Functional phyto-nanozymes for dual regulation of microbial metabolism and overinflammation microenvironment in diabetic wound

Abstract

Chronic wound management demands multifunctional therapeutic strategies that simultaneously address excessive inflammation and oxidative stress. To meet this challenge, we engineered a three-dimensional biomimetic scaffold (CSSTF) by integrating collagen-based thermosensitive hydrogel, a SiO₂-supported copper single-atom catalyst (Cu-SAC-SE), and tea tree oil-encapsulated liposomes (TTO@Lpo). This composite design enables sustained release of bioactive components, achieving synergistic ROS scavenging, mitochondrial protection, and suppression of NLRP3 inflammasome-mediated pyroptosis. Notably, CSSTF exhibits dual immunomodulatory effects by attenuating neutrophil extracellular trap (NET) formation and shifting macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype, thereby mitigating inflammation-associated tissue damage. Parallelly, TTO@Lpo orchestrates microbial remodeling by selectively inhibiting pathogenic bacteria while enriching beneficial commensals, coupled with elevated production of anti-inflammatory metabolites (e.g., short-chain fatty acids), establishing a self-reinforcing "microbiota-metabolism-inflammation" regulatory loop. In diabetic murine models, CSSTF significantly accelerated wound closure through coordinated mechanisms: (1) enhanced angiogenesis via VEGF upregulation, (2) NETosis suppression that dampens cytokine storms, and (3) ECM reconstruction facilitated by fibroblast activation. Beyond material innovation, this work pioneers a phyto-bionic therapeutic platform leveraging enzymatic catalysis and microbiome reprogramming, offering a paradigm shift in chronic wound treatment through simultaneous physical barrier restoration and dynamic biological modulation.

1. Introduction

Diabetic wounds (DW), a prevalent and debilitating complication of diabetes mellitus, present a substantial clinical burden due to their high incidence, susceptibility to infection, and persistent failure to proceed through orderly healing phases [1,2]. The effective management and treatment of chronic wounds remain a major clinical challenge, particularly due to their complex pathophysiology involving persistent inflammation, microbial infection, and impaired tissue remodeling.

Emerging evidence highlights the critical role of microbial community dynamics in wound repair [3]. As the body's first barrier, the skin harbors commensal microbiota that may colonize underlying tissues upon injury [4]. Proper host–microbiome interactions help regulate innate immune responses during wound healing [[5], [6], [7]]; however, the overgrowth of pathogenic microorganisms such as Staphylococcus aureus [8], Pseudomonas aeruginosa, and Enterococcus faecalis can disrupt this process by secreting toxins, forming biofilms, and inducing excessive inflammation [9]. These mechanisms impair re-epithelialization, delay granulation tissue formation, and ultimately hinder wound closure. Thus, targeted regulation of wound microbiota and their metabolic activity represents a promising yet underexplored therapeutic strategy.

Natural plant extracts—such as tea tree oil (TTO) [10], baicalin [11], and resveratrol [12]—have attracted interest as bioactive components in wound dressings owing to their favorable physicochemical properties, biocompatibility, and multifunctional benefits, including antioxidant, antimicrobial, and anti-inflammatory activities. Among these, TTO has shown particular promise for chronic wound management, supported by both preclinical and clinical evidence [10,13]. For instance, studies have demonstrated its potent broad-spectrum antimicrobial activity against common wound pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa [14], as well as its ability to attenuate excessive inflammation [15] and disrupt bacterial biofilms [16]. These attributes align well with the pathological features of chronic wounds, where biofilm formation and persistent infection often impede healing [17]. Specifically, TTO has been shown to enhance wound closure rates and reduce bacterial load in animal models of infected wounds, while clinical case reports have documented its utility in managing antibiotic-resistant wound infections [18,19]. This evidence provides a strong rationale for the selection of TTO as a functional agent in the present study. Nevertheless, its poor water solubility and low bioavailability remain major limitations, highlighting the need for advanced delivery strategies to maximize its therapeutic potential and facilitate clinical application.

DWs are characterized by a failure to progress through normal healing stages, primarily due to a self-perpetuating cycle of persistent inflammation, microbial infection, and dysregulated tissue remodeling [20]. A central mechanism driving this pathology involves NADPH oxidase (NOX) [21]-mediated overproduction of reactive oxygen species (ROS) [22,23], which depletes endogenous antioxidant defenses [24,25], damages critical extracellular matrix components such as collagen I/III and fibronectin [26], and synergizes with dysbiotic microbiome signals to perpetuate pathological immune activation [27]. This sustained oxidative-inflammatory milieu promotes neutrophil extracellular trap (NET) formation, dysregulated M1 macrophage polarization, and NLRP3 inflammasome-mediated pyroptosis [28], collectively stalling the inflammatory-to-proliferative phase transition [29]. Thus, combinatory therapeutic strategies targeting both ROS scavenging and immunomodulation represent a promising approach to disrupt this vicious cycle and facilitate wound resolution.

We designed an in situ-forming FHCC hydrogel (CSSTF) that locally delivers CSST, where CSST comprises tea tree oil-loaded liposomes (TTO@Lpo) assembled on a single-atom Cu nanozyme (Cu-SAC-SE), to concurrently address oxidative, microbial, and inflammatory barriers in chronic wounds. The catalytic core consists of Cu–N_X sites atomically dispersed in an N-doped carbon matrix (Cu-SAC-SE), produced via silica-assisted defect redistribution during 900 °C pyrolysis of Cu-doped ZIF-8-a choice that suppresses CuO_X nanoparticle formation and maximizes per-site redox activity. These Cu single atoms confer cascade SOD and CAT mimetic functions that sequentially remove O₂•^- and its product H₂O₂, thereby protecting ECM integrity. Tea tree oil is encapsulated in liposomes (TTO@Lpo) to stabilize and concentrate this broad-spectrum antimicrobial/anti-inflammatory agent at the catalytic interface and to augment CAT-like O₂ evolution, while the FHCC hydrogel ensures conformal wound coverage, moisture balance, and sustained, on-demand release. This architecture assigns distinct functions to each component: Cu-SAC mediates reactive oxygen species detoxification, TTO@Lpo provides antimicrobial activity and attenuates inflammatory responses, and the hydrogel ensures spatial localization with sustained, controlled dosing; together, these complementary mechanisms restore homeostasis within the wound metabolic-immune microenvironment and promote orderly regeneration, thereby providing a rigorous mechanistic rationale and underscoring the novelty of the materials system (see Scheme 1).

Scheme.

Schematic illustration of the structural units and fabrication process of CSSTF, along with its therapeutic mechanism in infectious diabetic wound management.

2. Results and discussion

2.1. Fabrication and characterization of CSST

Cu-doped ZIF-8 (Cu-MOF) was synthesized via stirring, followed by the formation of a SiO₂ outer layer (Cu-SAC-SiO₂) to facilitate defect redistribution of Cu atoms during pyrolysis at 900 °C. Subsequent etching in 10 % NaOH at 90 °C for 48 h yielded Cu-SAC-SE. For comparison, Cu-MOF without SiO₂ coating was also prepared. Transmission electron microscopy (TEM) revealed that Cu-SAC-SE exhibited uniform hexagonal morphology (Fig. 1A). High-resolution TEM (HRTEM) and energy-dispersive X-ray spectroscopy (EDS) confirmed homogeneous distributions of carbon, nitrogen, zinc, and copper (Fig. 1B). Aberration-corrected high-angle annular dark-field scanning TEM (AC-HAADF-STEM) images identified isolated bright spots, verifying atomic dispersion of Cu (Fig. 1C). XRD analysis confirmed successful ZIF-8 synthesis and structural evolution of metal atoms. Notably, neither Cu-SAC-SE nor Cu-SAC-C (control) showed diffraction peaks for Cu oxide nanoparticles; instead, only two characteristic graphite carbon peaks ((002) and (100)) were observed (Fig. 1D), confirming the single-atom nature of Cu in both materials.

Fig. 1.

Multistage characterization of SiO₂-assisted synthesized Cu single-atom catalysts loaded with tea resin plastids. (A) Transmission electron microscopy (TEM) image of ZIF-8-derived Cu-SAC-SE nanoparticles. (B) High-angle annular dark-field scanning TEM (HAADF-STEM) image of Cu-SAC-SE and corresponding TEM-energy-dispersive X-ray spectroscopy (TEM-EDS) elemental mapping. (C) Aberration-corrected (AC) HAADF-STEM image of Cu-SAC-SE, with isolated Cu atoms highlighted by red circles. (D) X-ray diffraction (XRD) patterns of ZIF-8, Cu-SAC-C, and Cu-SAC-SE. (E) Raman spectra of Cu-SAC-C and Cu-SAC-SE. (F, G) High-resolution XPS spectra of C 1s for (F) Cu-SAC-C and (G) Cu-SAC-SE. (H, I) High-resolution XPS spectra of N 1s for (H) Cu-SAC-C and (I) Cu-SAC-SE. (K) UV-Vis spectra comparison of TTO@Lpo, Cu-SAC-SE, and CSST. (L) Dynamic light scattering (DLS) analysis of hydrodynamic diameters for Cu-SAC-SE and CSST. (M) Zeta potentials of Cu-SAC-SE and CSST. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Raman spectroscopy further evaluated the sp²/sp³ hybridization of the carbon matrix derived from pyrolyzed ZIF-8. The G-band (∼1600 cm⁻¹, representing sp²-hybridized graphitic carbon) and D-band (∼1360 cm⁻¹, reflecting structural defects) exhibited similar intensity ratios (ID/IG) for both materials (Fig. 1E), indicating that SiO₂ protection did not significantly alter the carbon framework. X-ray photoelectron spectroscopy (XPS) elucidated the electronic structure of active sites. The C 1s spectra of Cu-SAC-C and Cu-SAC-SE (Fig. 1F and G) aligned with typical carbon-nitrogen bonding features. Deconvolution of N 1s spectra (Fig. 1H and I) revealed four peaks at 398.6 eV (pyridinic N), 399.8 eV (pyrrolic N), 401.2 eV (graphitic N), and 403.2 eV (oxidized N), where pyridinic/pyrrolic N serve as anchoring sites for single-atom metals [30].

For CSST preparation, Cu-SAC-SE was sonicated with tea tree oil-loaded liposomes (TTO@Lpo). TEM confirmed liposome coating on ZIF-8 surfaces (Fig. 1J), while UV-vis spectroscopy detected characteristic peaks of both ZIF-8 and TTO, verifying successful loading (Fig. 1K). Dynamic light scattering (DLS) showed hydrodynamic diameters of ∼100.00 nm (Cu-SAC-SE) and ∼121.65 nm (CSST), with zeta potentials of (−36.65 ± 1.03) mV and (−42.04 ± 1.67) mV, respectively. Collectively, these results demonstrate the successful synthesis of CSSTs, featuring atomically dispersed Cu in a carbon matrix with uniform size and stable surface charge.

2.2. Catalytic performance

The superoxide anion (O₂•^-), a highly reactive oxygen species with potent oxidative and destructive capabilities [31], is catalytically dismutated into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂) by superoxide dismutase (SOD) as the primary biological scavenger. When exposed to H₂O₂ to simulate oxidative microenvironment, CSST exhibited discernible structural deformation indicative of catalytic activity. Subsequent electron spin resonance (ESR) measurements using the spin-trapping agent 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) [32] quantitatively verified the O₂•^- scavenging efficacy (Fig. 2B). The ESR spectra at pH 7.4 demonstrated that CSST exhibited superior O₂•^- consumption compared to CSS, as evidenced by significantly attenuated characteristic peak intensities, confirming their SOD-like enzymatic activity [33].

Fig. 2.

Catalytic performance and cellular uptake of composite nanoparticles (CSST), along with the biosafety of hydrogel system CSSTF. (A) TEM images of CSST showing structural deformation in the presence of H₂O₂. (B) Catalytic performance of CSS and CSST in decomposing H₂O₂, measured by electron paramagnetic resonance (EPR). (C) Dissolved oxygen (DO) level changes in water after CSST-mediated catalase (CAT)-like activity. (D) Flow cytometry analysis of endothelial cell uptake of Rho-labeled CSST under co-culture conditions; (E) its corresponding quantitative data. (F) Confocal microscopy images of Rho-labeled CSST internalized by endothelial cells. (G) Macroscopic observation of FHCCGel solidification upon heating. (H) SEM image of the brittle fracture surface of thermally cured FHCCGel. (I) 3D confocal fluorescence microscopy imaging of cell proliferation within CSSTF scaffolds. (J) Cell viability of various repair cells co-cultured with CSSTF, assessed by CCK-8 assay. (K) Proliferation activity of endothelial cells co-cultured with CSSTF, measured via LDH assay. (L) in vivo fluorescence imaging tracking the retention of CSST and CSSTF at wound sites. (M) Degradation kinetics of CSSTF under varying pH and H₂O₂ conditions. (N) MTT assay evaluating the viability of repair-related cells in transwell co-culture with different concentrations of CSSTF. (O) H&E staining images of lung, kidney, and spleen tissues after CSSTF application at wounds.

Notably, while SOD provides crucial protection against O₂•^--mediated damage, its catalytic byproduct H₂O₂ paradoxically induces oxidative injury to biomacromolecules including DNA, proteins and lipids [34]. This necessitates subsequent H₂O₂ elimination by catalase (CAT), which decomposes it into water and O₂ [35]. Evaluation of CAT-mimetic activity through dissolved oxygen (DO) monitoring revealed that both CSST and CSS induced time-dependent DO elevation before reaching equilibrium (Fig. 2C). The significantly higher maximum DO level achieved by CSST suggests that TTO@Lpo contributes supplementary CAT-like catalytic activity beyond the Cu single-atom sites.

These findings collectively demonstrate that CSST, through its engineered Cu single-atom sites, possesses remarkable dual enzymatic activities mimicking both SOD and CAT. The synergistic combination of these activities enables comprehensive ROS scavenging through sequential elimination of O₂•^- and its downstream product H₂O₂, establishing CSST as a promising therapeutic agent for oxidative stress-related pathologies. The enhanced catalytic performance of CSST over CSS further highlights the functional contribution of TTO@Lpo in optimizing the antioxidant defense system.

2.3. Biocompatibility

To verify the cellular uptake of CSST by HUVECs, rhodamine-labeled nanoparticles (Nps) were co-cultured with cells and analyzed using flow cytometry (Fig. 2D and E) and confocal laser scanning microscopy (Fig. 2F). The results demonstrated detectable intracellular CSST accumulation within 6 h of co-culture, with a time-dependent increase in fluorescence intensity, indicating efficient cellular internalization. These findings not only confirm the rapid uptake of CSST by vascular endothelial cells but also suggest its potential for sustained intracellular delivery, a critical feature for promoting angiogenesis during wound healing.

To achieve optimal structural support for three-dimensional cell migration and tissue regeneration, we engineered a rigidity-enhanced thermosensitive hydrogel (FHCCGel) with a precisely optimized composition comprising 18 % F127, 0.5 % HPMC-100 M, 0.25 % chitosan, and 0.25 % type I collagen. This formulation builds upon established research demonstrating that HPMC-100 M effectively modulates the gelation temperature (Tgel) [36,37], while chitosan and collagen synergistically form a self-assembled crosslinked network to enhance both mechanical strength and biocompatibility. The resulting FHCCGel was further functionalized with CSST at a concentration of 125 μg/mL to ensure therapeutic efficacy (CSSTF). The thermosensitive hydrogel exhibited reversible sol-gel transition behavior when subjected to temperature modulation in vitro. At ambient temperature (25 °C), the precursor solution maintained low viscosity suitable for injection, while heating to physiological temperature (37 °C) induced rapid gelation. The molded hydrogel retained precise anatomical contours of the standard molds (Fig. 2G), demonstrating shape fidelity critical for biomedical applications. These results collectively verify that the material achieves injectable fluidity at room temperature while forming mechanically robust, shape-conforming gels under physiological conditions - a dual functionality essential for minimally invasive implantation and in situ defect repair.

Despite the promising potential of nanoparticle (NP)-based therapies, their clinical translation has been significantly hindered by safety concerns, particularly regarding systemic accumulation and local toxicity [38]. To evaluate the impact of CSSTF's post-curing three-dimensional architecture on cellular behaviors, HUVECs were seeded onto the scaffold surface, where they exhibited complete morphological spreading and successful migration through the interconnected porous network (Fig. 2I), demonstrating favorable biocompatibility. Subsequent Transwell assays coupled with CCK-8 and LDH analyses confirmed robust HUVEC proliferation (Fig. 2J) without detectable cytotoxicity (Fig. 2K), validating the scaffold's cellular compatibility.

For therapeutic delivery assessment, comparative IVIS imaging revealed that CSSTF (0.8 mL) achieved more sustained release of Rho-labeled CSST compared to non-fibrous CSST in wound beds (Fig. 2L), highlighting its structural advantage for controlled drug delivery. Degradation kinetics under simulated physiological conditions showed complete material dissolution across all tested pH levels, though alkaline (pH 8-8.8) and oxidative (100 μM saline) environments delayed degradation rates (Fig. 2M). Notably, this 14-day degradation profile in chronic wound-mimicking alkaline conditions (pH ∼8-8.8) aligns with optimal granulation tissue formation timelines [39]. MTT assays across multiple wound-relevant cell types established 125 μg/mL as the maximum safe concentration for CSSTF (Fig. 2N). Crucially, in vivo implantation studies confirmed the absence of nanoparticle accumulation or organ damage (Fig. 2O), addressing a critical translational concern for nanomaterial-based therapies. The internalization kinetics observed here align with previous reports on nanoparticle-endothelial cell interactions, where early uptake is often associated with enhanced therapeutic efficacy. Moreover, the absence of cytotoxicity (as supported by subsequent viability assays) further reinforces the biocompatibility of CSST, making it a promising candidate for wound dressing applications.

2.4. NLRP3 pathway downregulation mediates cellular proliferation and mitochondrial functional recovery

Significant efforts have been devoted to developing anti-inflammatory biomaterials, with composite hydrogels and biomimetic nanozymes emerging as prominent research focuses [40,41]. However, the anti-inflammatory efficacy of these approaches in endothelial cells and their specific mechanisms of action under highly oxidative microenvironments remain insufficiently explored. As demonstrated in Fig. 3A and B, the cell scratch assay was conducted in medium supplemented with 100 μM H₂O₂ to simulate oxidative stress conditions [42]. Time-lapse imaging revealed that HUVECs co-cultured with CSSTF exhibited significantly accelerated migration compared to CSSF and TF groups, with cells initiating movement into the wound area within 6 h post-scratching, while control groups displayed limited motility with persistent scratch borders. Immunofluorescence analysis confirmed that CSSTF, CSSF, and TF all effectively reduced intracellular and mitochondrial ROS levels, with no statistically significant difference observed between CSSF and TF (Fig. 3C–Q and R).

Fig. 3.

CSSTF mitigates endothelial cell pyroptosis by restoring mitochondrial function and suppressing pro-inflammatory factor production. (A) Scratch assay of endothelial cells under different co-culture conditions; (B) corresponding quantitative analysis. (C) Confocal microscopy images of endothelial cells co-stained with ROS- and mtROS-specific fluorescent probes. (D) Flow cytometry analysis of mitochondrial permeability transition pore (mPTP) opening in endothelial cells across treatment groups; (P) relative statistical analysis. Transcription levels of NETs-associated chemokines (E) CCL2 and (F) CXCL8 produced by endothelial cells. (G) Confocal laser scanning microscopy (CLSM) images of TFAM (mitochondrial transcription factor A) in HUVECs; white arrows indicate extracellular extrusion of oxidized DNA. (H) 8-OhdG and TOMM20 are immunofluorescently stained to locate damaged mitochondrial DNA via ultra high resolution confocal microscope. (J) Percentages of extra-mitochondrial DNA in macrophages with different treatments. (K) Cytosolic mtDNA detected by qPCR using D-loop primer. (L) qPCR analysis and (M) Western blot of NLRP3 inflammasome pathway proteins. (N) Statistical analysis of NLRP3 pathway protein expression (Western blot). (O) IL-1β transcription level detection via qPCR. (I) Bio-TEM images of mitochondrial ultrastructure in HUVECs across treatment groups. qPCR analysis of mRNA expression levels for chemotaxis-related factors (J) CXCL8 and (K) CCL2. Quantitative analysis of (Q) ROS and (R) mtROS levels measured by DCFH-DA fluorescence probe.

To investigate mitochondrial functional changes critical for cellular metabolism and proliferation [43], we assessed mitochondrial permeability transition pore (mPTP) dynamics. While physiological mPTP opening is essential for maintaining mitochondrial function and cellular homeostasis, excessive opening can trigger pathophysiological processes by disrupting mitochondrial and cellular ion/redox balance [44,45]. Using the calcein-cobalt quenching method, flow cytometry analysis demonstrated that CSSTF treatment resulted in significantly lower mPTP opening rates compared to all H₂O₂-exposed groups, indicating proper mPTP closure regulation (Fig. 3D and P). Moreover, a corresponding decrease in mitoSOX fluorescence was observed (Fig. S1, Supporting Information). Open mitochondrial function damage in mPTP is obvious, often accompanied by oxidative damage and leakage of mitochondrial DNA. CLSM images confirm that CSSTF can improve the DNA leakage to the extracellular space caused by hydrogen peroxide-induced mitochondrial damage (Fig. 3G). In Fig. 3H, TOMM20 and 8-OhdG staining for extramitochondrial DNA damage revealed markedly reduced fluorescence intensity in CSSTF-treated groups. Through quantitative analysis, it was confirmed that the damage mitochondrial DNA content in mitochondria and cytoplasm showed the same trend, and CSSTF significantly improved the mitochondrial DNA damage caused by peroxide microenvironment (Fig. 3J and K). Bio-TEM ultrastructural analysis further validated these findings, showing that while control HUVECs maintained normal mitochondrial morphology, CSSTF-treated cells exhibited mitochondrial structures more closely resembling untreated controls than H₂O₂-damaged cells (Fig. 3I, white arrows). Collectively, these results demonstrate that CSSTF provides superior protection against mitochondrial oxidative damage and facilitates functional recovery, while CSS and TF show comparable efficacy in these aspects.

Previous studies have demonstrated that Mitochondrial DNA (mtDNA) damage serves as a critical mediator of immune dysregulation by activating the NLRP3 inflammasome-pyroptosis axis, thereby disrupting immune homeostasis. Under conditions of oxidative stress or metabolic dysfunction (e.g., diabetes), mtDNA escapes into the cytosol due to mitochondrial permeability transition pore (mPTP) opening or BAX/BAK-mediated outer membrane permeabilization (MOMP). Cytosolic mtDNA is recognized by TLR9 and cGAS, initiating pro-IL-1β transcription via NF-κB. Concurrently, oxidized mtDNA directly binds and activates the NLRP3 inflammasome, triggering caspase-1-dependent pyroptosis-a lytic cell death characterized by gasdermin D (GSDMD) pore formation, IL-1β/IL-18 maturation, and DAMP release [46]. Mitochondria-mediated activation of the NLRP3 inflammasome pathway initiates cellular apoptosis while concurrently elevating the production of chemokines including IL-1β. This inflammatory cascade accelerates the recruitment of peripheral macrophages and other immune cells to wound sites, generating excessive pro-inflammatory cytokines that perpetuate a state of localized hyperinflammation [47]. Mechanistically, the assembly and activation of the NLRP3 inflammasome facilitates the oligomerization and proteolytic cleavage of pro-caspase-1 into its mature form, subsequently triggering the release of key inflammatory mediators such as IL-1β, CXCL8, and CCL2 [48]. Our PCR analysis demonstrated CSSTF's capacity to significantly suppress CXCL8 and CCL2 expression (Fig. 3E and F) - two chemokines known to potentiate neutrophil recruitment to injury sites through NADPH oxidase (NOX)-dependent mechanisms, thereby priming neutrophils for NETosis. Western blot validation further revealed markedly reduced NLRP3 protein levels in CSSTF-treated groups compared to hydrogen peroxide-exposed controls (Fig. 3L–O). Our ELISA analysis demonstrated CSSTF's capacity to significantly suppress IL-1β expression (Fig. S2, Supporting Information). These findings corroborate established literature demonstrating the critical involvement of NF-κB/NLRP3 signaling in chronic wound pathologies [49], while positioning CSSTF as a promising therapeutic agent capable of interrupting this maladaptive inflammatory cycle. Conventional anti-inflammatory biomaterials often fail to address the self-reinforcing nature of ROS-NLRP3 crosstalk [50], whereas nanozyme-based systems CSSTF demonstrate dual capacity for both ROS scavenging and inflammasome modulation. This dual functionality positions them as superior candidates for treating oxidative stress-associated vascular inflammation compared to single-mechanism materials.

2.5. CSSTF improves the immune microenvironment by inhibiting pro-inflammatory macrophage polarization and NET formation

Dysregulated macrophage phenotypic switching is critically implicated in the inflammatory imbalance of chronic wounds. Sustained pro-inflammatory (M1) polarization, driven by hyperglycemic conditions, advanced glycation end products (AGEs), and mitochondrial ROS (e.g., mtDNA leakage), promotes excessive secretion of IL-1β and TNF-α through the NF-κB/NLRP3 pathway, exacerbating tissue damage. This is further compounded by defective efferocytosis, creating a vicious cycle of "necrosis-inflammation." Additionally, macrophage crosstalk with other immune cells plays a pivotal role. In neutrophils, M1-derived IL-1β triggers NETosis, releasing proteases (e.g., MMP-9) that degrade the extracellular matrix (ECM).

To investigate this mechanism, we first assessed the effects of different treatments on macrophage polarization. Our results demonstrated that CSSTF not only suppressed M1 polarization under pro-inflammatory conditions but also promoted M2 phenotype expression. While both CSSF and TF exhibited similar effects, their efficacy was markedly inferior to CSSTF (Fig. 4A–D). Overall, CSSTF significantly reduced the secretion of pro-inflammatory cytokines (Fig. 4E–H) while enhancing the expression of anti-inflammatory factors (Fig. 4I).

Fig. 4.

CSSTF attenuates pro-inflammatory polarization of macrophages and neutrophil extracellular trap (NETs) formation induced by hyperinflammatory microenvironment sensing. (A) Flow cytometry and (D) immunofluorescence staining were used to analyze macrophage polarization; (B, C) related statistical analysis. Concentrations of pro- and inflammatory factors (E) IFN-γ, (F) IL-6, (G) IL-1β, (H) TNF-α and (I) IL-10 in supernatants (ELISA). (J) Schematic illustration of the experimental procedure: untreated neutrophils were incubated with conditioned medium from CSSTF-endothelial cell co-culture. (K) Confocal laser scanning microscopy (CLSM) images showing NETs formation (indicated by arrowheads) in neutrophils under different treatments. (L) Quantitative analysis of MPO⁺H2B⁺ double-positive areas (NETs) from (K). (M) MPO-DNA complex levels in supernatants measured by ELISA, reflecting NETs release.

In chronic wounds (such as diabetic foot ulcers and venous ulcers), the persistent release of neutrophil extracellular traps (NETs) may lead to excessive activation of proteases (e.g., elastase) and reactive oxygen species (ROS), which degrade growth factors and extracellular matrix (ECM), thereby delaying re-epithelialization [23,51]. Additionally, NETs sustain a pro-inflammatory state (e.g., elevated IL-1β and TNF-α) by activating the NLRP3 inflammasome or TLR signaling pathways (e.g., TLR4/9) [52], while also suppressing macrophage polarization toward the pro-repair M2 phenotype, further exacerbating the immune microenvironment. To investigate whether CSSTF modulates NETs formation under hyperinflammatory conditions, we first established an in vitro model in which untreated neutrophils were exposed to conditioned medium (CM) derived from CSSTF-endothelial cell co-cultures (Fig. 4J). Confocal laser scanning microscopy (CLSM) revealed robust NETs formation (indicated by arrowheads) in neutrophils stimulated with hydrogen peroxide-treated endothelial cell CM, characterized by extracellular DNA structures co-localized with myeloperoxidase (MPO) and histone H2B (Fig. 4K). However, CSSTF treatment significantly reduced NETs formation, as evidenced by decreased MPO⁺H2B⁺ double-positive areas (Fig. 4L, p < 0.01). Consistent with microscopic observations, ELISA quantification demonstrated that CSSTF markedly suppressed MPO-DNA complex release (Fig. 4M, p < 0.001), a hallmark of NETosis.

This study demonstrates that CSSTF effectively suppresses inflammatory macrophage polarization NETs formation triggered by a hyperinflammatory microenvironment, offering a potential therapeutic strategy for macrophage- and NETs-driven chronic inflammation. Notably, CSSTF also downregulated pro-inflammatory cytokines from macrophages known to amplify NETs release and sustain neutrophil activation. This dual action—direct NETs suppression and cytokine modulation—highlights CSSTF's pleiotropic anti-inflammatory effects.

2.6. CSSTF scaffold accelerates diabetic wound healing by enhancing angiogenesis and inhibiting NETs formation

To evaluate the therapeutic potential of CSSTF in vivo, we established a diabetic full-thickness wound model (Fig. 5A). To clarify the biodistribution of CSSTF, major organs and tumor tissues were collected and weighed at the indicated time points after treatment, and the levels of CSSTF within these tissues were quantified by ICP-MS. Our results showed that following CSSTF administration, both Zn and Cu were primarily detected in the liver, kidney, and tumor at normal physiological levels. This indicates that the amount of Zn and Cu absorbed through the wound was minimal, suggesting low systemic absorption of the main component (Cu-SAC-SE) and demonstrating the favorable in vivo safety profile of CSSTF (Fig. S3, Supporting Information). Macroscopic assessment revealed accelerated wound closure in the CSSTF-treated group compared to Control, CSSF, and TF groups (Fig. 5B). Heatmap analysis further confirmed that CSSTF significantly improved wound closure rates across all time points (Fig. 5C). Histological examination (H&E staining) demonstrated enhanced re-epithelialization and granulation tissue formation in CSSTF-treated wounds (Fig. 5D), with the shortest time-to-complete healing among all groups (Fig. 5E, p < 0.001). Immunohistochemical staining for CD31, a marker of endothelial cells, revealed a substantial increase in vascular density in CSSTF-treated wounds on Day 14 (Fig. 5F–G, p < 0.01), indicating robust angiogenesis.

Fig. 5.

CSSTF scaffold accelerates wound healing by promoting angiogenesis and suppressing NETs formation in vivo. (A) Schematic diagram of the animal experiment. (B) Macroscopic photographs of acute full-thickness diabetic wounds. (C) Heatmap of wound closure percentages across treatment groups (Control, CSSF, TF, CSSTF) at different time points. (D) Representative H&E-stained sections of wounds at various time points. (E) Time-to-complete healing (days) under five treatment regimens. (F) Immunohistochemical images of CD31⁺ vessels in wound tissues on Day 14; (G) Quantitative analysis of CD31⁺ area. (H) Immunofluorescence staining of CitH3 (green), MPO (red), and DAPI (blue) in wound sites at Day 3, demonstrating NETs formation. (I) qPCR analysis of NLRP3/caspase-1/IL-1β mRNA expression in wound tissues. (J) Serum MPO-DNA complex levels (ELISA), reflecting systemic NETs activity. (K, L) qPCR analysis of wound tissue mRNA levels for (K) IL-6 and (L) IFN-γ. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Immunofluorescence staining of wound tissues at Day 3 demonstrated reduced Citrullinated Histone H3 (CitH3) and myeloperoxidase (MPO) co-localization in the CSSTF group (Fig. 5H), suggesting diminished NETs formation. This was further supported by decreased serum MPO-DNA complex levels (Fig. 5J, p < 0.001). Additionally, qPCR analysis revealed that CSSTF downregulated key pro-inflammatory mediators, including NLRP3, caspase-1, and IL-1β (Fig. 5I, p < 0.01), as well as IL-6 (Fig. 5K, p < 0.05) and IFN-γ (Fig. 5L, p < 0.01), indicating attenuation of the hyperinflammatory response.

Our findings demonstrate that the CSSTF scaffold significantly accelerates diabetic wound healing through a dual mechanism: enhancing angiogenesis and suppressing pathological NETs formation. The accelerated wound closure and increased CD31⁺ microvessel density suggest that CSSTF creates a pro-regenerative microenvironment.

2.7. CSSTF scaffold modulates wound microbiota composition and metabolic profile

Tea tree oil (TTO) exhibits broad-spectrum antibacterial effects and modulates immune responses through its anti-inflammatory properties [53,54]. Existing studies suggest that in the regulatory mechanisms of microbiota-metabolism interactions [55], TTO can directly remodel the microbial community in chronic wounds by disrupting quorum sensing (QS) to suppress virulence factor secretion [56]. To further investigate whether CSSTF exerts similar effects in diabetic wounds, we collected wound exudates and surface swabs for transcriptomic and untargeted metabolomic analyses.

16S rRNA sequencing revealed significant alterations in wound surface microbiota following CSSTF treatment (Fig. 6A and B). At the phylum level, CSSTF-treated wounds exhibited increased abundance of beneficial Firmicutes (42.7 % vs 28.3 % in Sham) and decreased Proteobacteria (31.5 % vs 47.2 %). Genus-level analysis demonstrated enrichment of commensal Staphylococcus (18.4 % vs 9.7 %) and reduction in pathogenic Pseudomonas (12.1 % vs 22.3 %). Bray-Curtis dissimilarity analysis (Fig. 6C) and PCoA2 (3.9 %, p < 0.05) confirmed distinct clustering between CSSTF and Sham groups, indicating treatment-specific microbiome restructuring. Differential enrichment analysis (Fig. 6D) identified 27 significantly altered taxa (LDA score >3.5).

Fig. 6.

Regulatory effects of CSSTF scaffold on microbiota composition in DB treatment. (A, B) 16S transcriptomic analysis of wound surface bacteria (n = 8) in Sham and CSSTF groups. Relative abundance bar plots of microbial taxa at the phylum/genus level. (C) Sample clustering visualized by Bray-Curtis distance. Microbial community composition across groups was assessed using Principal Coordinate Analysis (PCoA) based on Bray-Curtis dissimilarity at the family level. (D) Differential enrichment analysis of microbial communities between CSSTF and Sham groups.

Untargeted metabolomics demonstrated clear separation between groups in PCA (Fig. 7A, PC1 = 36.41 %) and PLS-DA (Fig. 7B, R²Y = 0.89, Q² = 0.76) models. LC-MS/MS analysis (Fig. 7D) identified 146 differentially expressed metabolites (DEMs, FDR<0.05, VIP>1.5, |log2FC|>1), including 377 upregulated and 415 downregulated compounds. KEGG enrichmenthighlighted significant pathway modulation (Rich factor>0.3), especially in butanoic acid and ethyl ester pathway (Fig. 7E). Heatmap analysis revealed coordinated changes in wound-healing associated metabolites (Fig. 7F), with CSSTF treatment increasing anti-inflammatory SCFAs (glyceric acid −4.7-fold, p < 0.01) while decreasing pro-inflammatory leukotrienes (fasciculic acid +3.2-fold, p < 0.001; Guvacine +2.7-fold, p < 0.01; butanoic acid +2.1-fold, p < 0.001) (Fig. 7G). The increase in butyrate, a potent HDAC inhibitor with dual antimicrobial and anti-inflammatory properties, may explain both the microbiome restructuring and NETs suppression observed.

Fig. 7.

Elucidation of CSSTF scaffold's impact on differentially expressed metabolites in DB treatment through untargeted metabolomics. (A–C) Visualization of Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) results. PCA displays the metabolic profiles of untreated and CSSTF-treated wound tissues, with similar metabolite compositions within each group clustered together. (D) 2D-LC-MS/MS-based metabolomic analysis identified significantly altered metabolites between Sham and CSSTF groups using a screening threshold combining: 1) P-values from Student's t-test, 2) Variable Importance in Projection (VIP) scores from the OPLS-DA model's first principal component, and 3) absolute log2-fold change values. (E) KEGG pathway enrichment analysis. The Rich factor (ratio of differentially expressed metabolites annotated in a given pathway to total detected metabolites in that pathway) indicates enrichment degree, with higher values representing greater enrichment. (F) Heatmap visualization of significantly altered metabolites. Hierarchical clustering was performed using complete linkage method based on Euclidean distance matrices calculated from quantitative values of differential metabolites. (G) LC-MS-derived short-chain fatty acids associated with wound healing processes.

These results position CSSTF as a microbiome-metabolome modulator that creates a pro-healing wound ecosystem. The scaffold appears to function as a "metabolic scaffold", simultaneously: (i) fostering beneficial symbionts, (ii) suppressing virulence factor production, and (iii) shifting metabolic flux toward tissue-reparative pathways. Future studies should investigate whether CSSTF's effects are microbiota-dependent using germ-free models, and explore its potential to prevent diabetic wound recurrence through microbial niche engineering.

3. Conclusion

In this study, we developed a multifunctional CSSTF scaffold by integrating SiO₂-assisted Cu single-atom catalysts (Cu-SACs) with tea tree oil-loaded liposomes (TTO@Lpo) within a thermosensitive hydrogel matrix. Dual Catalytic Activity: The Cu-SACs exhibited robust SOD- and CAT-like enzymatic activities, effectively neutralizing ROS and mitigating oxidative damage, thereby preserving mitochondrial function and reducing NLRP3-mediated pyroptosis. In addition, CSSTF significantly attenuated NETs formation by downregulating pro-inflammatory cytokines (IL-6, IFN-γ) and suppressing the NLRP3/caspase-1/IL-1β pathway, thereby breaking the cycle of chronic inflammation. Last but not least, CSSTF restructured the wound microbiota, enriching beneficial Firmicutes while reducing pathogenic Proteobacteria. Concurrently, it elevated anti-inflammatory metabolites (e.g., butyrate) and suppressed virulence factors, creating a pro-healing microenvironment. in vivo, CSSTF accelerated wound closure, improved re-epithelialization, and increased CD31⁺ microvessel density, highlighting its potential to restore vascularization in chronic wounds. The CSSTF scaffold represents a paradigm shift in chronic wound therapy by simultaneously targeting oxidative stress, inflammation, microbial dysbiosis, and impaired angiogenesis. Its design as a "metabolic scaffold" offers a comprehensive solution to the multifactorial challenges of diabetic wound healing. Future studies should focus on translational validation in large-animal models and clinical scalability, with an emphasis on personalized microbiome-metabolome profiling to optimize therapeutic outcomes. This work not only advances the field of biomimetic enzyme-catalytic therapy but also provides a versatile platform for plant-derived drug delivery systems in regenerative medicine.

CRediT authorship contribution statement

Shan He: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Zhenhao Li: Writing – review & editing, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Wenguo Huang: Writing – review & editing, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation. Yujie Peng: Writing – review & editing, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation. Libin Niu: Visualization, Validation, Software, Resources, Formal analysis. Huangding Wen: Methodology, Investigation, Formal analysis. Youshan Xv: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Formal analysis, Data curation. Shuo Li: Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation. Zhiqing Li: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

Ethical statement

The care and use of all animals were approved by Laboratory Animal Ethics Committee of Nanfang hospital, Southern Medical University, Guangdong Province, China (approval number: NFYY-2023-1204) and was strictly compliant with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Author agreement

The work described has not been submitted elsewhere for publication. All authors have seen and approved the final version of the manuscript being submitted.

Funding

This research was supported, in whole or in part, by the National Natural Science Foundation of China (Zhiqing Li, Grant Number 82472556) and Guangdong Basic and Applied Basic Research Foundation (Zhiqing Li, Grant Number 2024A1515012825, 2025A1515011535).

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.