Revascularization-Driven Nanozyme Therapy: Disrupting the Vicious Cycle of ROS and Insufficient Vascularization for Chronic Wound Healing

Revascularization-Driven Nanozyme Therapy: Disrupting the Vicious Cycle of ROS and Insufficient Vascularization for Chronic Wound Healing | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 6 November 2025 V1 Latest version Share on Revascularization-Driven Nanozyme Therapy: Disrupting the Vicious Cycle of ROS and Insufficient Vascularization for Chronic Wound Healing Authors : Qiuxue Jiang , Meixia Zhang , Yuqi Yang , Mingzhu Jin , Junlong Zhao , Da Chen , Boqi Jiang , … Show All … , Mengfan Li , Jiahao Ma , Jie Li , Jianwei Zhao , Yongquan Qu 0000-0002-6202-1929 [email protected] , Ling Liu , Sanzhong Li , and Zhimin Tian Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176241997.77692386/v1 224 views 133 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The pathophysiology of chronic wound is characterized by a deleterious interplay between impaired angiogenesis and excessive generation of reactive oxygen species (ROS), which perpetuates a vicious cycle exacerbated by persistent inflammation. Current therapeutic strategies lack efficacy in addressing this multifaceted pathophysiology. To dismantle this vicious cycle, we design a composite nanozyme (TCC) by encapsulating ultrasmall ceria nanoclusters (CeNC) and introducing tryptophan as competitive ligands within a biocompatible cobalt-based zeolitic imidazolate framework (Co-ZIF). The spatial confinement of Co-ZIF matrix effectively suppresses the aggregation of highly defective CeNC, while facilitating the internal Co-to-Ce electron transfer. Collectively, this synergistic effect increases Ce 3+ fraction and modulates d-band center of CeNC, thereby enhancing its capability for catalytic decomposition of superoxide radicals. In vitro and in vivo investigations demonstrate that TCC exhibit exceptional ROS-scavenging capabilities. Concurrently, it effectively promotes angiogenesis via the released cobalt ions that stabilizing hypoxia-inducible factor-1α and further upregulating vascular endothelial growth factor expression. The dual functions of TCC synergistically disrupt the vicious pathogenic cycle of the malignant ROS accumulation and vascular insufficiency. Consequently, TCC significantly enhances pro-angiogenic outcomes in the wound microenvironment in vivo and in vitro . This work highlights a promising strategy of integrating nanozyme-based ROS-scavenging with vascular repair for comprehensive chronic wound management, offering translational potential for next-generation regenerative therapies targeting oxidative and vascular pathologies. Revascularization-Driven Nanozyme Therapy: Disrupting the Vicious Cycle of ROS and Insufficient Vascularization for Chronic Wound Healing ABSTRACT The pathophysiology of chronic wound is characterized by a deleterious interplay between impaired angiogenesis and excessive generation of reactive oxygen species (ROS), which perpetuates a vicious cycle exacerbated by persistent inflammation. Current therapeutic strategies lack efficacy in addressing this multifaceted pathophysiology. To dismantle this vicious cycle, we design a composite nanozyme (TCC) by encapsulating ultrasmall ceria nanoclusters (CeNC) and introducing tryptophan as competitive ligands within a biocompatible cobalt-based zeolitic imidazolate framework (Co-ZIF). The spatial confinement of Co-ZIF matrix effectively suppresses the aggregation of highly defective CeNC, while facilitating the internal Co-to-Ce electron transfer. Collectively, this synergistic effect increases Ce 3+ fraction and modulates d-band center of CeNC, thereby enhancing its capability for catalytic decomposition of superoxide radicals. In vitro and in vivo investigations demonstrate that TCC exhibit exceptional ROS-scavenging capabilities. Concurrently, it effectively promotes angiogenesis via the released cobalt ions that stabilizing hypoxia-inducible factor-1α and further upregulating vascular endothelial growth factor expression. The dual functions of TCC synergistically disrupt the vicious pathogenic cycle of the malignant ROS accumulation and vascular insufficiency. Consequently, TCC significantly enhances pro-angiogenic outcomes in the wound microenvironment in vivo and in vitro . This work highlights a promising strategy of integrating nanozyme-based ROS-scavenging with vascular repair for comprehensive chronic wound management, offering translational potential for next-generation regenerative therapies targeting oxidative and vascular pathologies. Keywords: angiogenesis | wound treatment | nanozyme | HIF-1α | antioxidant 1 | Introduction Angiogenesis ensures effective delivery of oxygen/nutrients to tissues while facilitating removal of metabolic wastes, which is essential for tissue repair and regeneration after traumatic injury or pathological insults [1,2]. Insufficient angiogenesis, causing delayed wound healing, is clinically classified as chronic wounds. They significantly impair patients’ life quality and may lead to mortality, imposing substantial healthy and economic burdens. Globally, ~500 million individuals annually sustain chronic wounds arising from diverse etiologies ( e.g. , traumatic injuries, burns, surgical incisions) [3-6]. Chronic wound pathogenesis can be initiated by multiple etiological factors, including aging, obesity, inflammation, infection, diabetes, and vascular insufficiency [7-10]. Current therapeutic strategies focus on debridement, infection control and local treatments for chronic wound healing. Unfortunately, for their complex pathological microenvironments, those clinical interventions are inadequate to meet the multi-dimensional demands for effective healing ­[11,12]. Moreover, angiogenesis critically underpins multiple tissue repair processes including brain injuries, bone defects, and post-transplantation organ regeneration [13-16]. Consequently, angiogenesis-promoting strategies (growth factors, gene therapy, cell therapy, etc. ) have been explored to enhance chronic wound healing. However, these approaches exhibit suboptimal efficacy and pose oncogenic risks [17,18]. Thus, there is an urgent need for innovative strategies to enhance angiogenesis for tissue regeneration and wound repair. Persistent inflammatory state in chronic wounds typically induces severe oxidative stress, which further impedes angiogenesis and establishes a vicious cycle characterized by excessive reactive oxygen species (ROS) accumulation and insufficient vascularization, severely compromising wound healing [19,20]. Consequently, breaking the vicious cycle of ROS and insufficient vascularization is imperative for revitalizing angiogenesis. Overall, targeting the pathological microenvironment of chronic wounds, a therapeutic strategy, integrating anti-inflammation, alleviation of ROS-induced damage, and promotion of angiogenesis, holds promise for effective healing. The hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) signaling pathway, a pivotal regulator of angiogenesis, is dysfunctional in chronic wounds due to the complex pathological microenvironment ( e.g ., high levels of glucose and ROS) [21-23]. HIF-1α, a crucial regulator of the cellular response to low O 2 levels following tissue injury, activates the expression of multiple proteins ( e.g ., VEGF) involved in cell proliferation, migration, and angiogenesis. Given its central role in revascularization, upregulating HIF-1α expression to restore angiogenic capacity represents a promising therapeutic strategy for chronic wound repair [22,24-26]. However, HIF-1α is inherently unstable in air-exposed chronic wounds [22]. Furthermore, previously reported chemical molecules as HIF-1α stabilizers are associated with side effects, such as liver damage [22,27]. Therefore, there is an urgent need to develop alternative strategies for HIF-1α activation in wound treatment. Recent studies have highlighted that extracellular vesicles, NO, H 2 S, CO, iridium complexes, Zn 2+ , Ca 2+ , copper and cobalt-based nanomaterials can up-regulate VEGF expression and enhance angiogenesis by promoting HIF-1α activity, thereby accelerating wound healing [22,25,26,28-40]. Additionally, an ideal nanomedicine for wound treatment should possess potent antioxidant activity. Recently, nanozymes, a class of nanocatalyst (metals, metal oxides, carbon-based materials, metal-organic frameworks (MOFs), etc. ) with enzyme-like activities, have garnered significant attention due to their designable biomimetic properties, facile preparation, high environmental stability and cost-effectiveness [41-47]. Particularly, MOFs, featured by uniform porosity and exceptionally high surface area, endowing them with superior catalytic potentials for biomedical applications [48-50]. Among MOF nanozymes, cobalt-based MOFs ( e.g ., ZIF-67) are widely reported as promising nanozymes due to their high catalytic activity and ease of preparation. More importantly, ZIF-67, serving as a cobalt source, holds potential for promoting angiogenesis, inducing anti-inflammation macrophage polarization to inhibit inflammation, and thereby accelerating wound healing [51-54]. However, its relatively low ROS scavenging capacity restricts its therapeutic efficacy. Nanoceria with mixed Ce 3+ /Ce 4+ valence states exhibits intrinsic antioxidant activity [55-60]. Nevertheless, their activity is significantly limited by agglomeration and low specific surface area, hindering their biomedical applications. Recently, ultrasmall ceria nanoclusters (CeNC) have demonstrated exceptional antioxidant performance for their large surface area, high Ce 3+ fraction, and optimized electronic structure. These features make CeNC particularly attractive for nanomedicine [61-63]. Unfortunately, synthesis of stable and uniform small nanoclusters remains challenging due to their thermodynamic instability and synthetic complexity [64]. SCHEME 1 | Schematic illustration of TCC nanozyme as an angiogenic agent for accelerated wound healing. (a) Synthetic scheme of TCC nanozyme. (b) Mechanism illustration of the enhanced antioxidant activity of TCC. (c) Wound healing mechanisms of TCC through the restored blood circulation, ROS scavenging and inflammation regulation. By activating HIF-1α/VEGF signaling pathway in vivo , TCC effectively disrupts the vicious pathogenic cycle of the malignant ROS accumulation and vascular insufficiency. Herein, we constructed a composite nanozyme (TCC) by integrating CeNC (~1.5 nm) and tryptophan into a Co-based ZIF-67 (Co-ZIF) framework to accelerate chronic wound healing by breaking the vicious cycle of the malignant ROS accumulation and vascular insufficiency (Scheme 1). Spatial confinement effect of Co-ZIF effectively immobilized highly defective CeNC within its framework, avoiding aggregation and preserving activity. Internal Co-to-Ce electron transfer enhanced the Ce 3+ fraction and modulated the d-band center of CeNC. These structural features significantly promoted the antioxidant efficacy of TCC, yielding ROS scavenging capability nearly 365 times (SOD-like activity) and 244 times (CAT-like activity) higher than that of CeO 2 . Furthermore, tryptophan reduced the TCC size from microns of Co-ZIF to ~200 nm, significantly enhancing its antioxidant activity and bioavailability for wound healing [65]. Both in vitro and in vivo experiments demonstrated that TCC enhanced wound healing by scavenging ROS, suppressing inflammation, and promoting angiogenesis through upregulated the level of HIF-1α/VEGF. Compared to the control group, TCC-treated wounds exhibited a 165% surge in vascular density by day 7 and a 56% elevation by day 14. Meanwhile, the wounds were almost completely healed on the 14th day, demonstrating its therapeutic superiority in chronic wound healing. Collectively, this TCC-enabled therapeutic strategy of reversing the malignant ROS accumulation-insufficient vascularization cycle establishes a promising paradigm for chronic wound management. 2 | Results and Discussion 2.1 | Synthesis and Characterizations As illustrated in Figure 1a, TCC was prepared via a one-pot synthetic protocol under mild conditions, enabling the spontaneous growth of ultrasmall CeNC and the introduction of tryptophan within the Co-ZIF framework [54]. Transmission electron microscopy (TEM) images adopted a hazelnut-like morphology of TCC with an average diameter of Co-ZIF alone (~1.0 μm) or CeNC@ZIF-67 (Ce/Co-ZIF) (~1.4 μm) (Figure 1b and Figures S1, S2). High-resolution transmission electron microscopy (HRTEM) images confirmed the uniform distribution of CeNC (~1.5 nm) on both the surface and within the mesoporous architecture of TCC. The measured lattice fringe spacing of 0.31 nm for the small clusters corresponded to the CeO 2 (111) crystallographic plane, further confirming the spontaneous formation of CeNC during synthesis (Figure 1c and Figures S3) [58]. Energy-dispersive spectroscopy (EDS) elemental mapping and spectra further confirmed the uniform distribution of O, C, N, Co and Ce throughout TCC, indicating homogeneous anchoring of CeNC in TCC (Figure 1d and Figure S4). Inductively coupled plasma optical emission spectrometry (ICP-OES) quantification revealed the cerium contents at 1.8 wt. % and 3.2 wt. % for Ce/Co-ZIF and TCC, respectively (Table S1). Ultraviolet-visible (UV-vis) spectrophotometry of TCC confirmed the successful introduction of tryptophan within the crystalline framework, as evidenced by a characteristic UV-vis absorption peak at 280 nm corresponding to tryptophan’s spectroscopic signature (the π→π* transition of indole ring) (Figure 1e). Quantitative analysis of UV-vis spectrophotometry at λ max = 280 nm revealed a tryptophan loading of 94 μg/mg in TCC (Figure S5) [65]. Meanwhile, a distinct color transition from pale purple (pristine Co-ZIF) to deeper purple in Ce/Co-ZIF and TCC provided complementary visual evidence for the integration of CeNC and tryptophan (Figure S6). N 2 adsorption-desorption isotherms were employed to evaluate the impact of their integration on the porous architecture on Co-ZIF. All samples exhibited Type I isotherm profiles, indicating the preserved crystallinity features after the introduction of CeNC and tryptophan into Co-ZIF (Figure S7a). The reduced porosity of TCC in comparison with that of pure Co-ZIF was attributed to partial pore occupation by the incorporated CeNC and encapsulated tryptophan molecules, as evidenced by its significantly weakened intensity of mesopores at 1-2 nm (Figure S7b). Brunauer-Emmett-Teller (BET) surface areas decreased from 1477 m 2 g -1 (Co-ZIF) to 1348 m 2 g -1 (Ce/Co-ZIF) and finally to 1023 m 2 g -1 (TCC), directing correlating to certain extent of pore occupation by CeNC and/or tryptophan (Figure S7c and Table S2). X-ray diffraction (XRD) analysis confirmed that all synthesized nanozymes preserved the framework of ZIF-67, with characteristic diffraction peaks exhibiting precise alignment to the simulated reference pattern. Notably, no diffraction peaks corresponding to CeO 2 phase were observed due to the ultrasmall size of CeNC (Figure 1f). Both TCC and Ce/Co-ZIF exhibited significantly attenuated XRD peak intensities relative to pristine Co-ZIF, indicating structural perturbations induced by the incorporated CeNC and encapsulated tryptophan [66]. Subsequently, electron paramagnetic resonance (EPR) spectroscopy was employed to examine their intrinsic structural defects. Compared to the pristine Co-ZIF, both Ce/Co-ZIF and TCC exhibited significantly enhanced EPR signals with near-symmetric profiles centered at g = 2.003, indicating the existence of abundant structural defects (Figure 1g) [67]. Quantitative EPR analysis revealed a 9.7-fold and 15.6-fold increases in EPR signal intensity for Ce/Co-ZIF and TCC, respectively, compared to Co-ZIF. These findings confirmed the most abundant structural defects in TCC, which correlated with its attenuated XRD peak intensity and reduced thermal stability (Figures 1f and Figure S8). The enhanced defect formation in TCC arises from two synergistic mechanisms: (i) disruptive effects of the CeNC incorporation on framework assembly, and (ii) competitive coordination between tryptophan and imidazole ligands during crystallization. Collectively, these perturbations disrupt the topological connectivity of the ZIF framework, thereby producing abundant structural defects. The resultant defect-rich architecture provides numerous active adsorption sites, potentially enhancing ROS scavenging activity of TCC. FIGURE 1 | Characterizations of TCC. (a) Schematic of synthesis process of TCC nanozyme. (b) TEM image of TCC nanozyme. (c) HRTEM image of TCC and the CeNC marked in white box. The inset shows the corresponding lattice fringe of CeNC. (d) High angle angular dark field-scanning transmission electron microscopy image and the corresponding EDS mappings (O, C, N, Co, Ce) of TCC. (e) UV-vis absorption spectra of Co-ZIF, Ce/Co-ZIF, TCC and tryptophan. (f) XRD patterns of Co-ZIF, Ce/Co-ZIF and TCC. (g) EPR spectra of Co-ZIF, Ce/Co-ZIF and TCC. FIGURE 2 | Electronic structures and coordination environments of TCC. (a) XPS survey of Co-ZIF, Ce/Co-ZIF and TCC. (b) XPS Co 2p spectra of Co-ZIF, Ce/Co-ZIF and TCC. (c) XPS Ce 3d spectra of Ce/Co-ZIF and TCC. (d) The Co k-edge XANES and (e) plots after range reduction of Co-ZIF, TCC and the references of CoO, Co foil and Co 3 O 4 . (f) The Ce L 3 -edge XANES of TCC and the references of CeO 2 and CeCl 3 . (g) Wavelet transform for the Co k-edge and Ce L 3 -edge EXAFS signals of Co-ZIF, TCC and CeO 2 . 2.2 | Electronic structures and coordination environments of TCC X-ray photoelectron spectroscopy (XPS) provided crucial insights into the surface composition and electronic structures of as-synthesized samples. Survey XPS spectra confirmed the presence of Co, Ce, C, N and O with no detectable contaminants, consistent with elemental mapping results (Figure 2a). High resolution C 1s XPS spectra showed multiple deconvoluted peaks at 284.8, 286.0, and 288.5 eV, corresponding to C-C/C=C, C-O/C-N, and O-C=O bonds, respectively, confirming the presence of carboxyl group in TCC (Figure S9). O 1s spectra exhibited the characteristic metal-oxygen bonds, validating the CeNC incorporation in both Ce/Co-ZIF and TCC (Figure S10). The Ce 3+ /Ce 4+ ratio profoundly influences nanozyme catalytic performance [55-60]. Quantitative analysis of Ce 3d XPS spectra identified ten resolved peaks, corresponding to mixed Ce 3+ (pink) and Ce 4+ (blue) oxidation states (Figure 2b and Table S3). Ce/Co-ZIF and TCC exhibited Ce 3+ /Ce 4+ ratios of 1.1 and 1.3, respectively, exceeding that of commercial CeO 2 nanoparticles (Figure S11 and Table S4). Compared to Co-ZIF, Co 2p 3/2 and 2p 1/2 XPS peaks of Ce/Co-ZIF revealed pronounced shifts to higher binding energies by 0.5 and 0.6 eV, respectively (Figure 2c and Table S3). The similar shifts were also observed for TCC. Concomitantly, peak area analysis showed the reduced Co 2+ /Co 3+ ratios in both Ce/Co-ZIF and TCC relative to pristine Co-ZIF (Table S4). These observations unambiguously illustrated the charge transfer from cobalt to cerium, leading to the enhanced electron density at Ce sites of CeNC confined within ZIF framework [68]. X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy were analyzed to investigate the internal coordination environment and electronic interactions of TCC. CoO, Co foil, Co 3 O 4 , CeCl 3 and CeO 2 were used as references. The Co K-edge XANES spectra revealed that the pre-edge peaks of TCC and Co-ZIF located between CoO and Co 3 O 4 , suggesting the mixed valence states of Co 2+ and Co 3+ (Figure 2d and 2e). The pre-edge of TCC fell between CeCl 3 and reference CeO 2 nanoparticles, which suggested the formation of CeO 2 clusters with mixed Ce 3+ /Ce 4+ valence states in TCC (Figure 2f). Comparative analysis of Co K-edge XANES spectra showed a slight pre-edge peak shift to higher energy in TCC relative to Co-ZIF, while Ce L-edge XANES spectra exhibited a shift to lower energy in TCC compared to reference CeO 2 . These spectral shifts provided strong evidence for charge redistribution in TCC via the Co 2+ to Ce 4+ electron transfer, aligning with XPS data [69]. Fourier-transform (FT) k 3 -weighted EXAFS spectra and wavelet analysis identified the formation of Co-N bonds at 1.4 Å in both TCC and Co-ZIF with an average coordination number of 4, indicating the ligand-coordinated Co ions in ZIF-67 framework (Figure 2g, Figure S12 and Table S5). EXAFS fitting further quantified the Ce-O coordination numbers as 2.4 for TCC, which was significantly lower than 5.2 for reference CeO 2 (Figure S13 and Table S6). This reduced coordination number in CeNC was attributed to synergistic effects of small size-induced defect formation and elevated Ce 3+ content [70,71]. Collectively, these findings demonstrated the internal Co-to-Ce electron transfer and elucidated the unique coordination environments of both Co cations and CeO 2 nanoclusters. This electronic/structural modulation facilitates enhanced ROS scavenging efficacy of TCC, disrupting the vicious cycle of ROS accumulation and inadequate vascularization to potentiate therapeutic outcomes in wound management. 2.3 | ROS Scavenging Properties Excessive ROS production disrupts the wound microenvironment. Effectively scavenging excessive ROS and restoring this microenvironment are critical for promoting vascularization and accelerating wound healing [19,20]. In various pathological processes, ROS primarily exists in the forms of superoxide radical (·O 2 - ), H 2 O 2 and hydroxyl radicals (·OH), etc . Superoxide dismutase (SOD) and catalase (CAT) catalytically decompose ·O 2 - and H 2 O 2 , respectively [58]. Figure 3a illustrated the catalytic pathways of ROS scavenging by TCC. For comparative analysis, commercial CeO 2 was used as a reference nanozyme. TCC demonstrated excellent SOD-like activity, showing 10.2-fold and 1.5-fold higher ·O 2 - scavenging ability over Co-ZIF and Ce/Co-ZIF, respectively, as quantified using a ·O 2 - assay kit (Figure 3b). Its intrinsic SOD-like activity was 365 times higher than that of commercial CeO 2 (Figure S14). CAT-like activity was assessed through H 2 O 2 decomposition assays. TCC achieved >80% H 2 O 2 elimination, significantly surpassing Ce/Co-ZIF (66.6%), Co-ZIF (7.6%), and commercial CeO 2 (244-fold higher) under identical conditions (Figure 3c and Figure S14). Oxygen generation was monitored in H 2 O 2 solutions with/without nanozymes. Minimal oxygen was generated in H 2 O 2 alone or with Co-ZIF. Conversely, O 2 concentrations increased from 0 to 7.4 mg L -1 (TCC) and 5.9 mg L -1 (Ce/Co-ZIF) within 180 s of mixing with H 2 O 2 , confirming their CAT-like activity and the highest capability of TCC for H 2 O 2 decomposition (Figure S15). Upon the addition of Co-ZIF, Ce/Co-ZIF and TCC into H 2 O 2 solution, distinct O 2 bubble formation was observed, with TCC exhibiting dramatically enhanced O 2 generation (Figure S16) [56]. EPR spectra provided the unambiguous evidences to confirm the superior ·O 2 - and ·OH scavenging capacities of TCC compared to Co-ZIF and Ce/Co-ZIF (Figure 3d and 3e) [63]. Dose-dependent studies demonstrated that TCC achieved 80% scavenging efficiency for ·O 2 - at a concentration of 100 μg mL -1 , while its hydrogen peroxide decomposition capacity reached 90% at 25 μg mL -1 (Figure S17). Collectively, these findings establish TCC as a highly potent antioxidant nanozyme that disrupts the self-perpetuating ROS-insufficient vascularization cycle, thereby potentiating wound healing efficacy. FIGURE 3 | ROS-scavenging and the catalytic mechanism of nanozymes. (a) Schematic diagram of enhanced multiple antioxidant activities for TCC. (b) SOD-like activities of Co-ZIF, Ce/Co-ZIF and TCC (nanozymes: 100 μg mL -1 , n = 3). (c) CAT-like activities of Co-ZIF, Ce/Co-ZIF and TCC (nanozymes: 25 μg mL -1 , n = 3). (d, e) EPR spectra of Co-ZIF, Ce/Co-ZIF and TCC for ·O 2 - and ·OH scavenging. (f) PDOS of Ce, O and Co orbital for [1] freestanding CeNC and [2] CeNC in Ce/Co-ZIF. (g) Proposed reaction pathways of ·O 2 - reduction to H 2 O 2 with optimized adsorption configurations on Ce/Co-ZIF. (h) Corresponding free energy diagram for SOD-like reaction on CeNC and Ce/Co-ZIF. Comparative analysis illustrated that Ce/Co-ZIF and TCC exhibited superior antioxidant activity in comparison with Co-ZIF. This enhancement primarily originates from the spontaneous incorporation of small-sized CeNC with abundant defects and the Co-to-Ce electron transfer to modulate the valence states of the incorporated CeNC with increased Ce 3+ fraction. Both small size-induced defects of CeNC and Co-to-Ce electron transfer enhance catalytic antioxidant performance of TCC through the promoted dynamic Ce 3+ /Ce 4+ redox cycling. Then, density functional theory (DFT) calculations were performed to investigate electronic structure modifications and elucidate catalytic enhancement mechanisms. Figure S18 illustrated the geometrically optimized structures of CeNC and CeNC-incorporated Co-ZIF (Ce/Co-ZIF), while Figure 3f representd projected density of states (PDOS) for freestanding CeNC and CeNC in Ce/Co-ZIF. Notably, substantial Co-Ce orbital hybridization enhanced electronic interactions between ZIF frameworks and CeNC. The d-band center for the spin-up electrons of CeNC encapsulated within Ce/Co-ZIF (-0.897 eV) located significantly closer to the Fermi level compared to freestanding counterpart (3.177 eV). This upward shift in the d-band center elevated the energy levels of unoccupied d-orbital, strengthening antibonding orbital overlap with ROS [72]. This configuration promotes ROS chemisorption and transformation on CeNC, effectively lowering activation barriers and enhancing catalytic efficiency for ROS degradation. As a major source of ROS generation, excessive accumulation of ·O 2 - not only triggers the oxidative cascades but also serves as the primary elimination target of the antioxidant system. Therefore, to elucidate how the incorporated CeNC within ZIF framework promoted the SOD-like activity, DFT calculations were performed to investigate the crucial intermediates, catalytic pathways and structure-activity relationship. Given that ·O 2 - is a Brønsted base (pK b = 9.12), it readily traps a proton from water to form the ·OOH radical, which was used to represent ·O 2 - in the DFT calculations. As shown in Figure 3g, 3h and Figure S19, the adsorption of the first ·OOH radical to form *OOH adsorbed and subsequent cleavage of O-H bond to generate *O 2 were exothermic processes, suggesting favorable energetics for these steps on both the freestanding CeNC and CeNC within Ce/Co-ZIF [59]. Notably, while the second ·OOH adsorption remained exothermic on freestanding CeNC, its adsorption became endothermic on CeNC within Ce/Co-ZIF, requiring an energy input of 0.35 eV. This disparity likely arose from the exceptionally high hydrogen affinity on the surface of CeNC within Ce/Co-ZIF, which stabilized the *H-adsorbed configuration and thereby raised the energy barrier for another ·OOH adsorption. Critically, although H 2 O 2 formation was endothermic on both freestanding CeNC (1.37 eV) and CeNC within Ce/Co-ZIF (0.29 eV), the substantially lower energy barrier for the latter indicated a thermodynamically favored pathway for the ·O 2 - disproportionation into H 2 O 2 and O 2 . These computational insights show remarkable consistency with experimental observations, demonstrating that CeNC within Ce/Co-ZIF exhibits favorable ROS adsorption and activation capability. These enhancements stem from the small size of CeNC and the electron acquisition from the ZIF framework, which induce an upward shift of the d-band center of CeNC. This electronic reconfiguration enhances the catalytic capability of CeNC for ·O 2 - decomposition, revealing the critical roles of the unique structural configuration of TCC in achieving exceptional SOD-like activity. 2.4 | Angiogenesis and Antioxidant in Vitro Angiogenesis is a critical determinant of successful wound healing. Previous investigations have attributed the protracted healing process of chronic wounds to the compromised angiogenesis stemming from inflammation-induced ROS [19,20]. Generally, angiogenesis requires the coordinated proliferation, migration, and tube formation of vascular endothelial cells. Given that Co 2+ is a well-known HIF-1α stabilizer, it promotes vascular regeneration by upregulating the HIF-1α/VEGF signaling pathway (Figure 4a) [53]. Consequently, the gradually increased release of cobalt ions from TCC through its decomposition into phosphate-buffered saline (PBS) solution was quantified over a period of 7-day at 37 ℃ (Figure S20). These released cobalt ions are anticipated to stabilize HIF-1α, creating an angiogenic microenvironment to facilitate wound repair. Subsequent investigations evaluated the biocompatibility and functional impact of TCC on endothelial cell proliferation, migration, and angiogenesis. Cytotoxicity remains a major obstacle to the clinical translation of nanomedicines. Using flow cytometry, we evaluated the cytotoxicity of various nanozymes (Co-ZIF, Ce/Co-ZIF and TCC) on human umbilical vein endothelial cells (HUVECs). As illustrated in Figure S21, these nanozymes exhibited excellent biocompatibility without obvious adverse effects on cell viability at concentrations up to 200 μg mL -1 , demonstrating their translational potential. To optimize therapeutic efficacy, HUVECs were co-cultured with H 2 O 2 in the presence of varying TCC concentrations for 24 h. Notably, TCC exhibited dose-dependent cytoprotective effects, with 25 μg mL -1 significantly attenuated H 2 O 2 -induced cellular damage and enhanced cell viability (Figure S22). This concentration was selected for subsequent angiogenesis studies. FIGURE 4 | In vitro pro-angiogenesis effect of nanozymes. (a) Schematic diagram of TCC-mediated pro-angiogenesis via HIF-1α/VEGF pathway modulation. (b) Tube formation capacity of HUVECs after different treatments. Scale bar: 200 μm. (c,d) Quantification of the total tube length and number of branch points ( n = 5). (e) Representative images of the scratch assay on HUVECs subjected to different treatments at 0 and 24 h. Scale bar: 200 μm. (f) Statistical analysis of the cell migration ratio ( n = 5). (g,h) Representative immunofluorescence images and quantitative statistical analysis of Ki67 in different treatment groups ( n = 5). Scale bar: 20 μm. (i-k) Relative expressions of VEGF, bFGF and HIF-1α after different treatments ( n = 5). (l) Fluorescent microscopy images of a ROS probe (DCFH-DA) in HUVECs after different treatment ( n = 5). Scale bar: 20 μm. (m) Statistical analysis of the ROS fluorescence intensity in HUVECs with indicated treatments ( n = 5). Experiment condition: Nanozymes: 25 μg mL -1 and H 2 O 2 100 μM. Data are expressed as the mean ± SD. Indication of significant differences is delineated by asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significance. Given that the formation of vascular networks is essential for successful skin regeneration, we then evaluated the angiogenic effects of nanozymes using in vitro tube formation assay and scratch assay [73,74]. Tube-forming capacity was assessed by co-incubating HUVECs with different treatments. Bright-field imaging revealed that Co-ZIF, Ce/Co-ZIF, and TCC groups exhibited enhanced tube formation, whereas the control group displayed sparse and immature structures (Figure 4b). Quantitative analysis of total tube length indicated that Co-ZIF, Ce/Co-ZIF, and TCC increased by 71%, 110% and 164%, respectively (Figure 4c). Compared to control, the branch point density showed 101%, 140% and 188% increases for Co-ZIF, Ce/Co-ZIF, and TCC, respectively (Figure 4d). The inferior in vitro tube-forming capacity of Co-ZIF relative to Ce/Co-ZIF and TCC correlated with its lower antioxidant capacity observed in earlier antioxidant assays. To validate this mechanism underlying these effects, we quantitatively examined the effects of nanozymes on the HUVECs migration and proliferation under oxidative stress using a scratch assay. As illustrated in Figure 4e and 4f, the H 2 O 2 treatment reduced cell migration to 27%, which was significantly lower than the normal cells. Notably, the cell migration rates in Ce/Co-ZIF and TCC groups increased by 63% and 67% above H 2 O 2 -treated group after 24 h, respectively, even surpassing normal cell migration. Conversely, no significant difference was observed between the Co-ZIF and H 2 O 2 -treated groups, consistent with its much weaker antioxidant activity. The comparative observations demonstrate that Ce/Co-ZIF and TCC effectively improve cell migration and promote angiogenesis via eliminating ROS. Afterward, the cell proliferation marker (Ki67) immunofluorescence was performed to investigate the effects of nanozymes on the proliferation of HUVECs under H 2 O 2 stimulation [12]. Ce/Co-ZIF and TCC treatments significantly increased the intensity of green Ki67 fluorescence cells in comparison with to the H 2 O 2 -treated group (Figure 4g and Figure S23). Compared to control, the fluorescence quantitative analysis demonstrated that the positive expression of Ki67 in HUVECs after the incubation with Ce/Co-ZIF and TCC were 81% and 106%, respectively, which was significantly higher than 37% of the H 2 O 2 alone group (Figure 4h). Comparatively, the Co-ZIF did not show any significant improvement in the reduction of cell proliferation rate caused by H 2 O 2 stimulation, similar to the cell migration experiment (Figure 4e and 4f). VEGF, a specific pro-vascular endothelial growth factor, critically regulates angiogenesis by inducing vascular permeability, endothelial migration, proliferation, and neovascularization [37]. Meanwhile, basic-fibroblast growth factor (bFGF) mediates the recruitment and repair of endothelial cells [15]. As HIF-1α-transcriptionally regulated factors secreted by endothelial cells, we hypothesized that nanozymes may enhance vascularization through activation of HIF-1α/VEGF signaling pathway. Hence, the expressions of angiogenic genes related to HIF-1α, VEGF and bFGF were further assessed by real-time quantitative polymerase chain reaction (RT-qPCR). As displayed in Figure 4i-4k, the H 2 O 2 -treated group exhibited the significantly reduced expression levels of angiogenic genes (HIF-1α, VEGF and bFGF) compared to the control group. In contrast, the Ce/Co-ZIF and TCC treatments effectively reversed the reduction in the expression of angiogenesis-related genes induced by H 2 O 2 . Especially, the expression levels of angiogenic genes after the TCC treatments were close to those of the control group, demonstrating the high capability of TCC for vascularization. As expected, the Co-ZIF treatments exerted minimal impacts on the expression of angiogenesis-associated genes, a phenomenon attributed to its inadequate antioxidant capacity. Notably, excessive ROS not only triggers oxidative damage but also serves as a key indicator of inflammatory response in skin injury. ROS accumulation further exacerbates inflammatory responses and induces endothelial dysfunction by disrupting the HIF-1α/VEGF signaling pathway, ultimately culminating in delayed wound healing. Consequently, these findings suggest a dual mechanism of TCC with high capability for angiogenesis: (i) the released cobalt ions stabilize HIF-1α; and (ii) the high antioxidant ability mitigates ROS-induced suppression of HIF-1α/VEGF signaling, thereby creating a favorable microenvironment for wound healing. To validate the above findings, we systematically evaluated the cytoprotective effects and intracellular ROS scavenging capacity of nanozymes. As demonstrated in Figure S24, the H 2 O 2 treatment reduced HUVEC viability to 83.3% of normal controls. After treatments of various nanozymes, the survival rates were 92.4%, 99.4% and 99.4% for the Co-ZIF, Ce/Co-ZIF and TCC groups, respectively, closely to the viability of normal HUVECs cells. Subsequently, the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect the levels of intracellular ROS [56]. H 2 O 2 treatment induced a 5.2-fold increase in the fluorescence intensity compared to the control group. As shown in Figure 4l, 4m and Figure S25, the pre-treatments with Co-ZIF, Ce/Co-ZIF and TCC could eliminate the excessive ROS in the presence of H 2 O 2 , reducing ROS levels by 30% (Co-ZIF), 66% (Ce/Co-ZIF) and 83% (TCC). Notably, the TCC-treated cells exhibited ROS levels approaching those of the control groups. Meanwhile, the fluorescence intensities of the Ce/Co-ZIF and TCC groups were significantly lower than that of the Co-ZIF group, manifesting superior ROS scavenging activity after loading CeNC. These findings demonstrate that TCC effectively alleviates ROS and reverses the functional limitations of HIF-1α/VEGF signaling pathway driven by ROS, thereby disrupting the vicious cycle of ROS-vascular insufficiency to accelerate wound treatment. 2.5 | Inflammatory Inhibitor in Vitro The regulation of the wound immune microenvironment plays a pivotal role in wound healing, requiring a delicate balance between pro-inflammation (M1) and anti-inflammation (M2) phenotype macrophages (Figure 5a) [19,20]. To further evaluate the nanzymes-mediated immune microenvironments, we investigated their effects on the surface markers of macrophages and the expression of M1 and M2 polarization-related genes. The levels of inflammatory markers were analyzed using mononuclear macrophage cell line (Raw 264.7) stimulated with lipopolysaccharide (LPS) as an inflammatory cell model [75]. The LPS stimulation induced a significant transformation from M1 to M2 phenotype, characterized by a significantly increased M1-related marker (CD86) and a dramatically decreased M2-related marker (CD206) compared to the control group (Figure 5b and 5c) [75]. Comparatively, the Ce/Co-ZIF and TCC treatments reversed this trend, in which the respective expressions of CD86 were significantly inhibited by 32% and 48% compared to that of the LPS group (Figure 5b,5d and Figure S26). On the contrary, the expressions of CD206 showed 1.8- and 1.9-fold increases relative to the LPS group for the Ce/Co-ZIF and TCC treatments, respectively (Figure 5c,5e and Figure S27). Consequently, the polarization rates of M2/M1 macrophages significantly increased after the treatments of Ce/Co-ZIF and TCC (Figure S28). Among them, the TCC treatment delivered the most pronounced inflammation inhibition activity, effectively inhibiting M1 polarization and promoting M2 polarization of macrophages. Moreover, the RT-qPCR analysis revealed that the Ce/Co-ZIF and TCC treatment groups markedly suppressed the transcription of pro-inflammatory factors ( e.g. , interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6), alongside enhanced anti-inflammatory cytokine expressions (IL-10 and IL-4), compared to the LPS group (Figure 5f and 5g) [76]. Notably, the TCC treatments delivered even higher anti-inflammatory efficacy than the Ce/Co-ZIF treatments. The RT-qPCR analysis was highly consistent with the immunofluorescence staining results (Figure 5b-5e), demonstrating the potentials of TCC for regulating the immune microenvironments of wound. FIGURE 5 | Capability of various nanozymes for inhibiting macrophagic inflammation induced by LPS in vitro . (a) Schematic depicting the effects of TCC on macrophages polarization. (b,c) Statistical analysis the expressions of CD86 and CD206 in different treatment groups ( n = 5). (d,e) Representative immunofluorescence images of CD86 and CD206 in Raw 264.7 cells. Scale bar: 20 μm. (f,g) RT-qPCR evaluating the expressions of IL-1β, TNG-α, IL-6, IL-10 and IL-4 in Raw 264.7 cells after various treatments ( n = 5). Experiment condition: Nanozymes 25 μg mL -1 and LPS 100 ng mL -1 . Data are expressed as the mean ± SD. Indication of significant differences is delineated by asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significance. LPS stimulations is known to induce the formation of intracellular ROS, further exacerbating inflammation [75]. Therefore, to elucidate the anti-inflammatory mechanism of the nanozymes, the intracellular ROS levels of macrophages were quantified under various treatments. As shown in Figure S29, both Ce/Co-ZIF and TCC demonstrated their potent ROS-scavenging capacity, significantly reducing intracellular ROS levels induced by the LPS stimulation. Meanwhile, the ROS-scavenging capability of both nanozymes were much stronger than that of Co-ZIF alone. These findings position Ce/Co-ZIF and TCC, with high antioxidant capacity and anti-inflammatory ability, as promising therapeutic agents for chronic wound management, demonstrating their potentials in improving the wound immune microenvironments and accelerating wound repair. 2.6 | In Vivo Wound Therapeutic Building upon the demonstrated antioxidant performance and angiogenesis ability of the biocompatible Ce/Co-ZIF and TCC, their therapeutic effect on chronic wounds was evaluated using a mouse whole skin defect model (1 cm diameter) [19]. Schematics outlined the experimental timeline for wound induction and treatment (Figure 6a). The treatments were divided into four treatment groups of control (PBS), Co-ZIF, Ce/Co-ZIF and TCC. Wound progression was monitored through digital photography at days 0, 3, 7 and 14 (Figure 6b). Representative optical images and quantitative analysis revealed a significant reduction in wound area across all three treatment groups compared to the control group by Day 3 (Figure 6c, d). At day 7, the TCC group achieved a wound closure rate of 72%, and the wound was almost healed and completely covered by new epithelial tissue. By day 14, the wound closure rates of the Co-ZIF, Ce/Co-ZIF and TCC groups reached 91%, 95% and 99%, respectively, significantly exceeding control (81%). Importantly, the healing rate of the TCC group was close to 100% after 14 days of treatment, showing the almost completely healed wound. FIGURE 6 | Capability of TCC for the promoted wound healing by improving tissue regeneration and promoting vascularization in vivo . (a) Scheme of the in vivo experiments process. (b) Photographs of the wound healing process after different nanozymes (0.5 mg mL -1 , 50 μL) treatment on day 0, 3, 7 and 14. (c) Overlaid images of the wound areas after various treatments on day 0, 3, 7 and 14 after surgery. (d) Quantitative analysis of the wound closure rates ( n = 5). (e) Representative H&E staining images of the skin wound healing and tissue regeneration on day 14. Scale bar: 500 μm. (f) Quantitative statistics of the epidermal thickness ( n = 5). (g) Masson staining of the wound areas on day 14. Scale bar: 500 μm. (h) Quantitative statistics of collagen volume fractions on day 14 ( n = 5). (i,j) DBFI and quantitative results on day 7 and day 14. Data are expressed as the mean ± SD. Indication of significant differences is delineated by asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significance. To further evaluate the wound treatment effects of various nanozymes, skin wound tissue specimens were collected on day 14 for histological assessments, including hematoxylin-eosin (H&E) staining and Masson staining [18,19]. The wound healing efficacy was evaluated by analyzing the epidermal thickness, residual wound length and collagen fraction. Among all groups, the TCC treatments demonstrated superior capacity for wound healing. As observed in Figure 6e, 6f and Figure S30, the TCC group exhibited not only a more reduction in epidermal thickness and residual wound length but also more orderly collagen deposition, indicating its superior structural restoration of the injured skin tissue. Besides, compared with the control group, Masson staining revealed more collagen fibers deposition in Ce/Co-ZIF and TCC, in which the collagen arrangement was much more orderly and denser (Figure 6g). Quantitative analysis results showed the collagen deposition density in the TCC group was the highest in all groups, which was 1.9 and 1.1 times higher than those of the control and Ce/Co-ZIF groups at day 14, respectively, indicating the highest capability of TCC for wound repair (Figure 6h). Finally, to further objectively evaluate the pro-angiogenesis efficacy of various nanozymes in vivo , we monitored the cutaneous microvascular blood flow by Doppler blood flow imaging (DBFI). As presented in Figure 6i, both the Ce/Co-ZIF and TCC groups delivered more mature vascular networks at early and later stage of treatment (7 and 14 days postoperatively). For the TCC group, the blood flow areas were 2.7-fold and 1.6-fold larger than those of the control group at 7 and 14 days, respectively (Figure 6j). Quantitative perfusion analysis further validated the superior in vivo pro-angiogenic capacity of TCC, as evidenced by its significantly larger blood flow areas compared to those of other groups. These findings were highly consistent with the in vitro analysis in tube-forming capacity (Figure 4b), in vivo observations in wound progression (Figure 6b-6d), and the histological analysis of skin wound tissues (Figure 6e-6h). Notably, this convergence of data from both in vitro and in vivo experiments collectively illustrated TCC as a potent pro-angiogenic agent for accelerated wound healing through enhanced vascularization. 2.7 | Angiogenesis and Anti-inflammatory Property in Vivo Previous in vitro results demonstrated that nanozymes enhanced both the angiogenic and immunoregulation capacity of cells. To further investigate the mechanism of nanozymes for the promoted wound repair capability of TCC, immunofluorescence staining was performed to evaluate their impacts on angiogenesis and inflammation in vivo during wound healing. As shown in Figure 7a, treatments with various nanozymes significantly upregulated the expression of angiogenesis-associated markers (HIF-1α, VEGF, bFGF and CD31), highly consistent with the corresponding i n vitro results (Figure 4i-4k) [15,19,77]. Specifically, the TCC treatment greatly increased the level of HIF-1α, demonstrating increases of 4.9-, 3.1- and 1.4-fold compared to the control, Co-ZIF and Ce/Co-ZIF groups, respectively (Figure 7b). The increase of HIF-1α was accompanied by a concomitant elevation in downstream angiogenic factors. The levels of VEGF and bFGF in the TCC group were 2.0-fold and 2.6-fold higher than those in the control group, respectively, and were significantly higher than those in other treatment groups (Figure 7c and 7d). VEGF and bFGF can induce the migration and proliferation of vascular endothelial cells, ultimately promoting angiogenesis. Then, CD31 as a neovascularization marker, was used to assess the degree of vascularization at the skin injury site [77]. As demonstrated in Figure 7a, CD31 immunofluorescence staining confirmed that a significantly denser distribution of CD31-positive cells in the TCC group than other groups, further indicating that dense microvascular structures formed in the wound tissues after TCC treatment. Quantitative assessment of the CD31-positive cells based on the immunofluorescence signal intensity revealed that CD31 expression in the TCC group was 2.4-fold, 1.8-fold and 1.2-fold higher than that in the control, Co-ZIF, and Ce/Co-ZIF groups, respectively (Figure 7e). These results established a clear mechanistic link between the stabilization of HIF-1α induced by the release of Co 2+ from TCC and the subsequent transcriptional activation of pro-angiogenic factors. Thereby, this validates the pivotal role of the HIF-1α/VEGF signaling pathway in mediating the angiogenic regenerative effect of TCC, which is consistent with the in vitro results (Figure 4). FIGURE 7 | Capability of nanozymes for the promoted angiogenesis and inflammation regulation in vivo . (a) Representative HIF-1α, VEGF, bFGF and CD31 immunofluorescence staining images of wound skin tissues on day 14. Scale bar: 20 μm. (b-e) Quantitative statistics of HIF-1α, VEGF, bFGF and CD31 ( n = 5). (f,g) Representative ROS fluorescence staining images of wound skin tissues on day 14 and the corresponding relative ROS level ( n = 5). Scale bar: 20 μm. (h) Representative images of TNF-α, IL-6 and IL-10 immunofluorescence staining of wound skin tissues on day 14. Scale bar: 20 μm. (i-k) Quantitative statistics of TNF-α, IL-6 and IL-10 ( n = 5). Data are expressed as the mean ± SD. Indication of significant differences is delineated by asterisks, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., no significance. Chronic inflammatory persistence in wound microenvironments critically impedes tissue regeneration, a pathological process mediated by sustained ROS accumulation and inflammatory response. Therefore, we further analyzed the effects of nanozymes on the ROS levels and inflammation conditions within the wound area. The results showed a reduction in wound tissue ROS levels in the three treatment groups (Figure 7f). The statistical analysis revealed that TCC exhibited highest ROS elimination rates exceeding 73%, significantly higher than those of Co-ZIF (29%) and Ce/Co-ZIF (61%), highlighting its superior effectiveness in clearing ROS of wound (Figure 7g). Further evaluation of inflammatory factors via immunofluorescent staining demonstrated that all treatment groups significantly reduced the expression of proinflammatory factor (TNF-α and IL-6), while the expression of anti-inflammatory cytokine (IL-10) was increased (Figure 7h) [77]. Compared to the control group, the TCC treatment induced the most pronounced downregulation of TNF-α (69.2%) and IL-6 (94.6%), with levels significantly lower than those observed in other treatment groups (Figure 7i, 7j). Oppositely, the TCC group showed the highest improved percentage of IL-10, which was 2.4-fold and 1.4-fold higher than the Co-ZIF and Ce/Co-ZIF groups (Figure 7k). Additionally, the significant attenuation of immunofluorescence staining for matrix metalloproteinases-13 (MMP-13) following the TCC treatment confirmed successful modulation of the wound protease, achieving an immune microenvironment approximating health skin (Figure S31). The expression profile of inflammatory cytokines was consistent with the results of antioxidant, angiogenesis and histological staining presented above, indicating that TCC possesses the highest anti-inflammatory activity. Given the crucial regulatory roles of ROS in modulating both vascularization and inflammatory processes, the inherent potent antioxidant activity of TCC, coupled with its pro-angiogenic and anti-inflammatory functionalities, is anticipated to enhance the therapeutic outcomes of existing wound management strategies. Taking all together, this work demonstrates that antioxidant capabilities enhanced TCC synergistically augment wound healing efficacy through disrupting the vicious cycle of pathological ROS accumulation and insufficient vascularization. 2.8 | In Vivo Biosafety Assessment The vigorous evaluation of the biosafety of nanozymes is a prerequisite for their clinical application. Consequently, we conducted a comprehensive in vivo biocompatibility assessment of the three nanozymes formulations in healthy mice. As shown in Figure S32, hematological parameters in mice treated with Co-ZIF, Ce/Co-ZIF and TCC showed no significant differences compared to the control group. Furthermore, hepatic and renal function indicators revealed no obvious abnormalities between the treated mice and the control group (Figure S33). Similarly, histological examination via H&E staining demonstrated the preserved tissue architectures in major organs (heart, liver, spleen, lung, kidney) across all treatment groups compared to the control group (Figure S34). Collectively, these results confirm the excellent biocompatibility and systemic safety profile of the evaluated nanozymes, supporting their potentials for clinical translation. 3 | Conclusion In summary, we developed a nanozyme-based therapeutic strategy to disrupt the pathological ROS accumulation-insufficient vascularization cycle in chronic wound healing. The composite nanozyme of TCC integrating ultrasmall CeNC and tryptophan within a Co-ZIF framework was designed to address the multifaceted challenges in complex pathological wound microenvironments. The immobilized defective CeNC within Co-ZIF framework against aggregation, coupled with internal Co-to-Ce electron transfer, delivered exceptional ROS-scavenging capabilities of TCC. Crucially, TCC simultaneously promoted angiogenesis by releasing cobalt ions to stabilize HIF-1α and activating the HIF-1α/VEGF pathway through induced macrophage polarization toward the anti-inflammatory M2 phenotype. 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Keywords angiogenesis nanozyme wound treatment Authors Affiliations Qiuxue Jiang Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Meixia Zhang Air Force Medical University View all articles by this author Yuqi Yang Air Force Medical University View all articles by this author Mingzhu Jin Air Force Medical University View all articles by this author Junlong Zhao Air Force Medical University View all articles by this author Da Chen Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Boqi Jiang Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Mengfan Li Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Jiahao Ma Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Jie Li Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Jianwei Zhao Air Force Medical University View all articles by this author Yongquan Qu 0000-0002-6202-1929 [email protected] Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Ling Liu Air Force Medical University View all articles by this author Sanzhong Li Air Force Medical University View all articles by this author Zhimin Tian Northwestern Polytechnical University School of Chemistry and Chemical Engineering View all articles by this author Metrics & Citations Metrics Article Usage 224 views 133 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Qiuxue Jiang, Meixia Zhang, Yuqi Yang, et al. Revascularization-Driven Nanozyme Therapy: Disrupting the Vicious Cycle of ROS and Insufficient Vascularization for Chronic Wound Healing. Authorea . 06 November 2025. DOI: https://doi.org/10.22541/au.176241997.77692386/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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