NIR-II-triggered copper single-atom catalyst depots coupling catalysis and drug release for infected chronic wounds with dysregulated inflammation

Abstract

Precise control of antimicrobial and immunomodulatory signals in both space and time is essential for addressing drug-resistant chronic wounds. Here, an NIR-II–responsive nanomedicine depot is created by integrating fasudil (FAS) into a copper single-atom catalyst scaffold (Cu SACs/FAS), which is formed through a coordination-constrained pyrolysis process. Under 1064-nm irradiation, Cu SACs/FAS couples photothermal heating with dual enzyme-mimicking activities (peroxidase-like catalysis and GSH depletion), producing reactive oxygen species (ROS) and copper-dependent bacterial stress consistent with cuproptosis-related injury. The porous framework enables on-demand, sustained FAS release, which promotes macrophage polarization toward a pro-angiogenic phenotype and mitigates excessive inflammation. The platform achieves broad-spectrum antibacterial activity against E. coli, S. aureus, and MRSA in vitro and accelerates closure with improved tissue quality in an MRSA-infected wound model in vivo with the supplementation of minimal exogenous H₂O₂. This study establishes a structure-function linkage between single-atom active sites, NIR-II-enhanced catalytic/photothermal coupling, and pore-regulated drug release, offering a materials-centric strategy for simultaneous infection control and immune-guided repair.

Graphical abstract

1. Introduction

Chronic wounds, particularly those associated with diabetes, infections, and aging, represent significant clinical challenges due to their prolonged healing processes and high susceptibility to bacterial infections [[1], [2], [3]]. The chronic wound environment is characterized by impaired angiogenesis, which limits oxygen and nutrient supply to the affected tissues, perpetuating a persistent inflammatory response [4,5]. Specifically, hypoxia resulting from inadequate blood vessel formation stabilizes hypoxia-inducible factor-1 alpha (HIF-1α), triggering an ongoing release of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which further impede effective tissue regeneration [[6], [7], [8]]. Additionally, bacterial colonization at wound sites exacerbates inflammation and hinders healing by promoting biofilm formation and dysregulating neutrophil activity, resulting in the degradation of essential extracellular matrix components like collagen and fibronectin [[9], [10], [11]]. These complexities underline the critical need for comprehensive therapeutic approaches capable of simultaneously addressing microbial infections, modulating inflammatory responses, and enhancing angiogenesis to restore healthy tissue function. Therefore, innovative treatments that integrate antimicrobial, anti-inflammatory, and pro-angiogenic functionalities are urgently required to improve clinical outcomes and effectively manage chronic wound healing.

Currently, advanced therapeutics such as fasudil (FAS) are widely employed in clinical practice to suppress inflammation and modulate immune responses [12]. However, its therapeutic potential is greatly limited by its short half-life and poor pharmacokinetics, necessitating frequent administration or high doses, which can compromise patient compliance and increase systemic side effects [13,14]. Therefore, the development of sustained-release delivery systems is critical to prolong the activity of FAS and improve its therapeutic efficacy in chronic wound environments [15]. In recent years, nanomaterials with large surface areas, tunable porosity, and controlled drug-loading capacities have emerged as ideal candidates for this purpose [[16], [17], [18]]. They offer not only extended release profiles but also the potential for targeted delivery, minimizing off-target effects [19]. Among these, single-atom catalysts (SACs) have attracted growing attention due to their multifunctionality and versatility in biomedical applications, especially as therapeutic carriers [20].

SACs are uniquely defined by their atomically dispersed metal centers, which enable exceptional catalytic efficiency, high atomic utilization, and tunable coordination structures [21]. These features allow SACs to exhibit enzyme-mimicking activities, such as peroxidase- and glutathione oxidase-like behavior, facilitating the generation of reactive oxygen species (ROS) for chemodynamic therapy (CDT), which is an approach particularly effective against biofilm-associated infections [22]. Among various transition metals, copper-based SACs (Cu-SACs) are of particular interest due to the good biocompatibility of copper and its ability to disrupt bacterial copper ion metabolism [23]. This novel mechanism involves Cu²⁺-mediated aggregation of lipoylated proteins and disruption of iron-sulfur clusters, causing lethal oxidative damage to bacteria [24]. Recent studies have explored Cu-SAC nanozymes in wound applications, where Cu single-atom sites on N-doped porous carbon have shown enhanced peroxidase-like activity, GSH depletion, and photothermal conversion, achieving near-complete antibacterial efficiency against E. coli and MRSA via synergistic CDT/PTT [25]. Cu-SACs embedded in g-C₃N₄ nanosheets have also scavenged excess ROS and promoted M2 macrophage polarization, attenuating wound inflammation alongside antibacterial action [26]. However, these platforms typically focus on antibacterial or anti-inflammatory effects separately, without incorporating a sustained drug delivery system to address all three pathological features of chronic wounds: infection, immune dysregulation, and impaired angiogenesis. Notably, SACs can serve as drug reservoirs, enabling the co-delivery of therapeutics like FAS. Integrating FAS into a Cu-SAC matrix combines ROS-mediated bacterial killing, copper-induced cuproptosis, and localized immune modulation, offering a promising multifunctional nanoplatform for precision wound therapy.

In this study, a nanomedicine depot strategy utilizing copper single-atom catalysts (Cu SACs) is presented for the synergistic treatment of infected chronic wounds. By coordinating FAS, a clinically approved ROCK inhibitor, onto Cu SAC scaffolds, a multifunctional therapeutic nanozyme (Cu SACs/FAS) was developed (Scheme 1A). This system integrates drug delivery and catalytic functionalities, enabling sustained FAS release alongside ROS-mediated bacterial eradication and immune modulation. Cu SACs/FAS exhibits dual enzyme-mimicking activity, catalyzing H₂O₂ to generate ROS that continuously attack bacteria in the wound microenvironment (Scheme 1B). The resultant oxidative stress depletes intracellular GSH and disrupts bacterial redox homeostasis. Additionally, the nanozyme demonstrates excellent photothermal conversion efficiency in the NIR-II region, where PTT-induced hyperthermia enhances Cu²⁺ turnover and increases bacterial membrane permeability, thereby amplifying the synergistic effect of CDT and PTT. Notably, the released Cu²⁺ disrupts bacterial metabolic homeostasis by penetrating the bacterial envelope from outside to inside, destabilizing intracellular proteins, and ultimately causing irreversible cellular damage. Beyond antimicrobial action, Cu SACs/FAS also achieves long-term anti-inflammatory regulation. Sustained FAS release polarizes macrophages toward the pro-angiogenic M2 phenotype, promoting neovascularization and tissue regeneration. Scheme 1C illustrates the complete wound repair process, from local administration and photothermal therapy to antibacterial effects, inflammatory regulation, collagen deposition, and angiogenesis. Overall, this study presents a robust and durable strategy that integrates antibacterial, anti-inflammatory, and angiogenic functions into a single SAC-based therapeutic platform, offering significant promise for treating drug-resistant infected wounds.

Scheme 1.

(A) Schematic illustration of preparation of Cu SACs/FAS. (B) Cu SACs/FAS with dual POD-like and GSH-like enzyme activities for photothermal-catalytic antibacterial therapy. (C) From local administration and photothermal therapy to antibacterial effect, inflammation regulation, and tissue repair.

2. Results and discussion

2.1. Characterization of Cu SACs

Transmission electron microscopy (TEM) characterization results showed that the Cu SACs possessed a uniformly distributed microsphere structure (Fig. 1A). The average diameter of Cu SACs was about 140 nm, which was further confirmed by dynamic light scattering (DLS) results (Fig. S1). The particle size distribution had no obvious outliers or multi-peak distribution, and the dispersion was stable and homogeneous. As displayed in the selected area electron diffraction (SAED) analysis (Fig. 1B), the diffraction pattern of Cu SACs presented typical amorphous diffuse scattering halo features, and no crystalline diffraction rings or spots of metallic Cu nanoparticles were detected. The high-resolution transmission electron microscopy (HRTEM) image revealed the absence of large Cu nanoparticles crystallines in the carbon framework (Fig. 1C). Aberration-corrected atomic resolution high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) image further confirmed that there the Cu species were present in atomically dispersed form without nucleation aggregation of metal crystals, which corroborated the previous results (Fig. S2). In addition, the HAADF-STEM image and the corresponding energy dispersive X-ray spectroscopy (EDS) mapping demonstrated that the C, N, and Cu elements were uniformly distributed in the carbon matrix of the nanospheres (Fig. 1D). The different elements illustrated good co-polymerization in the samples, with the Cu signal being rather strong, reflecting the high loading of Cu. The X-ray diffraction (XRD) pattern revealed broad diffraction peak shapes rather than sharp, narrow peaks, indicating that the Cu SACs are amorphous (Fig. S3). Additionally, the absence of Cu-related diffraction signals confirmed the atomic dispersion of Cu species. The Raman spectra of Cu SACs presented two typical peaks in graphitized carbon, the D-band (defects in the carbon structure) at 1350 cm⁻¹ and the G-band (vibrations of the sp²-hybridized carbon structural units) at 1580 cm⁻¹ (Fig. S4). The I_D/I_G value of Cu SACs was 0.958, indicating a high degree of graphitization and structural stability of carbon carriers. The graphitized carbon framework stabilized the Cu single atoms, preventing their migration and agglomeration during catalytic reactions [27]. The full spectrum of X-ray photoelectron spectroscopy (XPS) of Cu SACs included C, N, O, and Cu peaks, indicating the successful doping of Cu (Fig. S5). The valence, nitrogen content, and type of the Cu single atom are in good agreement with previously reported XPS results for related Cu-N4 SACs [28]. In addition, the XPS spectrum N 1s of Cu SACs was deconvolved into four peaks at 399.1, 399.9, 400.7, and 402.7, which were attributed to pyridinic N, pyrrolic N, graphitic N and protonated N, respectively (Fig. 1E). Among them, pyridinic N and pyrrolic N served as coordination sites for the Cu single atoms, graphitic N facilitated electrical conductivity, and protonated N reflected the surface chemical environment of Cu SACs [29]. Cu 2p XPS showed peaks at 934.4 and 954.9 eV, which are attributed to Cu 2p_3/2 and Cu 2p_1/2 of Cu²⁺, respectively. The peaks at 932.6 and 952.5 eV are related to the presence of Cu 2p_3/2 and Cu 2p_1/2 of Cu⁺ (Fig. 1F). X-ray absorption near-edge structure (XANES) spectroscopy was utilized as a powerful tool to investigate the precise atomic structure and coordination environment of Cu atoms in Cu SACs [30]. Fig. 1G displayed the Cu K-edge XANES profiles of Cu SACs, with Cu-foil (Cu⁰) and CuO (Cu²⁺) as references. The absorption edge of Cu SACs was located between those of Cu-foil and CuO, indicating a mixed-valence state with an average oxidation state between Cu⁰ and Cu²⁺. This intermediate oxidation state suggested a distinct coordination environment, which was further characterized by extended X-ray absorption fine structure (EXAFS) analysis. In the R-space FT-EXAFS spectrum of Cu SACs, a prominent peak at 1.59 Å corresponded to Cu-N scattering (Fig. 1H). In contrast, the spectrum of CuO exhibited a Cu-O scattering peak at a radial distance of approximately 1.62 Å. Additionally, the Cu-foil spectrum showed a prominent Cu-Cu back-scattering peak at 2.54 Å. These scattering features were distinct from those observed in the Cu SACs sample, suggesting a different Cu coordination environment. Notably, the absence of Cu-Cu scattering paths in Cu SACs (2.2-2.6 Å) conclusively confirmed the atomic dispersion of Cu species without clustering.

Fig. 1.

Morphological and structural characterization of Cu SACs. (A) TEM image of Cu SACs. Scale bar = 500 nm. (B) The magnified TEM (Scale bar = 50 nm) and corresponding SAED (Scale bar = 100 nm) of Cu SACs. (C) The HRTEM image of Cu SACs. Scale bar = 10 nm. (D) HAADF-STEM image and corresponding EDX mapping (C, N, and Cu) images of Cu SACs. Scale bar = 100 nm. The high-resolution XPS spectra in the (E) N 1s and (F) Cu 2p regions. (G) Normalized XANES spectra and (H) FT-EXAFS (R-space) spectra of Cu-foil, CuO, and Cu SACs samples. (I) N₂ sorption isotherms of Cu SACs.

In most SACs systems, the metal atoms were not randomly dispersed but highly homogeneously dispersed through the matrix [31]. Instead of being heavily enriched on the surface as in conventionally loaded catalysts, the metal atoms were cleverly “hidden” in the matrix, allowing the most active catalytic sites to be buried deep inside [32]. Consequently, the increase in catalyst-specific surface area (SSA) provided more space for the diffusion and penetration of reactant molecules, thus providing more active sites, which greatly improved the performance of the catalyst [33]. Therefore, N₂ adsorption and desorption experiments at 77 K were utilized to characterize the specific surface area and pore size distribution of the Cu SACs. As shown in Fig. 1I, the isotherms of Cu-SACs showed a rapid increase in adsorption at lower relative pressures, followed by a slower increase, which was consistent with the characteristics of type II isotherms [34]. This physical adsorption behavior was accompanied by the absence of a hysteresis loop, suggesting the predominance of non-porous or macroporous structures and weak gas-solid interactions [35]. Calculations using the Brunauer-Emmett-Taylor (BET) method yielded a SSA of 84.92 m² g⁻¹ for Cu SACs. This configuration allowed the Cu SACs to expose a greater number of active atomic sites, owing to their higher SSA and larger microporous volume [36]. When drug FAS was loaded onto Cu SACs, it occupied the pores and surface of the carrier, resulting in a decrease in the carrier's measurable specific surface area. As shown in Fig. S6 and Table S1, the BET specific surface area of Cu SACs/FAS was 53.51 m² g⁻¹, and we determined that the loading of FAS on Cu SACs was approximately 37.01 wt%. The microporous area plummeted from 31.52 m² g⁻¹ to 8.08 m² g⁻¹, representing a 74.4% reduction. This indicated that FAS molecules were not merely adsorbed onto the carrier's external surface but had extensively penetrated micropores smaller than 2 nm in diameter. The difference in the extent of change between the internal and external surface areas indicates the successful loading of the drug into the carrier's pores.

Although no additional chemical functionalization was introduced, FAS was effectively coordinated to Cu SACs, thereby reducing premature drug leakage. The UV-Vis spectra showed that the characteristic absorption profile of FAS changed after loading onto Cu SACs, with an evident red-shift and band perturbation relative to free FAS, indicating that the electronic environment of FAS was altered upon immobilization (Fig. S7). In parallel, the FT-IR spectra of Cu SACs/FAS exhibited noticeable changes in the characteristic vibrational regions associated with FAS, particularly in the broad stretching region around 3400 cm⁻¹ and the fingerprint region near 1000-1200 cm⁻¹, suggesting the involvement of FAS functional groups in intermolecular interactions after loading (Fig. S8). Specifically, the C=N stretching vibration of the triazole ring shifted from 1600 cm⁻¹ to 1550 cm⁻¹ and became broader. Similarly, the N-H stretching vibration of the sulfonamide group shifted from 3384 cm⁻¹ to 3284 cm⁻¹, with significant peak broadening. These spectral changes suggested that the nitrogen-containing functional groups of FAS participated in interactions with Cu SACs, likely through coordination. In addition, after FAS loading, the Cu 2p_3/2 main peak shifted by approximately 0.3 eV toward higher binding energy, accompanied by an increase in the Cu²⁺ fraction and a more pronounced Cu²⁺ satellite feature (Fig. S9). These changes indicated that the electron density around the Cu centers decreased after FAS incorporation and that the local electronic structure of Cu was significantly perturbed. Considering that FAS contains potential N/O donor atoms, the XPS results further supported the formation of Cu-N/O coordination interactions between FAS and the Cu single-atom sites. Overall, FAS loading on Cu SACs was primarily governed by coordination interactions, while hydrogen bonding and π-π stacking likely played auxiliary roles.

2.2. Photothermal property measurement of Cu SACs/FAS

A Cu SACs/FAS drug-carrying nanoplatform was successfully constructed through the nanomedicine depot strategy, and the photothermal properties of platform-mediated PTT were investigated (Fig. 2A). The normalized absorption was quantified by UV-Vis-NIR spectroscopy across diverse concentrations (20, 40, 60, 80, and 100 μg/mL) (Fig. 2B). A concentration-dependent absorption of Cu SACs/FAS at 1064 nm (NIR-II region) was observed, with a progressive increase from 20 to 100 μg/mL, which provided preliminary support for its application as an NIR-II photothermal agent.

Fig. 2.

(A) Schematic illustration of the photothermal performance of Cu SACs/FAS under 1064 nm NIR irradiation. (B) UV-Vis spectra of Cu SACs/FAS at different concentrations (20, 40, 60, 80, and 100 μg/mL). (C) Temperature profiles of different concentrations of Cu SACs/FAS under NIR irradiation (1.2 W/cm²) for 10 min. (D) Temperature profiles of Cu SACs/FAS (100 μg/mL) at diverse laser power densities (0.2, 0.6, 0.8, 1.0, and 1.2 W/cm²), and corresponding (E) Infrared thermal images. (F) Heating/cooling curve of Cu SACs/FAS (100 μg/mL) during four cycles under NIR irradiation (1.2 W/cm²). (G) A single heating/cooling curve of Cu SACs/FAS (100 μg/mL) followed by natural cooling after irradiation. (H) The system time constant determined by linear fitting of the cooling curve. (I) Photothermal conversion efficiency of Cu SACs/FAS compared with those of recently reported nanomaterials. Data are presented as mean ± SD (n = 3).

The photothermal-conversion performance of Cu SACs/FAS was systematically assessed by recording temperature changes with a thermal imaging camera during irradiation at diverse concentrations (20, 40, 60, 80, and 100 μg/mL, and control) under a 1064 nm NIR laser (1.2 W/cm²) (Fig. 2C). Owing to the high efficiency of NIR-to-thermal energy conversion, the temperature of the 100 μg/mL solution rapidly rose from 23 °C to 61.8 °C within 10 min, whereas the control exhibited only a minimal increase (23 °C to 26.2 °C). Subsequently, the photothermal performance under varying power densities (0.2, 0.6, 0.8, 1.0, and 1.2 W/cm²) was further evaluated at a fixed concentration of Cu SACs/FAS (100 μg/mL) (Fig. 2D and E). The solution temperature increased steadily with the rise of the NIR laser power during the 10 min of the test, indicating robust photothermal potential for antimicrobial applications. Furthermore, as shown in Fig. 2F, the temperature elevation curves of Cu SACs/FAS solutions (100 μg/mL, 1.2 W/cm² NIR irradiation) remained consistent even after four on/off cycles, confirming the material's exceptional photothermal stability. By further investigating the heating/cooling cycle, the photothermal conversion efficiency (η) of Cu SACs/FAS was calculated to be 61.3%, a significantly high value that highlighted its prominent photothermal conversion performance (Fig. 2G and H). As shown in Fig. 2I, this η value was considerably higher than those of other reported photothermal agents. These results demonstrated that Cu SACs/FAS exhibited excellent photothermal properties, making it a promising candidate for biomedical applications [[37], [38], [39], [40], [41]]. To examine whether ROS influenced the photothermal performance, we employed ascorbic acid (AA) as a ROS scavenger in the presence of H₂O₂. It was found that under 1064 nm NIR (0.8 W/cm²) irradiation, Cu SACs/FAS (100 μg/mL) could achieve the same warming effect even after adding AA, further confirming the relative independence of the photothermal mechanism (Fig. S10).

2.3. Chemodynamic evaluation and density functional theory calculations of Cu SACs/FAS

SACs-anchored catalysts are expected to provide efficient Fenton reaction kinetics, resulting in the production of a large number of ROS, while their intrinsic photothermal properties further amplify this catalytic process. Electron spin resonance (ESR) spectroscopy was employed to verify hydroxyl radical (·OH) generation, exhibiting a characteristic 1:2:2:1 quartet signal (Fig. 3A) upon treatment with Cu SACs/FAS and H₂O₂. Notably, Cu SACs/FAS + H₂O₂+NIR displayed very strong ESR signals compared with Cu SACs/FAS + H₂O₂, indicating the photothermal effect greatly promoted the generation of ·OH and provided preliminary evidence for the synergistic therapeutic effect of PTT on CDT. In general, TMB can be oxidized to blue oxidized TMB (ox-TMB) in the presence of a catalyst. The POD-like activity of Cu SACs/FAS in catalyzing the formation of ·OH from H₂O₂ was further studied using TMB as substrate. No significant absorbance signal at 652 nm was observed in the Cu SACs/FAS solution after the addition of TMB, suggesting the POD-like activity of Cu SACs/FAS was negligible. The absorbance intensity at 652 nm of Cu SACs/FAS + H₂O₂+NIR was markedly higher than that of Cu SACs/FAS + H₂O₂ alone (Fig. 3B). These results further support the rational design of Cu SACs/FAS as a synergistic CDT/PTT therapeutic platform. As shown in Fig. 3C, a time-dependent ox-TMB signal was observed after co-incubation of Cu SACs/FAS with H₂O₂, revealing the excellent POD-like catalytic ability of Cu SACs/FAS. Complementary validation was achieved through MB degradation assays, where ·OH-mediated oxidative decay exhibited time-dependent kinetics consistent with ESR and TMB results (Fig. 3D). To quantitatively assess the catalytic activity of Cu SACs/FAS, we performed a Michaelis-Menten kinetic analysis with Lineweaver-Burk linearization to investigate its H₂O₂ concentration-dependent activity (Fig. 3E and F). The Michaelis constant (K_m) and maximal reaction rate (V_max) of Cu SACs/FAS were determined to be 0.56 mM and 2.78 × 10⁻⁷ M s⁻¹, indicating favorable substrate affinity and catalytic efficiency. Based on the experimentally determined V_max and the Cu content (5.96 wt%), we estimated the apparent turnover number (k_cat) by approximating Cu atoms as active sites. The calculated kcat is approximately 2.96 × 10⁻² s⁻¹, and the catalytic efficiency (k_cat/K_m) is approximately 52.9 M⁻¹ s⁻¹. For comparison, natural horseradish peroxidase (HRP) typically exhibits Km ≈ 1.8 mM and kcat≈10² s⁻¹, corresponding to a catalytic efficiency in the range of 10⁴-10⁵ M⁻¹ s⁻¹. These values are significantly higher than those of nanozymes, consistent with the intrinsic differences between enzyme active sites and heterogeneous catalytic sites. We also compared our results with representative Cu-based nanozymes reported in the literature, which typically exhibit k_cat values in the range of 10⁻³-10⁻¹ s⁻¹ and catalytic efficiencies of 10-10³ M⁻¹ s⁻¹. Our system falls within the typical range of single-atom nanozymes (Table S2). Collectively, these findings demonstrate that Cu SACs/FAS operate via a dual-modality mechanism: (I) H₂O₂-triggered ·OH generation through POD-like catalysis and (II) photothermally amplified ROS production, thereby establishing a rationally designed platform for synergistic CDT/PTT applications.

Fig. 3.

Catalytic performances of Cu SACs/FAS. (A) ESR spectrum of DMPO/·OH adducts in different groups. (B) Changes in absorption spectra of different groups in TMB. (C) TMB and (D) MB degradation profile of Cu SACs/FAS + NIR when co-incubated with H₂O₂. (E) Michaelis-Menten kinetic and (F) Lineweaver-Burk plotting analysis of Cu SACs/FAS for POD activity at room temperature. (G) Levels of GSH depletion in different groups with increasing time. (H) Release profiles of Cu²⁺ from diverse groups after incubation. (I) FAS release profiles of FAS, Cu SACs/FAS, and Cu SACs/FAS + NIR (1064 nm, 0.8 W/cm²). (J) Free energy diagrams of Fenton-like processes on G and CuN₄-G at equilibrium potentials. Partial density of states of (K) G and (L) CuN₄-G. Data are presented as mean ± SD (n = 3, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

GSH, a thiol-containing tripeptide, acts as the primary endogenous antioxidant in bacterial systems, neutralizing ROS through its redox-active sulfhydryl group (-SH) to mitigate oxidative damage [42]. In the presence of oxidants, GSH is oxidized and converted to oxidized glutathione (GSSG). Therefore, the GSH-consuming capacity of Cu SACs/FAS was investigated by Ellman's assay, and the colorless DTNB was reduced by GSH (Fig. 3G). GSH in the negative control group was almost unchanged after 20 min. Notably, time-dependent GSH consumption was observed in both Cu SACs/FAS and Cu SACs/FAS + NIR groups, with depletion rates reaching 63.2% and 80.7% after 20 min, respectively. As a transition metal ion, Cu²⁺ can directly deplete GSH through redox reactions, which is one of the important reasons for the time-dependent consumption of GSH. Particularly in the Cu SACs/FAS + NIR group, NIR irradiation may increase the release of Cu²⁺ via photothermal effect, further accelerating GSH consumption. This stark contrast highlights the critical role of photothermal activation in amplifying oxidative stress. The Cu content in the Cu SACs/FAS nanosystem was 5.96 wt% by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The release of Cu²⁺ from Cu SACs/FAS, as presented in Fig. 3H, showed that only 41.4% of Cu²⁺ were released over a 5 h period. In contrast, the highest release of Cu²⁺, reaching 82.9%, was observed in Cu SACs/FAS + NIR. Previous studies confirmed that Cu²⁺ could scavenge endogenous bacterial GSH through redox reactions. In this process, Cu²⁺ was reduced to Cu⁺, while GSH was oxidized to GSSG. Furthermore, the generated Cu ⁺ could effectively catalyze H₂O₂ to produce ·OH, and at the same time, itself was oxidized back to Cu²⁺ [43]. A substantial fraction of Cu atoms remains stably coordinated as Cu-N_x sites within the carbon matrix, so the structural integrity of the single-atom active sites is preserved and the nanozyme maintains its intrinsic POD-like activity. Moreover, ROS generation can proceed through two parallel contributions. The retained Cu-N_x sites act as heterogeneous catalytic centers that continuously catalyze H₂O₂ decomposition, while the released Cu²⁺/Cu⁺ species can participate in localized Fenton-like reactions in the surrounding microenvironment. Importantly, the Cu²⁺/Cu⁺ redox cycle supports sustained catalytic turnover, because Cu²⁺ can be reduced to Cu⁺ and re-oxidized during ROS generation, preventing rapid loss of catalytic activity.

Due to bacterial invasion and severe inflammation, many drugs, including FAS, are rapidly inactivated in the wound microenvironment. Therefore, a slow-release system was constructed to improve the therapeutic efficacy of FAS. Under identical release conditions, comparison with a commercial sustained-release formulation provides a clinically relevant benchmark to evaluate whether the Cu SAC depot can achieve a prolonged and controllable release profile. As shown in Fig. 3I, Cu SACs/FAS exhibited a more sustained release behavior than the commercial FAS control, indicating that confinement within the porous Cu SAC framework retards FAS diffusion and reduces premature leakage. Upon NIR-II irradiation, the localized temperature rise can modulate both the drug–carrier interactions and mass transport within the porous matrix. First, FAS is immobilized on Cu SACs through a combination of Cu–N/O coordination and auxiliary noncovalent interactions (e.g., hydrogen bonding and π–π interactions) involving nitrogen-containing functional groups. Mild photothermal heating can weaken these interactions, facilitating partial desorption and release of FAS from the binding sites. Second, the elevated temperature increases molecular mobility and diffusion kinetics within the carbon-based framework, thereby reducing diffusion barriers and accelerating outward transport. Therefore, the enhanced FAS release under NIR-II irradiation is attributed to the combined effects of thermally weakened binding interactions and heat-accelerated diffusion, rather than a single mechanism such as irreversible structural changes of the carrier.

2.4. Density functional theory calculations

To unravel the atomic-level mechanism of Fenton-like catalysis, we leveraged the well-defined coordination structure of CuN₄-graphene (CuN₄-G) as a model system for density functional theory (DFT) calculations, with pristine graphene (G) serving as the control (Fig. S11). This approach enabled a comparative analysis of catalytic activity rooted in electronic structure differences. The experimental detection of DMPO/·OH spin adducts via ESR unequivocally supports ·OH as the dominant reactive intermediate. Accordingly, we proposed a four-step catalytic cycle for CuN₄-G. Before the reaction pathway analysis, all atoms were fully relaxed to remove internal stress from system. (I) The first step is the uptake of H₂O₂ molecules by the active site of CuN₄-G. Next, (II) The process of free ·OH generation was also the rate-limiting step in the overall reaction. (III) The residual ∗OH is able to bind protons (H⁺), which in turn form water (H₂O) molecules and are adsorbed by system. (IV) Eventually, the adsorbed H₂O molecules desorb, and the catalyst returns to its original state. The pathway and free energy of Fenton-like reaction in the active sites of G and CuN₄-G were shown in Fig. 3J. Upon Cu doping, the adsorption free energies of all products and intermediates were enhanced to varying degrees, indicating that the adsorption capacity of Cu toward these species was significantly stronger than that of C atoms. This result suggested that CuN₄-G, with Cu atoms incorporated into its structure, exhibited higher catalytic activity in the Fenton-like reaction.

Partial density of states (PDOS) analysis was performed for the active C and Cu centers to elucidate the mechanistic origins of the distinct catalytic activities between G and CuN₄-G. The PDOS profiles revealed that the C active site (primarily the C-p orbital) mediated adsorption interactions, while the Cu active site (dominated by the Cu-d orbital) governed electronic activation. Notably, near the Fermi level, the Cu-d orbitals exhibited significantly higher density compared to the C-p orbitals (Fig. 3K and L). This electronic contrast enhanced the binding energy between CuN₄-G and reaction intermediates and accelerated charge transfer during the Fenton-like process, aligning with predictions from frontier molecular orbital theory [44]. DFT calculations further demonstrated that CuN₄-G markedly strengthened single ∗OH adsorption, achieving synergistic enhancements in catalytic activity, structural stability, and charge conductivity. These findings collectively established a robust theoretical foundation and material platform for catalyst development.

2.5. Antibacterial property of Cu SACs/FAS in vitro

As illustrated in Fig. 4A, the antibacterial activity of Cu SACs/FAS operates through a multi-modal cascade. Under 1064 nm NIR irradiation, the nanodepot generates mild heat through the photothermal effect. This localized heating not only contributes directly to bacterial inactivation, but also accelerates the release of Cu ions and FAS from the nanospheres, thereby enhancing the overall therapeutic efficacy. In the presence of H₂O₂, both the retained catalytic Cu active sites and the released copper ions contribute to antibacterial activity through several synergistic processes. First, they promote ROS generation, which induces oxidative damage to bacterial membranes and other essential intracellular components. Second, copper ions deplete GSH, weakening the endogenous antioxidant defense of bacteria and making them more susceptible to ROS-mediated injury. Third, they interfere with bacterial central metabolic pathways, thereby inducing copper ion-mediated metabolic disturbance. Collectively, these synergistic effects endow Cu SACs/FAS with potent antibacterial activity against drug-resistant pathogens.

Fig. 4.

In vitro antibacterial properties of Cu SACs/FAS. (A) From sequential activation to synergistic amplification: engineering hyperthermia, ROS, ions, and drug quadruple systems with optimized bactericidal kinetics. Photographs of agar plates of bacterial colonies formed by (B) E. coli and (D) S. aureus after diverse treatments. The corresponding viabilities of (C) E. coli and (E) S. aureus. (F) Fluorescence images of surviving (green) and dead (red) bacteria of E. coli and S. aureus. Scale bar, 100 μm. (H) TEM images of E. coli and S. aureus after different treatments. Scale bar, 0.8 μm. Percentage of membrane damage in (G) E. coli and (I) S. aureus after irradiation. Data are presented as mean ± SD (n = 3, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

First, E. coli and S. aureus were selected as models of Gram-negative and Gram-positive bacteria, respectively, for this antibacterial test. In studying the antibacterial properties of FAS alone, we found that the drug alone did not exhibit a significant bactericidal effect. Plate experiments verified that FAS achieved an antibacterial rate of 6.7% against S. aureus and 7.8% against E. coli (Fig. S12). The wound microenvironment typically lacks sufficient H₂O₂ to support effective CDT. To overcome this limitation and evaluate the synergistic bactericidal effects of Cu SACs/FAS under combined photothermal and chemodynamic conditions, exogenous H₂O₂ was supplemented in the experiments. The single antimicrobial effect of H₂O₂ was first evaluated. Specifically, exogenous H₂O₂ was used as a well-defined substrate to assess the POD activity of Cu-SACs/FAS under controlled conditions, allowing us to quantitatively assess ·OH generation and to decouple the contribution of CDT from the photothermal effects. As shown in Fig. S13, bacterial survival showed a gradual decrease with increasing H₂O₂ concentration. However, when the H₂O₂ concentration was lower than 0.4 mM, its effect on the bacterial survival rate was minimal [45]. Therefore, in the subsequent experiments, the concentration of H₂O₂ was chosen to be 0.4 mM to avoid damage to normal tissues. The in vitro antibacterial efficacy of Cu SACs/FAS was systematically evaluated against E. coli and S. aureus under six treatment conditions: control, Cu SACS, Cu SACS + NIR, Cu SACS + H₂O₂, Cu SACS + NIR + H₂O₂, and Cu SACS/FAS + NIR + H₂O₂ (Fig. 4B–D). Colony-forming units (CFUs) on LB agar plates were quantified to determine bactericidal rates (Fig. 4C–E). The large number of bacterial colonies observed in the control and Cu SACs treatment groups suggested that Cu SACs alone did not affect the growth of E. coli and S. aureus. The Cu SACs + NIR group showed a pronounced antimicrobial effect (41.5% against E. coli and 37.5% against S. aureus), which was attributed to its higher photothermal conversion efficiency, yet insufficient for clinical requirements. The limited antibacterial efficacy of Cu SACs alone was likely due to insufficient endogenous H₂O₂ levels, which hindered the CDT effect. To overcome this limitation, exogenous H₂O₂ (0.4 mM) was supplemented to boost the in vitro CDT activity of Cu SACs. The antibacterial effect of the Cu SACs + H₂O₂ group was significant, with antibacterial rates of 37.3% and 32.8% for E. coli and S. aureus, respectively. Synergistic PTT/CDT (Cu SACs + NIR + H₂O₂) achieved eradication of 93.9% for E. coli and 92.3% for S. aureus, underscoring the necessity of dual-modality activation. The addition of FAS (Cu SACs/FAS + NIR + H₂O₂) further elevated bactericidal rates to 99.5% (E. coli) and 99.3% (S. aureus), demonstrating its effectiveness in sustained therapeutic delivery and CDT/PTT synergy. By testing a concentration gradient in the antimicrobial assays, we found that the antibacterial efficacy was concentration-dependent, and the minimum inhibitory concentration (MIC) was determined to be 85 μg/mL. This value indicates the concentration at which Cu SACs/FAS begins to show significant antibacterial activity against the tested pathogens (Fig. S14). Given the excellent antimicrobial activity of Cu SACs/FAS, we further investigated its broad-spectrum antibacterial efficacy against MRSA. As shown in Fig. S15, the killing effect of Cu SACs/FAS on MRSA without H₂O₂ and NIR irradiation was minimal. When NIR and H₂O₂ were added in a synergistic manner (Cu SACs + NIR + H₂O₂), the growth of MRSA was significantly inhibited. Almost complete bacterial inactivation was observed in the Cu SACs/FAS + NIR + H₂O₂ group, which corresponded with the preceding experimental findings. Bacterial colonies from different treatment groups were quantitatively analyzed, most encouragingly, the Cu SACs/FAS + NIR + H₂O₂ group achieved an antibacterial rate of 99.2%.

To further resolve the in vitro combined antimicrobial effect of Cu SACs/FAS, we performed live/dead bacterial staining experiments. Briefly, E. coli and S. aureus were treated differently and then stained by calcein-AM/PI double staining kit. As exhibited in Fig. 4F–S16, bacteria (E. coli and S. aureus) in the control and Cu SACs groups expressed a strong green fluorescence (live bacteria). In contrast, a relatively substantial red fluorescence (dead bacteria) was observed in the Cu SACs + NIR and Cu SACs + H₂O₂ treatment groups, implying excellent PTT and CDT antibacterial properties. In the Cu SACs/FAS + NIR + H₂O₂ treatment group, the red fluorescence almost entirely covered the fluorescence image, while the green fluorescence was barely visible. This indicated that the Cu SACs/FAS + NIR + H₂O₂ treatment could effectively destroy a large number of bacteria, and the combined therapy was sufficient to kill all of them. The fluorescence results were consistent with those of plate colony counting. To corroborate the antibacterial mechanisms inferred from colony counting assays, the surface morphological changes of the two bacterial strains under different treatments were observed using TEM (Fig. 4H). Bacterial membranes in the control group displayed preserved structural integrity with smooth surface morphology, suggesting absence of membrane compromise. In contrast, in the TEM images of E. coli and S. aureus, both the Cu SACs + NIR and Cu SACs + H₂O₂ treatment groups had bacterial surfaces with slight twisted deformation and folded features. Notably, the bacterial membrane structure of the Cu SACs/FAS + NIR + H₂O₂ group contracted more severely and even ruptured, which was attributed to the synergy effect of Cu SACs/FAS combined with multiple strategies. The bacterial membrane disruption rates of Cu SACs/FAS + NIR + H₂O₂ were 52.4% for E. coli and 48.5% for S. aureus (Fig. 4G–I). As shown in Table S3, Cu SACs/FAS was thoroughly compared with existing antimicrobial agents. In our system, Cu SACs generate ROS via a Fenton-like reaction, which induces oxidative damage in bacteria. The mesoporous structure of Cu SACs ensures the controlled and localized release of FAS at the infection site. The combination of Cu SACs and FAS creates a “double hit” effect: Cu SACs generate ROS to induce oxidative damage, while FAS simultaneously weakens the bacterial antioxidant defense by depleting GSH levels.

2.6. Differential gene expression in MRSA strains treated with Cu SACs/FAS

Given the outstanding anti-MRSA activity of Cu SACs/FAS, we investigated the underlying antibacterial mechanism of the Cu SACs/FAS system using RNA-sequencing-based transcriptomic analysis. The number of transcript fragments per kilobase per million mapped reads of a gene was analyzed to assess the level of gene expression (Fig. S17). As apparent in the volcano plot of Fig. 5A, there were 459 differentially expressed genes (DEGs) (256 up-regulated and 203 down-regulated) in the Cu SACs/FAS + NIR + H₂O₂-treated group compared with the control group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted to elucidate biological regulatory mechanisms associated with DEGs. The enrichment results indicated that the treatment significantly perturbed pathways related to fatty acid degradation and valine, leucine, and isoleucine biosynthesis, which are closely linked to the tricarboxylic acid (TCA) cycle. Additionally, pyrimidine metabolism and arginine biosynthesis were also affected, both of which are closely related to α-ketoglutarate, a key intermediate product in the TCA cycle [46]. Further analysis revealed that the treatment group affected the biosynthesis of secondary metabolites and the ribosome pathway, causing an imbalance in energy and material supply. Analysis of the KEGG pathways involving DEGs displayed that Cu SACs/FAS + NIR + H₂O₂ treatment interfered with core metabolic processes such as the TCA cycle. On one hand, it inhibited the ribosome pathway, leading to impaired protein synthesis; on the other hand, it blocked the synthesis of secondary metabolites, weakening bacterial adaptability, ultimately resulting in decreased bacterial division and proliferation capabilities [47].

Fig. 5.

Differential gene expression of Cu SACs/FAS + NIR + H₂O₂-treated MRSA. (A) Volcano plot of up-regulated and down-regulated differentially expressed genes (DEGs). (B) KEGG enriched functional terms of up-regulated DEGs in Cu SACs/FAS + NIR + H₂O₂ group. (C) KEGG classification map of DEGs. (D) GO enrichment analysis of DEGs. (E-G) qRT-PCR validation of copper ion-induced metabolic disturbance markers (FDX1, LIAS, DLAT). (H) Western blotting results of bacterial proteins involved with copper ion-induced metabolic disturbance. (I) The intracellular copper concentration quantified using ICP-AES.

Based on previous research indicating that copper-induced cell death (cuproptosis) relies on mitochondrial respiration and is triggered by copper binding to lipoylated components of the TCA cycle, we hypothesize that Cu SACs/FAS + NIR + H₂O₂ treatment may induce severe metabolic disturbance in drug-resistant bacteria [48,49]. These metabolic changes were visualized using heat maps, showing the consistency in metabolic alterations after treatment. Statistical analyses revealed that 152 DEGs in the Cu SACs/FAS + NIR + H₂O₂ group were associated with metabolism, which was the most numerous category. Of these DEGs, 39 were associated with amino acid metabolism and 33 with carbohydrate metabolism. This suggested that the antibacterial effect of photothermally/chemically activated Cu SACs/FAS against MRSA mainly stemmed from its interference with metabolic activities (Fig. 5C). Furthermore, Gene Ontology (GO) analysis was performed on the DEGs, which fall into categories like biological process, cellular component, and molecular function (Fig. 5D). DEGs in biological processes were mainly associated with metabolic processes, cellular processes, and localization. Cellular components of DEGs were mainly concentrated in cells, cell parts, and membranes and were also distributed in the extracellular region. The bacterial cell membrane served as a critical barrier against the external environment. Enrichment of DEGs in membrane-associated pathways indicated compromised membrane integrity and functionality. Furthermore, membrane damage impaired bacterial adhesion capacity to some extent, correlating with the observed enrichment of localization-related pathways. The relatively high percentage of DEGs in catalytic activity and binding indicated that key bacterial enzyme activity was inhibited and nutrient transport was blocked. In summary, the synergistic effect of Cu SACs/FAS + NIR + H₂O₂ triggers oxidative stress and disrupts energy metabolic homeostasis, which greatly disrupts bacterial proliferative behaviors through a variety of pathways, including bacterial structure, ribosome translation, DNA replication, and protein functional activity.

To investigate the copper-induced death mechanism mediated by materials, this study validated the expression levels of key marker genes and proteins within the relevant pathways. First, the transcriptional levels of copper death-associated genes (FDX1, LIAS, DLAT) were assessed via qPCR (Fig. 5E and F). Results indicated that following treatment with Cu SACs/FAS + NIR + H₂O₂, mRNA expression levels of these genes were significantly downregulated, suggesting preliminary evidence that the copper-induced lethal pathway was suppressed at the transcriptional level. Building upon this, we further validated the expression levels of the corresponding proteins through Western blot (WB) experiments. WB results consistently demonstrated significantly reduced protein expression levels of FDX1, LIAS and DLAT, consistent with the characteristics of copper ion-induced protein toxicity stress (Fig. 5G). Results from qPCR and WB collectively confirmed, at both the transcriptional and protein levels, that this treatment effectively downregulated the expression of and inhibited the activity of key molecules involved in bacterial copper homeostasis and core metabolic pathways, thereby inducing profound metabolic disruption in drug-resistant bacteria. The copper content within the bacterial cells was quantified using ICP-AES. The results indicated a significant increase in copper concentration in the Cu SACs/FAS + NIR + H₂O₂ treatment group. Consequently, the Cu SACs/FAS + NIR + H₂O₂ treatment induced copper ion overload in bacteria, leading to potent bactericidal activity through a copper ion-mediated metabolic disruption mechanism.

2.7. Cytotoxicity and anti-inflammatory activity of Cu SACs/FAS in vitro

In addition to its potent antimicrobial activity, favorable biocompatibility is essential for the biomedical application of Cu SACs/FAS in wound repair. Cytotoxicity was assessed using MTT, which detects cell viability after 24 h of incubation with Cu SACs/FAS at different concentrations (0, 20, 40, 60, 80, and 100 μg/mL). L929 cells maintained relatively high viability after treatment with various concentrations of Cu SACs/FAS, and the cell survival rate remained over 90% even at the highest concentration of 100 μg/mL, demonstrating excellent biocompatibility of the nanosystem (Fig. 6A). Calcein-AM staining revealed strong green fluorescence signals, consistent with high cell viability, further supporting the previous experimental results (Fig. 6D). The cell viability was assessed for all treatment groups, and it was found that cell viability remained above 80%, with no significant cell death observed (Fig. S18). This indicated that Cu SACs/FAS under H₂O₂ and NIR conditions exhibit good cellular safety. Hemocompatibility tests were performed by incubating red blood cells (RBCs) with various concentrations of Cu SACs/FAS. The hemolysis rate of Cu SACs/FAS (100 μg/mL) and Cu SACs/FAS + NIR + H₂O₂ under therapeutic conditions both remained well below the 5% safety threshold, confirming excellent hemocompatibility (Fig. 6B–S19). Optical imaging of RBCs from PBS, Cu SACs, Cu SACs/FAS and Cu SACs/FAS + NIR + H₂O₂ after treatment showed typical rounded morphology, indicating no significant hemolysis. In contrast, RBCs treated with DI Water exhibited fragmented morphology, confirming complete hemolysis (Fig. 6C).

Fig. 6.

In vitro cytocompatibility and anti-inflammatory activity of Cu SACs/FAS. (A) Cell viability of L929 cells with different concentrations (0, 20, 40, 60, 80, and 100 μg/mL) of Cu SACs/FAS treatments. (B) Hemolysis rate and (C) Microscopy images of RBCs with diverse materials. Scale bar, 50 μm. (D) Calcein-AM staining of L929 cells with different concentrations (0, 20, 40, 60, 80, and 100 μg/mL) of Cu SACs/FAS treatments. Scale bar, 50 μm. The gene expression of (E) anti-inflammation factor IL-10 and (F) pro-inflammation factor IL-1β. (G) The CD86 (M1)- or CD206 (M2)-positive RAW 264.7 macrophages were analyzed by flow cytometry after control (LPS) and Cu SACs/FAS treatments. Data are presented as mean ± SD (n = 3, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Wound healing is a multi-phase process encompassing antimicrobial, anti-inflammatory, and angiogenic stages [50]. Macrophages, as key orchestrators of inflammation, can polarize into classically activated (M1) or alternatively activated (M2) phenotypes depending on the local microenvironment [51]. To evaluate the anti-inflammatory potential of Cu SACs/FAS, we employed M1-polarized RAW 264.7 macrophages as an in vitro inflammation model. After 24 h of incubation, Cu SACs/FAS significantly suppressed IL-1β production and up-regulated IL-10 expression, indicating a shift toward an anti-inflammatory profile (Fig. 6E and F). To determine whether these changes were accompanied by altered macrophage polarization, flow cytometry was performed using CD86 and CD206 as markers of M1-and M2-like macrophages, respectively. As shown in Fig. 6G, Cu SACs/FAS markedly increased the proportion of CD206-positive cells, with the M2 population rising from 3.8% to 24.2% (6.4-fold). In contrast, the fraction of CD86-positive cells did not decrease significantly within the tested time window, suggesting that Cu SACs/FAS preferentially promoted M2-like polarization rather than rapidly reversing the established M1 population. Together, these results indicate that Cu SACs/FAS attenuates inflammatory activation and favors a pro-reparative immune microenvironment in vitro. The therapeutic benefit of sustained FAS release may extend beyond prolonged drug retention, potentially providing a sustained local immunomodulatory cue during healing. Such sustained exposure could help dampen excessive inflammatory signaling and support the emergence of a pro-reparative macrophage phenotype, thereby complementing the antibacterial action of Cu SACs. Given that FAS is a clinically used ROCK inhibitor, these observations are consistent with a possible involvement of ROCK-pathway modulation; however, direct pathway-level validation will be pursued in future work.

2.8. Impact of Cu SACs/FAS on wound healing

Leveraging the multi-modal antimicrobial activity, exceptional biocompatibility, and targeted immunomodulation of Cu SACs/FAS, we evaluated its therapeutic potential in MRSA-infected diabetic wounds as a clinically relevant model (Fig. 7A). This model was established to recapitulate delayed wound healing driven by persistent drug-resistant bacterial infection. It specifically served to evaluate the concurrent antibacterial and pro-healing efficacy of the Cu SACs/FAS therapeutic system. First, the in vivo photothermal performance was assessed (Fig. 7F–S20), revealing a rapid temperature increase from 27 °C to 46.8 °C within 10 min of 1064 nm NIR irradiation. Such heat treatment conditions were considered appropriate and generally resulted in a warming sensation when the skin was exposed to temperatures of about 45 °C for short periods of time, but usually no noticeable damage was caused [44]. It was worth noting that during the wound healing promotion process with Cu SACs/FAS, the temperature was maintained at approximately 45 °C, which was expected to have an inactivating effect on bacteria without causing damage to the surrounding dermal tissue.

Fig. 7.

Evaluation of the ability of Cu SACs/FAS to kill bacteria and promote wound healing in vivo. (A) Schematic diagram describing the experimental treatment. (B) Photographs of the infected wound area after different treatments on days 0, 2, 4, 6, 8, and 12. (C) Images and (D) corresponding quantitative calculations of surviving bacteria colonies on day 12 in the focal area. (E) Relative wound area of diverse groups within 12 days. (F) Temperature rise profiles of infected wounds after control and Cu SACs/FAS (100 μg/mL) with 1064 nm laser (0.8 W/cm²) irradiation for 10 min. (G) Body weight change curves of different groups of treated mice. (H) HE and Masson staining images of the wound tissues in each group on day 12. Scale bar, 100 μm. (red arrows: inflammatory cells) Data are presented as mean ± SD (n = 3, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Endogenous H₂O₂ in chronically infected wounds is highly variable and often insufficient to sustain efficient CDT. Therefore, exogenous H₂O₂ was supplemented in the in vivo study to evaluate the combined bactericidal efficacy of PTT and CDT under substrate-sufficient conditions. Local administration of 0.4 mM H₂O₂ was used to ensure adequate substrate availability for catalytic ROS generation, thereby enhancing ROS-mediated killing and facilitating clearance of drug-resistant bacteria. Mice were randomly assigned to six groups: control (PBS), Cu SACs, Cu SACs + NIR, Cu SACs + H₂O₂, Cu SACs + NIR + H₂O₂, and Cu SACs/FAS + NIR + H₂O₂. Representative photographs were recorded at designated time points and wound areas were quantified. Representative digital photographs of infected wounds were recorded for different days and relative wound areas were calculated. On day 4, control group wounds persisted with pronounced inflammatory edema and purulent exudate formation, while wounds treated with Cu SACs/FAS + NIR + H₂O₂ demonstrated only minimal edema and early-stage signs of crusting. On day 8, the wound sizes in the Cu SACs + NIR + H₂O₂ and Cu SACs/FAS + NIR + H₂O₂ groups were noticeably smaller than those in the control, Cu SACs, Cu SACs + NIR, Cu SACs + H₂O₂ groups. On day 12, the wound area of Cu SACs/FAS + NIR + H₂O₂ was significantly reduced to 3.8%, whereas the wound area of the control group remained large. The Cu SACs + NIR + H₂O₂ group also showed faster healing rates than the other groups, with a wound size of 8.5%. The photothermal and chemodynamic effects of Cu SACs were all dependent on the addition of NIR and H₂O₂, and its wound-healing efficacy was limited in the absence of both NIR irradiation and H₂O₂ supplementation. With the introduction of NIR or H₂O₂, the healing time of the wounds was greatly reduced and the scabs fell off earlier.

The antibacterial efficacy of various treatment groups against MRSA infection was quantified through bacterial culture and colony counting at wound sites on day 12. As shown in Fig. 7C, Cu SACs exhibited limited antibacterial effects throughout the treatment period in the absence of NIR irradiation and H₂O₂ treatment. Compared with the control group, the Cu SACs + NIR and Cu SACs + H₂O₂ groups showed fewer surviving bacteria. The Cu SACs + NIR + H₂O₂ group continued to maintain its antimicrobial advantages, while the Cu SACs/FAS + NIR + H₂O₂ group displayed the strongest antimicrobial capacity by killing almost all bacteria with an antimicrobial effect of over 98.9% (Fig. 7D). Therefore, benefiting from the multiple synergistic effects, the Cu SACs/FAS + NIR + H₂O₂ group demonstrated the highest anti-MRSA performance and the smallest wound area after 12 days of treatment. Furthermore, we also recorded the body weight changes in mice under different conditions. As presented in Fig. 7G, both the control and treatment groups showed no remarkable changes in the body weight of the mice after 12 days of treatment, indicating that the treatment process did not affect the normal growth of mice. The results indicated that the synergistic treatment using Cu SACs/FAS as a platform exhibited optimal antimicrobial efficacy and tissue repair for MRSA-infected chronic wounds.

2.9. Histopathologic analysis of wounds

In subsequent experiments, a range of biochemical indicators in the wounds were analyzed on day 12 after different treatments to identify the key factors contributing to wound healing. To evaluate inflammation and re-epithelialization, H&E and Masson's trichrome staining were performed (Fig. 7H). H&E staining revealed that the control and Cu SACs groups exhibited pronounced inflammatory infiltration and relatively few hair follicles (Fig. S21). In the control group, the wound edge was covered only by a thin and fragile epithelial layer. Compared with the corresponding non-irradiated groups, NIR irradiation modestly reduced inflammatory cell accumulation and accelerated re-epithelialization (Fig. S22). Notably, the Cu SACs/FAS + NIR + H₂O₂ group showed the most advanced tissue recovery, characterized by a thick, newly formed epidermis, minimal inflammatory cell infiltration, and abundant hair follicles. Appropriate collagen deposition during the remodeling phase is essential for restoring tissue integrity and mechanical strength. Therefore, Masson staining was used to assess collagen formation in the regenerated tissue. As shown in Fig. S23, the Cu SACs/FAS + NIR + H₂O₂ group displayed denser and more regularly organized collagen fibers than the other groups, indicating improved matrix deposition and tissue remodeling. Collectively, these histological results support the superior wound-healing performance of Cu SACs/FAS + NIR + H₂O₂, as evidenced by reduced inflammation, improved re-epithelialization, increased appendage regeneration, and improved collagen organization, consistent with a more favorable regenerative microenvironment.

To further support the ROS-associated mechanism, wound ROS levels were monitored in vivo at multiple time points (day 0, 1, 3, 7, and 12) using a hydroxyl radical–responsive HPF probe (Fig. S24). Baseline ROS signals were low at day 0 (pre-treatment). A marked increase was observed at day 1 after administration and NIR irradiation, consistent with activation of a Fenton-like process and enhanced ·OH generation. ROS levels then gradually declined from day 3 to day 7 as healing progressed, likely reflecting bacterial clearance and reduced inflammatory burden while maintaining moderate oxidative pressure against residual pathogens. By day 12, ROS levels returned to near baseline, consistent with infection resolution and tissue repair.

The two main obstacles to the healing of diabetic wounds are impaired angiogenesis and persistent excessive inflammation. Neovascularization delivers oxygen, nutrients, and immune cells to the wound site, which enables the wound to progress steadily toward healing [52]. In addition, persistent elevation of inflammatory factors can trigger excessive inflammation, damage newly formed tissue, and block the wound's transition from debridement to tissue regeneration. To elucidate the precise healing mechanisms and anti-inflammatory capabilities of Cu SACs/FAS, we conducted validation from two dimensions: promoting angiogenesis and suppressing excessive inflammation. The expression of CD31, a pivotal angiogenesis marker, was quantified using qPCR; its upregulation provides direct evidence of granulation tissue formation and wound revascularization. Furthermore, the expression levels of the classic pro-inflammatory cytokines TNF-α and IL-6 were evaluated, and their reduction represents a robust indicator of anti-inflammatory efficacy. Experimental data confirmed that, compared to the control group, CD31 expression was significantly increased in the Cu SACs/FAS + NIR + H₂O₂ treatment group, whereas IL-6 and TNF-α expression was markedly reduced (Fig. 8A–C). Subsequently, we sought to confirm that these transcriptional changes translated into alterations in functional protein output. WB analysis robustly validated our qPCR findings. A pronounced increase in CD31 protein abundance was evident in the Cu SACs/FAS + NIR + H₂O₂ group, unequivocally confirming the facilitation of neoangiogenesis. Mirroring the mRNA data, the protein levels of TNF-α and IL-6 were profoundly attenuated (Fig. 8D). This concordance between mRNA and protein levels firmly establishes the activation of angiogenic and anti-inflammatory processes at the functional proteomic level.

Fig. 8.

In vivo mechanistic analysis of wound healing in Cu SACs/FAS-treated wounds. (A-C) qPCR results of the expression of representative genes (CD31, TNF-α and IL-6) related to wound healing. (D) Western blotting results of CD31, TNF-α and IL-6. (E) Representative immunofluorescence staining images of CD31, TNF-α and IL-6 at wound margin. Scale bar, 100 μm. (F-H) Relative quantitative analysis of CD31, TNF-α, and IL-6. Data are presented as mean ± SD (n = 3, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

To visually verify the aforementioned pro-angiogenic and anti-inflammatory effects in terms of spatial distribution, we further employed immunofluorescence staining. The Cu SACs/FAS + NIR + H₂O₂ group exhibited the highest CD31 expression (green) relative to other groups. Quantitative statistics showed its CD31 level was 6.4-fold that of the control group, indicating the largest number of newly formed blood vessels and thus significantly aiding in wound healing (Fig. 8E and F). Conversely, the Cu SACs/FAS + NIR + H₂O₂ group displayed the lowest expression levels of the pro-inflammatory factors TNF-α (red) and IL-6 (green). The average fluorescence intensities of TNF-α and IL-6 in the Cu SACs/FAS + NIR + H₂O₂ group were markedly reduced to only 0.21-fold and 0.19-fold of the control group values, respectively, clearly demonstrating its potent anti-inflammatory effect (Fig. 8G and H).

In concert, the coordinated increase in CD31 and decrease in TNF-α and IL-6 across all analytical levels demonstrated that Cu SACs/FAS + NIR + H₂O₂ promoted diabetic wound repair through a synergistic dual-pathway mechanism. It effectively stimulated angiogenesis to restore oxygen and nutrient supply, while simultaneously ameliorating the hostile inflammatory microenvironment. This concerted regulation was pivotal to overcoming the fundamental barriers that impeded healing in diabetic wounds.

In vivo biological safety serves as a critical factor in evaluating the applicability of antimicrobial agents for infection therapy [53]. No significant differences were observed in hematological profiles or serum biochemistry between the experimental treatment group and the PBS control group, indicating minimal hemotoxicity and no evidence of hepatorenal dysfunction at the administered dose (Fig. S25). As shown in Fig. S26, IL-6 and TNF-α levels in the Cu SACs/FAS + H₂O₂+NIR group were comparable to those in healthy controls, with no significant differences. These results indicate that, at the therapeutic dose, Cu SACs/FAS predominantly acts locally at the infected wound site and does not elicit an apparent systemic inflammatory response, supporting its favorable systemic biocompatibility. To assess the long-term biosafety of Cu SACs/FAS, H&E staining of vital organs (heart, liver, spleen, lung, kidney) post-treatment was performed. Complementing these hematological findings, H&E-stained organ sections exhibited intact tissue architectures without pathological abnormalities such as necrosis, inflammatory infiltration, or fibrosis (Fig. S27). These biosafety validation results confirm that Cu SACs/FAS maintains excellent biocompatibility at the therapeutic dose. To assess the in vivo metabolism and clearance of the Cu SACs/FAS system, serum Cu levels were dynamically monitored in mice at multiple time points (Fig. S28). Because the formulation was administered locally to the wound, only a limited fraction of released Cu entered systemic circulation. In the Cu SACs/FAS group, serum Cu exhibited a mild and transient elevation at the early stage but remained within the physiological range throughout the 20-day observation period, with no significant difference from the control group at any time point. By the end of the study, serum Cu returned to baseline levels comparable to those of the control group. These results indicate that Cu released from Cu SACs/FAS does not cause systemic accumulation and can be effectively metabolized and cleared in vivo. Collectively, these results verify the good biocompatibility and biosafety of Cu SACs/FAS, highlighting its promising potential for bacterial infection treatment and wound healing promotion.

3. Conclusions

In summary, this work reports a nanomedicine-depot strategy in which the kinase inhibitor fasudil (FAS) was integrated into copper single-atom catalyst scaffolds (Cu SACs) to construct a multifunctional nanozyme (Cu SACs/FAS) with dual peroxidase-like and GSH-depleting activities for synergistic treatment of infected chronic wounds. Under exogenous H₂O₂ and NIR-II irradiation, Cu SACs/FAS activated complementary antibacterial processes, including ROS-mediated oxidative damage, localized hyperthermia-enhanced membrane disruption, and Cu²⁺-triggered collapse of the bacterial antioxidant defense system. Meanwhile, sustained FAS release promoted M2-like macrophage polarization and enhanced angiogenesis, thereby facilitating wound repair. In both in vitro and in vivo studies, Cu SACs/FAS exhibited potent broad-spectrum antibacterial activity, favorable biocompatibility, and effective wound-healing performance. These findings highlight the potential of this SAC-based therapeutic platform for combining infection control with immune-guided tissue regeneration. Looking ahead, further development of multistimuli-responsive SAC-based depots and in situ substrate-supply strategies is expected to enhance deep-tissue applicability, simplify clinical operation, and accelerate translation toward robust treatment of complex infected wounds.

CRediT authorship contribution statement

Mingqian Wang: Conceptualization, Data curation, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. Jiamu Xiao: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft. Songjie Han: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing – original draft. Wei Lu: Investigation, Validation, Visualization. Ziyi Li: Data curation, Investigation, Validation. Xu Dai: Methodology, Validation, Visualization. XueFeng Jiang: Investigation, Validation, Visualization. Jiale Zhang: Data curation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Haomiao Zhu: Conceptualization, Investigation, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Donglin Gan: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Declaration of competing interest

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

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.