Abstract The rapid emergence of drug‐resistant bacteria has outpaced the development of traditional antibiotics, necessitating the exploration of more effective therapeutic strategies. In this study, the design of a Cu[7]S[4] multifunctional nanozyme, activated by near‐infrared (NIR) light is presented, that demonstrates enhanced antibacterial activity. Cu[7]S[4] is synthesized with varying defect structures by utilizing different templates, which substantially optimize its absorption to H[2]O[2] and lipopolysaccharides (LPS) molecules. This process generates an optimal electronic structure, producing efficient antibacterial activity through photodynamic and photothermal synergetic processes. Specifically, the Cu[7]S[4] nanozyme with dual defects (VCu and VCuCuCuSSS) exhibits peroxidase‐like (POD), catalase‐like (CAT), and GSH‐depletion properties, effectively inactivating drug‐resistant bacteria such as Pseudomonas aeruginosa. Notably, in a mouse wound model infected with P. aeruginosa, the nanozyme demonstrates significant antibacterial efficacy, promoting wound healing under NIR light. This multifunctional Cu[7]S[4] nanozyme presents a promising new strategy for combating drug‐resistant bacterial infections. Keywords: Cu[7]S[4] nanozyme, dual‐defect, efficient antibacterial, suitable electronic structure, synergetic process __________________________________________________________________ Cu[7]S[4] nanozymes, exhibiting peroxidase‐like, catalase‐like, and GSH‐depletion activities, are synthesized through defect engineering. These nanozymes demonstrate rapid inactivation of Gram‐negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In a mouse skin wound model infected with P. aeruginosa, the nanozyme showcases exceptional bactericidal effect. graphic file with name ADVS-12-e03793-g007.jpg 1. Introduction Due to the diminishing supply of therapeutics targeting multidrug‐resistant (MDR) pathogens, drug‐resistant bacterial infections pose a pressing and escalating global public health crisis.^[ [54]^1 ^] Notably, the emergence of antibiotic‐resistant strains has rendered many infections untreatable, contributing to the deaths of 1.27 million people worldwide in 2019.^[ [55]^2 ^] Between 2019 and 2020, deaths from antimicrobial resistance (AMR) infections in the United States alone increased 15%, and it is projected that AMR will cause 10 million deaths annually by 2050.^[ [56]^3 ^] However, the discovery of new antibiotics has become increasingly challenging, with development costs exceeding $500 million and timelines extending over a decade.^[ [57]^4 ^] Alarmingly, the effective use period of new antibiotics is often less than three years due to the rapid emergence of resistance.^[ [58]^5 ^] Therefore, the urgent need for alternative therapeutic methods is evident. Nanomaterials, with their unique properties and enzyme‐like catalytic activities, offer a promising alternative.^[ [59]^6 ^] For instance, peroxidase mimics can effectively decompose hydrogen peroxide (H[2]O[2]) into hydroxyl radicals (·OH),^[ [60]^7 ^] while catalase mimics decompose H[2]O[2] into oxygen (O[2]), alleviating hypoxia and providing reaction substrates for photodynamic therapy.^[ [61]^8 ^] Bacterial infections typically create a proinflammatory microenvironment characterized by low pH, hypoxia, and abundant H[2]O[2], providing an excellent response environment for the enzymatic activity of nanoenzymes.^[ [62]^9 ^] Furthermore, Glutathione (GSH) is an important antioxidant that removes free radicals under physiological conditions, protecting the sulfhydryl groups in the proteins and enzymes.^[ [63]^10 ^] Thus, multifunctional nanozymes with peroxidase‐like (POD), catalase‐like (CAT), and GSH‐depletion activities can generate abundant ROS to destroy biomolecules on which drug‐resistant bacteria depend. Additionally, Antibacterial photodynamic therapy (aPDT), an environmentally friendly and efficient technology, has been extensively explored for inactivating drug‐resistant bacteria. It utilizes specific wavelengths of light to stimulate photosensitive nanomaterials to convert H[2]O and O[2] into large amounts of toxic reactive oxygen species (ROS), which kill pathogenic microorganisms through oxidative damage to biomolecules (e.g., phospholipids, enzymes, proteins, and DNA).^[ [64]^11 ^] Due to its beneficial mechanism of action, aPDT exhibits broad‐spectrum performance and a low potential for resistance to pathogenic microorganisms. However, the effectiveness of this therapy depends critically on the properties of photocatalysts, which must penetrate the skin and reach infected tissues while ensuring the presence of sufficient O[2]. The activity of photocatalysts is influenced by their electronic structure,^[ [65]^12 ^] which depends on the band edge positions necessary for target chemical reactions.^[ [66]^13 ^] Defects in crystalline structures, such as vacancies, can significantly alter the mechanical, electrical, chemical, and thermal properties of nanomaterials.^[ [67]^14 ^] For example, sulfur vacancies in MoS[2] nanosheets induce room‐temperature ferromagnetism,^[ [68]^15 ^] while oxygen vacancies in NiCo[2]O[4]@Pd nanozymes enhance catalytic activity.^[ [69]^16 ^] However, synthesizing photocatalysts with multifunctional nanozyme properties remains a significant challenge due to the complex interplay of composition, size, morphology, exposed crystal facets, and surface area. Moreover, the antibacterial method of photothermal therapy (PTT) relies on the physical heat of nanomaterials to inactivate bacteria.^[ [70]^17 ^] According to Mie's theory, the plasmon band of spherical particles is attributed to the dipolar oscillations of the free electrons in the conduction band,^[ [71]^18 ^] apt electronic structure plays a pivotal role in the process. Nevertheless, although photothermal therapy to inactivate bacteria has drawn widespread attention, it remains an enormous challenge that PTT agents of design and synthesis take on favorable properties such as high photothermal conversion efficiencies, photostabilities, and biocompatibility to obtain effective inactivation. It is an ingenious strategy that a suitable electronic structure can modulate an apt photothermal process, leading to effective photodynamic and photothermal synergetic antibacterial. In this study, we designed and synthesized two types of Cu[7]S[4] nanoparticles via a solvothermal route, each possessing distinct dual‐defect structures: VCu and VCuCuCuSSS (Cu[7]S[4]‐1) or VCu and VCuCuCuCuCuS (Cu[7]S[4]‐2). The presence of these defects gives the Cu[7]S[4] nanoparticles peroxidase (POD), catalase (CAT), and glutathione (GSH) depletion activities as well as excellent photocatalytic performance, which not only generates a large amount of ROS to induce bacterial oxidative damage but also generates O[2] as a substrate for its photocatalysis, which makes up for the defect that general photocatalysts are limited by low oxygen content. Meanwhile, density‐functional theory (DFT) calculations and experimental results showed that Cu[7]S[4]‐1 has a better uptake capacity for H[2]O[2], especially lipopolysaccharide (LPS), compared with Cu[7]S[4]‐2, which is significantly different from other reported nanozymes. In addition, Cu[7]S[4]‐1 possessed photodynamic and photothermal synergistic effects, which enabled effective inactivation of drug‐resistant bacteria such as E. coli and P. aeruginosa. Furthermore, in vivo antibacterial assays using a mouse wound model infected with P. aeruginosa confirmed that Cu[7]S[4]‐1, assisted by NIR light, possesses exceptional antibacterial efficacy, providing a promising strategy for combating MDR bacterial infections. 2. Results and Discussion 2.1. Characterization of Cu[7]S[4] The two synthesized Cu[7]S[4] samples were thoroughly characterized. X‐ray diffraction (XRD) patterns revealed that the Cu[7]S[4] nanoparticles predominantly grew along the (224) facet (Figure [72] 1a; Figure [73]S1a, Supporting Information), exhibiting a well‐indexed orthorhombic phase (Pnma (62)) with lattice parameters: a = 7.0965 Å, b = 7.8223Å, c = 11.078 Å (JCPDS No. 33‐0489). The absence of impurities or additional phase peaks in the XRD patterns confirmed the purity of the samples. The elemental mapping demonstrating the presence and uniform distribution of Cu and S across the nanoparticle surfaces (Figure [74]1b,c; Figure [75]S1b,c,d, Supporting Information). Low‐magnification scanning electron microscopy (SEM) image revealed a characteristic small 2D morphology (Figure [76]1d; Figure [77]S1e, Supporting Information), which was corroborated by transmission electron microscopy (TEM) image (Figure [78]1e; Figure [79]S1f, Supporting Information), showing particle diameters of ≈30–50 nm. High‐resolution TEM (HRTEM) images displayed lattice spacings of 0.19 nm (Figure [80]1f), consistent with the XRD analysis. Figure 1. Figure 1 [81]Open in a new tab a) XRD pattern of Cu[7]S[4]‐1. b, c) mapping images of Cu[7]S[4]‐1. b) show S element. c) show Cu element, d) SEM images of Cu[7]S[4]‐1. e) TEM image of Cu[7]S[4]‐1. (f) lattice spacings of the image of Cu[7]S[4]‐1. g–i) XPS images of Cu[7]S[4]‐1. j) defect VCu image of Cu[7]S[4]‐1. k) defect VCuCuCuSSS image of Cu[7]S[4]‐1. l) defect VCuCuCuCuCuS image of Cu[7]S[4]‐2. X‐ray photoelectron spectroscopy (XPS) was employed to analyze the elemental composition and surface chemical states of the Cu[7]S[4] sample. The high‐resolution XPS spectra for Cu[7]S[4]‐1 (Figure [82]1g,h,i) showed Cu 2p[1/2] and Cu 2p[3/2] binding energy peaks at 951.9 and 931.9 eV, respectively. For both samples, S 2p[1/2] and S 2p[3/2] peaks appeared at 162.3 and 161.2 eV, respectively. Notably, the sample exhibited shifts to lower energies,^[ [83]^19 ^] attributable to electron enrichment caused by the presence of defects.^[ [84]^20 ^] Positron annihilation spectroscopy was used to characterize defects within the Cu[7]S[4] samples. The results indicated four‐lifetime components (Tables [85]S1 and [86]S2, Supporting Information) for each sample. The two longest components (τ[3] and τ[4]) were associated with positron annihilation in large defect clusters or interfaces.^[ [87]^21 ^] The shorter components for Cu[7]S[4]‐1 (τ[1] = 221.6 ps, τ[2] = 355.0 ps) were attributed to the VCu and VCuCuCuSSS defect (Figure [88]1j,k), with relative intensities of 29.1% and 67.5%, respectively.^[ [89]^22 ^] Similarly, for Cu[7]S[4]‐2, the shorter components (τ[1] = 226.0 ps, τ[2] = 374.0 ps) corresponded to VCu and VCuCuCuCuCuS defects (Figure [90]1l), with relative intensities of 34.5% and 59.6%, respectively. Both Cu[7]S[4] samples exhibited dual defects. To investigate the influence of defects on the physicochemical properties of Cu[7]S[4], we employed first‐principles calculations. Based on the intact Cu[7]S[4] (224), we constructed two structures with different defect species to compare their bactericidal capabilities, namely Cu[7]S[4]‐1 (contains V[Cu] and V[3Cu3S] ) and Cu[7]S[4]‐2 (contains V[Cu] and V[5Cu1S] ), which shown in Figure [91] 2a,b. Figure 2. Figure 2 [92]Open in a new tab a) the optimized geometries of Cu[7]S[4]‐1. b) the optimized geometries of Cu[7]S[4]‐2. c) the band structure and e) DOS of Cu[7]S[4]‐1. d) the band structure and f) DOS of Cu[7]S[4]‐2. g) the optimized geometries of O[2], H[2]O, LPS and H[2]O[2] adsorbed on Cu[7]S[4]‐1. h) the optimized geometries of O[2], H[2]O, LPS and H[2]O[2] adsorbed on Cu[7]S[4]‐2. The effect of promotion is assessed via the comparison of adsorption energies, calculated as: [MATH: Eads=E(adsorbate/slab)−E(slab)−E(adsorbate) :MATH] (1) Where E[(adsorbate/slab)] is the energy of the slab with bound adsorbate, E[(slab)] is the energy of the fully relaxed slab, and E[(adsorbate)] is the energy of the free adsorbate placed in an empty cell of the same dimensions as that used to define the slab model. The band structure and density of states (DOS) were further analyzed to study the electronic properties of the two catalysts. For Cu[7]S[4]‐1, the band gap was only 0.02 eV (Figure [93]2c) and for Cu[7]S[4]‐2, the band gap was further reduced (Figure [94]2d). From Figure [95]2d, the conduction band and valence band were crossing at the Fermi level, resulting in a band gap of almost 0 eV. For the plot of DOS (Figure [96]2e,f), the Fermi level is mainly contributed by the d orbital of Cu and the p orbital of S. Moreover, the d band center of Cu[7]S[4]‐1 is −2.08 eV which was closer to the Fermi level than that of Cu[7]S[4]‐2 (−2.10 eV). Therefore, it can be speculated that Cu[7]S[4]‐1 has better catalytic performance. The O[2] molecules were bound to the Cu atoms through two O atoms with the O─O bond lengths were 1.45 and 1.46 Å, respectively. The adsorption energy for O[2] adsorbed on Cu[7]S[4]‐1 and Cu[7]S[4]‐2 were −0.70 and −0.92 eV. The H[2]O was physically adsorbed upon the defects with the adsorption energy of −0.17 and −0.37 eV, respectively. The LPS was binding to the Cu atoms through two O atoms with the adsorption energy of −3.44 and −2.71 eV. H[2]O[2] adsorbed upon the defects with the O─O bond lengths were 1.47 and 1.48 Å, respectively. And the adsorption energy of H[2]O[2] was −0.44 and −0.34 eV (Figure [97]2g,h). The adsorption results indicated that while Cu[7]S[4]‐2 demonstrated effective adsorption of H[2]O and O[2], Cu[7]S[4]‐1 exhibited superior adsorption of Lipopolysaccharide (LPS), a component of the cell wall of Gram‐negative bacteria.^[ [98]^23 ^] The presence of LPS provided an effective anchoring site for the nanozymes, thereby enhancing their efficacy against bacteria. Furthermore, compared to Cu[7]S[4]‐2, Cu[7]S[4]‐1 demonstrated a higher capacity to adsorb H[2]O[2], providing more substrates for its POD‐like activity. This enhanced generating capacity of ROS was consistent with the experimental results. As Cu[7]S[4]‐1 also exhibits excellent optical absorption properties in the first NIR‐I region (700–900 nm), its photothermal conversion properties under 808 nm laser irradiation were further investigated. As shown in Figure [99] 3 , the photothermal properties of Cu[7]S[4]‐1 aqueous solution under 808 nm laser illumination show an obvious enhancement trend with the increase of its concentration and the laser power density. In addition, the four cycles of heating and cooling curves indicate that Cu[7]S[4]‐1 has good photothermal stability in comparison with Cu[7]S[4]‐2. Therefore, the above results indicate that Cu[7]S[4]‐1 has the potential to be used as a new type of photothermal therapy (PTT) agent. Figure 3. Figure 3 [100]Open in a new tab a) Temperature curves of Cu[7]S[4]‐1, Cu[7]S[4]‐2 aqueous solution (50 µg mL^−1) and ddH[2]O under 808 nm laser (1.2 W cm^−2). b) Temperature curves of different concentration of Cu[7]S[4]‐1 (0, 25, 50, and 100 µg mL^−1) under 808 nm laser (1.2 W cm^−2). c) Temperature curves of Cu[7]S[4]‐1 aqueous solution (50 µg mL^−1) under different power densities of 808 nm laser irradiation (1.0, 1.2, and 1.4 W cm^−2). d) Heating and cooling curves of Cu[7]S[4]‐1 aqueous solution (50 µg mL^−1) under irradiation of 808 nm laser (1.2 W cm^−2). (e) Infrared thermal images of Cu[7]S[4] aqueous solution (50 µg mL^−1) under 808 nm laser irradiation (1.2 W cm^−2). 2.2. Antibacterial Performance of Cu[7]S[4] in E. coli The plate counting test was utilized to evaluate the antibacterial activity of Cu[7]S[4]. Two types of Cu[7]S[4] nanoparticles, each at a concentration of 50 µg mL^−1, were tested against E. coli for 30 min. A control group without Cu[7]S[4] was also included. Without NIR‐I (808 nm) irradiation, Cu[7]S[4]‐1 treatment caused an 80.42% killing effect, while Cu[7]S[4]‐2 treatment only caused 53.38%. However, under NIR‐I (808 nm) irradiation, the bacteria's functionality was significantly impaired. Experimental results demonstrated that Cu[7]S[4]‐1 achieved 99.99% E. coli inactivation, while Cu[7]S[4]‐2 inactivated only 85.13% of E. coli, as shown in Figure [101] 4a. To further investigate the antibacterial effects on E. coli, Confocal fluorescence microscopy was employed to examine cell membrane disruption. Using SYTO9 dye, live bacteria emitted green fluorescence, whereas dead bacteria emitted red fluorescence after propidium iodide (PI) staining. The results revealed that nearly all bacterial cell membranes were damaged following incubation with Cu[7]S[4]‐1 under NIR‐I (808 nm) irradiation. In contrast, only partial membrane disruption was observed in E. coli incubated with Cu[7]S[4]‐2 under the same conditions, as illustrated in Figure [102]4a. Our investigation revealed that Cu[7]S[4]‐1 possessed higher photothermal conversion and temperature increased higher than that of Cu[7]S[4]‐2 and exhibited more efficient antibacterial activity through the photodynamic and photothermal synergetic process under NIR‐I (808 nm) irradiation, although Cu[7]S[4]‐2 can form more ROS under the light. Figure 4. Figure 4 [103]Open in a new tab a) Antibacterial effects of Cu[7]S[4] on E. coli, Colony of E. coli exposed with Cu[7]S[4]‐1 and Cu[7]S[4]‐2, with or without 808 nm laser irradiation (1.2 W cm^−2) for 10 min and Live/dead fluorescence images of E. coli, scale bar, 20 µm. ESR spectra detection of b) ·OH, c) O[2·] ^−, d) ^1O[2], e) h^+. f) CAT‐like activities of Cu[7]S[4] for O[2] generation. The low amount of O[2] at 0 s is attributed to the dissolved O[2] in water. g) UV–vis absorption of TMB catalyzed by Cu[7]S[4] in the presence of air, H[2]O[2]. h) absorption spectra of consumed GSH after treatment with Cu[7]S[4]. To further explore the antibacterial mechanism and differences between the two Cu[7]S[4] samples, electron spin resonance (ESR) experiments were conducted to detect various reactive oxygen species (ROS), including hydroxyl radicals (·OH) (Figure [104]4b), superoxide anions (O[2] ^·−) (Figure [105]4c), singlet oxygen (^1O[2]) (Figure [106]4d), and holes (h^+) (Figure [107]4e) under NIR irradiation. Notably, Cu[7]S[4]‐2 produced higher levels of ·OH and h^+ compared to Cu[7]S[4]‐1. However, as previously stated, Cu[7]S[4]‐1 demonstrated more pronounced adsorption of LPS and H₂O₂. Consequently, Cu[7]S[4]‐1 exhibited a more pronounced bactericidal effect, as evidenced by the bacterial plate experiments and the measurements of enzyme‐like activities. CAT‐like activity was assessed by measuring the increase in dissolved oxygen concentration in H[2]O[2]‐treated solutions. Cu[7]S[4]‐1 demonstrated higher oxygen generation compared to Cu[7]S[4]‐2 (Figure [108]4f), indicating that Cu[7]S[4]‐1 has stronger CAT‐like activity, facilitating the decomposition of H[2]O[2] into O[2] molecules in the proinflammatory microenvironment of bacterial infection, thereby alleviating hypoxia. POD‐mimicking activity was characterized by monitoring the absorbance change at 652 nm, which corresponds to the efficient oxidation of 3,3′,5,5′‐tetramethylbenzidine (TMB) in an oxygen system. The results showed that Cu[7]S[4]‐1 exhibited superior POD‐like properties compared to Cu[7]S[4]‐2 (Figure [109]4g), suggesting that Cu[7]S[4]‐1 more efficiently decomposes H[2]O[2] into ·OH in the proinflammatory microenvironment of bacterial infections. Furthermore, we investigated its GSH‐consuming ability through Ellman's assay.^[ [110]^24 ^] The characteristic peaks of DTNB (5, 5′‐dithiobis (2‐nitrobenzoic acid)) at 412 nm were decreased significantly within 60 min post‐treatment with Cu[7]S[4] (Figure [111]4h). 2.3. Transcriptomics Analysis of E. coli Treated with Cu[7]S[4] Nanomaterials To systematically elucidate the mechanism by which Cu[7]S[4]‐1 nanomaterials affect E. coli, transcriptome RNA‐seq was performed. A volcano plot displayed the differentially expressed genes (DEGs) between the Cu[7]S[4]‐1‐treated and control groups, revealing 415 upregulated and 449 downregulated genes following a 5‐min exposure to Cu[7]S[4] nanomaterials (Figure [112] 5a). Notably, DNA damage and repair genes such as dinD and uvrC showed significant upregulation compared to the control group (Figure [113]5b). It has been posited that the presence of Cu[7]S[4] nanozymes generates a substantial quantity of ROS, which in turn causes damage to bacterial DNA, ultimately leading to its demise. Additionally, oxidative stress‐related genes, including sodA and hprS, were also upregulated (Figure [114]5c), further demonstrating that ROS production induces oxidative stress and damage in bacteria. Gene Ontology (GO) analysis indicated that exposure to Cu[7]S[4]‐1 nanomaterials led to specific enrichment of DEGs in biological processes such as oxidation‐reduction, locomotion, monocarboxylic acid metabolism, and biological regulation. Enrichment was also observed in cellular components like the periplasmic space and plasma membrane, as well as in molecular functions including protein binding, cation binding, and oxidoreductase activity, compared with the control group (Figure [115]5d), which was hypothesized to be possibly due to the disruption of biological macromolecules such as proteins, phospholipids and metabolism of the bacteria by the ROS‐induced oxidative stress. KEGG pathway enrichment analysis further revealed significant differences in sulfur metabolism, glyoxylate and dicarboxylate metabolism, bacterial chemotaxis, and selenocompound metabolism in the Cu[7]S[4]‐1‐treated group (Figure [116]5e). Finally, COG classifications of DEGs showed that the Cu[7]S[4]‐1‐treated group had more upregulated DEGs related to post‐translational modification, protein turnover, chaperones, signal transduction mechanisms, and transcription, while downregulated DEGs were enriched in carbohydrate transport and metabolism, as well as inorganic ion transport and metabolism (Figure [117]5f). Figure 5. Figure 5 [118]Open in a new tab Transcriptomics analysis of E. coli treated with Cu[7]S[4]‐1 nanomaterials. a) The volcano map displays DEGs between Cu[7]S[4]‐1 nanomaterials and control groups. b) The heatmap showing DEGs of DNA damage between Cu[7]S[4]‐1 nanomaterials and control groups. c) The heatmap showing DEGs of ROS‐related markers between Cu[7]S[4]‐1 nanomaterials and control groups. d) GO enrichment analysis of DEGs between Cu[7]S[4]‐1 nanomaterials and control groups. e) KEGG enrichment analysis of different pathways in Cu[7]S[4]‐1 nanomaterials compared to control in accordance with DEGs. f) COG classifications of the DEG set between Cu[7]S[4]‐1 nanomaterials and control groups. 2.4. Antibacterial Performance of Cu[7]S[4] in P. aeruginosa To further investigate the bacterial killing activity of the Cu[7]S[4] nanozyme in vitro, we examined its ability to inactivate P. aeruginosa using the colony counting method. Compared to Cu[7]S[4]‐2, Cu[7]S[4]‐1 nanozyme demonstrated superior antibacterial activity at a concentration of 50 µg mL^−1 (Figure [119] 6a,b). Additionally, an in vivo antibacterial model was established using mouse skin wounds infected with the P. aeruginosa standard strain PAO1. The infected wounds were treated with Cu[7]S[4]‐1 nanozyme and subjected to NIR irradiation. On Day 6 post‐treatment, bacterial burden and histopathological observations were assessed. As shown in Figure [120]6c, wounds treated with Cu[7]S[4]‐1 nanozyme exhibited significantly lower bacterial loads compared to the PBS control group. Correspondingly, hematoxylin and eosin (HE) staining images (Figure [121]6d) revealed a pronounced accumulation of immune cells in the control group (treated with PBS), indicating severe inflammation at the wound sites. In contrast, wounds treated with Cu[7]S[4]‐1 nanozyme showed a marked reduction in immune cell infiltration. These findings demonstrate that Cu[7]S[4] nanozyme possesses substantial anti‐inflammatory properties, likely attributable to its high antibacterial efficiency in treating P. aeruginosa‐infected wounds. Figure 6. Figure 6 [122]Open in a new tab a) Colony forming unit (CFU) counting assay of bacteria treated with PBS, Cu[7]S[4]‐1 nanozymes, and Cu[7]S[4]‐2 nanozymes. (n = 3 independent experiments). b) Plate counting test of bacteria treated with nanozymes. c) Bacteria loading in the wound tissue of control mice and mice treated with Cu[7]S[4] on day 6. n = 5 mice per group and d) HE‐stained images of wounds in the control group and Cu[7]S[4]‐1 group at 6 days of treatment, scale bar, 100 µm. ^** indicated a significant difference between the group exposed to irradiation (p < 0.01), ^*** indicated a significant difference between the group exposed to irradiation (p < 0.001). 3. Conclusion In summary, we designed and synthesized two Cu[7]S[4] nanozymes through defect engineering to inactivate drug‐resistant bacteria. By employing two types of template‐controlled synthetic processes, Cu[7]S[4] with distinct dual‐defect configurations was formed. Experimental results and density functional theory (DFT) computations revealed that the electronic structures of the Cu[7]S[4] nanozymes were effectively tuned, imparting them with multifunctional enzyme‐like properties, including peroxidase (POD)‐like, catalase (CAT)‐like, and glutathione (GSH)‐depletion activities. Compared to Cu[7]S[4]‐2 with dual defects of VCu and VCuCuCuCuCuS, the Cu[7]S[4]‐1 nanozyme with dual defects of VCu and VCuCuCuSSS exhibited superior adsorption properties for H[2]O[2]molecules, and lipopolysaccharides (LPS) and apt electronic structure, leading to the formation of suitable ROS and photothermal temperature, exhibited synergistically efficient antibacterial activity against drug‐resistant bacteria. Notably, the Cu[7]S[4] nanozyme with dual defects VCu and VCuCuCuSSS effectively treated P. aeruginosa‐infected wounds in vivo under NIR irradiation, as demonstrated in a mouse wound model. Our research provides valuable insights into the design of novel nanozymes with efficient antibacterial properties. Conflict of Interest The authors declare no conflict of interest. Supporting information Supporting Information [123]ADVS-12-e03793-s001.docx^ (3MB, docx) Acknowledgements