogen peroxide / Pt nanoparticle group, p (PtFeCuCoNi) < 0.001; p (PtFeCuCoNi) = 0.3177, p (hydrogen peroxide) < 0.001, compared with control group (n = 6 independent repeats); (c) Linear distribution of cytoskeleton staining fluorescence intensity; (d) Quantitative analysis of cell area (n = 12 independent repeats); compared with Pt group, p (PtFeCuCoNi) = 0.01; compared with hydrogen peroxide group, p (PtFeCuCoNi) = 0.002; compared with control group, p (PtFeCuCoNi) = 0.5358; compared with control group, p (hydrogen peroxide) < 0.001; (e) Quantitative analysis of main section of angiogenesis experiment of human umbilical vein endothelial cells (HUVEC) after different interventions (n = 3 independent biological repeats); compared with Pt group, p (PtFeCuCoNi) = 0.003; compared with hydrogen peroxide group, p (PtFeCuCoNi) = 0.0002; compared with control group, p (PtFeCuCoNi) = 0.044; compared with control group, p (hydrogen peroxide) < 0.001; Raw264.7Relative quantification of CCR7 (f) and CD163 (g) in cells relative to control groups, (f): p(PtFeCuCoNi) < 0.001 compared with LPS / Pt NPs group; p(PtFeCuCoNi) = 0.0752 compared with control group; p(hydrogen peroxide) < 0.001 compared with control group (n = 5 independent replicates); (g): p(PtFeCuCoNi) < 0.001 compared with control group / LPS / Pt NPs group; p(Pt) = 0.0365 compared with control group; p(LPS) = 0.556 compared with control group (n = 5 independent replicates). Control group: untreated cells; LPS: LPS-treated cells; Pt NPs and PtFeCuCoNi HEA NPs: cells co-incubated with LPS after pre-treatment with corresponding ROS scavengers. DETAILED DESCRIPTION

[0046] Inspired by the peroxisomal oxidative signaling, the present application developed a pH-adaptive high-entropy alloy (HEA) nanoparticle PtFeCuCoNi. This nanomaterial stabilizes the d-band center through a multi-metal "cocktail effect", optimizes the oxygen species binding capacity, and can seamlessly switch between different enzyme-mimetic activities. In an acidic microenvironment, these nanozymes act as potent oxidases, generating reactive oxygen species (ROS) to effectively kill methicillin-resistant Staphylococcus aureus (MRSA), a typical multi-drug resistant pathogen, and destroy biofilms with an ultra-low minimum inhibitory concentration of 8 μg / mL, far lower than the values reported for similar systems. However, under physiological pH conditions (7.4), they transform into superoxide dismutase and catalase-like functions, efficiently scavenging intracellular reactive oxygen species, alleviating oxidative stress, while maintaining the survival and proliferation of endothelial cells, fibroblasts, and macrophages with extremely low cytotoxicity. In MRSA-infected wound models, PtFeCuCoNi high-entropy alloy nanoparticles can accelerate bacterial clearance and promote tissue repair. Through mechanisms such as enhancing neovascularization and extracellular matrix remodeling, the wound ultimately heals faster. Mechanism studies show that this material can activate the Nrf2 antioxidant pathway, inhibit the secretion of pro-inflammatory cytokines, and promote the polarization of macrophages to the repair M2 phenotype, thus constructing an immune regenerative microenvironment. Therefore, by combining potent acid-activated antibacterial effects with effective neutral pH immune regulation on a single ultra-low dose platform, this pH-adaptive nanozyme addresses the dual challenges of drug resistance and chronic inflammation, providing a multifunctional strategy for combating persistent infections and promoting tissue regeneration.

[0047] The application will be further described in conjunction with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods, and equipment used in the application are conventional reagents, methods, and equipment in the technical field.

[0048] Materials and reagents: Iron acetylacetone (Fe(acac)2), copper nitrate hydrate (Cu(NO3)2·3H2O), cobalt nitrate hexahydrate (Co(NO3)2·6H2O), nickel nitrate hexahydrate (Ni(NO3)2·6H2O), N,N-dimethylformamide (DMF), ethylene glycol (EG), hydrogen peroxide (H2O2), 3,3',5,5'-tetramethylbenzidine (TMB), 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO), 2,2,6,6-tetramethylpiperidine (TEMP), tert-butanol (TBA), benzoquinone (BQ), sodium azide (NaN3), potassium superoxide (KO2), nitrotetrazole blue chloride (NBT), and 9,10-dibenzoanthraquinone (DPA) were all purchased from Aladdin Reagent Company (Shanghai, China). Platinum bis(acetylacetone) (Pt(acac)2) was purchased from Anhui Nengke Chemical Reagent Co., Ltd. Titanium sulfate (Ti(SO)4) was purchased from Mackintosh Reagents, Inc. (Shanghai, China).

[0049] Example: Preparation of PtFeCuCoNi high-entropy alloy nanoparticles

[0050] The synthesis process and structure of PtFeCuCoNi high-entropy alloy nanoparticles are shown in the figure below. Figure 1 As shown in a. This invention uses palladium acetylacetonate, iron acetylacetonate, copper nitrate trihydrate, cobalt nitrate hexahydrate, and nickel nitrate hexahydrate as metal precursors, and N,N-dimethylformamide (DMF) and ethylene glycol (EG) as solvents. An orange, finely dispersed raw material mixture is formed through ultrasonic treatment. As the temperature increases, the mixture gradually turns black, which may be due to the coordination reaction and co-reduction of the five metals. Subsequently, a single-phase high-entropy alloy is successfully formed.

[0051] Specifically, Pt(acac)₂ (20 mg), Fe(acac)₂ (17.96 mg), Cu(NO₃)₂·3H₂O (12.1 mg), Co(NO₃)₂·6H₂O (17.65 mg), and Ni(NO₃)₂·6H₂O (17.63 mg) were placed in a 100 mL polytetrafluoroethylene-lined autoclave, and a mixed solution of DMF (12 mL) and ethylene glycol (8 mL) was added. The mixture was sonicated for 30 minutes to completely dissolve all reactants. The resulting solution was heated at 200 °C for 8 hours. The final product was washed with ethanol and acetone and collected by centrifugation at 10,000 rpm.

[0052] Comparative example:

[0053] Using the same method as in the examples, single Pt particles were prepared as a control using Pt(acac)2 as the raw material via a solvothermal method.

[0054] Quaternary metal high-entropy alloy nanoparticles lacking Pt, Fe, Cu, Co, or Ni were also prepared using the same method as in the examples.

[0055] Test Example 1: Structural Characterization of PtFeCuCoNi High-Entropy Alloy Nanoparticles

[0056] To observe the morphology and size of high-entropy alloy nanoparticles (HEA NPs), we characterized the prepared products using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high-resolution transmission electron microscopy (HRTEM). The SEM and TEM characterization results are shown below. Figure 1 As shown in bd, it shows that HEA nanoparticles are uniformly distributed in small spherical shapes. HRTEM images further reveal that the PtFeCuCoNi high-entropy alloy nanoparticles have high crystallinity, and the clear lattice fringes of their (111) crystal plane can be verified by the corresponding fast Fourier transform (FFT) spectrum. Figure 1 e). Furthermore, the inverse FFT spectra and their corresponding integrated pixel intensities show that the average lattice spacing fluctuates between 0.212 nm and 0.215 nm. Figure 1 f) indicates that the PtFeCuCoNi high-entropy alloy nanoparticles exhibit significant lattice distortion and approximately 3.3% intrinsic tensile strain, which stems from factors such as differences in atomic size among components, indentation defects, and ultrathin thickness. Low-magnification, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) energy-dispersive X-ray spectroscopy (EDS) images show that platinum, iron, copper, cobalt, and nickel elements exhibit a uniform distribution. Figure 1 g).

[0057] The crystal structures of PtFeCuCoNi high-entropy alloy nanoparticles and materials prepared in comparison were characterized by powder X-ray diffraction (XRD). The XRD patterns revealed a typical platinum face-centered cubic structure with no obvious phase separation. With the formation of PtFeCuCoNi high-entropy alloy nanoparticles, the characteristic peak shifted to a higher angle, indicating that more atoms were successfully alloyed with the platinum nanoparticles to successfully prepare the high-entropy alloy. Figure 2 a). Simultaneously, the atomic ratios of Pt / Fe / Cu / Co / Ni in the PtFeCuCoNi high-entropy alloy nanoparticles were determined by inductively coupled plasma mass spectrometry (ICP-MS) to be 19.1 / 14.7 / 20.9 / 22.3 / 23.0 (a). Figure 2 b). X-ray photoelectron spectroscopy (XPS) was used as a surface-sensitive probe to conduct an in-depth analysis of the chemical state and composition of the surface of PtFeCuCoNi high-entropy alloy nanoparticles. For example... Figure 2As shown in the cg, experimental data confirm that this material contains platinum (Pt), iron (Fe), copper (Cu), cobalt (Co), and nickel (Ni). Specifically: platinum's 4f 7 / 2 and 4f 5 / 2 Characteristic peaks are located at 71.22 and 74.54 electron volts, respectively; the 2p group of iron... 3 / 2 and 2p 1 / 2 Characteristic peaks are located at 714.12 and 723.69 electron volts, respectively; the 2p peaks of copper... 3 / 2 and 2p 1 / 2 Characteristic peaks are located at 931.85 and 951.62 electron volts, respectively; the 2p peaks of cobalt are... 3 / 2 and 2p 1 / 2 Characteristic peaks are located at 778.14 and 792.83 eV, respectively; nickel's 2p... 3 / 2 and 2p 1 / 2 Characteristic peaks are located at 852.41 and 869.69 electron volts. Binding energy analysis shows that platinum, iron, copper, cobalt, and nickel exist in alloy form within the PtFeCuCoNi high-entropy alloy nanoparticles.

[0058] X-ray absorption near-edge structure (XANES) spectroscopy shows that the intensity of the "white line" (WL) on the L3 edge of platinum metal is completely consistent with that of the platinum foil and is significantly lower than that of PtO2, indicating that electrons are enriched on the platinum atoms. Figure 2 h). The high-entropy alloy (HEA) shows a peak at approximately 2.3 Å in the Fourier transform extended X-ray absorption fine structure (FT-EXAFS) spectrum in R space, corresponding to platinum-platinum interactions. This shifted peak indicates the presence of platinum-metal (M = Fe, Cu, Ni, Co) interactions within the alloy, which may be due to the synergistic effect of multiple elements in the alloy. Figure 2 i). No characteristic peaks of Pt-O bonds were detected in the FT-EXAFS spectrum of the high-entropy alloy, strongly indicating that the sample did not undergo oxidation, thus preserving its metallic properties. Furthermore, wavelet transform (WT) can further distinguish between Pt-O and Pt-Pt bonds oscillating on the L3 edge of platinum. Figure 2 j). Platinum foil in R space R = 2.7 Å and k space k = 10.8 Å. -1 The Pt-Pt bond spectrum is clearly displayed at the PtO2 value, and the R-space spectrum (R = 3.0 Å) and the k-space spectrum (k = 11.0 Å) are also observed. -1 The Pt-Pt bond spectrum at 7.8 Å was observed in the wavelet profile of the PtFeCuCoNi high-entropy alloy nanoparticles, consistent with the EXAFS spectral results. This finding supports the theoretical view that intermetallic coordination bonds exist within the platinum-metal matrix.

[0059] The test equipment and parameters used in this test example are as follows: scanning electron microscope (SEM) images were taken by Apreo S HiVoc system produced by Thermo Fisher Scientific. Transmission electron microscope (TEM) images and energy spectrum maps were obtained by FEI Talos F200x TEM at 200 kV accelerating voltage. Distortion correction scanning transmission electron microscope (AC-STEM) characterization was completed using FEI Titan Cubed Themis G2 300 type equipment for high angle annular dark field scanning transmission electron microscope (HAADF-STEM). Crystal phase was presented by DX-2700BH type X-ray diffractometer (using copper target, working voltage 40 kV). X-ray photoelectron spectroscopy (XPS) test used Thermo Fisher K-Alpha™+ X-ray photoelectron spectrometer, equipped with a hemispherical 180° bifocal analyzer and a 128-channel detector. Electron paramagnetic resonance (EPR) measurement used Bruker EMR XPlus spectrometer (Bruker Beijing Technology Co., Ltd., USA), working frequency 9.8 GHz (microwave power 1 mW). Raman spectrum was detected by Horiba HR Evolution Raman spectrometer (532 nm laser source). X-ray absorption spectrum (XAS) was collected on the BL07A1 beamline of the National Synchrotron Radiation Research Center, and monochromatized by silicon (111) double crystal monochromator. The processing and analysis of XANES (X-ray near edge structure) and EXAFS (extended X-ray absorption fine structure) data were completed using Athena software.

[0060] Test Example 2: pH-adaptive active oxygen catalytic activity test of PtFeCuCoNi high-entropy alloy nanoparticles

[0061] After determining the chemical structure and electronic structure of the PtFeCuCoNi high-entropy alloy nanoparticles, we further studied its enzyme-like activity, especially the pH-adaptive biological catalytic ability designed for bacterial infectious diabetic ulcers. In short, in an acidic environment, the PtFeCuCoNi high-entropy alloy nanoparticles convert harmless hydrogen peroxide into highly toxic active oxygen (•O2- and 1 O2) by simulating peroxidase (POD), thereby playing a bactericidal role; while in a neutral environment, the nanoparticles can simulate the function of superoxide dismutase (SOD), convert •O2- into hydrogen peroxide, and decompose excess hydrogen peroxide into non-cell-toxic O2 and water by means of catalase (CAT)-like activity to relieve intracellular oxidative stress, thereby promoting cell proliferation ( Figure 3 a}.

[0062] To verify the catalytic performance of PtFeCuCoNi HEA NPs, we first evaluated their ability to generate reactive oxygen species (ROS) in an acidic environment by 3,3,5,5-tetramethylbenzidine (TMB) colorimetric method. The oxidized TMB product, oxTMB, has a characteristic absorption peak at 652 nm. As shown in Figure 3 b and Figure 3 c, PtFeCuCoNi HEA NPs exhibited the best ROS generation performance, with the strongest luminescence intensity at 652 nm, and showed pH-adaptive catalytic activity, with almost no ROS production under neutral conditions. To further explore the synergistic effect of PtFeCuCoNi catalyst and clarify the role of each element in the POD-like biological catalyst, we synthesized a series of quaternary nanocatalysts with similar loading by removing each element (i.e., FeCuCoNi, PtCuCoNi, PtFeCoNi, PtFeCuNi, and PtFeCuCo) (see Comparative Examples). With the removal of Fe, Pt, Cu, Co, and Ni in turn, the reaction rate showed a decreasing trend: the largest decrease occurred when Fe was removed, and the smallest decrease occurred when Ni was removed. Notably, the reaction rates of these quaternary catalysts were lower than that of PtFeCuCoNi, indicating that the five elements synergistically regulate the reaction kinetics of ROS generation ( Figure 3 d).

[0063] To quantify the efficiency of PtFeCuCoNi HEA NPs in catalyzing the generation of ROS, we systematically analyzed their characteristic steady-state kinetic parameters, including the catalytic constant (Km / mM), the maximum reaction rate (Vmax / μM s-1), and the turnover rate (TON / second). As shown in Figure 4 a, PtFeCuCoNi HEA NPs (Vmax = 10.23, TON = 0.208) exhibited superior POD-like catalytic kinetic properties to platinum-based materials (Vmax = 7.67, TON = 0.03), fully demonstrating that the high-entropy effect significantly improves the catalytic performance of the material. In addition, its enzymatic indicators are comparable to mainstream POD-like biological catalysts, and even surpass most metal oxide and metal nanoparticle-based enzyme mimics reported in recent years (see Figure 4 b). Through free radical quenching experiments, we successfully identified the types of ROS generated by PtFeCuCoNi HEA NPs as ·O2- and 1 O2 ( Figure 4 c). Electron paramagnetic resonance (EPR) detection further confirmed that the main ROS in this material were ·O2- and 1O2(·) Figure 4 d). In summary, the PtFeCuCoNi material exhibits excellent active oxygen generation performance.

[0064] Since the residual active oxygen (ROS) after bacterial clearance can induce cellular oxidative stress, which in turn affects cell viability and function and hinders wound healing, we then investigated the ROS scavenging ability of PtFeCuCoNi high-entropy alloy nanoparticles (HEANPs) under neutral conditions. First, we evaluated its •O2-scavenging performance by nitro blue tetrazolium chloride (NBT) colorimetry. The results showed that the absorbance of PtFeCuCoNi HEA NPs at 680 nm was significantly lower than that of platinum nanoparticles (Pt NPs), indicating that it had a more excellent •O2-scavenging efficiency - reaching 90.42% within 5 minutes, which was significantly better than that of platinum nanoparticles (73.48%) (Fig. 4a). Figure 4 e).

[0065] In addition to the above free radicals, hydrogen peroxide is another important active oxygen molecule that can induce intracellular oxidative stress. Therefore, we further investigated the ability of PtFeCuCoNi high-entropy alloy (HEA) and platinum nanoparticles to scavenge hydrogen peroxide and their peroxidase-like activity in decomposing hydrogen peroxide into non-toxic substances water and O2. Time-adaptive hydrogen peroxide scavenging experiments showed that PtFeCuCoNi HEA nanoparticles performed significantly better than platinum nanoparticles, achieving a hydrogen peroxide scavenging rate of up to 85% within 30 minutes, indicating that they almost completely neutralized hydrogen peroxide (Fig. 4b). Figure 4 f). Simultaneous O2 generation experiments were consistent with hydrogen peroxide decomposition results (Fig. 4c). Figure 4 g), confirming that PtFeCuCoNi HEA nanoparticles can rapidly and efficiently decompose hydrogen peroxide into O2 and water. In addition, we also evaluated the binding affinity and reaction rate of PtFeCuCoNi HEA nanoparticles for hydrogen peroxide by calculating Vmax, Km, and TON values. As shown in Figure 4 h, the Vmax value of platinum nanoparticles was 51.53 μM s⁻¹, while the Vmax value of PtFeCuCoNi HEA nanoparticles increased to 82.14 μMs⁻¹ after the formation of high-entropy alloy by introducing multiple elements, with an increase of 1.6 times. The calculated TON value was 7.58 s⁻¹, which was particularly outstanding among current active oxygen scavenging materials based on metal compounds (Fig. 4d). Figure 4 i). These indicators fully demonstrate the ability of PtFeCuCoNi HEA nanoparticles to rapidly capture hydrogen peroxide, highlighting their excellent reaction kinetics.

[0066] Notably, the PtFeCuCoNi high-entropy alloy nanoparticles exhibit pH-adaptive reactive oxygen species (ROS) generation and scavenging characteristics: ROS generation is significantly enhanced in acidic environments, while scavenging is dominant under neutral conditions, fully validating their excellent microenvironment adaptability. The comprehensive research results demonstrate that by introducing multiple elements to construct a high-entropy alloy modification strategy, a dynamic pH-adaptive treatment protocol can be achieved—accelerating ROS generation in acidic environments and efficiently scavenging reactive oxygen species under neutral conditions, thereby effectively combating bacterial infections and promoting wound healing.

[0067] Test Example 3: PtFeCuCoNi high-entropy alloy nanoparticles eliminate MRSA under acidic conditions via ROS

[0068] After confirming the potent ability of PtFeCuCoNi alloy to promote the generation of reactive oxygen species (ROS) under acidic conditions, we systematically evaluated its antibacterial properties against methicillin-resistant Staphylococcus aureus (MRSA). MRSA, a common and widely drug-resistant pathogen in clinical practice, frequently causes skin and soft tissue infections. Experimental data showed that in an acidic environment, the ROS generated by the synergistic effect of PtFeCuCoNi and hydrogen peroxide mainly consists of ·O₂⁻ and singlet oxygen (…). 1 O2), while no peroxidase-like (POD) activity was observed in a neutral environment. OD and plate assays showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the PtFeCuCoNi alloy were only 8 μg / mL ( Figure 5 a and 5b), their doses were significantly lower than the typical doses reported in the literature for similar nanozyme-based antibacterial systems (a and 5b). Figure 5 c).

[0069] To evaluate the bactericidal effect of the alloy on planktonic bacteria, we quantified the amount of cytoplasmic contents released using a protein leakage experiment. The PtFeCuCoNi treatment group showed significantly increased membrane permeability and cytoplasmic leakage, indicating that the integrity of the bacterial envelope was severely disrupted. Figure 5 d). Colony forming unit (CFU) analysis consistently showed that MRSA was almost completely eliminated in samples treated with a combination of PtFeCuCoNi and hydrogen peroxide, while no significant bactericidal effect was observed with hydrogen peroxide alone. The antibacterial efficacy of this alloy is significantly superior to that of platinum alone, reducing the number of viable bacteria by only 21.49% under the same oxidizing conditions. Figure 5 (e, h) This indicates that PtFeCuCoNi nanozymes exhibit a significant synergistic antibacterial effect under oxidative conditions. Notably, these findings collectively demonstrate that PtFeCuCoNi can exert potent bactericidal activity against drug-resistant pathogens even at very low doses under physiologically challenging microenvironmental conditions.

[0070] Based on the observed reactive oxygen species-mediated lipid peroxidation in the biofilm matrix, we further evaluated the overall anti-biofilm effect of the alloy. Quantitative crystal violet staining results showed that the total biofilm biomass decreased by approximately 90% after PtFeCuCoNi treatment, confirming that the alloy possesses a strong biofilm scavenging ability. Figure 5 f, g, i).

[0071] Test Example 4: Reactive Oxygen Scavenging and Cell Protection Effects of PtFeCuCoNi High-Entropy Alloy Nanoparticles at Neutral pH

[0072] Based on its proven antioxidant enzyme-like activity, we further evaluated the ability of PtFeCuCoNi high-entropy alloy nanoparticles to scavenge oxidative stress and restore cellular homeostasis under physiological pH conditions (pH 7.4). Preliminary cell compatibility experiments showed that at a concentration of 4 μg mL⁻¹, the material had no significant toxicity to human umbilical vein endothelial cells (HUVECs), mouse renal cardiomyocytes (RSFs), and RAW264.7 macrophages; therefore, all subsequent experiments used a concentration of 2 μg mL⁻¹. Quantitative fluorescence analysis after 2,7-dichlorofluorescein diacetate (DCFH-DA) staining revealed that intracellular reactive oxygen species (ROS) levels significantly increased after hydrogen peroxide treatment, but cell viability essentially returned to baseline levels after treatment. Figure 6 a). The protective ability of HEA nanoparticles in a reactive oxygen species environment was evaluated using representative live / dead cell double staining fluorescence images. Quantitative results of live / dead cell staining showed that, unlike the untreated group or the platinum-only control group which exhibited extensive cell death, PtFeCuCoNi effectively inhibited H2O2-induced cell death (a). Figure 6 b).

[0073] To verify whether this cytoprotective effect translates into structural toughness, we examined the distribution of F-actin in human umbilical vein endothelial cells (HUVECs) and mouse renal cardiomyocytes (RSFs). Phalloidin staining showed that hydrogen peroxide exposure led to significant depolymerization of F-actin, while PtFeCuCoNi maintained the integrity of the cytoskeleton, with its cell area being no different from the unexposed control group. Figure 6 c, d). Tube formation experiments showed that HUVECs treated with PtFeCuCoNi formed more branching points and extended into capillary-like networks than cells exposed to hydrogen peroxide alone. Figure 6 e), which indicates that maintaining the cytoskeleton is fundamental to enhancing migration and angiogenesis.

[0074] In addition to cytoprotection, effective clearance of pro-inflammatory microenvironment is crucial for recovery after bacterial infection, which is mainly regulated by macrophage plasticity. To verify this, we studied the immunomodulatory effect of PtFeCuCoNi on macrophage polarization in LPS-stimulated RAW264.7 cells. Notably, pre-treatment with PtFeCuCoNi significantly down-regulated CCR7 expression to a much greater extent than LPS or Pt alone, which is a hallmark marker of pro-inflammatory M1 macrophages (Fig. 4a). Figure 6 f). Meanwhile, PtFeCuCoNi induced a much stronger effect on CD163 expression than the control group, confirming that it could significantly promote the transformation of macrophages to the repair M2 phenotype (Fig. 4c). Figure 6 g). These results collectively indicate that PtFeCuCoNi can effectively reprogram macrophages from an inflammatory state to a repair state, clear reactive oxygen species, and exert cytoprotective effects, thereby constructing an immune regenerative environment conducive to healing.

[0075] Summary and discussion:

[0076] In summary, inspired by the human innate immune-redox regeneration (IADS) mechanism, we innovatively synthesized PtFeCuCoNi high-entropy alloy (HEA) nanoparticles using a one-step solvothermal method. These high-entropy alloy nanoparticles mimic the dynamic enzymatic behavior of natural peroxisomes and exhibit excellent reactive oxygen species regulation characteristics in complex wound environments, enabling them to coordinate antibacterial activity and tissue repair functions.

[0077] Mechanism studies showed that the synergistic effect of multiple metal active sites, especially the synergistic effect between cobalt (Co) and iron (Fe) atoms, endows high-entropy alloy nanoparticles (HEA NPs) with pH-responsive enzyme-mimetic activity. Specifically, cobalt promotes hydrogen peroxide adsorption, while ruthenium regulates oxygen intermediate binding, both of which work together to significantly enhance the catalytic efficiency of reactive oxygen species (ROS), far exceeding the level of traditional platinum-based nanoparticles.

[0078] Notably, HEA nanoparticles not only exhibit excellent catalytic performance, but also show significant biological functions in MRSA-infected wound models. In the acid infection stage, they can effectively produce reactive oxygen species (ROS) to achieve bacterial clearance and biofilm destruction; in the neutral repair stage, they can alleviate oxidative stress-induced DNA damage and apoptosis of endothelial cells and fibroblasts by removing excess ROS, thereby promoting angiogenesis (CD31 / VEGF) and collagen-rich extracellular matrix deposition. Transcriptome analysis further confirmed that the antioxidant pathway (such as Nrf2, SLC7A11) was activated, while the pro-inflammatory genes (such as CHAC1, SESN2) were inhibited, which helped to maintain immune homeostasis and accelerate wound healing. In vitro experiments showed that HEA nanoparticles can enhance the migration ability of fibroblasts, promote the formation of tubular structures of human umbilical vein endothelial cells (HUVECs), and induce M2-like macrophage polarization (up-regulation of CD206 and Arg1 expression), indicating that they have the ability to adapt to the stage-specific wound repair of the microenvironment.

[0079] The comprehensive research results show that PtFeCuCoNi high-entropy alloy nanoparticles (HEA) as a new type of multifunctional biological catalyst show broad application prospects. Its strong active oxygen regulation ability not only makes it an ideal choice for combating drug-resistant infections and promoting tissue regeneration, but also has wide applicability in redox-related biomedical fields such as chronic inflammation, diabetic wound repair, ischemic disease treatment, and tumor microenvironment remodeling.

[0080] It should be noted that the preferred embodiments of the present application are given in the specification and drawings of the present application, but the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to be combined, forming various embodiments not listed above, which are considered to be within the scope of the present application. Furthermore, for those skilled in the art, the above description can be improved or modified, and all such improvements and modifications are within the scope of the appended claims of the present application.

Claims

1. Use of high-entropy alloy nanoparticles as biocatalysts, characterized in that, The high-entropy alloy nanoparticles comprise Pt, Fe, Cu, Co and Ni; The high-entropy alloy nanoparticles have the ability to promote the generation of active oxygen under acidic conditions and the ability to promote the elimination of active oxygen under neutral conditions. The application is not for the purpose of diagnosis or treatment.

2. Use according to claim 1, characterized in that, The high-entropy alloy nanoparticles have POD-like enzyme activity under acidic conditions and SOD-like and / or CAT-like enzyme activity under neutral conditions.

3. Use according to claim 1, characterized in that, The atomic ratio of Pt, Fe, Cu, Co and Ni in the high-entropy alloy nanoparticles is 19.1:14.7:20.9:22.3:23.

0.

4. Use according to claim 3, characterized in that, 5. Use of the high-entropy alloy nanoparticles as defined in any one of claims 1-4 in the preparation of a material for eliminating and destroying methicillin-resistant Staphylococcus aureus biofilm under acidic conditions.

6. The use according to claim 5, wherein the minimum inhibitory concentration of the high-entropy alloy nanoparticles is 8 μg / mL.

7. Use of the high-entropy alloy nanoparticles as defined in any one of claims 1-4 in the preparation of a product for treating wound infection caused by methicillin-resistant Staphylococcus aureus. ​

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