The synthesis method was improved on the basis of previous studies [33]. CuO nanoflowers were synthesized via a simple liquid-phase method, AgNO₃ solution was then added, and Ag-CuO nanozymes were finally successfully prepared (Fig. 2A). The morphology of the Ag-CuO nanozymes was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The diameter of the Ag-CuO nanozymes is approximately 460 nm, their size is uniform, and they have a flower-like morphology. The petals are composed of nanosheets (Fig. 2B to D). The elemental mapping images revealed that the Ag-CuO nanozymes were composed of Cu, O, and Ag and that Ag was uniformly loaded on the CuO nanoflowers (Fig. 2E). The energy-dispersive x-ray spectroscopy (EDS) spectra also show that the Ag-CuO nanozymes contain Cu, O, Ag, and other elements (Fig. S1). Furthermore, Ag-CuO nanozymes were observed via high-resolution TEM (HRTEM). The measured lattice spacing of 0.24 nm matches well with the plane of Ag (111), and the measured lattice spacing of 0.25 nm matches well with the plane of the monoclinic CuO phase (002). The selected area electron diffraction pattern also shows that the Ag-CuO nanozymes are polycrystalline (Fig. 2F and Fig. S2). The formation of Ag-CuO nanozymes was characterized by x-ray photoelectron spectroscopy (XPS). The results revealed obvious Cu 2p, Ag 3d, O 1 s, and C 1 s peaks in Ag-CuO nanozymes (Fig. 2G). The Cu 2p spectra show 4 peaks. The main peaks at 934.1 and 954.1 eV are attributed to 0Cu 2p_3/2 and Cu 2p_1/2, respectively. There is a band gap of 20 eV between the 2 peaks, which is consistent with previous studies [31]. The other 2 peaks are satellite peaks (Fig. 2H). Ag 3d has 2 peaks, one of which is Ag 3d_5/2 at 368.3 eV, and the other is Ag 3d_1/2 at 374.3 eV. There is a difference of 6 eV between the 2 peaks, which may be due to spin–orbit coupling (Fig. 2I). The O 1 s peak at 529.7 eV was attributed to the binding site between the Cu and O metals (Fig. 2J). Moreover, the crystal structures of the Ag-CuO nanozymes were analyzed via powder x-ray diffraction (XRD). The results show that the Ag-CuO nanozymes contain face-centered cubic (fcc) Ag (PDF No. 87-0720) and monoclinic CuO planes (PDF No. 04-3181) (Fig. 2K). The inductively coupled plasma optical emission spectrometry (ICP-OES) results revealed that the content of Cu was 79.56 WT% and that of Ag was 0.46 WT%. Through the above characterization techniques and results, Ag-CuO nanozymes were successfully synthesized.

The multienzyme activities of copper-based nanozymes have been verified [34,35]. To determine whether Ag doping affects the multienzyme activities of CuO, 3 probes, TMB (3,3′,5,5′-tetramethylbenzidine probe), OPD (1,2-bbenzylenediamine), and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), were used to detect the multienzyme activities (Fig. 3A). Moreover, the release ability of the metal ions from the Ag-CuO nanozymes at different pH values was detected via ICP. The antibacterial mechanism of Ag-CuO nanozymes can be better explained by fully characterizing the various enzyme activities of Ag-CuO nanozymes.

The color reaction of TMB oxidation is mainly due to the presence of benzidine, which contains 2 easily oxidized amino groups and can be oxidized into blue products by single-electron and 2-electron pathways. The oxidation process of benzidine is divided into 2 steps. The 371- and 652-nm bands are charge transfer complexes composed of diamine (TMB) as the donor and diimine ion (TMB²⁺) as the acceptor. Therefore, if Ag-CuO nanozymes have peroxidase activity (POD), they will catalyze hydrogen peroxide to form OH· under acidic conditions (pH = 5.5), and the reaction between OH· and TMB will produce blue ox-TMB. By setting different grouping control variables, we finally verified that Ag-CuO nanozymes can catalyze the oxidation of hydrogen to OH· under acidic conditions (Fig. 3B). With increasing hydrogen peroxide or Ag-CuO concentration, the absorption peak at 652 nm gradually increased (Fig. 3C and D), which indicated that Ag-CuO nanozymes have superior POD-like activity. Electron spin resonance spectroscopy with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trap was performed to confirm the generation of hydroxyl radicals (OH·) in the Ag-CuO nanozyme + H₂O₂ system. As shown in Fig. S3, a characteristic multiline EPR signal was detected in the Ag-CuO + H₂O₂ group, which was primarily assigned to the DMPO-OH adduct (the specific spin adduct of OH·), with a minor contribution from the DMPO-OOH adduct (the spin adduct of superoxide anion, O₂·⁻). These results directly demonstrated that Ag-CuO nanozymes could efficiently catalyze the decomposition of H₂O₂ to generate OH·, which served as the core bactericidal ROS in the synergistic antibacterial cascade.

OPD is a peroxidase substrate. After being oxidized, it will produce orange–brown ox-OPD, which can be read by a spectrophotometer at 435 nm. We also tested different concentrations of hydrogen peroxide or Ag-CuO nanozymes to verify that Ag-CuO nanozymes have superior POD-like activity (Fig. 3F and G). Interestingly, when different grouping control variables were used, even without the addition of hydrogen peroxide, the Ag-CuO nanozymes themselves would oxidize OPD to orange–brown ox-OPD (Fig. 3E), indicating that they also had OXD-like activity.

As a tripeptide, glutathione has a γ-amide bond and a sulfhydryl group, occurring in both reduced (GSH) and oxidized (GSSG) forms [36]. GSH has antioxidant effects. To detect whether Ag-CuO nanozymes can consume glutathione and reduce the removal of ROS by bacteria, a DTNB probe was used. DTNB reacts with GSH to produce orange–yellow TNB, which can be read by a spectrophotometer at 412 nm. The results show that GSH is gradually consumed with increasing time, and the time consumption becomes shorter with increasing Ag-CuO nanozyme concentration. When the concentration of Ag-CuO nanozymes was 10 μg/ml, it was depleted in only 60 min, which indicates that Ag-CuO nanozymes have superior glutathione-like peroxidase (GSH-Px-like) activity (Fig. 3H and I).

The activity of metal nanozymes is closely related to the concentration of metal ions that are dissociated. Three different pH values were used to detect the release ability of the Ag-CuO nanozyme metal ions. Compared with the other 2 pH values, the Ag-CuO nanozymes have the best ability to release Cu and Ag ions at pH = 5.5. At pH = 5.5, approximately 37% of the Cu ions and 12% of the Ag ions were released from the Ag-CuO nanozymes at 12 h (Fig. 3J and Fig. S4).

The above experimental results show that Ag-CuO nanozymes have excellent metal ion release ability at pH 5.5. It not only has OXD-like and POD-like activities to produce ROS but also has GSH-Px-like activity to reduce ROS clearance, resulting in the bidirectional accumulation of ROS, and has superior antibacterial potential.

The multienzyme activity of the Ag-CuO nanozymes was fully verified in vitro. To determine whether Ag-CuO nanozymes have superior bactericidal functions, plate tests and bacterial growth curves were used for verification. Although hydrogen peroxide is a commonly used disinfectant for treating wound infection in the clinic, when its concentration is high, it often causes oxidative damage to normal cells and affects wound healing [37]. On the one hand, a low dose of hydrogen peroxide (100 μM) can be used as an exogenous oxide donor; on the other hand, it will not cause damage to cells at a very low concentration, thus ensuring safety. The infection of burn wounds is often not caused by a single strain [38], so 2 gram-positive bacteria, MRSA and E. faecalis, and 2 gram-negative bacteria, P. aeruginosa and E. coli, were selected to test the antibacterial activity of the Ag-CuO nanozymes.

The results of the plate test revealed that the Ag-CuO nanozymes had antibacterial activity against 4 kinds of bacteria (Fig. 4A, C, E, and G). Compared with the control, exogenous hydrogen peroxide (100 μM) alone had no effect on the bacteria. In the group without hydrogen peroxide, the number of bacteria decreased gradually with increasing Ag-CuO nanozyme concentration, which was accompanied by a concentration dependence. However, after the addition of exogenous hydrogen peroxide, the bactericidal efficacy of the Ag-CuO group at the same concentration was markedly better than that of the group without hydrogen peroxide, which may be related to the POD-like activity of the Ag-CuO nanozymes (Fig. 4B, D, F, and H).

The optimum concentration of antibacterial agent was 8 μg/ml according to the plate test, so the growth inhibition curves of the 4 kinds of bacteria were generated by setting different groups. The results also revealed that the addition of exogenous hydrogen peroxide (100 μM) alone had no effect on bacteria. Compared with AgNO₃ or CuO alone, the Ag-CuO group presented better antibacterial effects, but after exogenous hydrogen peroxide was added, the effect was qualitatively improved (Fig. 4I to L). The above experimental results show that Ag-CuO nanozymes have excellent broad-spectrum antibacterial properties in vitro and superior prospects in the treatment of burn-infected wounds.

Burn wound infection is usually caused by mixed infection with many pathogens, and there are interactions between different kinds of bacteria [39]. Although E. faecalis is not the most common pathogen, it can increase the release of virulence factors and transmit drug resistance genes to other bacteria, and its cell wall is thick, resulting in inherent resistance to many antibiotics. If it progresses to VRE, it threatens the life of the body [40]. Therefore, this study focused on the antibacterial activity of E. faecalis. The killing effect of Ag-CuO nanozymes on E. faecalis was evaluated via plate tests, live and dead fluorescence staining, SEM, ROS fluorescence staining, flow cytometry, and lipid peroxidation (LPO) fluorescence staining. In previous work, different concentrations of Ag-CuO nanozymes were used for plate tests, so the best concentration of 8 μg/ml was used for the following experiments, and the effects of the Ag-CuO nanozymes were better than those of AgNO₃ or CuO according to the bacterial growth curve. To reduce the experimental cost, the experimental groups used were the control, H₂O₂, Ag-CuO, and Ag-CuO + H₂O₂ groups.

The results of the plate test revealed that Ag-CuO nanozymes alone showed germicidal ability, but at the same concentration, the Ag-CuO + H₂O₂ group had the best germicidal effect (Fig. 5A and B), which corresponded to the above results. A live bacteria/dead bacteria staining kit (N01/PI) can reliably stain and quantitatively distinguish live bacteria from dead bacteria within a few minutes via differences in the integrity of the bacterial cell membrane. The living bacteria staining probe (N01) is a green fluorescent-labeled living bacteria probe that can penetrate the plasma membrane with the complete structure of living bacteria. The dead bacteria staining probe with a damaged membrane is PI, which is a red fluorescent nucleic acid staining agent that can penetrate only dead bacteria with damaged membranes. Live/dead fluorescence staining revealed a large amount of green fluorescence and red fluorescence in the Ag-CuO group via confocal microscopy, which indicated that Ag-CuO nanozymes could dissociate Ag⁺ and Cu²⁺ and destroy the cell membrane of bacteria in a weakly acidic environment; however, the effect was limited, and the survival rate of bacteria in the Ag-CuO nanozymes group was 56.64% that of the control group. After H₂O₂ was added, almost all the bacteria emitted red fluorescence under a confocal microscope, and the survival rate of the bacteria in the H₂O₂-treated group was only 1.03% of that in the control group. We speculated that Ag-CuO nanozymes could catalyze low concentrations of H₂O₂ and produce more OH· to kill bacteria in weakly acidic bacterial environments (Fig. 5C and D). To further determine how the Ag-CuO nanozymes interact with bacteria directly, SEM was used to determine that the Ag-CuO nanozymes tightly adhered to the surface of the bacterial cell membrane and destroyed the bacterial cell membrane and that damage to the bacterial cell membrane clearly increased after the addition of H₂O₂ (Fig. 5E). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) exhibits no fluorescence and is membrane permeable. Upon their cellular uptake, esterases metabolize them into DCFH. ROS in cells can oxidize nonfluorescent DCFH to produce green DCF. To validate the hypothesis that Ag-CuO nanozymes can catalyze the production of more OH· at low concentrations of H₂O₂ in weakly acidic bacteria, we evaluated the ROS production ability via ROS fluorescence staining and flow cytometry. The results of confocal microscopy revealed that the Ag-CuO nanozymes produced sparse green light, whereas the Ag-CuO + H₂O₂ group produced more green light, which was approximately 1.4 times greater than that of the control group (Fig. 5F and G), and the results of the flow experiments were consistent with these findings (Fig. 5H). LPO is a key change in many pathological states [41]. Under conditions of diminished bacterial antioxidant capacity, peroxidative damage occurs to phospholipids containing unsaturated long-chain fatty acids in cellular and organelle membranes, ultimately leading to membrane disruption. To further evaluate the effects of Ag-CuO nanozymes on LPO damage to bacteria, we performed BODIPY 581/591 C11 fluorescence staining. The fluorescence confocal microscopy results revealed that the control and H₂O₂ groups were red, which is a reduced product, whereas the Ag-CuO nanozymes group produced sparse green light, and almost all the Ag-CuO + H₂O₂ groups produced green lipid peroxides, which were approximately 79.69% of those of the control group (Fig. 5I and J). The results indicated that the antioxidant capacity of the Ag-CuO + H₂O₂ treatment decreased markedly, which may be related to the GSH-Px-like activity of the Ag-CuO nanozymes.

The above experimental results show that Ag-CuO nanozymes can adhere to the bacterial membrane and destroy the bacterial membrane structure through ion release and the generation of ROS through the activities of multiple enzymes. Moreover, GSH consumption promotes the collapse of the bacterial antioxidant system, resulting in bacterial death.

The killing effect of Ag-CuO nanozymes on E. faecalis has been verified, but the bacteria present in burn wounds are not single, and E. faecalis often exists in the form of biofilms [42]. The formation of biofilms can be roughly divided into the following stages: initial adhesion, stable adhesion, early maturity, maturity, and separation and diffusion. After the formation of biofilms, the drug resistance of bacteria markedly improves, which leads to difficulty in curing and prolonging infection and further aggravates the economic and psychological burdens of patients [43]. Because the characteristics of biofilms cause great problems in clinical treatment, targeted therapy for biofilm infection is particularly important [44]. In this study, we used crystal violet staining, confocal fluorescence staining, and SEM to verify the effects of Ag-CuO nanozymes on the inhibition or destruction of biofilm formation.

Crystal violet staining revealed that Ag-CuO nanozymes alone inhibited and disrupted the formation of biofilms to some extent, whereas H₂O₂ addition markedly enhanced the inhibition and destruction of biofilms. Interestingly, we found that after biofilm formation, when the same concentration of drug was used, the effect of destruction was significantly less pronounced than the effect of inhibition, which corresponds to the clinical resistance of bacteria after biofilm formation (Fig. 6A to C). Similarly, we used confocal fluorescence staining to observe the planar and 3-dimensional structure of the biofilm, and the results were consistent with those of crystal violet staining (Fig. 6D and E). The structure of the biofilm was further observed via SEM. The results revealed that the biofilms of the control and H₂O₂ groups were very compact, and the bacteria presented obvious adhesion proteins and hyphae. In the Ag-CuO + H₂O₂ group, obvious biofilm destruction and residual damaged bacteria were observed (Fig. 6F). On the basis of the above results, Ag-CuO nanozymes have superior effects on both the inhibition of biofilm formation and the destruction of E. faecalis biofilms and have good potential for burn-infected wounds.

A full study of antibacterial mechanisms will provide a key scientific basis and technological breakthrough for the research and development of new targeted therapeutic drugs [45]. Therefore, we analyzed the transcriptome sequences of the control group and the Ag-CuO + H₂O₂ group to further explore the killing mechanism of Ag-CuO nanozymes on E. faecalis. Differentially expressed genes (DEGs) thermography revealed that there was a markedly difference in gene set enrichment between the 2 groups (Fig. 7A). The results of the volcano map revealed that 418 genes were down-regulated and that 401 genes were up-regulated in E. faecalis treated with Ag-CuO + H₂O₂ (Fig. 7B). The circular thermogram revealed representative genes with obvious differences, such as the ptsP, atpF, atpA, pfkA, and lpdA genes (Fig. 7C). Gene Ontology (GO) enrichment analysis revealed that the DEGs were enriched mainly in carbohydrate transmembrane transporter activity, lipopolysaccharide metabolic process, carbohydrate transmembrane transport, ATP biosynthesis process, acetyl-CoA carboxylase complex, and other pathways (Fig. 7D). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the down-regulated DEGs were enriched mainly in the PTS, fructose, and mannose metabolism and tricarboxylic acid (TCA) cycle pathways, accompanied by the down-regulation of key genes such as manX/Y/Z, fruA/B/K, and lpdA (Fig. 7E). Gene set enrichment analysis (GSEA) revealed that after Ag-CuO + H₂O₂ treatment, carbohydrate transmembrane transport and fructose and mannose metabolism pathways were down-regulated, whereas the oxidative phosphorylation pathway was up-regulated (Fig. 7F to H).

After the analysis of the above transcriptome sequencing results, we preliminarily proposed that the killing mechanism of Ag-CuO nanozymes against E. faecalis is as follows: Ag-CuO nanozymes not only have various enzyme activities to accumulate ROS for sterilization but can also cooperate with the antibacterial activity of the starvation-cuproptosis-like death cascade (Fig. 8A).

First, the POD-like, OXD-like, and GSH-Px-like activities of the Ag-CuO nanozymes were verified via various probe experiments and confocal fluorescence staining. It can produce ROS and reduce ROS clearance, thus destroying the bacterial cell membrane and causing LPO damage.

Second, PTS exists in bacteria, fungi, and some archaea, and the basic composition of PTS in the studied species is similar. PTS, which is the key system of cell growth and biofilm formation and can mediate virulence regulation and the stress response, mediates glucose transport and phosphorylation. Interestingly, microorganisms have acquired a self-regulatory mechanism in the evolutionary process to adapt to survive. For example, as long as glucose, fructose or sucrose exist in large quantities in the growth medium, it inhibits the synthesis of enzymes necessary for the transportation and metabolism of nonpreferred carbon sources. This phenomenon is called carbon catabolite inhibition [46]. Therefore, the fructose and mannose metabolism pathways are the preferred carbon sources for E. faecalis. After treatment with Ag-CuO + H₂O₂, the decrease in genes related to this pathway affected carbon uptake, ATP synthesis, and tolerance to external stimuli. In other words, Ag-CuO nanozymes down-regulate the PTS regulated by manX/Y/Z and fruA/B/K genes, reduce the carbohydrate intake required for energy synthesis, down-regulate the TCA cycle, and reduce ATP synthesis, thus inhibiting their own protection against external stimuli. This process is called starvation antibacterial activity [47,48]. RT-qPCR was used to validate the manX/Y/Z and fruA/B/K gene pairs (Fig. 8B and C), and ATP synthesis decreased after Ag-CuO treatment and was more obviously inhibited after the addition of exogenous H₂O₂ (Fig. 8E).

Finally, owing to the destruction of the bacterial cell membrane by metal ions released by Ag-CuO nanozymes, especially the interaction between Ag ions and -SH groups on the membrane [49] and the inability of bacterial starvation to resist external stimuli, Cu²⁺ influx and Cu⁺ accumulation increased, thus amplifying the cuproptosis-like death characteristics of bacteria. LpdA is a key protein in the TCA cycle that serves as the E3 component of the α-ketoglutarate dehydrogenase complex and connects to the mitochondrial electron transport chain [50]. Mutation of the lpdA gene causes excessive accumulation of Cu⁺ in cells, which induces copper death [51]. We detected the key gene lpdA of cuproptosis-like death via RT-qPCR and confirmed that the lpdA gene level was down-regulated after Ag-CuO + H₂O₂ treatment, that is, the occurrence and existence of cuproptosis-like death (Fig. 8D).

To determine which component plays a role in promoting cuproptosis-like death, we set up 6 groups to detect Cu⁺ in bacterial cells. The results showed that CuO alone could cause partial Cu⁺ accumulation, while the Ag-CuO nanozymes had a better effect than CuO, indicating that the existence of Ag⁺ promoted Cu⁺ accumulation, and the amount of Cu⁺ was greater in the Ag-CuO + H₂O₂ group, which proved that Ag⁺ and CuO cooperated with antibacterial activity (Fig. 8F and G). Specifically, Ag⁺ can disrupt the integrity of bacterial cell membranes, thereby facilitating the rapid entry of Cu²⁺ into bacterial cells and accelerating its reduction to Cu⁺. Moreover, the catalytic activity of CuO nanozymes can be enhanced by Ag⁺, which further promotes the production of ROS and synergistically amplifies the antibacterial effect with Cu⁺-mediated cuproptosis-like death.

The above experimental results show that Ag-CuO nanozymes can not only accumulate the antibacterial activity of ROS but also down-regulate genes (manX/Y/Z and fruA/B/K) to regulate the PTS transferase system, which reduces the protection of the bacteria itself, promotes an increase in Cu²⁺ influx and Cu⁺ accumulation, and amplifies the cuproptosis-like death of bacteria (down-regulation of the lpdA gene). Previous studies have developed diverse antibacterial nanozymes, including Ru single-atom catalysts with NIR-II-triggered oxidase-peroxidase cascades, trimetallic Pt-PdAu alloy nanozymes, and monometallic Cu-based nanozymes that induce bacterial cuproptosis-like death [52–54]. Among them, Ru single-atom nanozymes mainly rely on photothermally enhanced ROS generation to kill bacteria, while trimetallic Pt-Pd-Au nanozymes achieve synergistic sterilization through multicomponent ROS cascade amplification. Although the latest Cu-based nanozymes have proposed the concept of cuproptosis-like death in bacteria, they only focus on single copper-mediated metabolic damage and lack the intervention of bacterial energy metabolism and synergistic effects of other metals. Compared with single-metal or single-pathway antibacterial nanozymes, Ag-CuO nanozymes integrate oxidative stress, metabolic starvation, and death-like cuproptosis into a continuous, self-enhanced amplification effect. This design not only improves the sterilization efficiency of drug-resistant bacteria but also fundamentally reduces the possibility of bacterial resistance. This new antibacterial mechanism of ROS starvation/cuproptosis could provide new ideas for the treatment of burn-infected wounds.

Burn wounds are generally divided into 3 degrees according to depth, and a 3-degree quartering method (I degree, shallow II degree, deep II degree, and III degree) is adopted. The reticular layer of the dermis in deep degree II scalding is necrotic, forming exudates rich in protein, which easily becomes bacterial culture medium [55]. To simulate deep II degree burn wounds in the clinic, we used a metal probe at 90 °C to contact the back skin of the mice for 8 s, thus establishing deep II degree burn wounds in the mice. After 24 h, the necrotic tissue was removed, and 20 μl of the standard suspension of E. faecalis (concentration of 1 × 10⁶ CFU/ml) was gently scraped and smeared on the scald area for 1 min until the bacterial mixture was absorbed, simulating common bacterial infection after burn. At present, many reports on MRSA and P. aeruginosa infection in burn wounds have been published, and the mechanism involves long-term inflammation and inability of the wound to heal. There are few studies on the mechanism by which E. faecalis causes burn wound healing, so we established 5 groups: the blank group (burn), the control group (burn + infection), the H₂O₂ treatment group (burn + infection + H₂O₂), the Ag-CuO treatment group (burn + infection + Ag-CuO), and the Ag-CuO + H₂O₂ treatment group (burn + infection + Ag-CuO + H₂O₂). Wound healing was evaluated by the wound healing rate, wound colony formation, hematoxylin and eosin (H&E) staining, and Masson’s trichrome staining. Inflammation- and extracellular matrix-related indicators were detected by immunofluorescence (Fig. 9A).

The results showed that by strictly controlling the intensity, time, and temperature, the scalding depth of all the wounds was deep II. According to the photos and wound size data, on the 14th day, the healing rate of the control group was only 71.1%, that of the Ag-CuO treatment group was 84.4%, and that of the Ag-CuO + H₂O₂ treatment group was 94.0%, which was close to that of the blank group (95.4%). Compared with the blank group, the control group healed more slowly after E. faecalis infection. Viscosity exudation and slight redness were observed in the wounds of the control group and H₂O₂ group. Ag-CuO nanozymes alone can accelerate wound healing, but the Ag-CuO + H₂O₂ treatment has the best effect, which may be due to the chemical kinetics effect of the Ag-CuO nanozymes combined with H₂O₂ (Fig. 9B and C). On the 14th day, secretions from the wound surface were coated. The results revealed that there were more bacteria in the control group and H₂O₂ group than in the other groups, and the Ag-CuO + H₂O₂ treatment had the greatest effect (Fig. 9D and E).

To further evaluate the histology of the wounds, we used H&E and Masson staining. H&E staining revealed that, compared with the blank group, the control group and the H₂O₂ group exhibited markedly inflammatory cell infiltration and epithelial rupture around the wound. After Ag-CuO + H₂O₂ treatment, the number of inflammatory cells in the wound decreased significantly, and the regeneration of skin appendages such as hair follicles and blood vessels was obvious (Fig. 9F and Fig. S6). Compared with those in the blank group, the number of collagen fibers around the wounds in the control group and H₂O₂ group decreased significantly, and the degree of collagen fiber deposition in the wounds increased significantly after Ag-CuO + H₂O₂ treatment (Fig. 9G and Fig. S7). To further verify that Ag-CuO nanozymes can reduce wound inflammation, promote angiogenesis, and remodel the extracellular matrix, we evaluated these effects via immunofluorescence staining. Compared with those in the blank group, the levels of IL-1β, IL-6, and TNF-α around the wounds in the control group and H₂O₂ group increased significantly, whereas the levels of inflammatory factors in the wounds decreased after Ag-CuO nanozyme treatment, and the effect in the Ag-CuO + H₂O₂ treatment group was the greatest (Fig. 10A to C and G to I). Compared with the blank group, the control group and H₂O₂ group presented less angiogenesis and increased angiogenesis after Ag-CuO nanozyme treatment, which may be related to the effect of Cu²⁺ on angiogenesis, whereas the Ag-CuO + H₂O₂ treatment group presented the greatest effect because inflammation was relieved (Fig. 10D and J). COL I is the most abundant collagen protein in the human body and the main mechanical supporting component of the ECM, which provides a structural framework for fibroblast migration and proliferation [56]. COL I can promote the formation of granulation tissue, and if its synthesis is reduced, it will lead to delayed healing and a fragile wound surface. Fibronectin (FN) and fibrin form a “temporary ECM” in early wounds, which guides the location of repair cells, provides a template for the deposition of COL I and COL III, and affects fiber arrangement [57]. Compared with those in the blank group, the levels of COL I and FN in the control group and H₂O₂ group decreased significantly, whereas the levels of COL I and FN increased significantly after Ag-CuO + H₂O₂ treatment, which may be related to the function of E. faecalis as a matrix metalloproteinase (MMP) [58] (Fig. 10E, F, K, and L).

To further explore the mechanism by which E. faecalis causes slow healing of burn wounds and by which Ag-CuO nanozymes promote wound healing, we selected fresh wound samples for transcriptome sequencing. The blank and control groups and the control and Ag-CuO + H₂O₂ groups were compared for comparative analysis. DEG thermography revealed that there was a significant difference in gene set enrichment between the blank group and the control group (Fig. 11A). After treatment with Ag-CuO + H₂O₂, the Ag-CuO + H₂O₂ group and the control group also presented significant differences in gene set enrichment (Fig. 11D). The results of the volcano map revealed that, compared with those of simple burn wound samples, the sequencing of wound tissue samples infected with E. faecalis resulted in the down-regulation of 2,137 genes and the up-regulation of 1,325 genes (Fig. 11B). After treatment with Ag-CuO + H₂O₂, 264 genes were down-regulated, and 1,092 genes were up-regulated (Fig. 11C). GO enrichment analysis revealed that, compared with those in the blank group, the down-regulated DEGs were enriched mainly in the immune response, defense response, and collagen-containing extracellular matrix (Fig. 11E). KEGG enrichment analysis revealed that the cytokine–cytokine receptor interaction and ECM–receptor interaction signaling pathways were down-regulated in the control group compared with those in the blank group (Fig. S5). Compared with those in the control group, the genes whose expression was up-regulated in the Ag-CuO + H₂O₂ group were enriched mainly in cell adhesion molecules, the extracellular matrix, and the cytokine signaling pathway (Fig. 11F). GSEA revealed that after treatment with Ag-CuO + H₂O₂, the granulocyte migration, angiogenesis, and cytokine interaction pathways were up-regulated (Fig. 11, G to I), which was consistent with the immunofluorescence results.

On the basis of the above experimental results, we preliminarily determined that once a burn wound is infected with E. faecalis, E. faecalis acts as an MMP after it forms a biofilm on the wound, which overwhelms the collagen and fibronectin needed for wound healing, making healing difficult. In addition, the immune escape of E. faecalis makes the body unable to effectively remove bacteria, resulting in an inflammatory state for a long period of time [59]. After Ag-CuO nanozyme treatment, it can effectively clear the bacterial biofilm of infected wounds, restore inflammatory homeostasis and eliminate the state of overdecomposition of proteins, thus remodeling the ECM [60]. In addition, the Ag-CuO nanozymes effectively promoted vascular growth and accelerated wound healing (Fig. 11J).

Owing to ethical limitations and the particularity of burn wounds, it is difficult to carry out human experiments to understand the antibacterial activity of Ag-CuO nanozymes in the human body and their ability to promote burn wound healing. We conducted experiments in mice to preliminarily verify its effectiveness. However, the mouse epidermis is thinner, the human epidermis is thicker (multiple layers, especially the stratum corneum, are more developed), and the density of collagen and accessory structures (such as hair follicles and sebaceous glands) in the mouse dermis is different from that in humans, which may affect the speed of wound healing. The limitations of these studies cannot be ignored. The thickness of the epidermis and dermis (especially the back skin) of New Zealand rabbit skin is between that of mouse skin and human skin, which is closer to that of some parts of human skin (such as limbs) and is suitable for simulating II degree scald skin. The healing cycle (2 to 3 weeks) of some deep scalds (such as those with a depth of II degrees) is similar to that of humans. Therefore, we established a New Zealand rabbit scald infection model to further verify the antibacterial activity of Ag-CuO nanozymes in vivo and their ability to promote burn wound healing (Fig. 12A).

The results revealed that the scalding depth of all the wounds was deep II degrees. According to the photos and wound size data, on the 14th day, the wound healing rate of the control group was only 74.3%, and that of the Ag-CuO + H₂O₂ treatment group was 95.4%, which was close to that of the blank group (95.2%). Unlike the wounds of the control group of New Zealand rabbits, the wounds of the control group of New Zealand rabbits presented obvious redness and exudation, which is very similar to the state of human skin infection. In the Ag-CuO + H₂O₂ treatment group, there was no obvious redness or swelling around the wound, and the wound was clean and free of exudate (Fig. 12B and C). On the 14th day, the secretions were removed from the wound surface and coated. The results revealed that there were more bacteria in the control group, accompanied by miscellaneous bacteria, which was consistent with the clinical mixed infection of the wound surface. The Ag-CuO + H₂O₂ treatment effectively eliminated bacteria, but some bacteria were still present (Fig. 12D and E). The results of H&E staining revealed that, compared with those in the blank group, there was obvious inflammatory cell infiltration around the wounds in the control group, but no obvious inflammatory cells were found in the wounds after Ag-CuO + H₂O₂ treatment, accompanied by angiogenesis (Fig. 12F and Fig. S8). Masson staining revealed that, compared with the control, Ag-CuO + H₂O₂ effectively promoted collagen deposition (Fig. 12G and Fig. S9). These results are consistent with those in mice. This series of progressive animal experiments (mice-New Zealand rabbits) will strengthen the verification results; that is, Ag-CuO nanozymes can not only eliminate bacteria and reduce inflammation but also promote angiogenesis and collagen deposition and accelerate wound healing.

Before the in vivo experiments, we tested the biosafety of these compounds using cell counting kit-8 (CCK8) and a hemolysis assay in human umbilical vein endothelial cells (HUVECs). CCK8 experiments revealed that when the concentration of Ag-CuO nanozymes was 10 μg/ml, the cell activity was 91.6% of that of the control group; when the concentration of Ag-CuO nanozymes was 40 μg/ml, the cell activity was 84.2% of that of the control group (Figs. S10 and S11). The results of the hemolysis experiments revealed that when the concentration of Ag-CuO nanozymes was less than 30 μg/ml, the hemolysis rate was less than 5%. According to in vitro probe efficacy detection and antibacterial tests, our selected concentration (8 μg/ml) is within the safe range. On the 14th day of the experiment in vivo, we performed H&E staining on the heart, liver, spleen, lung, and kidney of all groups of mice, and the results revealed no obvious lesions (Fig. S12). The routine blood and biochemical indices of the mice were all within the normal range (Figs. S13 and S14). The above results indicated that 8 μg/ml Ag-CuO nanozymes had good biosafety in vivo. The negligible in vivo toxicity can be attributed to the rational dosage selection within the safety threshold, the pH-dependent catalytic behavior that confines ROS generation mainly to acidic infectious microenvironments rather than healthy tissues, and the good biocompatibility and nonaccumulative nature of the Ag-CuO nanozymes in major organs.

The efficacy of Ag-CuO nanozymes in the treatment of infected wounds after burn injury has been verified by the above experimental results. To further explore the feasibility of its clinical application, we comprehensively compared its efficacy with that of clinical medication through in vivo and in vitro experiments. This research has focused mainly on the biological safety, minimum inhibitory concentration (MIC), drug resistance, and treatment effect of drug-resistant bacterial infection and other aspects of comprehensive evaluation.

Resazurin staining for the determination of the MIC is a widely used and very classical microbiological method. The core of this principle is to detect the metabolic activity of bacteria by using resazurin as a redox indicator. We incubated 100 μl of the negative control group (phosphate-buffered saline [PBS]), positive control group (vancomycin [Van], 1 mg/ml), or drug control group (equal gradient concentration) with 100 μl of the E. faecalis standard strain (optical density [OD] = 0.1) for 18 h and then incubated the mixture with resazurin staining solution for 2 h. The results revealed that the MICs of the Ag-CuO nanozymes, Van, and the hydrofiber dressing with silver (Hydrofiber@Ag) were 8, 2, and 5 μg/ml, respectively (Fig. S15). Among the 3 drugs, Van has the lowest dosage and the least toxicity to cells. Even when the concentration of Van was 256 μg/ml, the toxicity and side effects on cells were not obvious, which may be related to the bactericidal mechanism by which Van inhibits the synthesis of the bacterial cell wall (Figs. S16 and S17).

To detect the ability of drugs to kill drug-resistant bacteria, we first cultured VRE (256 μg/ml) through 16 days of rapid screening and then established an antibacterial experimental model to verify its efficacy. The results of the inhibition zone revealed that Van at a mass of 32 μg had no obvious inhibitory effect on VRE, which met the international general standards and verified the successful establishment of VRE strains. The Ag-CuO + H₂O₂ mixture at equal concentrations had excellent inhibitory effects on VRE. When the mass of Ag-CuO was 32 μg, the average antibacterial diameter of VRE was 1.9 cm, and VRE was sensitive to the Ag-CuO + H₂O₂ group. To compare the effects of Ag-CuO and Hydrofiber@Ag more accurately, we controlled the silver ion content to compare the bacteriostatic ring effect (the mass ratio of Ag in Hydrofiber@Ag was 1.2%). The results showed that the Ag-CuO + H₂O₂ group had better antibacterial effects than the Hydrofiber@Ag group at the same silver ion content (Fig. S18). To more intuitively reflect the killing effect of the 3 drugs on VRE, we continuously sampled the incubated bacterial solution within 120 min and then coated the plate to observe bacterial growth. The results showed that Van had no obvious killing effect on VRE. The Ag-CuO + H₂O₂ group and the Hydrofiber@Ag group with the same content of silver ions showed excellent killing effects. Among them, the Ag-CuO + H₂O₂ group had the best effect and almost completely killed the VRE at 60 min (Fig. 13A and B). In addition, to further compare the resistance of standard strains of E. faecalis caused by the 3 drugs, we tested the MIC by screening Van-resistant E. faecalis strains at equal concentrations. Even when the resistance concentration of E. faecalis to Van reached 256 μg/ml, Ag-CuO and Hydrofiber@Ag still killed E. faecalis, and the fold change in the MIC was within 5 (Fig. 13C). The unique bactericidal mechanism of Ag-CuO and hydrofibers@Ag makes bacterial resistance difficult.

To compare the efficacy of the 3 drugs in vivo, we selected BALB/c mice to establish a burn wound model with VRE infection. The results showed that the Van group had no obvious effect on promoting wound healing in vivo, and there was no significant difference in wound healing time between the Van group and the PBS group. Compared with the PBS and Van groups, the Ag-CuO + H₂O₂ and Hydrofiber@Ag groups presented excellent wound healing properties. At the same silver ion content, the plants in the Ag-CuO + H₂O₂ group healed faster than those in the Hydrofiber@Ag group on days 4, 7 and 10, but both groups completed healing on day 14. This may be due to the role of sodium carboxymethyl cellulose in Hydrofiber@Ag, which quickly completes epithelization during the remodeling phase (Fig. 13D and F). On the fourth day, the antibacterial effect of the Ag-CuO + H₂O₂ group was greater than that of the Hydrofiber@Ag group (Fig. 13E and G). The results of HE and Masson staining revealed that the PBS group and the Van group contained many inflammatory cells, the granulation tissue was missing, and the degree of collagen synthesis and deposition was insufficient. Complete epithelial regeneration was observed in the Ag-CuO + H₂O₂ and Hydrofiber@Ag groups, without continuous or massive inflammatory cell infiltration. Extensive, bright blue-stained areas were observed in the wound area, indicating that a large amount of collagen was synthesized and deposited (Fig. 13H and I and Figs. S19 and S20). In particular, the antiangiogenic effect of Ag-CuO + H₂O₂ was greater than that of the Hydrofiber@Ag group, which may be related to the role of copper ions in Ag-CuO (Fig. 13J to L). In addition, the organs of the mice treated with the drug for 2 weeks were examined, and the results revealed that the 3 drugs had no obvious toxicity or side effects in the short term (Fig. S21).

The above experimental results show that although Van has high biosafety and is effective for standard strains, it easily induces drug resistance, resulting in its ineffectiveness for VRE, and systemic medication has difficulty reaching an effective concentration in wounds, so it is not suitable for local wound treatment. With the same silver content, the bactericidal speed and efficacy of the Ag-CuO nanozyme are better than those of Hydrofiber@Ag, and it is not easy to resist and has the unique advantage of promoting angiogenesis. However, it is difficult for simple nanoparticles to effectively remain in wounds. Although the direct bactericidal ability of Hydrofiber@Ag in clinical application is slightly worse than that of Ag-CuO, its carboxymethylcellulose sodium matrix can provide an ideal moist environment for wound epithelization, and the comprehensive healing effect is excellent. Therefore, if Ag-CuO nanozymes are combined with advanced dressing matrices such as hydrofibers, the development of next-generation wound dressings with strong antibacterial, nonresistant, and active healing functions is expected [61–63]. Such composite dressings can not only effectively address the clinical challenges of drug-resistant bacterial infections in wounds but also avoid the side effects caused by excessive use of antibiotics or traditional silver-based dressings. Moreover, the unique dual-metal synergistic effect of Ag-CuO nanozymes can maintain long-term antibacterial activity while promoting wound tissue regeneration, making them highly suitable for the treatment of various difficult-to-heal wounds, such as burn infections and diabetic ulcers.

The synthesis was initiated by dissolving CuCl₂·H₂O (1.3400 g) and PEG (1.0000 g) in 30 ml of distilled water, followed by the addition of 2 ml of hydrogen peroxide under vigorous stirring for 20 min. A 30-ml aliquot of 2 M NaOH solution was then introduced dropwise over a 30-min period. After the reaction mixture was heated to 90 °C for 30 min, 50 ml of AgNO₃ solution (0.1 g in water) was added dropwise with continuous stirring. The resulting precipitate was isolated by centrifugation at 18,000 rpm for 30 min, subjected to 3 washing cycles with deionized water and ethanol under ultrasonication, and finally vacuum-dried at 60 °C for 12 h [33].

The morphology and particle size of the Ag-CuO nanozymes were observed via SEM (ZEISS Gemini SEM 300, Germany), TEM (Japan-JEOL-JEM 2100 F), and HRTEM. The lattice price was observed via XRD (Bruker, D8 Advanced Diffractometer) and selected area electron diffraction. The valence state and elemental analysis were carried out via XPS (USA—Thermo Fisher + ESCALAB Qxi). The elemental content was quantified via ICP (EXPEC 6500).

The POD-like and OXD-like activities of Ag-CuO nanozymes were detected with TMB and OPD probes. The concentrations of H₂O₂ (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM), Ag-CuO nanozymes (0, 2, 4, 6, 8, and 10 μg/ml), and different groups were determined. The pH of all the solutions was 5.5. The activity of GSH-Px-like was detected by a DTNB probe, and the consumption rates of the Ag-CuO nanozymes at 4 μg/ml were detected at different time points (0, 10, 30, 60, 120, 180, 240, and 300 min). When the concentration of the Ag-CuO nanozymes was 10 μg/ml, the consumption rates of the Ag-CuO nanozymes during different time periods (0, 5, 10, 30, and 60 min) were measured. The pH of all the solutions was 5.5, n = 3.

The data are presented as the means ± SDs from a minimum of 3 independent experimental replicates. For comparisons between 2 groups, Student t test was employed, whereas 1-way or 2-way analysis of variance with Tukey’s post hoc test was used for multiple group comparisons. Significance thresholds were set at *P < 0.05, **P < 0.01, and ***P < 0.001.