High-entropy alloy Janus artificial enzymes for pH-gated sequential redox therapy of drug-resistant bacterial infection

Domain: chemistry
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
Drug-resistant bacterial infections in chronic wounds remain a critical challenge, particularly under persistent inflammation. Here, we report the de novo design of high-entropy alloy (HEA, PtFeCuCoNi)-based Janus artificial enzymes with pH-gated redox biocatalysis for sequential antibacterial and repair functions. The multi-metal synergy stabilizes the d-band center, allowing acidic oxidase/peroxidase-like activity and neutral antioxidase-like activity. In infection, the enzymes generate bactericidal reactive oxygen species (ROS) to eliminate methicillin-resistant Staphylococcus aureus (MRSA) and biofilms at ultralow concentrations (8 μg/mL). During healing, they scavenge ROS, alleviate oxidative injury and support cellular proliferation. In MRSA-infected wounds, this dual-action system clears bacteria and then accelerates regeneration through enhanced neovascularization and matrix remodeling. Mechanistic analyses reveal PFKFB3-mediated metabolic reprogramming, suppression of pro-inflammatory cytokines, and macrophage polarization toward the M2 phenotype. Integrating pH-gated antimicrobial and immunomodulatory repair within one nanoplatform, this strategy addresses the conflicting demands of infection control and tissue healing.

Introduction
Chronic non-healing wounds infected with antibiotic-resistant bacteria present a growing global health challenge, compounded by the complex pathophysiology of these persistent lesions1–4. The wound microenvironment exhibits dynamic pH evolution: initial bacterial proliferation and biofilm formation create an acidic milieu through organic acid secretion, which simultaneously compromises host immunity and diminishes antibiotic effectiveness5. When sterilization is completed, the wound will undergo chronic inflammation with a near-neutral pH condition, sustained pro-inflammatory macrophage (M1) phenotype activity, and pro-inflammatory cytokine production, which will facilitate the production of massive reactive oxygen species (ROS) that impair vascular and stromal cell function, stalling tissue repair6,7. Current approaches, including the antibiotics, peptides, and charged polymers, fail to achieve effective microbial eradication and anti-inflammatory action concurrently8. The development of non-antibiotic and pH-responsive agents that can sequentially eliminate pathogens in acidic environments and then achieve anti-inflammatory and immunomodulatory modes to promote wound healing, therefore, represents a critical therapeutic frontier.

Peroxisome-mediated ROS regulation plays a pivotal role in innate immunity, orchestrating precisely controlled oxidative bursts that eliminate pathogens while protecting host tissues9. These organelles employ peroxisomal oxidases (OXD) and peroxidases (POD) to generate targeted ROS attacks that disrupt bacterial membranes, oxidize vital biomolecules, and degrade protective biofilms10,11. Following pathogen clearance, antioxidases, such as catalase (CAT), decompose residual hydrogen peroxide (H2O2) into water and oxygen, restoring redox equilibrium and safeguarding tissue integrity12–14. However, chronic inflammatory conditions disrupt this delicate balance, leading to sustained ROS production that overwhelms endogenous defenses and perpetuates tissue damage through excessive oxidative stress and amplified inflammatory signaling. While peroxisomal enzymes exemplify nature’s sophisticated ROS management system, their therapeutic potential is constrained by poor pharmacokinetic profiles and immunogenic risks15. This limitation has spurred the development of engineered ROS-modulating platforms as next-generation anti-infective strategies. Current nanotechnology approaches, spanning inorganic nanoparticles (NPs) to metal-organic frameworks16,17, attempt to recapitulate enzymatic ROS generation and scavenging capabilities18. Yet most synthetic systems still lack the precision of their biological counterparts, often exhibiting suboptimal catalytic efficiency and spatiotemporal control that result in either insufficient antimicrobial activity or off-target oxidative damage.

High-entropy alloy (HEA) represents an emerging paradigm in enzyme-mimetic catalysis19,20, leveraging its unique multi-element synergy to overcome limitations of conventional catalysts21. These alloys incorporate five or more transition metals in near-equiatomic ratios (5–35%), where the high configurational entropy, pronounced lattice distortion, and sluggish diffusion kinetics collectively stabilize single-phase solid solutions22–24. This distinctive “cocktail effect” generates an exceptionally diverse array of active sites while enabling fine-tuning of the d-band center and oxygen adsorption characteristics, which may enable superior catalytic performance and stability compared to monometallic systems25,26. While HEAs have made significant progress in the fields of energy conversion and environmental catalysis, their application in biomedical redox regulation remains largely unexplored, constrained by demanding activation conditions, aqueous instability, and unresolved biocompatibility issues27,28. Realizing their therapeutic potential will necessitate precise engineering of HEA with optimized size distributions29, tailored surface chemistries30,31, and intelligent pH-responsive behavior to achieve spatiotemporal control over ROS modulation in physiological environments32.

Drawing inspiration from the dynamic redox regulation of peroxisomes, we report the de novo design of HEA, PtFeCuCoNi-based Janus artificial enzymes with pH-gated redox biocatalysis for sequential therapy of drug-resistant bacteria and inflammatory wounds. The precisely balanced incorporation of five transition metals with comparable atomic radii enables synergistic electronic effects while minimizing precious metal content. Density functional theory (DFT) reveals strong d-electron interactions within the lattice HEA NPs that redistribute electron density near the Fermi level, optimizing oxygen intermediate adsorption/desorption at catalytic sites (Fig. 1a). In acidic infected wounds, these NPs exhibit potent OXD/POD-mimetic activity, generating bactericidal ROS bursts that eliminate methicillin-resistant Staphylococcus aureus (MRSA) and biofilms at ultralow concentrations (8 μg/mL). As infection resolves and pH normalizes, the particles switch to superoxide dismutase (SOD)/CAT-like functions, quenching excess ROS and mitigating oxidative damage (Fig. 1b). Kinetic analyses demonstrate exceptional catalytic efficiency (POD-like activity: the maximum reaction rate (Vmax) = 10.23 μM s−1; CAT-like activity: Vmax = 74.69 μM s−1), outperforming existing ROS-catalytic materials while maintaining minimal cytotoxicity at therapeutic doses. Crucially, the platform orchestrates dual-phase immunomodulation, which directs macrophage polarization in a stage-dependent manner, first enhancing M1-mediated bacterial clearance and then inducing anti-inflammatory macrophage (M2)-driven regenerative responses. In vivo studies confirm rapid pathogen clearance coupled with protection of endothelial cells, fibroblasts, and macrophages, resulting in accelerated wound closure. This HEA-based Janus artificial enzymes with pH-gated redox biocatalysis represents a programmable, antibiotic-free approach that concurrently addresses antimicrobial resistance and chronic inflammation while promoting regeneration (Fig. 1c).

Results
Synthesis and structural characterization
The HEA-based Janus artificial enzymes were synthesized via a one-step solvothermal approach using Pd(acac)2, Fe(acac)3, Cu(NO3)2·3H2O, Co(NO3)2·6H2O, Ni(NO3)2·6H2O as metal precursors (Fig. 2a), N,N-dimethylformamide (DMF) and ethylene glycol (EG) as the solvents. A mauve, finely dispersed mixture of the raw materials was formed through ultrasonication. As the temperature increased, the mixture turned black, possibly due to the coordination and reduction of the five metals. Subsequently, the single-phase HEA formed after the diffusion and rearrangement of atoms. Transmission electron microscope (TEM) and high-resolution transmission electron microscopy (HRTEM) were used to characterize the obtained products. The HEA NPs revealed spherical NPs with uniform morphology (Fig. 2b and Supplementary Fig. 1). Meanwhile, the number-averaged size of PtFeCuCoNi HEA NPs is measured to be ≈122.4 nm in dynamic light scattering, indicative of a stable aqueous phase with low agglomeration tendency. (Supplementary Fig. 2).

X-ray diffraction (XRD) analysis confirmed a face-centered cubic structure (Fig. 2c), with characteristic peaks shifted to higher angles relative to pure Pt (PDF#35–1358), consistent with alloy formation. We also investigated the effect of precursor ratio on the crystal structure of the HEA NPs (Supplementary Fig. 3). It was observed that the PtFeCuCoNi HEA NPs formed from an equimolar precursor ratio exhibited the most intense XRD diffraction peaks. This is attributed to the near-random distribution of the five constituent elements among the fcc lattice sites, which allows the crystal to maintain superior periodicity and thereby generate strong XRD diffraction peaks. Conversely, when the proportion of any single element was doubled, the excessive metal atoms introduced more intense and localized lattice expansion and strain, thereby disrupting the periodic arrangement of the crystal lattice and ultimately leading to a significant reduction in XRD diffraction peak intensity33.

Inductively coupled plasma-mass spectrometry (ICP-MS) quantification established a Pt/Fe/Cu/Co/Ni atomic ratio of 19.1:14.7:20.9:22.3:23.0 (Fig. 2d). HRTEM imaging revealed well-defined (111) lattice planes (Fig. 2e), with fast Fourier-transform (FFT) analysis confirming crystalline perfection. Inverse FFT reconstruction showed lattice spacings ranging from 0.212 to 0.215 nm (Fig. 2f and Supplementary Fig. 4), corresponding to ~3.3% intrinsic tensile strain arising from atomic-size mismatch and the ultrathin particle morphology. The low-magnification, high-angle annular dark-field scanning TEM (HAADF-STEM) energy-dispersive X-ray spectroscopy (EDS) images show a homogeneous distribution of Pt, Fe, Cu, Co, and Ni throughout the NPs (Fig. 2g).

Surface chemical states of the PtFeCuCoNi HEA NPs were analyzed by X-ray photoelectron spectroscopy (XPS) (Fig. 2h and Supplementary Fig. 5)34–36, and distinct photoelectron peaks confirmed the presence of all five metallic components. The Pt 4f spectrum exhibited characteristic doublets at 71.22 eV (4f7/2) and 74.54 eV (4f5/2), while Fe 2p peaks appeared at 714.12 eV (2p3/2) and 723.69 eV (2p1/2). Similarly, Cu 2p signals were observed at 931.85 eV (2p3/2) and 951.62 eV (2p1/2), with Co 2p peaks at 778.14 eV (2p3/2) and 792.83 eV (2p1/2), and Ni 2p emissions at 852.41 eV (2p3/2) and 869.69 eV (2p1/2). The systematic shifts in binding energies relative to their pure metallic states, coupled with relatively weak oxide signatures, provides strong evidence for alloy formation rather than phase-segregated or metal oxides.

X-ray absorption near-edge structure (XANES) analysis at the Pt L3-edge reveals a characteristic white line intensity comparable to Pt foil and markedly reduced relative to PtO2, confirming the predominantly metallic state of Pt atoms in the HEA NPs (Fig. 2i). Fourier-transform extended X-ray absorption fine structure (FT-EXAFS) analysis demonstrates a primary coordination peak at ≈2.3 Å, corresponding to Pt-Pt interactions with a distinct shoulder attributable to Pt-M (M=Fe, Cu, Ni, Co) bonding (Fig. 2j). The complete absence of Pt-O scattering paths provides definitive evidence against surface oxidation37. Complementary wavelet transform (WT) analysis of the Pt L3-edge oscillations resolves the spatial distribution of atomic interactions, with PtFeCuCoNi HEA NPs exhibiting a prominent Pt-M bonding signature at 2.36 Å in R-space that is absent in both Pt foil and PtO2 references (Fig. 2k). These results verify the formation of a homogeneous multimetallic coordination environment within the HEA matrix.

Experimental and theoretical studies on pH-gated ROS-catalytic activities
With the structural and electronic characterization established, we examined the pH-gated enzymatic activities of HEA NPs for treating bacterially infected chronic wounds. In acidic microenvironments of infected wounds, the HEA-based Janus artificial enzymes exhibit potent POD-like activity, catalyzing the conversion of H2O2 into cytotoxic •O2− and 1O2 that effectively eliminate bacterial pathogens. As the wound pH normalizes during healing, the HEA-based Janus artificial enzymes transition to a protective mode, sequentially mimicking SOD to dismutase •O2− to H2O2 and CAT to decompose excess H2O2 into harmless O2 and H2O (Fig. 3a). This dual-phase catalytic switching not only eradicates infection but also creates a redox-balanced microenvironment conducive to tissue regeneration38.

Experimental validation of the predicted ROS-catalytic properties began with a quantitative assessment of ROS generation under acidic conditions using a 3,3,5,5-tetramethylbenzidine (TMB) chromogenic assay. The PtFeCuCoNi HEA NPs demonstrated superior POD-mimetic activity, as evidenced by intense oxTMB absorption at 652 nm (Fig. 3b). To elucidate the compositional synergy, we systematically synthesized and evaluated quaternary nanocatalysts (FeCuCoNi, PtCuCoNi, PtFeCoNi, PtFeCuNi, and PtFeCuCo) with controlled elemental stoichiometries (Supplementary Fig. 6). Kinetic analysis revealed a hierarchical dependence of catalytic activity on elemental composition: Fe removal caused the most pronounced activity reduction, followed by Pt, Cu, Co, and Ni. The consistent underperformance of all quaternary systems relative to the pentametallic HEA confirms the essential cooperative interactions among all five elements in optimizing ROS-generation kinetics.

Subsequently, we systematically analyzed the characteristic steady-state kinetic constants, including the catalytic constant (Km/mM), Vmax/μM s−1, and the turnover number (TON/s−1). As shown in Fig. 3c and Supplementary Fig. 7, PtFeCuCoNi HEA NPs (Vmax = 10.23, TON = 0.208) exhibited superior POD-like catalytic kinetics compared to the low activity of Pt (Vmax = 7.67, TON = 0.03), highlighting the beneficial impact of the high entropy effect. Furthermore, the enzymatic performance of PtFeCuCoNi HEA NPs surpasses that of most recently reported metal oxides and metal nanoparticle-based POD mimics (Fig. 3d and Supplementary Table 1). We conducted free radical quenching experiments to qualitatively identify the ROS species, which could be identified as •O2− and 1O2 (Fig. 3e and Supplementary Figs. 8 and 9). Subsequently, the 1O2 species can be detected by the 9,10-diphenanthraquinone (DPA) in a time-dependent manner (Fig. 3f). In addition, adopting 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) as the specific spin trap reagents, respectively, the electron paramagnetic resonance (EPR) detection also confirms that the major ROS in HEA NPs is •O2− and 1O2 (Fig. 3g and Supplementary Fig. 10). In-situ Fourier-transform infrared spectroscopy (FTIR) is utilized to investigate the intermediates’ evolution during the POD-like reaction process, indicating the formation of *OOH intermediate and •O2− product (Fig. 3h, i). Together, these results demonstrate the superior ROS-generating capacity of PtFeCuCoNi HEA NPs, attributable to their optimized electronic structure and multi-element synergy.

Recognizing that persistent ROS after bacterial eradication may exacerbate oxidative damage and impair tissue regeneration, we evaluated the antioxidant capacity of PtFeCuCoNi HEA NPs at physiological pH (neutral). Using a nitrotetrazolium blue chloride (NBT) assay, the HEA NPs demonstrated significant ROS scavenging efficiency, achieving 90.42% •O2− elimination within 5 min, which significantly surpasses the performance of Pt NPs (73.48%) (Fig. 3j). Besides •O2− radicals, H2O2 is another important ROS molecule that contributes to intracellular oxidative stress. Thus, we examined the CAT-like activity of HEA NPs in neutralizing H2O2, a key mediator of oxidative stress. The PtFeCuCoNi HEA NPs demonstrated rapid H2O2 decomposition, achieving 85% scavenging within 30 min, significantly surpassing the performance of Pt NPs (Fig. 3k). Oxygen evolution measurements confirmed complete conversion of H2O2 to H2O and O2 (Fig. 3l), with kinetic analysis revealing a 1.6-fold enhancement in Vmax (Vmax = 82.14 μM s−1) compared to pure Pt (51.53 μM s−1) (Fig. 3m and supplementary Figs. 11 and 12). The exceptional TON (TON = 7.58 s−1) further positions these HEA NPs among the most efficient metal-based ROS-scavenging systems (Fig. 3n and Supplementary Table 2).

The long-term activity and stability of PtFeCuCoNi HEA NPs as a CAT-like antioxidant have also been evaluated, revealing consistent maintenance of high activity with no observable decline after five cycles of testing (Supplementary Fig. 13). It was found that the HEA synthesized with an equimolar precursor ratio exhibited the optimal enzyme-mimicking performance (Supplementary Fig. 14). This is attributed to the optimal d-band center and lattice structure of the equimolar PtFeCuCoNi HEA NPs, which strikes an optimal balance between the adsorption and desorption of reactants39. Importantly, PtFeCuCoNi HEA NPs exhibited pH-dependent ROS production and ROS scavenging, with a predominance of ROS production in acidic environments and a predominance of ROS scavenging in neutral conditions, confirming their microenvironmental adaptability (Supplementary Fig. 15).

The PtFeCuCoNi HEA NPs demonstrate significant pH-gated redox biocatalysis, preferentially generating bactericidal ROS in acidic microenvironments while switching to antioxidant functions at physiological pH. This dynamic behavior stems from the HEA’s unique electronic configuration, which enables microenvironment-adaptive ROS biocatalysis. By simultaneously addressing pathogen clearance and oxidative stress resolution via pH-gated mechanisms, the HEA-based Janus artificial enzymes enable sequential therapy for infection control and tissue regeneration, thereby overcoming a fundamental challenge in chronic wound therapy.

To explore the structural advantages of PtFeCuCoNi HEA NPs, DFT calculations have been employed to reveal the ROS-catalytic reaction process. The optimized full geometry structure of the PtFeCuCoNi HEA NPs is modeled based on the fcc lattice, then a surface model is built with Pt:Fe:Cu:Co:Ni composition ratio close to the experimental ratio 19.1:14.7:20.9:22.3:23.0 by ICP-MS characterization (Fig. 4a). We evaluated the overall catalytic activity and catalytic sites of the PtFeCuCoNi HEA NPs using electron localization function (ELF) map (Fig. 4b) and the electrostatic potential (ESP) simulation calculations (Fig. 4c) before determining the enzymatic activity reaction path40. Notably, the Pt center in PtFeCuCoNi HEA NPs, with a higher charge density, plays a role in providing electrons in catalytic processes. Accordingly, the Fe and Co metal centers with lower ESP are obvious nucleophilic reaction sites, which have strong interactions with oxygen intermediates. Therefore, we have chosen the part circled in black in Fig. 4b as the main reactive site. The partial projected density of state (pDOS) for the HEA NPs is presented in Fig. 4d to illustrate their electronic structures. Distinct d-orbital overlaps among the different metals were clearly observed, demonstrating that the elements in the alloy were strongly bonded to each other. Notably, Fe and Co exhibit more unoccupied orbitals above the Fermi level, suggesting that Fe and Co atoms in PtFeCuCoNi are more susceptible to interaction with oxygen intermediates during the catalytic process, which is consistent with the calculated results of ESP. Bader charge analysis further corroborates this mechanism, showing substantial electron transfer from Fe (the highest positive charge) to Pt (the electron reservoir) (Fig. 4e and Supplementary Fig. 16). These electronic structure features enable efficient oxygen intermediate binding and activation at the Fe/Co sites while maintaining electron supply through Pt centers.

Analysis of spin-polarized d-orbitals reveals critical electronic structure modifications in the HEA NPs (Fig. 4f, g and Supplementary Fig. 17). According to the d-band center theory and Sabatier principle, a rational up-shift of the d-band center position relative to the Ef generally enhances the bond strengths between the metal atoms and oxygen intermediates41, thus facilitating the POD-like activity. As shown in Fig. 4f, in PtFeCuCoNi HEA NPs, Fe and Co atoms exhibit the highest d-band centers among the constituent metals. Consequently, due to their favorable electronic structure for the adsorption of oxygen intermediates, Fe and Co atoms serve as the primary ROS-catalytic sites. In contrast, Pt possesses the lowest d-band center, which promotes desorption of oxygen intermediates, thereby rendering Pt atoms the primary desorption sites. An excessively elevated d-band center can hinder product desorption. Therefore, in PtFeCuCoNi HEA NPs, Cu and Ni atoms also play crucial roles, effectively modulating the d-band center and facilitating the production of ROS.

Subsequently, we compared the d-band centers of individual metals with those in the HEA NPs (Fig. 4g). We found that upon alloy formation, the d-band centers of Fe and Co exhibited a significant upward shift, which facilitated the adsorption of oxygen intermediates. Conversely, the d-band centers of Pt, Cu, and Ni shifted downward, indicating enhanced desorption of final products during the POD-like reaction, thereby preventing active-site poisoning and enhancing ROS-catalytic efficiency. In summary, the d-electron structure of the HEA NPs was regulated by different constituent metals, which guaranteed efficient and stable ROS-catalytic activity.

Take the POD-like ROS generation process as a representative example; Fig. 4h presents the key intermediate structures and corresponding Gibbs free energy profiles. The catalytic pathway initiates with H2O2 adsorption at Fe sites (step i), followed by either homolytic or heterolytic cleavage (step ii). DFT calculations reveal favorable adsorption energetics for both dissociation pathways, with binding energies of −3.075 eV for *OH+*O and −2.760 eV for 2*OH, indicating that 2*OH and *OH+*O were adsorbed stably (Fig. 4f), which confirms the spontaneous stabilization of these species. The Fe sites facilitate this process, enabling strong interfacial interactions between the HEA surface and adsorbed *OH/*O radicals, as evidenced by pronounced orbital hybridization in pDOS analysis and substantial electron transfer (0.89 |e| to *OH; 0.56 |e| to *O) in Bader charge calculations (Supplementary Figs. 18 and 19). Notably, the homolytic pathway demonstrates particular thermodynamic preference, requiring only a 0.174 eV energy barrier for •OOH formation from *OH+*O intermediates (step iii). This low activation energy highlights the kinetic advantage of homolytic H2O2 dissociation on the HEA surface. The catalytic proficiency arises from the collective effects of multiple transition metals. While Fe dominates the initial activation, the surrounding metal atoms provide essential coordination environments and electron donation capacity that collectively enhance the intrinsic POD-like activity. Pt primarily functions as an electron reservoir, and Cu/Ni atoms play a key role in modulating the d-band center. These computational insights comprehensively characterize the electronic structure-catalytic function relationship in HEA NPs, demonstrating how elemental synergy enables their exceptional biomimetic performance.

Furthermore, according to our previous studies, the structure of adsorbed O* is a key intermediate in compartmentalizing the POD-like and CAT-like pathways42. Therefore, to demonstrate the continuous pH-switchable catalytic activity of PtFeCuCoNi HEA NPs, we complemented the DFT studies of the free energy of the structure of PtFeCuCoNi HEA NPs adsorbing O* (Structure 1) for the adsorption of H+ or H2O2 under acidic and neutral conditions. As shown in Supplementary Fig. 20, the free energies of structure 1 for the adsorption of both H+ or H2O2 are negative, indicating that both processes can occur spontaneously. However, compared with adsorbing H2O2 (−0.384 eV), the free energy of adsorbing H+ by structure 1 is lower (−0.436 eV), indicating that structure 1 will preferentially combine with H+ to form *OH to complete the POD-like activity pathway in an acidic environment with the presence of H+. Furthermore, under neutral or slightly alkaline conditions, structure 1 is more prone to adsorb H2O2 to carry out the CAT-like activity pathway to realize the continuous pH-switchable catalytic activity.

Acid-triggered ROS catalysis to eradicate MRSA and disrupt biofilms
Having established the robust pH-responsive ROS generation of PtFeCuCoNi HEA NPs, we next evaluated their antimicrobial performance against MRSA, a clinically relevant, extensively drug-resistant pathogen implicated in skin and soft-tissue infections43–45. In acidic media containing H2O2, the NPs predominantly produced •O2− and 1O2, whereas at near-neutral pH they exhibited no detectable POD-like activity (Fig. 5a). To directly verify ROS generation in bacteria, intracellular ROS levels were assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Bacteria treated with PtFeCuCoNi HEA NPs exhibited strong green fluorescence, indicative of pronounced intracellular ROS accumulation and presenting direct evidence for the ROS-mediated antibacterial mechanism of the artificial enzyme (Supplementary Fig. 21). Notably, the particles achieved a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 8 μg/mL (Fig. 5b and Supplementary Fig. 22), a dosage substantially lower than those typically reported for comparable ROS-catalytic antimicrobial systems (generally >100 μg/mL) (Fig. 5c). Thus, HEA NPs are capable of maintaining potent bactericidal activity against drug-resistant pathogens even under physiologically challenging microenvironmental conditions46–49.

To delineate the cellular basis of the antibacterial activity, we first quantified ROS-augmented membrane disruption in planktonic MRSA. A protein leakage assay, which measures cytoplasmic release from damaged cells50, revealed markedly increased permeability and leakage in HEA-treated bacteria (Fig. 5d and Supplementary Fig. 23), consistent with envelope disruption. In line with this, colony-forming unit (CFU) analysis showed almost complete eradication of MRSA when HEA NPs were combined with H2O251–54, whereas H2O2 alone had a negligible effect. Under identical oxidative conditions, monometallic Pt NPs reduced viable counts by only 21.49% (Fig. 5e, f and Supplementary Fig. 24), underscoring the pronounced synergistic antibacterial effect of the HEA formulation. Quantitative crystal-violet assays further demonstrated an approximate 90% reduction in biofilm biomass following HEA NPs treatment (Fig. 5g–i and Supplementary Fig. 25)55,56.

Scanning electron microscopy (SEM) also revealed minor surface damage with abundant extracellular polymeric substances (EPS) in Pt-treated bacteria57,58, whereas HEA-exposed group displayed marked structural disruption (red arrows) and visible internalization of HEA NPs (pink circles). 3D CLSM images (l) and ratio (m) of Live/Dead ratios of planktonic MRSA, Live: ***p(PtFeCuCoNi)=0.000000061 (n=3 independent replicates). n C11-BODIPY581/591 stained confocal images of MRSA biofilms. t LPO statistics (n=3 independent replicates). v Schematic illustration of pH-dependent ROS-mediated antibacterial action of HEA NPs (Created in BioRender. Chen, T. (2025) https://BioRender.com/hs7ivcs). Data are presented as mean ± SD, ***p<0.001, statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test, all tests were two-sided. Experiments were repeated independently (d, e, g, h, j, k, l, n, p, r, s, u) at least three times. Source data are provided as a Source data file.

Afterward, we have rigorously evaluated the solution stability of HEA NPs. The PtFeCuCoNi HEA NPs exhibit exceptional dispersibility, with no detectable precipitation or phase separation following static incubation at room temperature for 12 days (Supplementary Fig. 29). Moreover, assessments of particle size and zeta potential of PtFeCuCoNi HEA NPs across various intervals (days 3, 6, 9, and 12) indicate no significant dimensional alterations or aggregation (Supplementary Fig. 30). Therefore, PtFeCuCoNi HEA NPs exhibit robust stability and hold promise for enhanced therapeutic efficacy in forthcoming in vivo and in vitro studies.

To visualize bacterial viability at high spatial resolution, we acquired three-dimensional reconstructions by fluorescence microscopy and confocal laser scanning microscopy (CLSM)59–62. In the PtFeCuCoNi HEA NPs-treated group, bacterial clusters showed dominant red fluorescence, indicating extensive membrane compromise and effective killing of MRSA (Fig. 5l, m and Supplementary Fig. 31). In contrast, monometallic Pt NPs control retained strong green fluorescence signals, reflecting a high proportion of viable bacteria under comparable conditions. To determine whether ROS-induced lipid damage contributed to this effect, we assessed membrane lipid peroxidation (LPO) using the ratiometric probe C11-BODIPY581/591. A pronounced shift toward green fluorescence was observed by CLSM in the HEA NPs-treated group, corresponding to elevated lipid peroxide accumulation and confirming extensive oxidative degradation of bacterial membrane lipids (Fig. 5n, o and Supplementary Figs. 32 and 33).

Accordingly, we then evaluated the biofilm eradication efficacy62,63. Live/Dead fluorescence imaging revealed a substantial loss of viability and pronounced architectural degradation in HEA NPs-treated biofilms, accompanied by a significant decrease in matrix thickness (Fig. 5p, q and Supplementary Figs. 34 and 35). Conversely, Pt NPs-treated biofilms largely preserved a compact, densely packed morphology with minimal thickness change (Fig. 5r). Consistently, CLSM-based LPO analysis showed a strong green fluorescence shift in HEA NPs-exposed biofilms64,65, particularly within deeper layers, suggesting extensive phospholipid oxidation and ROS-induced damage throughout the biofilm matrix (Fig. 5s–u and Supplementary Figs. 36 and 37)66–68. In line with this, malondialdehyde (MDA) quantification further confirmed enhanced lipid peroxidation in MRSA treated with PtFeCuCoNi HEA NPs compared with Pt NPs and controls (Supplementary Fig. 38). To place these findings in context, we propose a conceptual model in which PtFeCuCoNi HEA NPs facilitate oxidative degradation of biofilm EPS and bacterial membranes69,70, followed by membrane penetration and intracellular accumulation, thereby exerting multifaceted bactericidal effects (Fig. 5v).

To elucidate how HEA-catalyzed ROS reshape MRSA biology under acidic conditions, we performed transcriptomic sequencing on MRSA exposed to H2O2 alone (as a control) or H2O2 with PtFeCuCoNi HEA NPs. A total of 401 differentially expressed genes (DEGs) were identified, including 226 upregulated and 175 downregulated genes (Fig. 6a). Hierarchical clustering of global expression profiles revealed a clear distinction between treated and control groups (Fig. 6b), indicating a treatment-induced transcriptional shift. Gene Ontology (GO) enrichment analysis comparing HEA+H2O2 with H2O2 alone highlighted terms related to envelope and nucleoprotein architecture in the Cellular Component (CC) category11, including “cell wall”, “external encapsulating structure”, and “protein-DNA complex” (Fig. 6c). Together with the downregulation of Biological Process (BP) terms such as “response to oxygen-containing compound” and “response to starvation”, these enrichments are consistent with ROS-induced structural damage accompanied by a starvation-like stress program.

Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed broad perturbation of metabolic networks, with repression of sulfur metabolism71, cysteine72 and methionine metabolism73, histidine metabolism74, and glycerophospholipid metabolism73, indicating disruption of thiol-based antioxidant defenses and membrane-lipid homeostasis (Fig. 6d). Notably, gene set enrichment analysis (GSEA) further showed positive enrichment of “oxidative phosphorylation” and “ATP-dependent activity”75, suggesting an early compensatory surge in energy metabolism, likely reflecting transient activation of bacterial respiratory and ATP-generating pathways in response to acute ROS stress42,76. In contrast, “ATP-binding cassette (ABC) transporters”77 and “histidine metabolism”78 were negatively enriched (Fig. 6e), indicating impaired substrate trafficking and amino-acid biosynthesis12. Collectively, these transcriptomic data support a model in which HEA-driven ROS inflict envelope and macromolecular damage, trigger maladaptive energy overactivation, and erode transport and biosynthetic capacity, culminating in a dual hit of oxidative injury and metabolic exhaustion that underlies efficient bacterial killing (Fig. 6f).

Neutral-pH ROS scavenging preserves cellular function and reprograms macrophages
Given its demonstrated antioxidase-like activity79–81, we evaluated PtFeCuCoNi HEA NPs for the mitigation of oxidative stress and restoration of cellular homeostasis at physiological pH (7.4)41,82. Cytocompatibility testing showed no appreciable toxicity of PtFeCuCoNi HEA NPs or Pt NPs to human umbilical vein endothelial cells (HUVEC), rat skin fibroblasts (RSF), or murine macrophages (Raw264.7) up to 4 μg mL−1 (Supplementary Figs. 39–41); therefore, 2 μg mL−1 was used in subsequent assays. Upon H2O2 challenge, intracellular ROS rose sharply by DCFH-DA readout, whereas HEA NP pretreatment reduced fluorescence to near-baseline across all three cell types (Fig. 7a and Supplementary Fig. 42), with quantification confirming significant suppression of ROS (Fig. 7b). In agreement, Live/Dead staining showed marked protection from H2O2-induced cytotoxicity in the HEA NPs group compared with untreated or Pt NPs-treated cells (Fig. 7c, d). To determine whether this protection extends to cell structure and function, we examined F-actin organization83. Phalloidin staining revealed that H2O2 exposure led to pronounced cytoskeletal collapse and cell rounding, whereas HEA NPs preserved cortical actin and cell spreading comparable to unstressed controls (Fig. 7e–g and Supplementary Fig. 43). Functionally, we found that HEA NPs significantly accelerated scratch-wound closure by both endothelial cells and fibroblasts under oxidative challenge (Supplementary Figs. 44 and 45). Likewise, in tube-formation assays, HEA NPs-treated HUVEC formed denser, more extensive capillary-like networks than H2O2-exposed controls, indicating that cytoskeletal preservation underpins enhanced migratory and angiogenic capacity (Fig. 7h, i).

Beyond cytoprotection, effective resolution of the pro-inflammatory milieu is essential for recovery from bacterial infections, a process critically governed by macrophage plasticity84,85. To assess this, we examined macrophage polarization under lipopolysaccharide (LPS) stimulation. Of note, HEA NPs pretreatment significantly reduced C-C chemokine receptor type 7 (CCR7) expression, a signature marker of M1 macrophages, relative to both LPS alone and Pt treatment (Fig. 7j, k and Supplementary Fig. 46). In parallel, CD163 expression was strongly upregulated (Fig. 7l, m), suggesting a phenotypic shift from a pro-inflammatory M1 phenotype toward a reparative M2 state. Furthermore, RT-qPCR analysis revealed that, compared to Pt NPs, PtFeCuCoNi HEA NPs exerted a stronger inhibitory effect on the mRNA expression of pro-inflammatory cytokines, such as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), while concurrently enhancing genes associated with anti-inflammatory activity, including arginase-1 (Arg-1), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β) (Supplementary Figs. 47 and 48). Together, these results demonstrate that, at neutral pH, PtFeCuCoNi HEA NPs extinguish intracellular ROS, preserve cytoskeletal architecture, sustain angiogenic function, and reprogram macrophages toward tissue repair, thus maintaining redox homeostasis and protecting cells from oxidative injury (Fig. 7n).

To delineate how PtFeCuCoNi HEA NPs restore cellular redox homeostasis at neutral pH, we performed RNA sequencing (RNA-seq) on cells treated with PBS, H2O2, or H2O2 + HEA NPs. Principal component analysis (PCA) separated the three groups, with the co-treatment clustering closer to control than to H2O2 (Fig. 8a), consistent with a partial reversal of oxidative stress-induced transcriptional alterations. Differential expression analysis identified significant upregulation of protective genes, including PFKFB386 (glycolysis) and THBS187 (extracellular matrix remodeling), together with downregulation of stress-associated genes such as SLC7A1165, PSAT188, CHAC1, MTHFD2, and SESN2 (Fig. 8b), suggesting a transcriptional switch from injury signaling to programs supporting metabolic recovery and structural repair. To validate these transcriptomic trends, we further performed RT-qPCR analysis of representative genes involved in glycolytic activation and oxidative stress response. Consistent with the RNA-seq results, PtFeCuCoNi HEA NPs treatment markedly upregulated PFKFB3, THBS1, and HSPA8, while significantly downregulating CHAC1, ASNA, and PSAT compared with both H2O2 and Pt NPs groups (Supplementary Fig. 49). These results confirm that HEA NPs transcriptionally activate glycolysis- and repair-related programs while suppressing stress-associated pathways, thereby supporting the restoration of redox homeostasis and cellular recovery. Hierarchical clustering corroborated a shift toward a homeostatic expression profile after HEA NPs treatment (Fig. 8c).

Furthermore, GO enrichment analysis highlighted the upregulation of terms linked to cell proliferation and angiogenesis, as well as dioxygenase activity (Fig. 8d), suggesting engagement of pro-repair programs. GSEA showed positive enrichment trends for tube development and cytoskeleton organization, alongside significant negative enrichment of apoptosis signaling and a trend toward reduced cellular response to oxygen-containing compounds (Fig. 8e and Supplementary Fig. 50). KEGG pathway analysis further revealed broad attenuation of inflammatory and death-related cascades, including TNF87, IL-1789, NF-kB86, p5388, and MAPK90,91 signaling as well as the ferroptosis and necroptosis pathways, which were all diminished in the HEA NP group relative to H2O2 alone (Fig. 8f). Together, these data support a model in which ROS scavenging by PtFeCuCoNi shifts the transcriptome from injury and inflammatory signaling toward pathways associated with cytoskeletal maintenance, cell proliferation, and vascular development, thereby facilitating the transition from damage to repair (Fig. 8g).

To evaluate pH-gated redox biocatalysis of PtFeCuCoNi HEA NPs in vivo92–94, a murine full-thickness wound model was inoculated with MRSA, with intradermal vancomycin serving as a positive control (Fig. 9a)95. Treatments were administered in two phases to reflect the evolving niche: an initial acid-activated bactericidal window (days 1–3) and a subsequent neutral-pH regenerative phase (days 4–12)96. Digital imaging and planimetric analysis revealed that PBS and H2O2 controls remained inflamed and largely unclosed through day 6, whereas both vancomycin and HEA NPs achieved minimal swelling and near-complete closure by day 12 (Fig. 9b–e and Supplementary Fig. 51)97,98. Consistently, CFU assays on day 3 showed extensive bacterial growth in PBS and H2O2 groups. In contrast, vancomycin and HEA NP treatment yielded few or no colonies, confirming potent antimicrobial activity during the acidic phase (Fig. 9f, g)99,100.

Reflecting the transition from infection control to tissue repair, hematoxylin and eosin (H&E) staining on day 12 revealed persistent neutrophil infiltration and necrosis in controls101, whereas vancomycin- and HEA-treated wounds displayed well-organized epidermal regeneration with minimal inflammation (Fig. 9h). Masson’s trichrome staining showed dense, uniformly aligned collagen only in treated wounds102, in contrast to the sparse, disordered matrices in controls (Fig. 9i–k). Angiogenesis, assessed by α-smooth muscle actin (α-SMA)18, CD31103,104, and vascular endothelial growth factor (VEGF)105 immunostaining, was most pronounced in the HEA group, which exhibited the highest densities of α-SMA+ vessels and CD31+ capillaries together with the strongest VEGF signal (Fig. 9l–o and Supplementary Figs. 52 and 53). Interestingly, the HEA NPs treatment group displayed even better pro-angiogenic activity than that seen in vancomycin-treated wounds, which only offer antibacterial effect, suggesting that PtFeCuCoNi HEA NPs not only eradicate pathogens but also actively stimulate vascular formation, potentially associated with reduced levels of inflammatory factors such as interleukin-1 beta (IL-1β) (Supplementary Fig. 54).

Moreover, peripheral blood analysis on day 12 revealed that neutrophil and white blood cell counts in the HEA NPs-treated rats were comparable to those of healthy controls, indicating attenuation of systemic inflammation. In addition, lymphocyte levels were higher and close to normal, suggesting recovery of immune homeostasis (Supplementary Fig. 55). Furthermore, macrophage dynamics at the wound edge were examined by immunofluorescence on days 3 and 7. Early after infection, HEA-treated wounds recruited significantly more F4/80+ iNOS+ M1-like macrophages than controls, supporting rapid bacterial clearance (Fig. 9p–s and Supplementary Figs. 56 and 57). By day 7, M1 markers decline while F4/80+ CD206+ M2-like macrophages increased, indicating a timely transition to a reparative phenotype that was not observed in untreated lesions106–109. Major-organ histology, hemolysis test, and serum biochemistry showed no adverse effects following HEA NPs administration (Supplementary Figs. 58–60). Taken together, these findings demonstrate that HEA NPs execute a programmed, microenvironment-adaptive therapy: they enable rapid MRSA eradication in the acidic phase and subsequently promote inflammation resolution, angiogenesis, and matrix remodeling as pH normalizes, thereby accelerating wound repair in an antibiotic-independent manner.

Methods
Material and reagents
The iron acetylacetonate (Fe(acac)3), copper nitrate hydrate (Cu(NO3)2·3H2O), cobaltous nitrate hexahydrate (Co(NO3)2·6H2O), nickel nitrate hexahydrate (Ni(NO3)2·6H2O), DMF, EG, H2O2, TMB, DMPO, and TEMP, TBA, BQ, sodium azide (NaN3), potassium superoxide (KO2), NBT and DPA were purchased from Aladdin reagents (Shanghai, China). The platinum bis(acetylacetonate) (Pt(acac)2) was purchased from Energy Chemical (Anhui, China). The titanic sulfate (Ti(SO4)2) was purchased from Macklin Reagents (Shanghai, China).

Synthesis of PtFeCuCoNi HEA NPs
Pt(acac)2 (20 mg), Fe(acac)3 (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 dissolved in a 100 mL Teflon-lined autoclave with DMF (12 mL) and EG (8 mL) mixed solution and ultrasonic for 30 min to make all reactants completely dissolved. The resulting solution was heated at 200 °C for 8 h. The final product was washed and collected via centrifugation (10,000 rpm) with ethanol and acetone. Moreover, Pt metal one was prepared under typical conditions using only the Pt(acac)2 as a metal precursor. The preparation conditions of FeCuCoNi, PtCuCoNi, PtFeCoNi, PtFeCuNi, and PtFeCuCo NPs are similar to those of PtFeCuCoNi HEA NPs except that one of the metal precursors is absent.

In-situ FTIR tests
Operando infrared spectroscopy was recorded on an infrared spectrometer (Thermo Scientific, iS50 FTIR) equipped with an operando spectrum cell (SPECEL-III). To be specific, dissolve 10 mg of the catalyst in a 10 mL mixture solution (210 μL isopropyl, 750 μL H2O, and 40 μL Nafion) to prepare the test sample. Then, adding 40 μL of the above catalyst solution into 5 mL acetic acid buffer (pH = 4.5) solution with 0.5 M H2O2 at room temperature for testing.

Structural characterization
SEM images were obtained by using an Apreo S HiVoc (Thermo Fisher Scientific, FEI). Transmission electron microscopy (TEM) images and mapping were obtained via a Talos F200x TEM microscope (FEI Ltd., USA) operated at 200 kV. Aberration-corrected scan transmission electron microscopy (AC-STEM) characterization (FEI Titan Cubed Themis G2 300) was used for magnified high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). X-ray diffraction (XRD) pattern presented the crystal phase state via a DX-2700BH X-ray diffractometer with Cu radiation at a voltage of 40 kV. XPS spectra were measured using a K-Alpha™+ X-ray Photoelectron Spectrometer System (Thermo Scientific) equipped with a Hemispherical 180° dual-focus analyzer and a 128-channel detector. EPR measurements were carried out using a Bruker EPR EMX Plus spectrometer (Bruker Beijing Science and Technology Ltd, USA) at a frequency of 9.8 GHz (microwave power: 1 mW). Inductively coupled plasma-Mass Spectrometry (ICP-MS) was carried out using an Agilent 7850 (Agilent Technologies, CA, USA). In-situ FTIR measurements were conducted using an infrared spectrometer (Thermo Scientific, iS50 FTIR) equipped with an in-situ spectrum cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-lll). The X-ray absorption spectra (XAS) were collected on the beamline BL07A1 at the NSRRC, and the radiation was monochromatized using a Si (111) double-crystal monochromator. XANES (X-ray absorption near-edge structure) and EXAFS (extended X-ray absorption fine structure) data reduction and analysis were handled via Athena software.

POD-like activity
The PtFeCuCoNi NPs solution (4 mg mL−1, 10 μL), H2O2 (0.1 M, 25 μL), and TMB (10 mg mL−1, 24 μL) were added into a NaOAc-HOAc buffer (100 mM, pH 4.5). The final volume of the mixture was adjusted to 2 mL with NaOAc-HOAc buffer. Then, a portion of the mixture was used for UV-vis spectroscopy measurements at an absorbance of 652 nm.

Steady-state enzyme dynamic studies of POD-like activity
The Michaelis-Menten constant was calculated based on the Michaelis-Menten saturation curve. For each H2O2 concentration, the initial reaction rates (V0) were calculated from the absorbance variation using the Beer-Lambert Law (Eq. 1) (ε = 39000 M−1 cm−1 for oxTMB; c indicates the oxTMB concentration; l cm for the length of the solution in the light path). The reaction rates were then plotted against their corresponding H2O2 concentration and then fitted with the Michaelis-Menten curves (Eq. 2). Furthermore, a linear double-reciprocal plot (Lineweaver-Burk plot, Eq. 3) was used to determine the maximum reaction velocity (Vmax) and Michaelis constant (Km). Furthermore, the turnover number (TON) was calculated according to Eq. (4). The amount of H2O2 was 15 μL, 25 μL, 50 μL, 100 μL, 200 μL (0.1 M), and 40 μL, 60 μL, 100 μL (1 M) respectively.

A = εlc (1)
Steady-state dynamic parameters
v0 = Vmax * [S] / (Km + [S]) (2)
1/v0 = Km / Vmax * 1/[S] + 1/Vmax (3)
TON = Vmax / [E0] (4)
[S] is the concentration of H2O2, and [E0] is the molar concentration of metal in enzyme mimics.

Free radical quenching test
6 μL biocatalysts (4 mg mL−1), 25 μL TMB (10 mg mL−1), 25 μL H2O2 (0.1 M), and appropriate quenching agent (200 μL TBA), 100 μL BQ (10 mg mL−1), and 100 μL NaN3 (10 mg mL−1) were added to NaOAc-HOAc buffer (100 mM, pH 4.5) to reach 2 mL. The whole color rendering process was evaluated by measuring the absorbance of oxTMB at 652 nm (UV-Vis spectroscopy, L6S, INESA, China). Among them, •OH quenched by TBA, •O2− quenched by BQ, and 1O2 quenched by NaN3 in the catalytic oxidation process of TMB.

Detection of 1O2 by 9,10-diphenanthraquinone (DPA)
DPA was a specific probe that could react with 1O2 to produce 9,10-diphenanthraquinone dioxide (DPO2), which tested the absorbance around 378 nm. 25 μL PtFeCuCoNi NPs (4 mg mL−1), 25 μL H2O2 (0.1 M), and 100 μL DPA-Dimethyl sulfoxide (DMSO) solution (1 mg mL−1) were added to 1850 μL NaOAc-HOAc buffer (pH 4.5) to reach 2 mL. Then, the entire spectrum was evaluated by measuring the absorbance at 378 nm (UV-Vis spectroscopy, L6S, INESA, China).

EPR measurements
5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect •O2−. 10 μL of PtFeCuCoNi NPs solutions (10 mg mL−1), 10 μL of DMPO, and 10 μL of 10 M H2O2 were added into 500 μL of DMSO. EPR measurements were carried out via the Bruker EPR EMX Plus (Bruker Beijing Science and Technology Ltd, USA) at a frequency of 9.8 GHz (microwave power: 1 mW).

2,2,6,6-tetramethylpiperidine (TEMP) was used to detect 1O2. 10 μL of PtFeCuCoNi NPs solutions (10 mg mL−1), 10 μL of TEMP, and 10 μL of 10 M H2O2 were added into 500 μL of NaOAc-HOAc buffer (pH 4.5). The EPR was performed using the Bruker EPR EMX Plus, as mentioned above.

•O2− scavenging test
1 mg Potassium superoxide (KO2) was dissolved into 1 mL dimethyl sulfoxide solution (DMSO, containing 3 mg/mL 18-crown-6-ether) to generate and stabilize •O2−. Then, the biocatalysts were dispersed into the above KO2/DMSO solution at a final concentration of 50 μg/mL. After reaction for 5 min, the remnant •O2− will be trapped by nitroblue tetrazolium (NBT)-DMSO solution (10 μL, 10 mg/mL). The absorbance of the solution at 680 nm was measured and then compared with the original concentration of •O2− to ensure the •O2− scavenging ability.

CAT-like test: H2O2 elimination
A total of 10 mM of H2O2 and 50 μg/mL of biocatalyst were mixed in PBS (pH 7.4) to 2 mL. Then, 50 μL of the above solution was mixed with Ti(SO4)2 solution (100 μL, 13.9 mM), and the absorbance value at 405 nm was recorded every 5 min for 30 min.

O2 generation assay
A total of 100 mM of H2O2 and 10 μg/mL of biocatalysts were mixed in PBS (pH = 7.4) to 20 mL, followed by measuring the O2 concentration using a dissolved oxygen meter (INESA, JPSJ-605F) every 5 s until 300 s.

To analyze the biocatalytic kinetics of O2 generation, 10 μg/mL of biocatalysts and different concentrations of H2O2 were mixed in PBS to obtain 20 mL, and then the O2 concentration was measured every 5 s for 100 s. The values of Vmax and Km were calculated based on the Michaelis-Menten saturation curve. The specific calculation formula was referred to in section-Steady-state enzyme dynamic studies of POD-like activity.

Theoretical calculation
All theoretical calculations were performed using the Density functional theory (DFT) method, as implemented in the Vienna ab initio simulation package (VASP)117,118. The core electrons were described using the spin-polarized projector augmented wave (PAW) method119, and the electron exchange and correlation energy was treated within the generalized gradient approximation in the Perdew-Burke-Ernzerhof functional (GGA-PBE)120. The valence states of all atoms were expanded in a plane-wave basis set with a cutoff energy of 450 eV. The convergence criteria for the electronic self-consistent iteration and force were set to 10−5 eV and 0.02 eV/Å, respectively, with a Gamma-centered 1×1×1 K-points. Additionally, Van der Waals interactions were included in all calculations using DFT-D3121,122. The HEA (111) slab was modeled by three layers of slabs. During the HEA structure simulation, the two bottom layers of the substrates were fixed, while the top layer was kept fully relaxed. A slab model was constructed with a vacuum layer of 18 Å in the z-direction to avoid interaction between neighboring images. And during the *O intermediate structures simulation, the three bottom layers of the substrates were fixed, while the rest of the layers were kept fully relaxed. A slab model was constructed with a vacuum layer of 18 Å in the z-direction to avoid interaction between neighboring images. The charge density differences were evaluated using the formula Δρ = ρA+B − ρA − ρB, where ρX is the electron density of X. Atomic charges were computed using the atom-in-molecule (AIM) scheme proposed by Bader123,124.

To explore the catalytic effect, the change of Gibbs free energy (ΔG) was calculated (Eq. 5), which is defined as:
ΔG = ΔE + ΔZPE + ΔH 0→298K − TΔS (5)
where ΔE is the energy change obtained from DFT calculations; ΔZPE, ΔH, and ΔS denote the difference in zero-point energy, enthalpy, and entropy due to the reaction, respectively. The enthalpy and entropy of the ideal gas molecule were obtained from standard thermodynamic tables, and some of the calculation results were analyzed using the VASPKIT package125.

Antibacterial experiments
MRSA (ATCC 43300, Gram-positive) was used as a representative pathogenic bacterium to investigate the bacterial capture and eradication abilities of the PtFeCuCoNi HEA NPs. The bacteriostatic property was studied by MIC values, MBC, the absorbance at 600 nm (OD600), and agar plate counting. Bacteria were incubated separately with a series of final concentrations (2, 4, 6, 8, 16, 32, 64, 96, and 128 μg/mL) of ROS-generation biocatalysts (Pt and PtFeCuCoNi HEA NPs). The system, which contains H2O2 (0.2 mM) and bacteria (106 CFU mL−1), was cultured at 37 °C for 12 h. Then, OD values of the bacterial suspensions treated by the systems at different concentrations (2, 4, 6, 8, 16, 32, 64, 96, and 128 μg/mL) were recorded to calculate the MIC values. The ROS-generation biocatalysts (Pt and PtFeCuCoNi HEA NPs) systems (8 μg/mL) with H2O2 (0.2 mM) were cultured with 1 mL of bacterial suspensions (106 CFU mL−1) for 12 h at 37 °C. Then, the cultured suspensions were diluted 105 times and taken for agar plate counting. The agar plates were cultured for another 12 h at 37 °C and counted to obtain the final colony quantities and calculate the MBC values. The antibacterial properties were estimated by comparing the OD600 of different systems with that of the control. Bacterial protein leakage was quantified using a bicinchoninic acid (BCA) protein assay kit (Solarbio). Briefly, MRSA were incubated with PBS, H2O2, Pt+H2O2, PtFeCuCoNi+H2O2 under the indicated conditions. After incubation, supernatants were collected by centrifugation, and protein content was determined following the manufacturer’s protocol. A standard curve was established using bovine serum albumin (BSA, 0–0.5 mg/mL), and the corresponding optical density at 562 nm (OD562) was recorded. The calibration equation (y = 1.698x + 0.166, R2 = 0.992) was used to convert OD values into absolute protein concentrations. Representative images of the gradient color reaction were also obtained. All measurements were performed in triplicate.

Extraction and Gel Electrophoresis of EPS from MRSA Biofilms
Biofilm Culture and Treatment: 10 mL of MRSA suspension (OD600 ≈ 0.05) was incubated at 37 °C for 24 h to form biofilms. The biofilms were then treated with PBS (control), H2O2, PtNPs+H2O2, or PtFeCuCoNiNPs+H2O2 for 6 h. EPS Extraction: After treatment, the biofilms were scraped and centrifuged to remove bacterial cells. Agarose Gel Electrophoresis: 20 µL EPS was mixed with 4 µL loading buffer, heated at 95 °C for 5 min, and loaded onto a 1% agarose gel. Electrophoresis was carried out at 100 V for 60 min. Staining and Imaging: The gel was stained with SYBR Green I for 30 min in the dark, and images were captured using a fluorescence imaging system. Degradation effects were assessed by comparing band migration and fluorescence intensity.

EPS nucleic acid electrophoresis
MRSA biofilms were treated with PBS (control), H2O2, PtNPs+H2O2, or PtFeCuCoNi NPs+H2O2 for 6 h. EPS was extracted by centrifuging scraped biofilms at 12,000 × g for 20 min. The supernatant was mixed with DNA loading buffer, heated at 95 °C for 5 min, and analyzed on a 1% agarose gel. Nucleic acids were visualized using SYBR Green I. DNase I treatment confirmed nucleic acid identity.

Observation of bacterial morphology
The bactericidal performance of these systems was visualized via SEM, TEM, and fluorescence microscopy. After treating different systems, the bacterial suspensions were fixed with a 2.5% glutaraldehyde-containing PBS solution for 12 h at 4 °C and dehydrated using a gradient of ethanol/water. Then, SEM and TEM images were obtained to assess the bacterial capture ability and morphology.

Crystal violet staining of bacterial biofilms
The treated biofilms from each group were washed three times with PBS and then fixed with 2.5% glutaraldehyde for 20 min. Then the liquid in each well was discarded. Crystal violet test solution (0.5%) was transferred into each well for 30 min, and excess dye was removed with PBS. Furthermore, the coverslips were allowed to dry. Next, absolute ethyl alcohol was used to dissolve the crystal violet bound to the biofilm. Finally, the value of Optical Density (OD) 570 nm of the dissolved solution was examined by a microplate reader (SAF-6801, Bajiu Corporation, Shanghai, China).

Live/Dead and LPO staining
Regarding MRSA biofilm culturing, the MRSA suspension (100 μL, 108 CFU mL−1) and lysogeny broth medium (LB, 100 μL) were placed in ibidi 8-well slides, and then they were cultured at 37 °C. After 24 h, the medium was removed, and the unattached bacteria were gently washed away with sterile PBS three times. The resulting biofilm on the ibidi 8-well slides was then harvested. Moreover, the killing behaviors of different samples were stained by Live/Dead BacLight Viability Kits (SYTO-9 for live cells and PI for dead cells) for observation using CLSM (St5, Leica). The LPO was detected by C11-BODIPY581/591 (Thermo Fisher, D3861). In general, pure medium, Pt, and PtFeCuCoNi HEA NPs were co-cultured with MRSA for 12 h, respectively. After washing, MRSA was stained by C11-BODIPY581/591 solution at a concentration of 5 mM for 20 min. Then, the CLSM (St5, Leica) was used to obtain the images. EPS were detected by Dextran Alexa Flour 647 (Thermo Fisher, D22914). In general, pure medium, H2O2, Pt, and PtFeCuCoNi HEA NPs were co-incubated with MRSA for 12 h. After washing, MRSA was stained with Dextran Alexa Flour 647 solution at a concentration of 10 mM for 30 min. A CLSM (St5, Leica) was then used to obtain images.

Transcriptome sequencing of MRSA and data analysis
For RNA-seq analysis, MRSA was co-cultured with PBS, PtFeCuCoNi HEA NPs+H2O2 at 37 °C for 3 h. After different treatments, bacteria were collected for RNA-seq analysis by Shanghai Personalbio Co., Ltd on the Illumina platform. GO (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genome (http://www.genome.jp/kegg/) were used to analyze the gene functions. Differential gene expression analysis was performed using the R package, and those genes conformed to |log Foldchange| > 1 (P-value < 0.05) were considered to be DEGs.

Cell culture
HUVEC and Raw264.7 were purchased from the American Type Culture Collection (ATCC). HUVEC were cultured in the medium of Dulbecco’s Modified Eagle Medium (DMEM, 6123045, Gibco) containing 10% fetal bovine serum (FBS), and 1% antibiotics mixture (10,000 U penicillin and 10 mg streptomycin) at 37 °C under 5% CO2. Raw264.7 were cultured in high-glucose DMEM supplemented with 10% FBS at 37 °C in a humidified incubator with 5% CO2. RSF were isolated from the skin of the Sprague-Dawley (SD) rats. Briefly, Primary dermal fibroblasts were isolated from the dorsal skin of SD rats by enzymatic digestion, in which excised dermal tissue was minced and digested with collagenase type I, followed by filtration and culture in α-MEM supplemented with 10% FBS. The collected cells were cultured in α-MEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. After 24 h of culture, the culture medium was replaced with fresh complete α-MEM medium to remove the nonadherent cells. The adherent cells were cultured for another 3–5 days and passaged until reaching 90% confluence.

Cellular protection assay
Cells were pretreated with the corresponding biocatalyst (2 µg mL−1) for 24 h and then co-incubated with 200 µM H2O2 for 2 h. Cells without any treatment were regarded as the Control group. Cells were treated with 200 μM H2O2, and those without biocatalyst were regarded as the ROS Condition group. After stimulation, the cell viability rate was detected with Cell Counting Kit-8, and the Live/Dead ratio was determined by Calcein acetoxymethyl ester/Propidium Iodide (Calcein AM/PI) Kit.

After stimulation, cell morphology was detected via cytoskeletal staining. Cells were seeded at low density and treated before fixation with 4% paraformaldehyde (#BL539A, Biosharp, China) for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 (#T8200, Solarbio, China). Rhodamine phalloidin (#A12379, Invitrogen, China) was used for F-actin (red) staining for 30 min, and DAPI (#P0131, Beyotime, China) was used for DNA (blue) staining for 5 min. Fluorescence photographs were taken using a confocal laser endomicroscope (FV2000, Olympus, Japan). After stimulation, DCFH-DA (S0033S, Beyotime, China) was used to detect the residual ROS level, according to manufacturers’ instructions. Briefly, probes were preloaded by incubating cells with the DCFH-DA working solution for 30 min. After washing with PBS three times, the intracellular ROS level was determined through a CLSM (FV3000 and Nikon, A1R MP+, Olympus, Japan). After stimulation, Calcein AM/PI staining was performed according to the instructions (C2012, Beyotime, China). Briefly, 2 μM Calcein AM solutions in PBS were used to stain live cells, and then 4.5 μM PI solutions were used to stain the dead. The cells were captured by a CLSM and counted by the Celigo Image Cytometer (Nexcelom Bioscience LCC., America).

Cell wound scratch assay
Cells were seeded in 6-well plates and treated as before. Draw a line vertically with the 200-microliter gun tip over the ruler. The cells were washed three times with PBS, photographed under the microscope (FV2000, Olympus, Japan), and then placed at 37 °C in 5% CO2. HUVEC and RSF were photographed at 12 and 24 h to observe the healing. The wound closure rates were accounted for via the ImageJ program (Media Cybernetics, Rockville, USA).

Matrigel tube formation
The possibility of tube formation of HUVEC was tested using Matrigel (ABW, 082706, China) according to the manufacturer’s instructions. Briefly, the matrix gel was thawed overnight at 4 °C, and 50 μL was added to each well of a 96-well plate. The petri dish with Matrigel was incubated at 37 °C for 30 min to allow the Matrigel to solidify. The cells were trypsinized and inoculated into Matrigel at 1×105 cells/cm2 for 4 h. Then, observed under an automatic inverted microscope. The vascular network was analyzed by the ImageJ angiogenesis analyzer.

Macrophage polarization
Raw264.7 cells were seeded at a density of 5×104/well and processed as above. Primary antibodies: anti-CCR7 (ab-196640, abcam, USA, 1:400 dilution) and anti-CD163 (ab-182422, abcam, USA, 1:400 dilution). Secondary antibodies: Fluor 647 donkey anti-rabbit (A31573, Life Tech., 1:500 dilution).

Transcriptome sequencing and data analysis
HUVEC (5×106 cells mL−1) were cultured in 100 mm dishes and were treated with different samples for 24 h. Then, the cells were cultured overnight in a standard culture medium. After being treated with different samples, the HUVEC were lysed by TRIzolTM reagent (15596026, Invitrogen, CA, USA), and cell lysates were stored at −80 °C before sequencing. RNA concentration and purity were measured using NanoDrop 2000. The integrity of RNA samples was assessed by the DNF-471 kit, and the system was an Agilent 5300. The libraries were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads, according to the manufacturer’s instructions. DEGs were defined by DESeq2/edgeR with thresholds of |log FC| ≥ 1 and FDR < 0.05. The PCA, Volcano plot, Heatmap, KEGG pathway enrichment, Chordal plots of KEGG, GO term enrichment, and GSEA pathway enrichment analyses were performed using the online platform Majorbio (www.majorbio.com).

Real-time quantitative polymerase chain reaction (RT-qPCR)
HUVEC were pre-incubated with the corresponding biocatalyst (2 µg mL−1) for 24 h, followed by co-incubation with 200 µM H₂O₂ for an additional 3 h. After stimulation, total mRNA was extracted from HUVEC, and the expression levels of PFKFB3, THBS1, HSPA8, CHAC1, ASNS, and PSAT1 were determined by RT-qPCR. Similarly, Raw264.7 cells were pre-incubated with the corresponding biocatalyst (1 µg mL−1) for 24 h, and then followed by co-incubation with 500 µg/mL lipopolysaccharide (LPS) for an additional 24 h. After stimulation, total mRNA was extracted from Raw264.7 cells, and the expression levels of iNOS, IL-6, TNF-α, Arg-1, IL-10, and TGF-β were determined by RT-qPCR. The primer sequences are listed in Supplementary Table 3.

Hemolysis test
The hemolysis test was performed on the red blood cells (RBCs) of the rat according to the reported method. The 2 mL of blood was collected from the rat, and RBCs were immediately washed with PBS and centrifuged at 5000 rpm. After washing, 1 mL of RBCs was dissolved in 25 mL of PBS. Then 300 µL of RBC was added separately to a vial containing 8, 16, 32, 64 µL of PtFeCuCoNi HEA NPs dispersed in PBS. The positive and negative controls were prepared by dispersing RBCs in 0.1% Triton X-100 and PBS, respectively. After 1 h of incubation at 37 °C, samples are centrifuged at 5000 rpm, and 200 µL of the supernatant is collected for absorbance measurement at 570 nm. The percentage of hemolysis is calculated as follows: P = (AS−AN)/(AP−AN)×100%. Where AS is the absorbance resulting from the mixture of NPs and RBCs, AN and AP represent the absorbance of negative and positive controls, respectively.

In vivo wound disinfection experiments
Rats were selected as models in animal experiments. The random assignment was conducted under conditions where there was minimal variation in the health status of all samples/organisms. The protocols of all the animal experiments were permitted and carried out as requested. After being anesthetized with 3% sodium pentobarbital, a hole around 1 cm in diameter in the epidermis was created. Then 200 μL of MRSA suspension (1×108 CFU mL−1) was dropped onto the wound and incubated for 1 day. After that, 200 μL of liquid containing PtFeCuCoNi HEA NPs (8 μg/mL) and H2O2 (0.2 mM) was added to treat the infected wound. Normal saline, H2O2 (0.2 mM), and vancomycin (80 μg/mL) were also used as contrasts. After 12 days, the treated wounds were cut off and fixed with 10% formaldehyde solution for histological H&E, Masson trichrome (Solarbio, G1015).

Immunofluorescence staining
Skin samples from SD rats were fixed with 4% buffered paraformaldehyde overnight. After dehydration, samples were embedded in paraffin and sectioned. The paraffin sections were dewaxed in xylene and then rehydrated with a gradient of alcohols. After the slides were immersed in antigen retrieval solution for 30 min at 95 °C, they were washed with PBS twice and fixed in 4% paraformaldehyde for 10 min at room temperature, then permeabilized in PBS containing 0.1% Triton X-100 for 10 min and blocked in PBS with 1% bovine serum albumin (BSA) for 1 h. Primary antibodies were diluted in PBS with 1% BSA and incubated overnight at 4 °C. Then, the cells were incubated with secondary antibodies with the required fluorophore in the dark at room temperature for 2 h. Before imaging, cells were counterstained with 10 μg mL−1 DAPI and mounted in the antifading mounting medium (S2110, Solarbio, China). Samples were washed with PBS three times between each step. A CLSM (FV3000 and Nikon, A1R MP+, Olympus, Japan) was used to capture images.

Primary antibodies and corresponding concentrations used in this study were α-SMA (ab5694, abcam, USA, 1:100 dilution), CD31 (ab24590, abcam, USA, 1:100 dilution), iNOS (sc-7271, Santa Cruz Biotechnology, USA, 1:200 dilution), CD206 (sc-58986, Santa Cruz Biotechnology, USA, 1:200 dilution), F4/80 (sc-25830, Santa Cruz Biotechnology, USA, 1:100 dilution), IL-1β (sc-52012, Santa Cruz Biotechnology, USA, 1:200 dilution), TNF-α (sc-52746, Santa Cruz Biotechnology, USA, 1:200 dilution), VEGF (ab46154, abcam, USA, 1:200 dilution). The secondary antibodies were Alexa Fluor 647 goat anti-mouse IgG (ab150115, Abcam, USA, 1:200 dilution), Alexa Fluor 647 donkey anti-rabbit IgG (ab150075, Abcam, USA, 1:200 dilution) and Alexa Fluor 488 goat anti-rabbit IgG (ab150077, Abcam, USA, 1:200 dilution), Alexa Fluor 555 goat anti-mouse IgG (A21422, Invitrogen, Thermo Fisher Scientific, USA, 1:200 dilution).

In vivo toxicity assessment
To assess the systemic toxicological profile of synthetic biocatalytic constructs, comprehensive histopathological analyses were conducted on SD rat models following a 2-week post-operative period. Major organ systems, specifically the heart, liver, spleen, lung, and kidneys, were harvested and subjected to H&E staining protocols for microscopic evaluation of tissue architecture and cellular morphology.

Statistical analysis
ImageJ (version 1.8.0) and Image-Pro Plus (version 6.0) were used for in vitro and in vivo imaging analysis. Data were analyzed using GraphPad Prism 10.0 software (GraphPad Prism, San Diego, California, USA). Figures were formed using Origin 2022. Sample size (n), probability (p) value, data normalization, and specific statistical tests for each experiment were clarified in the figure legends. All of the data were presented as the mean ± SD from a minimum of three independent experiments. The comparison of mean values across multiple groups was performed using one-way ANOVA followed by Tukey’s post-hoc test; all tests were two-sided. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ns represents a non-significant difference. A value of p < 0.05 was considered significant.

Ethical statement
Animal experiments and procedures, including euthanasia, were conducted following protocols approved by the Institutional Animal Care and Use Committee at Sichuan University (Number: WCHSIRB-AT-2025-513). Additionally, the Laboratory Animal Welfare and Ethics Committee of West China Hospital of Stomatology reviewed and approved the study. All experiments involving animal use were performed in accordance with the ARRIVE guidelines.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript introduces an interesting high-entropy alloy (PtFeCuCoNi)-based Janus artificial enzymes with pH-responsive dual enzymatic activities for sequential treatment of drug-resistant bacterial infections and chronic inflammatory wounds. Under acidic conditions, the biocatalytic materials demonstrates potent POD-like activity, effectively eradicating biofilms and killing bacteria. At neutral pH, it switches to antioxidant (SOD/CAT-like) functions, alleviating oxidative stress and promoting tissue regeneration. Through comprehensive in vitro and in vivo validation, the authors show that the Janus artificial enzymes not only eliminates MRSA and biofilms at ultralow concentrations but also reprograms macrophage polarization and enhances angiogenesis, offering a promising antibiotic-free strategy for managing complex wound microenvironments. Compared with earlier works, this study shows a new and interesting biocatalytic pathway to treat infected wounds, the data are sufficient and can fully validate the concepts, which offers a promising antibiotic-free strategy for managing complex wound microenvironments and is recommended for publication after addressing the following points.\n1. Since the high-entropy alloys (PtFeCuCoNi) are based on different elements, I am wondering how the precursor ratios affect HEA NP crystal structure and enzyme-like activity. This will validate the optimality and improve the reproducibility of the synthesis protocol. Has the authors tried with different ratios? Maybe more discussion is needed in this part.\n2. Will these materials also show SOD- and CAT- activities under acidic conditions? It is suggested to supplement more information on these catalytic activities to further verify the precision and response thresholds of pH gating effects.\n3. The description on page 9, first paragraph, does not correspond to Figure 3f and 3g.\n4. The catalytic activity and stability of HEA NPs over multiple reaction cycles require further investigation.\n5. The writing could be improved. For example:\nThe horizontal axis of Figure 7b.\nThe p(H2O2) values in Figures 7 and 9.\nThe large or lowercase of first letter of the references title should be unified.\n6. In Supplementary Fig. 38, the authors provided the detection data of the inflammatory factor IL-1β at the skin defect site on day 10, which supports the claim that the PtFeCuCoNi HEA NPs reduces tissue inflammation during the late stage of infection. However, data on systemic inflammation are lacking and should be supplemented with relevant experiments.\n7. The antimicrobial section is comprehensive, addressing both planktonic and biofilm MRSA. However, the opening paragraph of the Discussion could be further strengthened. The statement “Infections caused by antibiotic-resistant pathogens represent an escalating clinical challenge due to antibiotic treatment failure, compounded by complex inflammation and dynamic microenvironmental alterations.” would be more compelling if expanded with relevant examples of severe infections caused by antibiotic-resistant bacteria (e.g., endocarditis, intra-abdominal infections, or bacteremia). This would situate the work within the broader clinical landscape.", "reviewer_2": "This manuscript reports the design of bioinspired Janus artificial enzymes based on high-entropy alloys (PtFeCuCoNi) for sequential therapy of drug-resistant bacterial infections and inflammatory wounds. The study is well-motivated, drawing on the biphasic microenvironment of chronic wounds, and demonstrates that the proposed HEA biocatalysts can achieve pH-dependent switching between oxidase/peroxidase-like ROS generation under acidic conditions and antioxidase-like scavenging at neutral pH. The combination of theoretical calculations and experimental analyses provides a coherent picture of the catalytic mechanism, and the in vivo data indicate antimicrobial efficacy against MRSA as well as immunomodulatory and pro-regenerative effects. The concept of exploiting multi-metal synergy in HEAs for redox modulation is novel and of potential interest for the field of nanomedicine. The manuscript is scientifically sound and is suitable for publication in Nature Communications. I therefore recommend its rapid publication after the following minor revisions are addressed:\n(1) In the Supplementary Methods, it was mentioned that “Raman spectra were examined via the HORIBA HR Evolution Raman spectrometer with a 532 nm laser source”, but this characterization technique was not included in the main manuscript. Is it a mistake?\n(2) The synthesis methods for the mentioned FeCuCoNi, PtCuCoNi, PtFeCoNi, PtFeCuNi, and PtFeCuCo alloys are not provided in the manuscript, which should be supplemented.\n(3) While the study highlights the advantages of HEA materials, the current text provides limited discussion of prior biomedical research on HEAs. The authors are encouraged to expand the Introduction or Discussion with additional references and a more explicit consideration of potential toxicity concerns associated with high-entropy alloys.\n(4) In Supplementary Fig. 28, the cytocompatibility of PtFeCuCoNi HEA NPs is presented. However, as Pt is a key constituent metal, the cytotoxicity of Pt NPs alone should also be discussed, which would strengthen the argument that the alloy formulation indeed reduces toxicity relative to single-metal systems.\n(5) In Fig. 6, the transcriptional responses provide valuable mechanistic insight. However, the current text would benefit from the inclusion of supporting references for some of the highlighted pathways. Including key citations would help place these findings within the broader context of bacterial stress responses.", "reviewer_3": "This manuscript describes the development of high-entropy alloy (HEA) (PtFeCuCoNi) as bioinspired nanocatalysts for the treatment of infected and non-healing wounds. The work addresses an important clinical challenge by targeting both the antimicrobial and tissue-regenerative phases of wound healing. The authors provide convincing evidence that the HEA nanozymes exhibit robust catalytic adaptability, functioning as ROS-generating oxidase/peroxidase mimics in acidic environments while switching to ROS-scavenging antioxidase activity at neutral pH. The integration of transcriptomic profiling, mechanistic studies, and in vivo validation provides strong support for the dual regulatory role of these materials in modulating oxidative stress and inflammation. The concept of tailoring redox activity through entropy-driven alloy design is innovative and of clear significance to the biomedical nanomaterials community. It is suggested that this study is well executed and suitable for publication in Nature Communications, pending minor revisions to improve clarity and contextual discussion.\n1. The mechanism for the synthesis of the PtFeCuCoNi HEA NPs via the hydrothermal method should be discussed in the main manuscript.\n2. Some earlier references regarding the HEA materials for antibacterial studies should be carefully compared and discussed included in the manuscript.\n3. The contents of the elements measured by XPS and ICP in the manuscript are inconsistent. Please check the data and explain the difference.\n4. In Fig. 6f, an interesting mechanistic illustration is presented; however, the results differ from previous sequencing studies on active bactericidal processes, where a decrease in ATP activity was observed. The authors are encouraged to further explain the reasons for this discrepancy and provide relevant literature support.\n5. Fig. 7n presents a schematic illustration of cellular effects under high-concentration H2O2 treatment and the protective role of HEA NPs. However, the labeling of the cell types is missing, making it unclear which three cells are being represented.\n6. Fig. 8d and Fig. 8f present GO and KEGG analyses, respectively. It is noted that Fig. 8d compares PtFeCuCoNi vs H2O2, while Fig. 8f shows the opposite comparison, H2O2 vs PtFeCuCoNi. As the rationale for this discrepancy has not been explained in the manuscript, the authors are advised to provide further discussion.\n7. Supplementary Figs. 31 and 32 show scratch assays of HUVECs and RSFs, respectively. However, unlike Figs. 7a and 7c, the scratch assay for RAW264.7 cells is missing. The authors are advised to either provide the corresponding experiment or explain the reason for its absence.", "reviewer_4": "Gao et al. designed a high-entropy alloy-based Janus artificial enzyme targeting chronic wound infections. This enzyme leverages the unique microenvironment characteristics at the infection site to catalyze antimicrobial and anti-inflammatory actions, demonstrating excellent antimicrobial efficacy both in vitro and in vivo. Additionally, the study found that it can promote the PFKFB3 pathway, inhibit the expression of inflammatory cytokines, and accelerate wound healing. The results are very interesting; however, the manuscript still has some issues to address. It is recommended that the authors make further additions and revisions.\n1. Through TEM and SEM observations, the Janus artificial enzyme exhibits a nanostructure. The authors should further supplement the DLS and material stability data of the artificial enzyme, particularly regarding its bioavailability. It is also recommended that the authors include stability data of the artificial enzyme under different environmental conditions.\n2. The HEA nanoenzyme demonstrated different catalytic activities under acidic and neutral pH conditions, which is very interesting and differs from other reported HEA-based artificial enzymes. However, the underlying mechanism is not sufficiently discussed. Strengthening this part would improve the manuscript.\n2. Fig. 5C is used to detect bacterial protein leakage, typically assessed by the BCA assay. However, the manuscript does not provide a detailed description of this method. Given the limited bacterial protein content, the detection results showed a deep purple color and lacked a standard curve. It is recommended that the authors provide further clarification on this issue.\n3. The authors demonstrated the reduction of EPS through SEM characterization, but this claim has certain limitations. It is recommended that the authors include a biofilm EPS gel electrophoresis experiment to further validate this result.\n4. The authors demonstrated lipid peroxidation in MRSA after treatment with the artificial enzyme by the increase in LPO. It is recommended that the authors include MDA detection at the bacterial level to further confirm this phenomenon.\n5. The antibacterial mechanism of the artificial enzyme primarily involves the generation of ROS. However, the manuscript lacks relevant experiments at the bacterial level. It is recommended that the authors include ROS generation at the bacterial level after labeling with the DCFH-DA probe.\n6. At the cellular level, the artificial enzyme exhibits anti-inflammatory effects. The authors confirmed its ROS scavenging ability using the DCFH-DA probe; however, ROS scavenging is only one step in the anti-inflammatory process. It is recommended that the authors supplement the study with the polarization trends of M1 and M2 macrophages at the cellular level and concurrently assess the release of inflammatory cytokines TNF-α, IL-6, and IL-10 at the cellular level.\n7. The authors indicate that the artificial enzyme can activate the PFKFB3 pathway at the transcriptomic level. However, the authenticity of the activation results based solely on transcriptomic data remains to be further verified. It is recommended that the authors supplement the study with RT-qPCR testing of relevant factors to further confirm these results."}, "author_response": "Point-by-point response to the detailed comments by reviewers of “H igh-Entropy Alloy-based Ja nus Artificial Enzymes with pH-Gated Redox Biocatalysis fo r Sequential Therapy of Drug-Re sistant Bacteria and Inflammatory Wounds”\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\n“This manuscript introduces an interesting high-entropy alloy (PtFeCuCoNi)-based Janus artificial\nenzymes with pH-responsive dual enzymatic activities for sequential treatment of drug-resistant\nbacterial infections and chronic inflammatory wounds. Under acidic conditions, the biocatalytic\nmaterials demonstrates potent POD-like activity, effectively eradicating biofilms and killing bacteria.\nAt neutral pH, it switches to antioxidant (SOD/CAT-like) functions, alleviating oxidative stress and\npromoting tissue regeneration. Through comprehensive in vitro and in vivo validation, the authors\nshow that the Janus artificial enzymes not only eliminates MRSA and biofilms at ultralow\nconcentrations but also reprograms macrophage polarization and enhances angiogenesis, offering a\npromising antibiotic-free strategy for managing complex wound microenvironments. Compared with\nearlier works, this study shows a new and interesting biocatalytic pathway to treat infected wounds,\nthe data are sufficient and can fully validate the concepts, which offers a promising antibiotic-free\nstrategy for managing complex wound microenvironments and is recommended for publication after\naddressing the following points.”\nResponse to the general comment:\nWe sincerely thank you for recognizing the novelty and translational potential of our pH-\nresponsive, ROS-catalytic high-entropy alloy nanozymes. In response to the reviewers’ constructive\ncomments, we have carried out additional experiments, refined data interpretation, and thoroughly\nrevised the manuscript and Supplementary Information to address all concerns. New data and\nanalyses have been incorporated where necessary to reinforce our conclusions. We believe that these\nsubstantial revisions have further strengthened the rigor, clarity, and overall impact of our work. We\ngreatly appreciate your valuable time and insightful feedback, which have been instrumental in\nimproving the quality of our study.\n(1) Since the high-entropy alloys (PtFeCuCoNi) are based on different elements, I am wondering\nhow the precursor ratios affect HEA NP crystal structure and enzyme-like activity. This will validate\nthe optimality and improve the reproducibility of the synthesis protocol. Has the authors tried with\ndifferent ratios? Maybe more discussion is needed in this part.\nResponse to comment:\nThank you for your valuable comments. We acknowledge the limited data in our research on\nhow precursor ratios affect the HEA NP crystal structure and enzyme-like activity. In response to\nyour feedback, we have prepared HEA NPs with different precursor ratios (Pt FeCuCoNi,\n2 2 2 2\nenzymatic catalytic properties. It was found that the HEA synthesized with an equimolar precursor\nratio exhibited the strongest XRD diffraction peaks and the optimal enzyme-mimicking performance.\nWe appreciate your thoughtful input, which has significantly informed our work. The corresponding\ndetails can be found in the revised manuscript and revised supplementary information, as also shown\nbelow:\nPage 6 in the revised manuscript: “We also investigated the effect of precursor ratio on the\ncrystal structure of the HEA NPs (Supplementary Fig. 3). It was observed that the PtFeCuCoNi HEA\nNPs formed from an equimolar precursor ratio exhibited the most intense XRD diffraction peaks.\nThis is attributed to the near-random distribution of the five constituent elements among the fcc\nlattice sites, which allows the crystal to maintain superior periodicity and thereby generate strong\nXRD diffraction peaks. Conversely, when the proportion of any single element was doubled, the\nexcessive metal atoms introduced more intense and localized lattice expansion and strain, thereby\ndisrupting the periodic arrangement of the crystal lattice and ultimately leading to a significant\nreduction in XRD diffraction peak intensity33.”\nSupplementary Fig. 3 X-ray diffraction (XRD) of HEA NPs with different precursor ratios.\nReferences:\nSci. Adv. 9, eadf9931 (2023).\nPage 10 in the revised manuscript: “It was found that the HEA synthesized with an equimolar\nprecursor ratio exhibited the optimal enzyme-mimicking performance (Supplementary Fig. 14). This\nis attributed to the optimal d-band center and lattice structure of the equimolar PtFeCuCoNi HEA\nNPs, which strikes an optimal balance between the adsorption and desorption of reactants39.”\nSupplementary Fig. 13 a POD-like activity, b Time-dependent CAT-like activity via TiSO -based UV-vis\n4\nspectra in the presence of biocatalytic materials and H O , and c SOD-like activity of HEA NPs with different\n2 2\nprecursor ratios (n=3 independent experiments, data are presented as mean values ± SD). Source data are\nprovided as a Source Data file.\nReferences:\ndriven discovery. Science 376, eabn3103 (2022).\n(2) Will these materials also show SOD- and CAT- activities under acidic conditions? It is suggested\nto supplement more information on these catalytic activities to further verify the precision and\nresponse thresholds of pH gating effects.\nResponse to comment:\nThank you for your valuable and constructive comments for enhancing the quality of our\nmanuscript. In response to your suggestions, we supplemented the CAT performance under acidic\nconditions. However, because SOD was tested with DMSO solvent and its pH could not be\nmodulated, we used CAT to assess its ROS-scavenging ability under acidic conditions. Your\nfeedback has greatly strengthened our paper, and we appreciate your thoughtful review. The\ncorresponding details can be found in the revised manuscript and revised supplementary information,\nas shown below:\nPage 10 in the revised manuscript: “Importantly, PtFeCuCoNi HEA NPs exhibited pH-dependent\nROS-production and ROS-scavenging activities, with a predominance of ROS production in acidic\nenvironments and a predominance of ROS scavenging in neutral conditions, confirming its excellent\nmicroenvironmental adaptability (Supplementary Fig. 15).”\nSupplementary Fig. 15 PtFeCuCoNi NPs show a and b peroxidases (POD)-mimetic and c catalase\n(CAT) activities in a pH-dependent manner (n = 3 independent experiments, data are presented as\nmean ± SD). a.u. indicates the arbitrary units. Source data are provided as a Source Data file.\n(3) The description on page 9, first paragraph, does not correspond to Figure 3f and 3g.\nResponse to comment:\nWe appreciate the reviewer’s insightful comment. Your careful examination is greatly\nappreciated, and the updated corrections are included in the revised manuscript, as also illustrated\nbelow:\nPage 10 in the revised manuscript: “Subsequently, the 1O species can be detected by the 9,10-\n2\ndiphenanthraquinone (DPA) in a time-dependent manner (Fig. 3f). In addition, adopting 5,5-\ndimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) as the specific\nspin trap reagents, respectively, the electron paramagnetic resonance (EPR) detection also confirms\nthat the major ROS in HEA NPs is •O − and 1O (Fig. 3g and Supplementary Fig. 9).”\n2 2\n(4) The catalytic activity and stability of HEA NPs over multiple reaction cycles require further\ninvestigation.\nResponse to comment:\nThank you for your good comments and helpful suggestions. We have supplemented the\ncatalytic activity and stability of HEA NPs over multiple reaction cycles. The corresponding\nsupporting literature has been added in the revised manuscript, as also shown below:\nPage 10 in the revised manuscript: “The long-term activity and stability of PtFeCuCoNi HEA\nNPs as a CAT-like antioxidant have also been evaluated, revealing consistent maintenance of high\nactivity with no observable decline after five cycles of testing (Supplementary Fig. 13).”\nSupplementary Fig. 13 Cyclic stability test of H O decomposition of PtFeCuCoNi HEA NPs.\n2 2\n(5) The writing could be improved. For example: The horizontal axis of Figure 7b. The p(H2O2)\nvalues in Figures 7 and 9.The large or lowercase of first letter of the references title should be unified.\nResponse to comment:\nThank you for your valuable comments. In response, we have further refined the text throughout\nthe manuscript, including standardizing the horizontal axis label in Figure 7b, clarifying the p(H O )\n2 2\nannotations in Figure 9 with explanations and relevant references, and unifying the capitalization of\nthe first letters in reference titles to ensure consistent formatting and clear presentation.\n1) The horizontal axis label in Figure 7b has been corrected and standardized.\nFig. 7 b3 Quantitative analysis of DCFH-DA fluorescence across various cell types subjected to\ndistinct treatments.\n2) The p values in Figures 7 and 9 have been clarified with appropriate annotations and\n(H2O2)\nexplanatory notes in the figure captions. The entire content following “p” is already presented in\nsubscript format, and the number “2” in “ ” cannot be further subscripted due to typographical\nH2O2\nconstraints. This presentation is consistent with conventions used in prior publications (Nat.\nCommun., 2024, 15, 9592; Nat. Commun., 2025, 16, 856).\n3) The large or lowercase of the first letter of the reference title has been unified. We have identified\nthe first letter of the title of the reference as larger and the rest as lowercase.\n(6) In Supplementary Fig. 38, the authors provided the detection data of the inflammatory factor IL-\n1β at the skin defect site on day 10, which supports the claim that the PtFeCuCoNi HEA NPs reduce\ntissue inflammation during the late stage of infection. However, data on systemic inflammation are\nlacking and should be supplemented with relevant experiments.\nResponse to comment:\nWe sincerely thank the reviewer for this insightful comment. We fully agree that assessment of\nsystemic inflammatory responses is critical for comprehensively evaluating the therapeutic efficacy\nof the material. Accordingly, we have supplemented the revised manuscript with peripheral blood\nanalyses from rats at day 12 post-treatment. The results show that neutrophil (NEU) and white blood\ncells (WBC) counts in the HEA NP-treated group were comparable to those in healthy controls,\nindicating substantial attenuation of systemic inflammation. In addition, the lymphocyte (LYM)\npercentage was higher and approached normal levels, suggesting recovery of immune function and\nrestoration of immune homeostasis. These findings further corroborate that PtFeCuCoNi HEA NPs\neffectively alleviate both local and systemic inflammatory responses during infection resolution\n(Supplementary Fig. 55).\nPage 29 in the revised manuscript: “Moreover, peripheral blood analysis on day 12 revealed that\nneutrophil and white blood cell counts in the HEA NPs-treated rats were comparable to those of\nhealthy controls, indicating attenuation of systemic inflammation. In addition, lymphocyte levels\nwere higher and close to normal, suggesting recovery of immune homeostasis (Supplementary Fig.\n55).”\nSupplementary Fig. 55 The number of WBC (White Blood Cell), NEU (Neutrophil), and the\npercent of LYM (Lymphocyte) in the peripheral blood of rats on day 12. n = 3 independent replicates.\nData are presented as means ± SD, ns, not significant, *p < 0.05, **p < 0.01; one-way ANOVA with\nmultiple comparisons test, all tests were two-sided. Source data are provided as a Source Data file.\n(7) The antimicrobial section is comprehensive, addressing both planktonic and biofilm MRSA.\nHowever, the opening paragraph of the Discussion could be further strengthened. The statement\n“Infections caused by antibiotic-resistant pathogens represent an escalating clinical challenge due to\nantibiotic treatment failure, compounded by complex inflammation and dynamic\nmicroenvironmental alterations.” would be more compelling if expanded with relevant examples of\nsevere infections caused by antibiotic-resistant bacteria (e.g., endocarditis, intra-abdominal infections,\nor bacteremia). This would situate the work within the broader clinical landscape.\nResponse to comment:\nWe followed the reviewer's advice and agree that incorporating representative examples of\nsevere antibiotic-resistant infections would better contextualize our study within the broader clinical\nframework. Accordingly, we have revised the opening paragraph of the discussion to include specific\nexamples, such as chronic wound infections, pulmonary infections, intra-abdominal infections,\nurinary tract infections, and infective endocarditis, which underscore the serious clinical impact and\ntherapeutic challenges posed by multidrug-resistant pathogens. The revised text now reads as follows:\nPage 32 in the revised manuscript: “Infections caused by antibiotic-resistant pathogens\nrepresent an escalating clinical challenge due to antibiotic treatment failure. They can lead to hard-to-\nheal wound infections110, urinary tract infection111, pulmonary infections, intra-abdominal\ninfections112, and infective endocarditis113. Compounded by complex inflammation and dynamic\nmicroenvironmental alterations, these factors greatly increase the difficulty of clinical management.”\nReferences:\nbacteriological profile of wound infections in Ukraine. Commun. Med-London 5, 394 (2025).\nassociated urinary tract infection. Nano Today 56, 102271 (2024).\nIncreasing capsular polysaccharide biosynthesis via the PTS-CRP axis. Nat. Commun. 16, 6057\n(2025).\nendocarditis over two decades in a Thai tertiary care setting. Sci. Rep. 15, 13746 (2025).\nReviewer #2 (Remarks to the Author):\n“This manuscript reports the design of bioinspired Janus artificial enzymes based on high-entropy\nalloys (PtFeCuCoNi) for sequential therapy of drug-resistant bacterial infections and inflammatory\nwounds. The study is well-motivated, drawing on the biphasic microenvironment of chronic wounds,\nand demonstrates that the proposed HEA biocatalysts can achieve pH-dependent switching between\noxidase/peroxidase-like ROS generation under acidic conditions and antioxidase-like scavenging at\nneutral pH. The combination of theoretical calculations and experimental analyses provides a\ncoherent picture of the catalytic mechanism, and the in vivo data indicate antimicrobial efficacy\nagainst MRSA as well as immunomodulatory and pro-regenerative effects. The concept of exploiting\nmulti-metal synergy in HEAs for redox modulation is novel and of potential interest for the field of\nnanomedicine. The manuscript is scientifically sound and is suitable for publication in Nature\nCommunications. I therefore recommend its rapid publication after the following minor revisions are\naddressed:”\nResponse to the general comment:\nThank you for the outstanding summary and encouraging feedback on our manuscript. We have\ncarefully revised the manuscript and supplementary materials, addressing all queries and concerns in\ndetail. We greatly appreciate your valuable guidance, which has helped us refine the study, and we\nbelieve these revisions have further strengthened the paper's quality and impact.\n(1) In the Supplementary Methods, it was mentioned that “Raman spectra were examined via the\nHORIBA HR Evolution Raman spectrometer with a 532 nm laser source”, but this characterization\ntechnique was not included in the main manuscript. Is it a mistake?\nResponse to comment:\nWe sincerely thank the reviewer for identifying the errors in our supporting information. We\nhave corrected and deleted it, and added new instruments for structural characterization. Thanks\nagain for your kind reminder, and the updated corrections are included in the revised supporting\ninformation, as also illustrated below:\nPage 53 in the revised supporting information: “Inductively coupled plasma-Mass Spectrometry\n(ICP-MS) was carried out using a Agilent 7850 (Agilent Technologies, CA, USA). In-situ FTIR\nmeasurements were conducted using an infrared spectrometer (Thermo Scientific, iS50 FTIR)\nequipped with an in-situ spectrum cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-lll).”\n(2) The synthesis methods for the mentioned FeCuCoNi, PtCuCoNi, PtFeCoNi, PtFeCuNi, and\nPtFeCuCo alloys are not provided in the manuscript, which should be supplemented.\nResponse to comment:\nThanks for your important and helpful comments on improving the quality of our manuscript.\nWe have added to the problem, and the updated corrections are included in the manuscript, as also\nillustrated below:\nPage 34 in the revised manuscript: “The preparation conditions of FeCuCoNi, PtCuCoNi,\nPtFeCoNi, PtFeCuNi, and PtFeCuCo NPs are similar to that of PtFeCuCoNi-HEA NPs except that\none of the metal precursors is absent.”\n(3) While the study highlights the advantages of HEA materials, the current text provides limited\ndiscussion of prior biomedical research on HEAs. The authors are encouraged to expand the\nIntroduction or Discussion with additional references and a more explicit consideration of potential\ntoxicity concerns associated with high-entropy alloys.\nResponse to comment:\nThank you for your valuable suggestion regarding the discussion of previous biomedical studies\nand potential toxicity concerns of high-entropy alloys (HEAs). We will address your comments in\ndetail from the following two aspects: 1) prior biomedical research on HEAs; 2) evaluation of\ncytotoxicity.\n1) Prior biomedical research on HEAs:\nPage 32 in the revised manuscript: “Various high-entropy alloys have been explored for\nantibacterial applications. For example, MnFeCoNiCu nanozymes showed antibacterial activity\nagainst S. enteritidis and E. coli at 40 μg/mL114; the Al CoCrCu FeNiSi alloy, designed as a low-\n0.6 0.1 0.2\ncopper AHEA, achieved complete E. coli elimination by day 3115; Zhou et al. reported a Cu-Ag HEA\nwith strong antibacterial effects, though the high cost of Ag may limit its broader application116. In\nsummary, most previously reported HEAs exhibit single-mode antibacterial activity and lack the\nmultifunctional or biocompatible properties required for effective biomedical use.”\nReferences:\nhigh-efficiency peroxidase mimics. Adv. Sci. 10, 2303078 (2023).\nsynergistic effect of multiple elements. J. Mater. Res. Technol. 38, 581-596 (2025).\nagainst corrosive marine biofilms. J. Mater. Sci. Technol. 46, 201-210 (2020).\n2) Evaluation of cytotoxicity: To comprehensively evaluate the biosafety of PtFeCuCoNi HEA\nnanoparticles, we conducted both in vitro and in vivo assessments. In vitro cytocompatibility and\nhemolysis assays demonstrated negligible toxicity toward HUVECs, RSFs, and Raw264.7 cells at\nconcentrations up to 4 μg·mL-1, and no significant hemolysis even at 64 μg·mL-1. In vivo, peripheral\nblood biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase\n(AST), urea (UREA), and creatinine (CREA), revealed no abnormalities compared with untreated\ncontrols (Supplementary Fig. 60). Histopathological examination of major organs (heart, liver, spleen,\nlung, and kidney) by H&E staining also showed normal tissue architecture at the therapeutic dose.\nCollectively, these findings confirm that PtFeCuCoNi HEA nanoparticles possess excellent\nbiocompatibility in addition to potent antibacterial efficacy, highlighting their potential for safe and\neffective biomedical applications.\nPage 29 in the revised manuscript: “Major-organ histology, hemolysis test, and serum\nbiochemistry showed no adverse effects following HEA NPs administration (Supplementary Figs. 58,\n59, 60).”\nSupplementary Fig. 60 Biochemical analysis of the rat serum after treatments with different\nsamples. n = 3 independent replicates. Data are presented as means ± SD; ns, not significant; *p <\n0.05, **p < 0.01; one-way ANOVA with multiple comparisons test; all tests were two-sided. Source\ndata are provided as a Source Data file.\n(4) In Supplementary Fig. 28, the cytocompatibility of PtFeCuCoNi HEA NPs is presented. However,\nas Pt is a key constituent metal, the cytotoxicity of Pt NPs alone should also be discussed, which\nwould strengthen the argument that the alloy formulation indeed reduces toxicity relative to single-\nmetal systems.\nResponse to comment:\nWe took the advice and have supplemented the cytocompatibility evaluation of monometallic Pt\nNPs under identical experimental conditions. The additional results show that Pt NPs exhibited no\nstatistically significant effect on the viability of HUVEC and RSF at concentrations up to 10 μg·mL-1,\nand no notable cytotoxicity toward Raw264.7 cells at concentrations up to 4 μg·mL-1\n(Supplementary Figs. 40, 41). These data indicate that both PtFeCuCoNi HEA NPs and Pt NPs\nmaintain excellent cytocompatibility at concentrations below 4 μg·mL-1. The revised manuscript now\nreads as follows:\nPage 23 in the revised manuscript: “Cytocompatibility testing showed no appreciable toxicity of\nPtFeCuCoNi HEA NPs or Pt NPs to human umbilical vein endothelial cells (HUVEC), rat skin\nfibroblasts (RSF), or murine macrophages (Raw264.7) up to 4 μg·mL-1 (Supplementary Fig. 39-41)”\nSupplementary Fig. 40 Viability of human umbilical vein endothelial cells (HUVEC), rat skin\nfibroblasts (RSF), and murine mononuclear macrophage cells (Raw264.7), after incubation with Pt\n(n = 6 independent biological replicates). Data are presented as means ± SD, *p < 0.05, **p < 0.01,\n***p < 0.001, ns, not significant; one-way ANOVA with multiple comparisons test, all tests were\ntwo-sided. Source data are provided as a Source Data file.\nSupplementary Fig. 41 Representative Live/Dead dual-stained fluorescence images of human\numbilical vein endothelial cells (HUVEC), rat skin fibroblasts (RSF), and murine macrophages\n(Raw264.7) after incubation with Pt NPs.\n(5) In Fig. 6, the transcriptional responses provide valuable mechanistic insight. However, the current\ntext would benefit from the inclusion of supporting references for some of the highlighted pathways.\nIncluding key citations would help place these findings within the broader context of bacterial stress\nresponses.\nResponse to comment:\nThank you for the helpful suggestions. We have incorporated additional references in the\nrevised manuscript to strengthen the interpretation of the transcriptional mechanisms and\ncontextualize the observed pathways within the broader framework of bacterial stress responses. The\ncorresponding citations and revised text are provided below:\nPage 21 in the revised manuscript: “Furthermore, Kyoto Encyclopedia of Genes and Genomes\n(KEGG) analysis revealed broad perturbation of metabolic networks, with repression of sulfur\nmetabolism71, cysteine72 and methionine metabolism73, histidine metabolism74, and\nglycerophospholipid metabolism73, indicating disruption of thiol-based antioxidant defenses and\nmembrane-lipid homeostasis (Fig. 6d). Notably, gene set enrichment analysis (GSEA) further\nshowed positive enrichment of “oxidative phosphorylation” and “ATP-dependent activity”75,\nsuggesting an early compensatory surge in energy metabolism, likely reflecting transient activation\nof bacterial respiratory and ATP-generating pathways in response to acute ROS stress42,76. In contrast,\n“ATP-binding cassette (ABC) transporters”77 and “histidine metabolism”78 were negatively enriched\n(Fig. 6e), indicating impaired substrate trafficking and amino-acid biosynthesis12.”\nReferences:\nBactericidal Effect of Antibiotics by Inhibition of Enzymes Involved in Production of Hydrogen\nSulfide in Bacteria. Mol. Biol. 56, 638-648 (2022).\ninfection and immunity (vol 23, pg 1, 2021). Eur. J. Clin. Nutr. 75, 1319–1327 (2021).\n10, 213-224 (2015).\nvariants links protein dynamics to active-site electrostatic preorganisation. Nat. Commun. 13,\n7607 (2022).\nMG1655 to nine representative classes of antibiotics. Microbiol. Spectr. 11, e00317-00323 (2023).\nCell Bio. 10, 218-227 (2009).\n(2017).\nReviewer #3 (Remarks to the Author):\n“This manuscript describes the development of high-entropy alloy (HEA) (PtFeCuCoNi) as\nbioinspired nanocatalysts for the treatment of infected and non-healing wounds. The work addresses\nan important clinical challenge by targeting both the antimicrobial and tissue-regenerative phases of\nwound healing. The authors provide convincing evidence that the HEA nanozymes exhibit robust\ncatalytic adaptability, functioning as ROS-generating oxidase/peroxidase mimics in acidic\nenvironments while switching to ROS-scavenging antioxidase activity at neutral pH. The integration\nof transcriptomic profiling, mechanistic studies, and in vivo validation provides strong support for\nthe dual regulatory role of these materials in modulating oxidative stress and inflammation. The\nconcept of tailoring redox activity through entropy-driven alloy design is innovative and of clear\nsignificance to the biomedical nanomaterials community. It is suggested that this study is well\nexecuted and suitable for publication in Nature Communications, pending minor revisions to\nimprove clarity and contextual discussion.”\nResponse to the general comment:\nWe sincerely thank the reviewer for the positive evaluation and thoughtful feedback. We are\ngrateful for your recognition of the clinical significance and scientific innovation of our work on\nbioadaptive high-entropy alloy (HEA) nanozymes. We have conducted additional systematic\nexperiments and made extensive revisions throughout the manuscript to improve clarity, precision,\nand contextual discussion. All essential data have been included to support our conclusions, and each\npoint has been carefully addressed in both the main text and the Supplementary Information. We\nbelieve that these revisions have further strengthened the scientific rigor and overall quality of the\nwork, and we appreciate your constructive review and recommendation for publication.\n(1) The mechanism for the synthesis of the PtFeCuCoNi HEA NPs via the hydrothermal method\nshould be discussed in the main manuscript.\nResponse to comment:\nWe are grateful to the reviewers for this valuable suggestion and We are very grateful for your\ncomments and have supplemented the synthesis mechanism of the HEA NPs prepared via the\nhydrothermal method in the manuscript, as also illustrated below:\nPage 5 in the revised manuscript: “The HEA-based Janus artificial enzymes were synthesized\nvia a one-step solvothermal approach using Pd(acac) , Fe(acac) , Cu (NO ) ·3H O, Co (NO ) ·6H O,\n2 3 3 2 2 3 3 2\nNi (NO ) ·6H O as metal precursors (Fig. 2a), N,N-dimethylformamide (DMF) and ethylene glycol\n3 3 2\n(EG) as the solvents. A mauve, finely dispersed mixture of the raw materials was formed through\nultrasonication. As the temperature increased, the mixture turned black, possibly due to the\ncoordination and reduction of the five metals. Subsequently, the single-phase HEA formed after the\ndiffusion and rearrangement of atoms.”\n(2) Some earlier references regarding the HEA materials for antibacterial studies should be carefully\ncompared and discussed included in the manuscript.\nResponse to comment:\nThank you for these constructive suggestions. As mentioned above, we have incorporated\nadditional references on previous studies of HEA materials with antibacterial properties to provide\nclearer context and a comparison with our work. The corresponding citations and discussion have\nbeen added in the revised manuscript, as shown below:\nPage 32 in the supporting information: “Various high-entropy alloys have been explored for\nantibacterial applications. For example, MnFeCoNiCu nanozymes showed antibacterial activity\nagainst S. enteritidis and E. coli at 40 μg/mL114; the Al CoCrCu FeNiSi alloy, designed as a low-\n0.6 0.1 0.2\ncopper AHEA, achieved complete E. coli elimination by day 3115; Zhou et al. reported a Cu-Ag HEA\nwith strong antibacterial effects, though the high cost of Ag may limit its broader application116. In\nsummary, most previously reported HEAs exhibit single-mode antibacterial activity and lack the\nmultifunctional or biocompatible properties required for effective biomedical use.”\nReferences:\nModulated High-Efficiency Peroxidase Mimics. Adv. Sci. 10, 2303078 (2023).\nsynergistic effect of multiple elements. J. Mater. Res. Technol. 38, 581-596 (2025).\nagainst corrosive marine biofilms. J. Mater. Sci. Technol. 46, 201-210 (2020).\n(3) The contents of the elements measured by XPS and ICP in the manuscript are inconsistent. Please\ncheck the data and explain the difference.\nResponse to comment:\nThank you for your attentive review and valuable feedback. We have carefully checked the data\nand earlier literature. The discrepancy arises from the inherent differences between XPS (a surface-\nsensitive technique) and ICP (a bulk analysis technique). The most probable cause is the surface\nlayer composition of the nanoparticles, different from the bulk composition. We have double-\nchecked our ICP data and are confident in its reliability. A brief explanation to this effect has been\nadded to the manuscript.\n(4) In Fig. 6f, an interesting mechanistic illustration is presented; however, the results differ from\nprevious sequencing studies on active bactericidal processes, where a decrease in ATP activity was\nobserved. The authors are encouraged to further explain the reasons for this discrepancy and provide\nrelevant literature support.\nResponse to comment:\nWe sincerely appreciate the reviewer’s constructive comment. We fully agree that the\nexpression pattern of ATP synthase and oxidative phosphorylation–related genes reflects the\nmetabolic state of bacteria under oxidative stress, and we thank you for highlighting this important\nmechanistic aspect.\nIn the study of Co–Ru atomic pair catalysts (APCR) (Adv. Mater. 2024, 36, 2408787),\ntranscriptomic analysis revealed the suppression of oxidative phosphorylation and disruption of\nredox homeostasis, consistent with the metabolic exhaustion observed during the late bactericidal\nphase. In contrast, our transcriptomic profiling was performed at an early stage after HEA treatment,\nwhen a portion of bacteria remained metabolically active and were undergoing adaptive responses to\nHEA-catalyzed ROS stimulation, rather than irreversible oxidative damage.\nAt this early phase, bacteria typically initiate a transient compensatory metabolic activation to\nsustain energy production and counteract oxidative imbalance. This transient upregulation of ATP\nsynthesis and oxidative phosphorylation has been reported in several studies. For instance, a\ncomparative transcriptomic analysis of E. coli exposed to nine classes of antibiotics demonstrated\nthat genes involved in oxidative phosphorylation, such as NADH: quinone oxidoreductase I,\ncytochrome oxidases, and ATP synthase, were upregulated under ciprofloxacin, erythromycin, and\ntetracycline treatment (mBio 2022, 13, e03645-21), suggesting that early oxidative stress may trigger\nenhanced ATP production as a compensatory mechanism. Similarly, a ribosome profiling study\nunder acidic stress showed significant upregulation of ATP-dependent copper-exporting enzymes\n(CopA and CusA) (mSystems 2021, 6, e01234-20), reflecting increased ATP-dependent activity to\nmaintain metal ion homeostasis and survival under stress conditions.\nTherefore, the differences among these studies likely arise from the temporal dynamics of\nbacterial energy metabolism. During the early phase of oxidative or acidic stress, bacteria exhibit\nmetabolic activation characterized by transient upregulation of ATP-dependent pathways and\noxidative phosphorylation, whereas in the later energy-depletion phase, ATP production declines and\nmetabolic collapse ensues, leading to cell death.\nIn summary, our findings most likely capture the early adaptive phase of bacterial response to\nHEA-induced ROS, while the APCR study reflects the late metabolic exhaustion stage. This\ninterpretation suggests that bacterial energy metabolism under oxidative stress is dynamic and time-\ndependent, and our results provide complementary insight into the temporal evolution of stress-\nresponsive metabolism, representing a novel mechanistic observation in ROS-mediated antibacterial\nresearch.\nPage 21 in the revised manuscript: “Notably, gene set enrichment analysis (GSEA) further\nshowed positive enrichment of “oxidative phosphorylation” and “ATP-dependent activity”75,\nsuggesting an early compensatory surge in energy metabolism, likely reflecting transient activation\nof bacterial respiratory and ATP-generating pathways in response to acute ROS stress42,76, while\n“ATP-binding cassette (ABC) transporters”77 and “histidine metabolism”78 were negatively enriched\n(Fig. 6e), indicating impaired substrate trafficking and amino-acid biosynthesis12.”\nReferences:\nwith synergetic Co-Ru pair centers for programmed diabetic ulcer therapy. Adv. Mater. 36,\n2408787 (2024).\nMG1655 to nine representative classes of antibiotics. Microbiol. Spectr. 11, e00317-00323 (2023).\n(5) Fig. 7n presents a schematic illustration of cellular effects under high-concentration H O\n2 2\ntreatment and the protective role of HEA NPs. However, the labeling of the cell types is missing,\nmaking it unclear which three cells are being represented.\nResponse to comment:\nThank you for pointing this out. We apologize for the omission. In Fig. 7n, the three\nrepresentative cell types correspond to vascular endothelial cells (VEC), anti-inflammatory\nmacrophages (M2), and rat skin fibroblasts (RSF), which are the principal cell populations involved\nin anti-inflammation regulation, angiogenesis, and tissue remodeling during wound healing. The\ncorresponding labels have now been added, and the schematic illustration has been updated in the\nrevised figure to improve clarity and interpretability.\nFig. 7(n) Schematic illustration of cell effects upon the addition of high-concentration H O and\n2 2\nHEA NPs’ protection.\n(6) Fig. 8d and Fig. 8f present GO and KEGG analyses, respectively. It is noted that Fig. 8d\ncompares PtFeCuCoNi vs H2O2, while Fig. 8f shows the opposite comparison, H2O2 vs\nPtFeCuCoNi. As the rationale for this discrepancy has not been explained in the manuscript, the\nauthors are advised to provide further discussion.\nResponse to comment:\nThank you for pointing this out. The inconsistency in the comparison directions of the GO and\nKEGG analyses arises from our intention to capture two complementary biological dimensions—the\nprotective mechanisms activated by the HEA NPs and the molecular injuries caused by oxidative\nstress. Specifically, the GO enrichment analysis (PtFeCuCoNi vs. H O ) was designed to highlight\n2 2\nthe functional restoration and metabolic protection afforded by the HEA nanozyme, focusing on\nbiological processes associated with cellular homeostasis, cytoskeletal organization, and angiogenic\nrepair. In contrast, the KEGG analysis (H O vs PtFeCuCoNi) emphasized oxidative stress-induced\n2 2\nmolecular disruption, revealing pathways linked to apoptosis, inflammatory signaling, and energy\nimbalance. This bidirectional comparison framework provides a holistic “damage-protection”\nperspective, clarifying both the pathophysiological impact of oxidative stress and the restorative\neffects of HEA nanozyme.\nTo further strengthen this interpretation, we integrated the GSEA results (based on GO-BP gene\nsets) within the main text. Notably, “tube development” and “cytoskeleton organization” were\nsignificantly enriched in the PtFeCuCoNi group, indicating enhanced structural integrity and pro-\nregenerative activity. Conversely, “apoptotic signaling pathway” was suppressed compared with the\nH O group, confirming the material’s anti-apoptotic and antioxidative functions. Enrichment of\n2 2\n“cellular response to oxygen-containing compounds” further aligned with the ROS and cell viability\ndata, validating the redox-protective behavior of the HEA nanozyme.\n(7) Supplementary Figs. 31 and 32 show scratch assays of HUVECs and RSFs, respectively.\nHowever, unlike Figs. 7a and 7c, the scratch assay for RAW264.7 cells is missing. The authors are\nadvised to either provide the corresponding experiment or explain the reason for its absence.\nResponse to comment:\nWe sincerely thank the reviewer for this careful and constructive comment. Scratch assays are\nprimarily used to evaluate the migration and proliferative capacity of cells involved in tissue\nregeneration. In the context of wound healing, HUVECs and RSFs are the principal effector cells\nthat drive angiogenesis and extracellular matrix remodeling. Therefore, we performed scratch assays\nfor these cell types to assess the regenerative potential of the HEA nanoparticles (Supplementary\nFigs. 44, 45). In contrast, Raw264.7 macrophages primarily serve immunomodulatory rather than\nmigratory roles during skin repair, orchestrating inflammatory resolution through M1/M2\npolarization. Moreover, due to their growth characteristics, which require frequent subculture to\nmaintain viability, they are unsuitable for conventional scratch assays, as prolonged culture after\nscratching often leads to proliferation arrest and apoptosis. Hence, migration assays were not\nperformed for Raw264.7 cells.\nTo comprehensively assess the effect of PtFeCuCoNi HEA NPs on macrophage functionality,\nwe instead conducted RT-qPCR analyses of M1 (iNOS) and M2 (Arg1) markers, along with\ncytokines (TNF-α, IL-6, IL-10, and TGF-β). The results revealed that the HEA NPs suppressed pro-\ninflammatory mediators while enhancing anti-inflammatory gene expression (Supplementary Figs.\n47, 48). Together with the immunofluorescence data showing decreased CCR7 and increased CD163\nexpression (Fig. 7j–m and Supplementary Fig. 46), these findings indicate that the HEA\nnanoparticles effectively reprogram macrophages toward an anti-inflammatory phenotype.\nPage 23 in the revised manuscript: “To evaluate macrophage polarization under\nlipopolysaccharide (LPS) stimulation, real-time quantitative polymerase chain reaction (RT-qPCR)\nwas performed. Compared with Pt NPs, PtFeCuCoNi HEA NPs were found to exert a stronger\ninhibitory effect on the mRNA expression levels of pro-proinflammatory cytokines, such as\ninducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-\nα), while concurrently augmenting genes associated with anti-inflammatory activity, including\narginase-1 (Arg1), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β)\n(Supplementary Figs. 47, 48). Furthermore, immunofluorescence analysis revealed that HEA NP\npretreatment significantly reduced C-C chemokine receptor type 7 (CCR7) expression, a signature\nmarker of M1 macrophages, relative to both LPS alone and Pt treatment (Fig. 7j, k and\nSupplementary Fig. 46).”\nSupplementary Fig. 47 a The relative mRNA expression of iNOS, TNF-α, and IL-6 in Raw264.7\ncells was measured by Real-time quantitative polymerase chain reaction (RT-qPCR) after 24 h of\nLPS stimulation (n = 3 independent biological replicates). b The relative mRNA expression of Arg-1,\nIL-10, and TGF-β in Raw264.7 cells following 24 h of LPS stimulation (n = 3 independent biological\nreplicates). Data are presented as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001,\nns, not significant; one-way ANOVA with multiple comparisons test, all tests were two-sided. Source\ndata are provided as a Source Data file.\nSupplementary Fig. 48 Schematic depicting the effects of PtFeCuCoNi HEA NPs on macrophage\npolarization. M0 indicates macrophages, M1 indicates pro-inflammatory macrophages, M2 indicates\nanti-inflammatory macrophages, and LPS indicates lipopolysaccharide.\nReviewer #4 (Remarks to the Author):\n“Gao et al. designed a high-entropy alloy-based Janus artificial enzyme targeting chronic wound\ninfections. This enzyme leverages the unique microenvironment characteristics at the infection site to\ncatalyze antimicrobial and anti-inflammatory actions, demonstrating excellent antimicrobial efficacy\nboth in vitro and in vivo. Additionally, the study found that it can promote the PFKFB3 pathway,\ninhibit the expression of inflammatory cytokines, and accelerate wound healing. The results are very\ninteresting; however, the manuscript still has some issues to address. It is recommended that the\nauthors make further additions and revisions.”\nResponse to comment:\nWe sincerely appreciate the reviewer’s recognition of our high-entropy alloy-based Janus\nartificial enzyme as a promising bioadaptive platform for infection control and tissue repair. In\nresponse to your insightful and constructive comments, we have conducted additional experiments\nand comprehensively revised the manuscript to address all the concerns raised. In particular, we have\nincluded systematic evaluations of material stability, ROS generation and lipid peroxidation at the\nbacterial level, EPS degradation, macrophage polarization and cytokine expression profiling, as well\nas RT-qPCR validation of transcriptomic results. All newly obtained data and corresponding\ndiscussions have been incorporated into the revised main text and Supplementary Information. We\nbelieve that these revisions have substantially strengthened the mechanistic depth, scientific rigor,\nand overall clarity of the manuscript. We are deeply grateful for your thoughtful feedback, which has\ngreatly enhanced the quality, coherence, and impact of our work.\n(1) Through TEM and SEM observations, the Janus artificial enzyme exhibits a nanostructure. The\nauthors should further supplement the DLS and material stability data of the artificial enzyme,\nparticularly regarding its bioavailability. It is also recommended that the authors include stability\ndata of the artificial enzyme under different environmental conditions.\nResponse to comment:\nThank you for your insightful comments and helpful suggestions. In response to your feedback,\nwe supplemented the PtFeCuCoNi HEA NPs with DLS. Additionally, the stability of the material\nwas evaluated over a twelve-day period in four different environments: PBS (pH 4.5), PBS (pH 7.4),\nDulbecco's Modified Eagle Medium (DMEM) cell culture medium, and Fetal Bovine Serum (FBS).\nThe corresponding supporting literature has been added in the revised manuscript, as also shown\nbelow:\nPage 6 in the revised manuscript: “Meanwhile, the number-averaged size of PtFeCuCoNi HEA\nNPs is measured to be ≈122.4 nm in dynamic light scattering, indicative of a stable aqueous phase\nwith low agglomeration tendency. (Supplementary Fig. 2).”\nSupplementary Fig. 2 Dynamic light scattering (DLS) of PtFeCuCoNi HEA NPs.\nPage 16 in the revised manuscript: “Afterward, we rigorously evaluated the solution stability of\nHEA NPs. The PtFeCuCoNi HEA NPs exhibit exceptional dispersibility, with no detectable\nprecipitation or phase separation following static incubation at room temperature for 12 days\n(Supplementary Fig. 29). Moreover, assessments of particle size and zeta potential of PtFeCuCoNi\nHEA NPs across various intervals (days 3, 6, 9, and 12) indicate no significant dimensional\nalterations or aggregation (Supplementary Fig. 30). Therefore, PtFeCuCoNi HEA NPs exhibit robust\nstability and hold promise for enhanced therapeutic efficacy in forthcoming in vivo and in vitro\nstudies.”\nSupplementary Fig. 29 Digital photographs of PtFeCuCoNi HEA NPs in four physiologically\nrelevant solvents: PBS (pH 4.5), PBS (pH 7.4), Dulbecco's Modified Eagle Medium (DMEM) cell\nculture medium, and fetal bovine serum (FBS).\nSupplementary Fig. 30 Analysis of the long-term stability of PtFeCuCoNi HEA NPs in terms of\nzeta potential values and hydrodynamic diameter in four physiologically relevant solvents: a PBS\n(pH 4.5), b PBS (pH 7.4), c DMEM cell culture medium, and d FBS.\n(2) The HEA nanoenzyme demonstrated different catalytic activities under acidic and neutral pH\nconditions, which is very interesting and differs from other reported HEA-based artificial enzymes.\nHowever, the underlying mechanism is not sufficiently discussed. Strengthening this part would\nimprove the manuscript.\nResponse to comment:\nWe are grateful to the reviewers for this valuable suggestion and positive comment on our\nconcept of pH-controlled bioadaptive ROS regulation. We agree that investigating the underlying\nreasons for the different catalytic activities observed in acidic and neutral environments is highly\nuseful for further understanding catalytic mechanisms and validating the originality of our study.\nFurther details will be provided in the subsequent discussion.\nDensity functional theory (DFT) calculations indicate that variations in hydrogen ion (H+)\nconcentration, particularly under acidic and neutral conditions, lead to distinct reaction pathways and\nproducts when the catalyst interacts with H O . Initially, the Fe active site adsorbs an H O molecule,\n2 2 2 2\nleading to the release of H O and the subsequent formation of an *O intermediate. In a neutral\n2\nenvironment, H O interacts with *O to result in the creation of *OH and *OOH, thereby exhibiting\n2 2\ncatalase-like activity. Conversely, in an acidic environment, H+ exhibits a greater propensity to\ninteract with *O (with a Gibbs free energy barrier of -0.436 eV) compared to H O (with a Gibbs\n2 2\nfree energy barrier of -0.384 eV), giving rise to *OH intermediates. Subsequent re-adsorption of\nH O leads to the generation of *H O and *OOH, thereby showing POD-like activity. Notably, OOH\n2 2 2\nis the precursor to produce •O - and 1O (Adv. Mater., 2024, 36, 2408787; Nat. Commun., 2024, 15,\n2 2\n1010; J. Am. Chem. Soc., 2023, 145, 8965-8978).\nIn summary, the proton source in neutral conditions is solely H O , leading the active sites to\n2 2\npredominantly interact with H O and display catalase-like activity, whereas under acidic conditions,\n2 2\nthe proton sources include both H+ and H O , and H+ has a higher affinity for the *O intermediates\n2 2\nthan H O , thus resulting in peroxidase-like activity. The corresponding mechanism of different\n2 2\ncatalytic activities triggered by acidic and neutral environments has been added in the revised\nmanuscript and revised supplementary information, as also shown below:\nPage 14 in the revised manuscript: “Furthermore, according to our previous studies, the\nstructure of adsorbed O* is a key intermediate in compartmentalizing the POD-like and CAT-like\npathways42. Therefore, to demonstrate the continuous pH-switchable catalytic activity of\nPtFeCuCoNi HEA NPs, we complemented the DFT studies of the free energy of the structure of\nPtFeCuCoNi HEA NPs adsorbing O* (Structure 1) for the adsorption of H+ or H O under acidic and\n2 2\nneutral conditions. As shown in Supplementary Fig. 20, the free energies of structure 1 for the\nadsorption of both H+ and H O are negative, indicating that both processes can occur spontaneously.\n2 2\nHowever, compared with adsorbing H O (−0.384 eV), the free energy of adsorbing H+ by structure\n2 2\n1 is lower (−0.436 eV), indicating that structure 1 will preferentially combine with H+ to form *OH\nto complete the POD-like activity pathway in an acidic environment with the presence of H+.\nFurthermore, under neutral or slightly alkaline conditions, structure 1 is more prone to adsorb H O\n2 2\nto carry out the CAT-like activity pathway to realize the continuous pH-switchable catalytic activity.”\nSupplementary Fig. 20 Free energy analysis of further adsorption of H+ and H O by PtFeCuCoNi\n2 2\nHEA NPs O* intermediates.\n(3) Fig. 5C is used to detect bacterial protein leakage, typically assessed by the BCA assay. However,\nthe manuscript does not provide a detailed description of this method. Given the limited bacterial\nprotein content, the detection results showed a deep purple color and lacked a standard curve. It is\nrecommended that the authors provide further clarification on this issue.\nThanks for this insightful comment, and we have added a detailed description of the\nbicinchoninic acid (BCA) assay used to quantify bacterial protein leakage in the revision (Fig. 5C).\nTo ensure methodological transparency and data reliability, a standard curve was established using\ngradient concentrations of bovine serum albumin (BSA, 0-0.5 mg/mL) solutions. The corresponding\noptical density (OD ) values were recorded and photographed, yielding a linear calibration\n562\nequation of y = 1.698x + 0.166 (y = OD , x = protein concentration, mg/mL) with R2 = 0.992,\n562\nconfirming high linearity.\nIn addition, we have clarified that the “protein leakage (a.u.)” in the original figure represents\nrelative OD values that reflect the extent of bacterial membrane disruption. Following the reviewer’s\nsuggestion, all OD data have now been converted to absolute protein concentrations using the\nstandard curve to achieve quantitative accuracy.\nThe observed deep purple coloration in the reaction system possibly resulted from the high\nbacterial load (108 CFU/mL, 1 mL total volume). Under these conditions, the strong ROS-mediated\nmembrane disruption induced by the PtFeCuCoNi HEA NPs led to substantial cytoplasmic protein\nrelease, producing an intensified colorimetric response.\nPage 35 in the revised manuscript: “Antibacterial experiments. Bacterial protein leakage was\nquantified using a bicinchoninic acid (BCA) protein assay kit (Solarbio). Briefly, MRSA were\nincubated with PBS, H O , Pt + H O , PtFeCuCoNi + H O under the indicated conditions. After\n2 2 2 2 2 2\nincubation, supernatants were collected by centrifugation, and protein content was determined\nfollowing the manufacturer’s protocol. A standard curve was established using bovine serum albumin\n(BSA, 0-0.5 mg/mL), and the corresponding optical density at 562 nm (OD ) was recorded.The\n562\ncalibration equation (y = 1.698x + 0.166, R2 = 0.992) was used to convert OD values into absolute\nprotein concentrations. Representative images of the gradient color reaction were also obtained. All\nmeasurements were performed in triplicate.”\nSupplementary Fig. 23 a Representative image of Bicinchoninic Acid (BCA) reaction solutions\nwith gradient bovine serum albumin (BSA) concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5\nmg·mL-1), showing the increasing purple color intensity corresponding to measured OD values for\n562\nstandard curve quantification. b Standard curve of BSA for protein quantification using the BCA\nassay. Gradient concentrations of BSA (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg·mL-1) were\nmeasured at OD , and the resulting data were fitted to obtain the standard curve (y = 1.698x +\n562\n0.166, R2 = 0.992). c Protein leakage of MRSA after incubation with PBS, H O , Pt + H O ,\n2 2 2 2\nPtFeCuCoNi + H O , quantified as absolute protein concentrations using the BSA standard curve\n2 2\nfrom the BCA assay. (n = 3 independent replicates). Data are presented as means ± SD; ns, not\nsignificant, ***p < 0.001; one-way ANOVA with multiple comparisons test, all tests were two-sided.\nSource data are provided as a Source Data file.\n(4) The authors demonstrated the reduction of EPS through SEM characterization, but this claim has\ncertain limitations. It is recommended that the authors include a biofilm EPS gel electrophoresis\nexperiment to further validate this result.\nResponse to comment:\nWe sincerely thank you for this valuable comment. As pointed out, relying solely on SEM to\ndemonstrate EPS reduction indeed has certain limitations. The extracellular polymeric substances\n(EPS) of bacterial biofilms mainly consist of proteins, polysaccharides, and small amounts of\nextracellular DNA (eDNA), among which proteins and polysaccharides form the structural backbone\nthat determines the stability and function of the biofilm. To systematically evaluate the destructive\neffects of PtFeCuCoNi nanozymes on MRSA biofilms, we employed multiple complementary\nanalytical methods.\nIn our initial submission, we had already conducted SEM observation and Dextran Alexa Fluor\n647 staining. SEM provides direct morphological information on the overall biofilm architecture and\nthe spatial distribution of major EPS components, including proteins and polysaccharides. The\nresults showed that biofilms treated with PtFeCuCoNi exhibited pronounced membrane roughening,\nlocalized collapse, and a substantial reduction in EPS coverage. In contrast, only mild changes were\nobserved in the Pt-treated group. The control biofilms remained intact (Fig. 5j and Supplementary\nFig. 26). Meanwhile, Dextran Alexa Fluor 647 staining, which specifically labels polysaccharides\nwithin the EPS matrix through its dextran-based structure that embeds into the polysaccharide\nnetwork, allowed visualization of polysaccharide distribution under confocal microscopy. The\nfluorescence intensity of EPS polysaccharides was markedly reduced in the PtFeCuCoNi-treated\ngroup compared with the Control, H O , and Pt groups (Supplementary Fig. 34, 35), indicating a\n2 2\nsignificant decrease in polysaccharide abundance.\nFollowing the reviewer’s suggestion, we further supplemented protein level analyses to\nstrengthen the compositional evidence. SDS-PAGE analysis revealed that EPS proteins in the\nPtFeCuCoNi-treated samples were almost completely degraded, with only low-molecular-weight\nfragments remaining. In contrast, the Pt group showed partial degradation, while the control group\nretained intact protein bands. In addition, we performed agarose gel electrophoresis to examine total\nDNA extracted from biofilm samples, which includes both extracellular DNA and a small fraction of\nintracellular DNA. The results showed that DNA bands in the PtFeCuCoNi-treated group were\nalmost completely absent, with only faint signals near the loading wells; partial degradation was\nobserved in the Pt group, whereas clear intact bands remained in the control group. These findings\nindicate that PtFeCuCoNi treatment severely disrupts the structural integrity of the biofilm, leading\nto oxidative degradation of both extracellular and intracellular components.\nCollectively, the evidence from SEM morphology, Dextran Alexa Fluor 647 polysaccharide\nstaining, and protein/DNA electrophoresis provides comprehensive, multi-level validation of the\npronounced disruption of biofilm structure by PtFeCuCoNi. These results support the idea that ROS-\nmediated oxidative degradation of multiple biofilm components, including polysaccharides, proteins,\nand DNA, leads to the effective collapse of biofilm architecture and loss of integrity. This multi-\nmethod verification not only strengthens the logical completeness of our results but also fully\naddresses and supports the reviewer’s valuable suggestion.\nPage 16 in the revised manuscript: “Scanning electron microscopy (SEM) also revealed minor\nsurface damage with abundant extracellular polymeric substances (EPS) in Pt-treated bacteria57,58,\nwhereas HEA-exposed group displayed marked membrane roughening, localized collapse, and\nvisibly reduced EPS coverage (Fig. 5j and Supplementary Fig. 26). Consistently, Dextran Alexa\nFluor 647 staining showed markedly decreased fluorescence intensity of polysaccharides within the\nEPS after HEA treatment, confirming substantial matrix degradation (Supplementary Figs. 34, 35).\nFurthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agarose\ngel electrophoresis of EPS proteins and total DNA (including extracellular DNA) demonstrated\nextensive degradation following PtFeCuCoNi treatment, whereas Pt treatment induced only partial\ndegradation, and controls remained largely intact (Supplementary Figs. 27, 28), corroborating the\nSEM observations. TEM images revealed highly fragmented membranes and clear internalization of\nHEA NPs following incubation with H O (Fig. 5k), indicative of a ROS-potentiated, membrane-\n2 2\ndisruptive bactericidal process.”\nSupplementary Fig. 27 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)\nanalysis of the proteins in the extracellular polymeric substances (EPS) of MRSA biofilm after\nincubating with PBS, Pt + H O , and PtFeCuCoNi + H O (n = 3 independent replicates).\n2 2 2 2\nRepresentative images are shown. Data are presented as means ± SD, *p < 0.05, **p < 0.01; one-\nway ANOVA with multiple comparisons test, all tests were two-sided. Source data are provided as a\nSource Data file.\nSupplementary Fig. 28 Agarose gel electrophoresis analysis of DNA extracted from MRSA biofilm\nafter incubation with PBS, Pt + H O , and PtFeCuCoNi + H O (n = 3 independent replicates).\n2 2 2 2\nRepresentative gel images are shown. Data are presented as means ± SD, *p < 0.05, ***p < 0.001;\none-way ANOVA with multiple comparisons test, all tests were two-sided. Source data are provided\nas a Source Data file.\n(5) The authors demonstrated lipid peroxidation in MRSA after treatment with the artificial enzyme\nby the increase in LPO. It is recommended that the authors include MDA detection at the bacterial\nlevel to further confirm this phenomenon.\nResponse to comment:\nWe sincerely thank you for this insightful and constructive suggestion. We fully agree that\ndetecting malondialdehyde (MDA), a well-recognized marker of lipid peroxidation, would provide\nmore direct evidence of oxidative membrane damage in bacteria. Accordingly, we have performed an\nadditional MDA assay to evaluate the lipid peroxidation levels in MRSA after different treatments\n(PBS, H O , Pt NPs, and PtFeCuCoNi HEA NPs).\n2 2\nAs shown in Supplementary Fig. 38, the PtFeCuCoNi HEA NPs group exhibited a markedly\nelevated MDA content (≈0.045 nmol/104 cells), which was significantly higher than the PBS, H O ,\n2 2\nPt NPs, and PtFeCuCoNi HEA NPs groups. These findings clearly indicate that the HEA NPs\ninduced stronger oxidative lipid damage in MRSA, corroborating our previous LPO results and\nfurther confirming the enhanced ROS‐driven antibacterial mechanism of the material.\nPage 17 in the revised manuscript: “Consistently, CLSM-based LPO analysis showed a strong\ngreen fluorescence shift in HEA NPs-exposed biofilms64,65, particularly within deeper layers,\nsuggesting extensive phospholipid oxidation and ROS-induced damage throughout the biofilm\nmatrix (Fig. 5s-u and Supplementary Figs. 26, 27)66-68. In line with this, MDA quantification further\nconfirmed enhanced lipid peroxidation in MRSA treated with PtFeCuCoNi HEA NPs compared with\nPt NPs and controls (Supplementary Fig. 38).”\nSupplementary Fig. 38 Malondialdehyde (MDA) levels in MRSA after different treatments.\nQuantitative analysis of MDA production in MRSA following treatment with PBS, H₂O₂, Pt NPs, or\nPtFeCuCoNi HEA NPs (n = 3 independent biological replicates). Data are presented as means ± SD,\n***p < 0.001, ns, not significant; one-way ANOVA with multiple comparisons test, all tests were\ntwo-sided. Source data are provided as a Source Data file.\n(6) The antibacterial mechanism of the artificial enzyme primarily involves the generation of ROS.\nHowever, the manuscript lacks relevant experiments at the bacterial level. It is recommended that the\nauthors include ROS generation at the bacterial level after labeling with the DCFH-DA probe.\nResponse to comment:\nWe sincerely appreciate your valuable suggestion to improve the quality of our manuscript. We\nunderstand your concern that the antibacterial mechanism of artificial enzymes mainly relies on the\ngeneration of reactive oxygen species (ROS), while the current version lacks direct experimental\nevidence at the bacterial level. To evaluate ROS generation at the bacterial level, we applied 2′,7′-\ndichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe to assess intracellular ROS\nproduction in MRSA under different treatment conditions. As shown in Supplementary Fig. 28, both\nthe PBS and H O groups exhibited weak green fluorescence, whereas the PtFeCuCoNi HEA NPs-\n2 2\ntreated group displayed markedly enhanced green fluorescence intensity, indicating substantial\nintracellular ROS accumulation. The Pt NP-treated bacteria also showed increased fluorescence,\nthough significantly weaker than that of the PtFeCuCoNi group. These findings demonstrate that\nunder acidic conditions (pH = 4.5), PtFeCuCoNi HEA nanozymes effectively catalyze H O\n2 2\ndecomposition to generate ROS, thereby elevating bacterial intracellular ROS levels and inducing\noxidative damage. Our data provides direct evidence supporting the ROS-mediated antibacterial\nmechanism of the artificial enzyme. The corresponding details can be found in the revised\nmanuscript and revised supplementary information, as shown below:\nPage 16 in the revised manuscript: “In acidic media containing H O , the NPs predominantly\n2 2\nproduced •O – and 1O , whereas at near-neutral pH they exhibited no detectable POD-like activity\n2 2\n(Fig. 5a). To verify ROS generation in bacteria directly, intracellular ROS levels were assessed using\n2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Bacteria treated with PtFeCuCoNi\nHEA NPs exhibited strong green fluorescence, indicative of pronounced intracellular ROS\naccumulation and presenting direct evidence for the ROS-mediated antibacterial mechanism of the\nartificial enzyme (Supplementary Fig. 21).”\nSupplementary Fig. 21 Top: Two-dimensional (2D) images from confocal laser scanning\nmicroscope (CLSM) images of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence\non the planktonic MRSA after incubating with PBS, H O , Pt + H O , PtFeCuCoNi + H O . The scale\n2 2 2 2 2 2\nbar represents 50 μm. Experiments were repeated independently three times with similar results.\nBottom: Quantitative analysis of DCFH-DA fluorescence after different treatments (n = 3\nindependent replicates). Data are presented as means ± SD; *p < 0.05, **p < 0.01; ns, not significant;\none-way ANOVA with multiple comparisons test; all tests were two-sided. Source data are provided\nas a Source Data file.\n(7) At the cellular level, the artificial enzyme exhibits anti-inflammatory effects. The authors\nconfirmed its ROS scavenging ability using the DCFH-DA probe; however, ROS scavenging is only\none step in the anti-inflammatory process. It is recommended that the authors supplement the study\nwith the polarization trends of M1 and M2 macrophages at the cellular level and concurrently assess\nthe release of inflammatory cytokines TNF-α, IL-6, and IL-10 at the cellular level.\nResponse to comment:\nThanks so much for this insightful comment. As rightly noted, ROS scavenging constitutes only\none aspect of the anti-inflammatory process, and a more comprehensive understanding requires\nevaluating macrophage polarization and inflammatory cytokine regulation at the cellular level.\nAccordingly, we performed additional experiments to assess macrophage polarization trends and\ninflammatory cytokine expression following treatment with PtFeCuCoNi HEA NPs (Supplementary\nFig. 47). The results showed a clear phenotypic transition from the pro-inflammatory M1 type to the\nreparative M2 phenotype, as evidenced by significant downregulation of the M1 marker iNOS and\nupregulation of the M2 marker Arg1. In addition, secretion of pro-inflammatory cytokines (TNF-α,\nIL-6) was markedly decreased, whereas secretion of anti-inflammatory cytokines (IL-10, TGF-β)\nwas significantly increased. These results were complemented by a schematic illustration\nsummarizing the immunomodulatory effects of PtFeCuCoNi HEA NPs on macrophage polarization\nand cytokine balance (Supplementary Fig. 48).\nPage 23 in the revised manuscript: “In parallel, CD163 expression was strongly upregulated (Fig.\n7l, m), suggesting a phenotypic shift from a pro-inflammatory M1 phenotype toward a reparative M2\nstate. Furthermore, RT-qPCR analysis revealed that, compared to Pt NPs, PtFeCuCoNi HEA NPs\nexerted a stronger inhibitory effect on the mRNA expression of pro-inflammatory cytokines, such as\nsuch as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha\n(TNF-α), while concurrently enhancing genes associated with anti-inflammatory activity, including\narginase-1 (Arg1), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β)\n(Supplementary Figs. 47, 48). Together, these results demonstrate that, at neutral pH, PtFeCuCoNi\nHEA NPs extinguish intracellular ROS, preserve cytoskeletal architecture, sustain angiogenic\nfunction, and reprogram macrophages toward tissue repair, thus maintaining redox homeostasis and\nprotecting cells from oxidative injury (Fig. 7n).”\nSupplementary Fig. 47 a By Real-time quantitative polymerase chain reaction (RT-qPCR), the\nrelative expression of iNOS, TNF-α, and IL-6 mRNA in Raw264.7 cells was measured after 24 h of\nLPS stimulation (n = 3 independent biological replicates). b RT-qPCR measurements of mRNA\nexpression (Arg-1, IL-10, and TGF-β) in Raw264.7 cells following 24 h of LPS stimulation (n = 3\nindependent biological replicates). Data are presented as means ± SD, *p < 0.05, **p < 0.01, ***p <\n0.001, ****p < 0.0001, ns, not significant; one-way ANOVA with multiple comparisons test, all tests\nwere two-sided. Source data are provided as a Source Data file.\nSupplementary Fig. 48 Schematic depicting the effects of PtFeCuCoNi HEA NPs on macrophage\npolarization. M0 indicates macrophages, M1 indicates pro-inflammatory macrophages, M2 indicates\nanti-inflammatory macrophages, and LPS indicates lipopolysaccharide.\n(8) The authors indicate that the artificial enzyme can activate the PFKFB3 pathway at the\ntranscriptomic level. However, the authenticity of the activation results based solely on\ntranscriptomic data remains to be further verified. It is recommended that the authors supplement the\nstudy with RT-qPCR testing of relevant factors to further confirm these results.\nResponse to comment:\nWe appreciate the reviewer’s insightful comment highlighting the need for experimental\nvalidation of the PFKFB3 pathway activation beyond transcriptomic evidence. To strengthen the\nreliability of our findings, we performed RT-qPCR analysis to confirm the expression patterns of key\ngenes associated with glycolytic metabolism and oxidative stress regulation. Six representative genes\nwere selected for validation, including upregulated (PFKFB3, THBS1, HSPA8) and downregulated\n(CHAC1, ASNA, PSAT1) targets. The RT-qPCR data showed that treatment with PtFeCuCoNi HEA\nNPs significantly increased PFKFB3, THBS1, and HSPA8 expression, consistent with enhanced\nglycolytic and reparative activity, while CHAC1, ASNA, and PSAT1 were significantly suppressed,\nindicating alleviation of oxidative stress-related responses. These results are in strong agreement\nwith our RNA-seq findings and provide direct evidence that PtFeCuCoNi HEA nanozymes\ntranscriptionally activate PFKFB3-mediated glycolysis while alleviating redox imbalance\n(Supplementary Fig. 49).\nPage 26 in the revised manuscript: “Differential expression analysis identified significant\nupregulation of protective genes, including PFKFB378 (glycolysis) and THBS179 (extracellular\nmatrix remodeling), together with downregulation of stress-associated genes such as SLC7A1164,\nPSAT180, CHAC1, MTHFD2, and SESN2 (Fig. 8b), suggesting a transcriptional switch from injury\nsignaling to programs supporting metabolic recovery and structural repair. To validate these\ntranscriptomic trends, we further performed RT–qPCR analysis of representative genes involved in\nglycolytic activation and oxidative stress response. Consistent with the RNA-seq results,\nPtFeCuCoNi HEA NPs treatment markedly upregulated PFKFB3, THBS1, and HSPA8, while\nsignificantly downregulating CHAC1, ASNA, and PSAT1 compared with both H O and Pt NPs\n2 2\ngroups (Supplementary Figs. 49). These results confirm that HEA NPs transcriptionally activate\nglycolysis- and repair-related programs while suppressing stress-associated pathways, thereby\nsupporting the restoration of redox homeostasis and cellular recovery. Hierarchical clustering\ncorroborated a shift toward a homeostatic expression profile after HEA NPs treatment (Fig. 8c).”\nSupplementary Fig. 49 a Volcano plots of DEGs (gray: not significantly different genes; red:\nupregulated genes; blue: downregulated genes). b RT-qPCR data, the relative expression of PFKFB3,\nTHBS1, HSPA8, CHAC1, ASNS, and PSAT1 mRNA in HUVEC cells was measured after\nincubating with PBS (Control), H O , Pt + H O , PtFeCuCoNi + H O . (n = 3 independent biological\n2 2 2 2 2 2\nreplicates). Data are presented as means ± SD; ns, not significant; *p < 0.05, **p < 0.01, ***p <\n0.001, ****p < 0.0001; one-way ANOVA with multiple comparisons test; all tests were two-sided.\nSource data are provided as a Source Data file."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns.", "reviewer_2": "The authors have replied to all my question, made revisions according to my suggestions, and agreed to the publication.", "reviewer_3": "The authors have appropriately addressed the requests and concerns of the reviewers in a very detailed and thorough manner.\nPublication of the manuscript can be recommended in the present form.", "reviewer_4": "The very detailed revison from the authors have addressed all of my concerns. I recommend the manuscipt is accepted as it is."}, "author_response": ""}]

Decision letter