A mitochondria-targeted nanozyme for myocardial ischemia/reperfusion injury with synergistic antioxidant and anti-inflammatory properties

A mitochondria-targeted nanozyme for myocardial ischemia/reperfusion injury with synergistic antioxidant and anti-inflammatory properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A mitochondria-targeted nanozyme for myocardial ischemia/reperfusion injury with synergistic antioxidant and anti-inflammatory properties Congcong Li, Zhengfeng Zhang, Chao Luo, Wanqi Lan, Chen Liu, Wu Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7445253/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 14 You are reading this latest preprint version Abstract Acute myocardial infarction (AMI) remains the leading cause of mortality worldwide, posing a significant threat to global public health. Although revascularization strategies such as percutaneous coronary intervention represent the standard treatment for AMI, myocardial cell death caused by myocardial ischemia/reperfusion injury (MI/RI)significantly compromises clinical efficacy. The clinical application of anti-inflammatory and antioxidant therapeutic strategies for MI/RI is confronted with critical limitations due to poor targeting and low bioavailability. This study successfully constructed a new mitochondria-targeted nanozyme, VB@MOF/TA, in which tannic acid (TA) mediates specific mitochondrial targeting, and the metal-organic framework (MOF) serves as a carrier to synergistically enhance the antioxidant and anti-inflammatory effects of verbascoside (VB). Cellular experiments demonstrate that VB@MOF/TA co-localizes with mitochondria, exerts potent antioxidant effects, significantly suppresses oxygen-glucose deprivation/reoxygenation-induced cardiomyocyte apoptosis, and effectively modulates macrophage polarization. In vivo studies confirm that, compared with VB monotherapy, the VB@MOF/TA group exhibits a 2.59-fold reduction in apoptosis rate, a 7.72% ± 3.71% improvement in left ventricular ejection fraction, and a 2.50-fold increase in vascular density. These findings indicate that VB@MOF/TA significantly mitigates MI/RI and promotes myocardial tissue remodeling through its targeted antioxidant and synergistic anti-inflammatory mechanisms, highlighting its substantial clinical translational potential. myocardial ischemia/reperfusion injury mitochondria-targeted nanozymes reactive oxygen species inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Acute myocardial infarction (AMI) remains the leading cause of mortality worldwide, posing a significant threat to global public health[ 1 ]. The pathological process involves cardiomyocyte necrosis secondary to acute coronary occlusion, with timely reperfusion therapy serving as the standard clinical strategy to salvage ischemic myocardium[ 2 ]. However, reperfusion exhibits a “double-edged sword” effect while restoring blood flow, it paradoxically aggravates myocardial damage through ischemia/reperfusion injury (MI/RI)[ 3 , 4 ]. Despite therapeutic advances, current MI/RI management faces several critical limitations, including poor drug target specificity, safety concerns with combination therapies, and inadequate long-term prevention of myocardial fibrosis[ 5 ]. Effective MI/RI mitigation is crucial for minimizing cardiomyocyte death, preserving post-reperfusion cardiac function, and preventing adverse ventricular remodeling[ 6 – 8 ]. Mitochondrial reactive oxygen species (ROS) overload and the ensuing inflammatory cascade are now regarded as the core drivers of MI/RI[ 9 ]. This positions mitochondria as a rational therapeutic target to fine-tune ROS output, safeguard organelle quality, rebalance metabolism, and curb maladaptive inflammation. Addressing these challenges, this study presents the first construction of VB@MOF/TA, a mitochondria-targeting nanozyme, with potent antioxidant and anti-inflammatory activities for the prevention and treatment of MI/RI. Leveraging the exceptional porosity and tunable pore distribution of metal-organic framework (MOF)-based nanozymes[ 10 – 12 ], high-efficiency encapsulation of verbascoside (VB) was achieved, yielding the VB@MOF nanozyme (Scheme 1 A). VB, a phenylethanoid glycoside with established antioxidant and anti-inflammatory properties, is a bioactive constituent derived from Plantago asiatica L. , a traditional Chinese herb native to central China[ 13 – 15 ]. The MOF carrier system was specifically engineered to overcome clinical limitations of VB[ 16 – 18 ]: (1) Chemical instability due to its polyhydroxy structure, (2) Poor lipophilicity, (3) Pronounced first-pass effect (oral bioavailability as low as 0.12%)[ 17 ]. To enable mitochondrial targeting, we functionalized the VB@MOF nanoparticles with tannic acid (TA), which facilitates charge-independent mitochondrial accumulation[ 19 ]. As illustrated in Scheme 1 A, this surface engineering approach yielded the mitochondria-targeting nanozyme VB@MOF/TA, which was then applied to alleviate MI/RI (Scheme 1 B). The nanozyme selectively accumulates in mitochondria via TA-mediated tropism, where it efficiently scavenges ROS, restores mitochondrial membrane potential (Scheme 1 C-I), and enhances adenosine triphosphate (ATP) production (Scheme 1 C-II), thereby precisely regulating mitochondrial quality. Consequently, deoxyribonucleic acid (DNA) damage induced by ROS (Scheme 1 C-III) and inflammatory cytokine release (Scheme 1 C-IV) are significantly reduced, enabling intracellular targeted antioxidant protection against reperfusion injury. Furthermore, the dual antioxidant and anti-inflammatory action of the nanozyme promotes M2 macrophage polarization while suppressing pro-inflammatory M1 polarization, leading to the downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the upregulation of the anti-inflammatory cytokine (IL-10), thereby ameliorating the inflammatory microenvironment (Scheme 1 D). Ultimately, this approach attenuates adverse cardiac remodeling (Scheme 1 E). In summary, this multifunctional nanoplatform, which integrates mitochondrial targeting, antioxidant, and anti-inflammatory properties, represents a promising translatable strategy for MI/RI therapy. Subsequent experiments were conducted to validate the feasibility of this system. Experimental Methods Materials 2-methylimidazole (2-MI) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O), tannic acid (TA), and verbascoside (VB) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and the 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Phygene Life Sciences Co., Ltd. (Fuzhou, China). Inhibition and produce superoxide anion assay kit, hydroxyl free radical assay kit, hydrogen peroxide assay kit, lactate dehydrogenase (LDH) assay kit, creatine kinase-myocardial band (CK-MB) isoenzyme assay kit, alanine aminotransferase (ALT) assay kit, aspartate aminotransferase (AST) assay kit, blood urea nitrogen (BUN) assay kit, and creatinine (CRE) assay kit were purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China). Malondialdehyde (MDA) assay kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kits of mouse TNF-α, IL-1β, IL-6, and IL-10 were purchased from BOSTER Biological Technology Co., Ltd. (Wuhan, China). 4,6-diamidino-2-phenylindole (DAPI) solution, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), mitochondrial membrane potential assay kit with JC-1, catalase activity assay kit, superoxide dismutase activity assay kit, and CCK-8 cell proliferation and cytotoxicity assay kit were purchased from Solarbio Technology Co., Ltd. (Beijing, China). YF®488 TUNEL cell apoptosis assay kit was purchased from UElandy (Suzhou, China). Annexin V-FITC/PI double-stained cell apoptosis detection kit and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Key GEN BioTECH (Nanjing, China). Fetal bovine serum (FBS) was purchased from Abcell (Beijing, China). The Calcein-AM/PI live/dead cell double staining kit was purchased from BIOPRIMACY (Wuhan, China). Adenosine triphosphate (ATP) assay kit, Mtio-Tracker Green, and Lyso-Tracker Green were purchased from Beyotime Biotechnology (Shanghai, China). Synthesis of VB@MOF/TA Firstly, 2-MI (6.568 g, 80 mmol) and Zn(NO 3 ) 2 ·6H 2 O (2.9749 g, 10 mmol) were fully dissolved in methanol solution[ 20 ]. The reaction solution was stirred for 3 h at room temperature and then allowed to stand for 12 h. The samples in the centrifuge tube were washed 3 times with methanol, and the metal organic framework (MOF) nanoparticles were obtained after centrifuging at 1000 rpm for 10 min. The sample was dried and then stored at room temperature. Afterward, the MOF methanol solution, followed by ultrasonication, was mixed with 20 mg of VB, the mixture was stirred for 24 h, and the VB@MOF nanomedicine was collected by centrifugation. Subsequently, the VB@MOF methanol solution was added to the TA solution (12 mM, 5 mL, pH 7.5), and the mixture was further stirred for 5 min. Then, VB@MOF/TA was washed 3 times with methanol and collected by centrifugation. Finally, the VB@MOF/TA nanomedicines were obtained by vacuum drying. Characterization of VB@MOF/TA The morphology was observed using scanning electron microscopy (SEM, ZEISS Sigma 300, Germany) and transmission electron microscopy (HT7800, Hitachi Ltd., Japan). A particle size and ζ potential analyzer (NanoBrook, Brookhaven, USA) was used to measure size distribution and ζ potential. X-ray diffraction (XRD, Bruker, D8ADVANCE, Germany) was employed to confirm the crystalline structure. The chemical structure and composition were characterized using Fourier-transform infrared spectroscopy (FTIR) (Thermo Nicolet, Nicolet 5700, USA) and X-ray photoelectron spectroscopy (XPS, ESCALAB25OXI, Thermo Fisher, USA), respectively. The spectrum of VB aqueous solution was obtained using a Ultraviolet-visible (UV-Vis) spectrophotometer (UV-2600, SHIMADZU, Japan) and plotted as a standard curve. The VB content in VB@MOF and VB@MOF/TA was quantified by UV-Vis spectrophotometry at 334 nm, with concentrations determined using the pre-established VB standard curve. SOD-like activity The SOD-like activity was measured with SOD activity assay kit (Solarbio). Different concentrations of samples (20 µL) were incubated with the working solution in a 37 ℃ water bath for 30 min, following the manufacturer’s instructions. Absorbance changes were monitored via Multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 450 nm. CAT-like activity The CAT-like activity was measured with the CAT activity assay kit (Solarbio). Different concentrations of samples (10 µL) were mixed with the working solution (190 µL) prepared according to the manufacturer’s instructions. Absorbance changes were monitored immediately via Multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 240 nm. POD-like activity The POD-like activity was performed according to the mechanism in which colorless 3,3′,5,5′-Tetramethylbenzidine (TMB) is oxidized in the presence of hydrogen peroxide (H 2 O 2 ) to produce blue oxidized TMB (oxTMB) and H 2 O. The HAc/NaAc buffer (0.1 M, pH 4.5) was mixed with TMB (Aladdin, ≥ 99%) (1.0 mM), H 2 O 2 (1.0 mM), and different concentrations of MOF, VB@MOF, and VB@MOF/TA. The reaction was performed using TMB (Aladdin, ≥ 99%) (1.0 mM), H 2 O 2 (1.0 mM), and varying concentrations of MOF, VB@MOF, and VB@MOF/TA, all at room temperature. Absorbance at 652 nm was monitored by a UV-Vis spectrophotometer (UV-2600, SHIMADZU, Japan). DPPH free radical scavenging activity The mixed solution of different concentrations of VB@MOF/TA (50 µL), PBS (150 µL), and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) ethanol solution (0.3 mM, 200 µL) was incubated in the dark. The UV-Vis absorbance was monitored every 2 min for 30 min at 517 nm, and UV-Vis spectra were recorded between 400 nm and 700 nm at 30 min. The standard curve of DPPH• was calculated using UV-Vis absorbance at 517 nm to estimate DPPH scavenging capacity. ABTS• + free radical scavenging activity The ABTS• + working solution was prepared 24 h in advance. Briefly, an equal volume of ABTS (7.4 mM) and K 2 S 2 O 8 (2.6 mM) was mixed in the PBS (pH 7.4) in the dark. The ABTS• + working solution was obtained by diluting the concentrated solution with PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Then, different concentrations of VB@MOF/TA (20 µL) and PBS buffer (80 µL) were added to the ABTS• + working solution (400 µL). The UV-Vis absorbance was monitored every 2 min for 30 min at 734 nm, and UV-Vis spectra of 500–900 nm were detected at 30 min. The standard curve of ABTS• + was calculated using UV-Vis absorbance at 734 nm to estimate ABTS• + scavenging capacity. Hydroxyl radical (•OH) and superoxide radical (•O 2 − ) scavenging activity The hydroxyl radical (•OH) and superoxide Radical (•O 2 − ) scavenging activity of VB@MOF/TA was measured by using the hydroxyl free radical assay kit and the inhibition and produce superoxide anion assay kit, respectively, according to the manufacturer’s instructions. Hemolysis The centrifuged erythrocytes were obtained from fresh blood samples, and the erythrocytes (1.00 mL) were diluted with saline (3.67 mL). The diluted erythrocytes (100 µL) were added to physiological saline solution (1 mL) containing different concentrations of MOF, VB@MOF, and VB@MOF/TA. The mixture was incubated at 37 ℃ for 3 h, and then centrifuged at 12000 rpm for 15 min. The absorbance of supernatants was monitored by a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 540 nm. The rate of hemolysis was measured, with ddH 2 O serving as a positive control and saline as a negative control. Cell culture RAW 264.7 macrophages, H9C2 cells, and HUVECs were cultured in DMEM with 1% (V/V) penicillin/streptomycin (Key GEN BioTECH) and 10% (V/V) FBS (Abcell) in a 5% (V/V) carbon dioxide (CO 2 ) incubator at 37 ℃ (standard conditions). Cytocompatibility assay H9C2 cells, RAW 264.7 macrophages, and HUVECs were seeded into 96-well plates (5 × 10 3 cells per well) and incubated for 24 h, allowing the cells to adhere fully to the wall. The cells were washed with PBS and replaced with fresh medium (100 µL/well) containing various concentrations of different materials. The cells were then incubated for 24, 48, and 72 h. Subsequently, the cells were rinsed twice with PBS, and then the CCK-8 assay (Solarbio) was performed according to the manufacturer’s instructions. The absorbance was monitored at 450 nm using a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA). The formula calculated cell viability: A is the absorbance of the cells treated with materials, A control is the absorbance of the cells treated with PBS, and A blank is the absorbance of the medium containing CCK-8 without cells. Cellular Uptake and mitochondria-targeting ability To evaluate whether VB@MOF/TA can be taken up by macrophages and cardiomyocytes, rhodamine B (RhB), a fluorescence probe, was used instead of VB in RhB@MOF/TA to track the uptake process. RAW 264.7 cells and H9C2 cells were cultured in a confocal culture dish at a density of 1 × 10 4 cells and incubated for 24 h. After the cells were adherent, fresh medium containing RhB@MOF/TA was added. The cells and RhB@MOF/TA were co-cultured for 0 h, 1 h, 4 h, 8 h, and 12 h in CO 2 incubator at 37 ℃, RhB@MOF/TA solution was sucked away and washed thrice with PBS. Subsequently, the nuclei were stained withDAPI (Solarbio) for 5 min. A laser scanning confocal microscope (LSM980, ZEISS, Germany) was used to visualize, followed by three washes. To evaluate whether the VB@MOF/TA can effectively escape from lysosomes and target mitochondria. The H9C2 cells were cultured in a confocal dish (1 × 10 4 cells) for 24 h, and then treated with RhB@MOF (red) and RhB@MOF/TA (red) in cell culture medium for 1 h, respectively. The cells were washed three times with PBS and stained with Mito-Tracker Green (Beyotime) and Lyso-Tracker Green (Beyotime) for 30 min in a CO 2 incubator at 37 ℃. After being washed thrice with PBS, the cells were photographed using a laser scanning confocal microscope (LSM980, ZEISS, Germany). Extracorporeal oxygen–glucose deprivation and reperfusion (OGD/R) process Myocardial cell hypoxia/reperfusion injury was simulated using an in vitro oxygen-glucose deprivation (OGD/R) model constructed according to reported protocols[ 21 ]. H9C2 cells were seeded in 6-well plate at a density of 1 × 10 4 cells per well, when the density of the cells reached 70–80%, the H9C2 cells were exposed to 0.1% oxygen in serum-free no glucose DMEM at 37 ℃ to achieve OGD treatment for 10 h, followed by culturing under standard DMEM added VB@MOF/TA (25 µg/mL), or VB (25 µg/mL), or same dose of PBS for recovery oxygenation 12 h. The cells treated with the OGD/R process were used to detect ROS, assess mitochondrial function, evaluate cell apoptosis, and investigate inflammation. Live/dead cell stain After the H9C2 cells were treated with OGD/R process, the cells were stained with calcein acetoxymethyl ester/propidium iodide (Calcein-AM/PI) using a Calcein-AM/PI live/dead cell double staining kit (BIOPRIMACY, China), and visualized by fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany) after incubating for 15 min in the dark. Mitochondrial membrane potential H9C2 cells were seeded in a 6-well plate at a density of 1 × 10 4 cells per well. The mitochondrial membrane potential of cells after the OGD/R model was measured using the mitochondrial membrane potential assay kit with JC-1 (Solarbio) under the protocol. Fluorescently labeled cells were photographed using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany). The ratio between the monomer (green) and aggregate (red) mean fluorescence intensities was calculated using ImageJ software as a measure of mitochondrial membrane potential. Intracellular MDA and ATP The levels of intracellular MDA and ATP were detected with the MDA assay kit (Shanghai Enzyme-linked Biotechnology) and the ATP assay kit (Beyotime), respectively. Cells that had undergone the OGD/R procedure were collected and processed following the manufacturer’s protocol. For the determination of MDA, absorbance at 532 nm and 600 nm was detected by a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA), and protein concentration was tested by BCA protein assay kit (Beyotime). The level of ATP was detected by a luminometer (LB960, Berthold Technologies, Germany). Polarization regulation of RAW 264.7 macrophages RAW 264.7 macrophages were seeded into 6-well plate (1 × 10 5 cells per well) and incubated for 12 h. To mimic inflammatory microenvironments, RAW 264.7 macrophages of experiment group were treated with LPS (300 ng/mL), the cells in the control group were treated with same dose of PBS, the cells of VB group were treated with LPS (300 ng/mL) + VB (25 µg/mL), the cells of VB@MOF/TA group were treated with LPS (300 ng/mL) + VB@MOF/TA (25 µg/mL), all cells were co-cultured for another 24 h. The impact of VB and VB@MOF/TA on regulating macrophage phenotype and inflammation was demonstrated through immunofluorescence staining, Western blot, qPCR, flow cytometry, and ELISA. Intracellular ROS scavenging The intracellular levels of total ROS were assessed using DCFH-DA. DCFH-DA (10 µM) was incubated with OGD/R-treated H9C2 cells and LPS-induced RAW 264.7 macrophages for 30 min. The cells were then observed using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany). Moreover, flow cytometry was also used to collect quantitative data about the intracellular ROS level of H9C2 cells. The cells were subjected to the OGD/R process and then incubated with DCFH-DA (10 µM) for 30 min. After trypsinization, the cells were harvested, and the fluorescence intensity was measured using flow cytometry (ID7000, Sony Group Corporation, Japan). Data was analyzed using Flow Jov_10.9.0. The gating strategy was presented in Figure S27a. Quantitative real-time polymerase chain reaction(qPCR) The qPCR was used to detect the relative mRNA expression levels of pro-inflammatory cytokine ( TNF-α , IL-1β , IL-6 ) and anti-inflammatory cytokine ( IL-10 ). The total RNA of macrophages was extracted by SteadyPure Quick RNA Extraction Kit (Accurate Biology, China) according to the manufacturer’s instructions, and reverse transcription of mRNA was performed utilizing the Hifair ® AdvanceFast 1st Strand cDNA Synthesis SuperMix for qPCR (DNA digester plus) (YEASEN, China). QPCR was conducted using a TB Green Premix Ex Taq II on a ViiA 7 Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA). The relative gene expression levels in all samples were normalized using the 2 −ΔΔCt method, with GAPDH as the internal control for comparison. Flow cytometry of CD86 and CD206 RAW 264.7 macrophages were resuspended with PBS (100 µL) after being collected and placed in flow cytometry tubes from different groups. PE Anti-Mouse CD86 Antibody (Elabscience) or APC Anti-Mouse CD206/MMR Antibody (Elabscience) was used for staining, and the cells were detected by flow cytometry. Myocardial infarction injury model in mouse C57BL/6C mice (6–8 weeks, male) were obtained from Charles River Laboratories (Beijing, China) and were bred in a specific pathogen-free (SPF) environment. All animals were handled in accordance with protocols approved by the Nanchang University Laboratory Animal Center and its internal ethics committee (NCULAE-20250120001). The mice were housed in a 12-h photoperiod with standard temperature and humidity, and given free access to water and food. The mice were acclimated for 1 week before the operations. Mice were anesthetized and sustained with 1.5% isoflurane, the left open thoracotomy was performed to visualize the heart, the left anterior descending artery (LAD) of the coronary artery was temporarily ligated with a 7–0 suture for 45 min. The saline, VB, and VB@MOF/TA were treated (dose: 1 mg/kg) via tail vein injection at 40 min, followed by suture removal for reperfusion after 5 min. The mice of the control group underwent the same surgical process, and the LAD was passed with a suture without ligation; an equal amount of saline was injected after 40 min. Surgical wounds of all mice were sutured using sterile 4–0 sutures. Transthoracic echocardiography Transthoracic echocardiography was performed using an Animal ultrasound imaging system (VINNO6 LAB, VINNO, China), in a blinded manner. Mice were anesthetized and sustained with isoflurane after depilation of the chest area skin. Hearts were visualized from short-axis and long-axis views. The images of B-mode and M-mode were captured. The EF and FS were calculated automatically by the software in the Animal Ultrasound Imaging System. Myocardial enzyme spectrum Mouse blood was collected at 24 h post-surgery and centrifuged to obtain serum. The culture medium from H9C2 cells after OGD/R was collected and centrifuged to obtain the supernatant in different groups. The myocardial enzyme spectrum in serum and cell supernatant was detected using the LDH assay kit and the CK-MB isoenzyme assay kit, according to the manufacturer’s instructions. Detection of cytokines The ELISA kits were used to detect the secretion of pro-inflammatory and anti-inflammatory cytokines. For H9C2 cells and RAW 264.7 macrophages, the medium was collected after various treatments and centrifuged to obtain the supernatants. The serum of mice was obtained at 24 h after surgery. The concentrations of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-10) in serum and cell supernatant were measured according to the manufacturer’s protocol. 2,3,5-Triphenyltetrazolium chloride (TTC) staining The hearts of mice were obtained after perfusing with PBS at 24 h post-surgery and stored in − 80 ℃. The 1-mm-thick cross sections of the heart were incubated with 2% TTC (Solarbio, China) solution at 37 ℃ for 15 min in the dark. After that, the slices were fixed with 4% paraformaldehyde, and photographs of the slices were taken. Western blot The radioimmunoprecipitation assay lysis buffer (RIPA, Solarbio, China), which contained proteinase and phosphatase inhibitors (Salorbio, China), was used to extract protein from cells and heart tissue. Protein concentration was measured by the BCA Protein Assay Kit (Beyotime, China). Proteins were separated by 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to 0.2–0.45 µm Polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk dissolved in Tris-buffered saline/Tween 20 (TBST) for 1–2 h. Primary antibodies were incubated overnight at 4 ℃. After being washed with TBST, secondary antibodies were incubated for 2 h at room temperature. The membranes were immersed in enhanced chemiluminescence (ECL) and analyzed using an automated gel imaging system (Tanon 5200, Shanghai, China). Protein levels were quantified with the ImageJ software. The following primary antibodies were used: β-actin (1:5000, Proteintech), Bax (1:3334, Proteintech), C-casp 3 (1:1000, Abmart), Bcl-2 (1:1000, Abmart), iNOS (1:1000, Abmart), Arg-1 (1:1000, Servicebio), γ-H2AX (1:1000, Abmart). The secondary antibodies used were HRP-conjugated IgG antibody (Anti-Rabbit IgG (H + L) antibody, 1:10000, Proteintech, or anti-Mouse IgG (H + L) antibody, 1:10000, Proteintech). Immunofluorescence analysis For cell samples, cells were fixed with 4% paraformaldehyde and permeabilized with a 0.5% Triton X-100 solution in PBS. 5% bovine serum albumin (BSA, Solarbio) was used to block, and then cells were incubated with primary antibodies (γ-H2AX, 1:200, Abmart; CD86, 1:400, Proteintech; CD206, 1:500, Proteintech; iNOS, 1:200, Abmart; Arg-1, 1:200, Servicebio) overnight at 4 ℃. After washing with PBS, cells were incubated with fluorescently coupled secondary antibodies (CoraLite ® 488-Conjugated Goat Anti-Rabbit IgG (H + L), 1:1000, Proteintech; Multi-rAb ™ CoraLite ® Plus 555-Goat Anti-Rabbit Recombinant Secondary Antibody (H + L), 1:1000, Proteintech). 4,6-diamidino-2-phenylindole (DAPI, Solarbio) was used to visualize nuclei, followed by washing with PBS. The cells were visualized using a laser scanning confocal microscope (LSM980, ZEISS, Germany). Cardiac tissue samples were cut into 7-µm tissue sections, and fixation and permeabilization blocking were carried out. The tissues were then washed and incubated overnight at 4 ℃ with primary antibodies (CD86, 1:400, Proteintech; CD206, 1:500, Proteintech; CD31, 1:200, Protentech; α-SMA, 1:500, Proteintech). The tissues were incubated with fluorescently coupled secondary antibodies for 1 h at room temperature after rinsing, and then stained with DAPI. The slides were visualized using a laser scanning confocal microscope (LSM980, ZEISS, Germany). Apoptosis assays For Annexin V/PI staining of cells, an Annexin V-FITC/PI double-stained cell apoptosis detection kit (Key GEN) was used. Cells with OGD/R disposal were digested with EDTA-free trypsin (Solarbio) to form a single-cell suspension (500 µL). 5 µL of Annexin V-FITC and 5 µL of Propidium Iodide were gently mixed with the suspension, and the mixture was incubated for 10 min at room temperature in the dark. Samples were assessed using a flow cytometer (ID7000, Sony Group Corporation, Japan), and the data were analyzed using FlowJo 10.9.0. The gating strategy was presented in Figure S27b. For TUNEL staining of cells and tissue, cells and cardiac tissue sections were fixed with 4% paraformaldehyde and permeabilized with a 0.5% Triton X-100 solution. They were then stained using a TUNEL kit (Uelandy) and visualized using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany). Wheat germ agglutinin (WGA) staining Cardiomyocyte area was presented with WGA staining. Cardiac tissue sections were washed and then fixed with 4% paraformaldehyde. WGA (50 µg/mL) was applied to the tissue and incubated for 30 min at room temperature. Short-axis imaging of the heart was taken. In vivo safety evaluation For the detection of hepatic and renal function, blood was collected 28 days post-I/R injury, centrifuged at 3000 rpm for 10 min at 4 ℃ to obtain serum. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured by the ALT assay kit, the AST assay kit, the BUN assay kit, and the CRE assay kit. Statistical analysis All the data were presented as mean ± standard deviation. Comparisons of two groups were performed using a t-test. One-way ANOVA or Two-way ANOVA was used for the comparisons of more than two groups. The significance level was set at p < 0.05. Calculations were performed using GraphPad Prism 9.0 (GraphPad Prism Software, USA). Results and Discussion Synthesis and characterization of VB@MOF/TA As illustrated in Fig. 1 A, VB@MOF/TA nanoparticles were synthesized via a three-step fabrication process. First, MOF nanoparticles were prepared through a self-assembly reaction using 2-methylimidazole (2-MI) and zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O) as precursors. Subsequently, VB was encapsulated within the MOF nanoparticles via physical embedding, yielding the VB@MOF composite. Finally, TA was conjugated onto the surface of VB@MOF to obtain the VB@MOF/TA nanoparticles. Scanning electron microscopy (SEM) analysis (Figure S1 ) and transmission electron microscopy (TEM) imaging (Figs. 1 B, 1 C, and S2) demonstrated that pristine MOF nanoparticles maintained a well-defined dodecahedral morphology, with structural integrity preserved even after VB encapsulation and TA surface modification. Dynamic light scattering (DLS) measurements (Figure S3) yielded hydrodynamic diameters of approximately 90 nm, 250 nm, and 350 nm for MOF, VB@MOF, and VB@MOF/TA, respectively, which is consistent with the electron microscopy observations. Zeta potential (ζ) measurements (Fig. 1 D) showed that TA modification induced a significant negative shift in surface charge, with VB@MOF/TA exhibiting a ζ potential of − 29.49 ± 0.33 mV, compared to 28.26 ± 0.37 mV for MOF and − 11.39 ± 0.59 mV for VB@MOF. This negative surface charge has been demonstrated to concurrently enhance both the colloidal stability and biocompatibility of the material[ 22 ]. X-ray diffraction (XRD) analysis (Fig. 1 E) confirmed that both VB@MOF and VB@MOF/TA retained the characteristic diffraction peaks of the MOF, indicating that neither VB encapsulation nor TA conjugation disrupted the crystalline structure. Fourier-transform infrared (FTIR) spectroscopy (Fig. 1 F) further validated the chemical composition of VB@MOF/TA. The key absorption bands of MOF, including the C = N stretching vibration at 1460 cm − 1 , Zn − O coordination bond at 780 cm − 1 , and Zn − N coordination at 695 cm − 1 were observed in VB@MOF and VB@MOF/TA[ 23 ]. The FTIR spectrum of VB@MOF/TA exhibited absorption bands of C − O−C stretching vibrations of ester/ether bonds at 1110 cm − 1 and 1270 cm − 1 , indicating VB encapsulation. The FTIR spectrum of VB@MOF/TA exhibited a significantly weakened bonding absorption peak between 3200 cm − 1 and 3600 cm − 1 , which was attributed to the O − H stretching vibrations of TA, confirming the successful surface modification of VB@MOF with TA. X-ray photoelectron spectroscopy (XPS) provided additional evidence of successful synthesis, with the Zn 2p 3 peak persisting in both VB@MOF and VB@MOF/TA (Fig. 1 G). The C 1s spectra (Figs. 1 H, 1 I, and S4) revealed a distinct C = O peak at 288 eV in VB@MOF and VB@MOF/TA, absent in pristine MOF, directly confirming VB encapsulation. UV-Vis spectrophotometry (Fig. 1 J) demonstrated a redshift in the absorption peak of VB from 332 nm to 372 nm post-encapsulation. Multi-enzyme mimicking VB@MOF/TA for broad-spectrum free radical scavenging This study systematically evaluated the multienzyme-mimicking properties and free radical scavenging efficacy of VB@MOF/TA in vitro . Firstly, we evaluated the free radical scavenging capacity of VB@MOF/TA against various free radical. Initially, standard radical reagents, specifically 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•⁺) and 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH•), were incubated with VB@MOF/TA at different concentrations (6.25, 12.5, 25, 50, 100, and 200 µg/mL) for 30 min to assess their scavenging capability. The ABTS•⁺ solution exhibited a blue coloration with a characteristic UV-Vis absorption peak at 734 nm[ 24 ] (Fig. 2 A). The characteristic absorption peak at 734 nm (Fig. 2 B) demonstrated that VB@MOF/TA scavenged ABTS•⁺ in a concentration-dependent manner, with the blue ABTS•⁺ solution decolorizing to colorless (Figure S5a). The radical scavenging kinetics and rate also displayed concentration-dependent and time-dependent characteristics (Fig. 2 C). At 30 min post-reaction, VB@MOF/TA (200 µg/mL) achieved an ABTS•⁺ scavenging rate of about 79.84%. Similarly, in the DPPH• scavenging assay, the DPPH• ethanol solution was observed to be deep purple (Fig. 2 D) with a characteristic UV-Vis absorption peak at 517 nm[ 25 ]. The absorption peak intensity decreased proportionally with increasing VB@MOF/TA concentration in the DPPH• solution (Fig. 2 E), accompanied by a color transition from purple to yellow (Figure S5b). The DPPH• scavenging efficiency of VB@MOF/TA exhibited both concentration-dependence and time-dependence, reaching about 66.12% at 200 µg/mL after 30 min of reaction (Fig. 2 F). Superoxide dismutase (SOD) activity was measured using the water-soluble tetrazolium salt-1 (WST-1) method, which is based on SOD-catalyzed dismutation of superoxide anions (•O 2 − )[ 26 ]. The results showed that both VB@MOF and VB@MOF/TA exhibited comparably high •O 2 − inhibition percentages, significantly outperforming the MOF alone (Fig. 2 G), This confirmed VB@MOF/TA’s superior SOD-like activity, primarily attributed to VB loading, while TA surface modification preserved the enzymatic performance without interference. Hydrogen peroxide (H 2 O 2 ) displays a characteristic absorption peak at 240 nm. Catalase (CAT)-like activity was assessed by monitoring the time-dependent decrease in absorbance at 240 nm, reflecting H 2 O 2 decomposition. The results demonstrated that VB@MOF/TA exhibited excellent CAT-like activity, with H 2 O 2 scavenging efficiency showing a positive correlation with nanoparticle concentration (Fig. 2 H). Furthermore, the peroxidase (POD)-like activity of VB@MOF/TA was evaluated using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H 2 O 2 chromogenic system. In this reaction, colorless TMB is oxidized by H 2 O 2 to generate blue oxidized TMB (oxTMB) and H 2 O, with POD-like activity quantified by monitoring the characteristic oxTMB absorption at 652 nm[ 27 ]. The VB@MOF/TA-treated solution showed significantly reduced absorbance at 652 nm, indicating that VB@MOF/TA displayed superior POD-like catalytic activity compared to MOF alone (Fig. 2 I). The catalytic performance showed a concentration-dependent enhancement, with higher concentrations yielding greater activity (Figure S6). Finally, we evaluated the scavenging efficacy of VB@MOF/TA against •O 2 − and hydroxyl radicals (•OH). Using the superoxide anion radical inhibition and generation assay kit, we observed a dose-dependent decrease in characteristic absorbance at 550 nm with increasing concentrations of VB@MOF/TA (Fig. 2 J). Quantitative analysis revealed that at a concentration of 200 µg/mL, the •O 2 − scavenging capacity reached about 82.90 U/L (Fig. 2 K). For evaluation of •OH scavenging capacity using the hydroxyl radical detection kit, the experimental results showed a concentration-dependent attenuation of the 550 nm absorbance signal (Figs. 2 L and S7), correlating with a visible transition from pink to colorless solution. At the concentration of 200 µg/mL, the •OH scavenging efficiency achieved about 31.88 U/mL (Fig. 2 M). Comprehensive analysis revealed that the VB@MOF/TA nanocomposite possessed multiple enzyme-mimetic activities (SOD, CAT, and POD). A quantitative comparison showed that its activity intensity was similar to VB@MOF but superior to MOF, confirming that the incorporation of VB enhanced the antioxidant properties, while TA surface modification did not affect the multi-enzyme activities. Biocompatibility of VB@MOF/TA Hemolysis assays were performed to assess the blood compatibility of the nanomaterials. Fresh red blood cells were incubated with varying concentrations of MOF, VB@MOF, and VB@MOF/TA. All experimental groups showed hemolysis rates below 5%, confirming excellent hemocompatibility of the three nanomaterials (Figure S8). Subsequently, the cytocompatibility of MOF, VB@MOF, and VB@MOF/TA was evaluated in rat cardiomyocyte cells (H9C2 cells), mouse macrophages (RAW 264.7 macrophages), and human umbilical vein endothelial cells (HUVECs) using the cell counting kit-8 (CCK-8) assay. The results demonstrated that cell viability remained above 80% at concentrations ≤ 25 µg/mL, establishing this as the biocompatible threshold (Figure S9). Cellular uptake and mitochondrial targeting of VB@MOF/TA Effective cellular internalization is critical for VB@MOF/TA to exert its intracellular functions. We investigated both the uptake capability and temporal dynamics of VB@MOF/TA in H9C2 cells. For visual tracking of cellular uptake, the fluorescent probe Rhodamine B (RhB) was encapsulated in RhB@MOF/TA as a substitute for VB. Confocal laser scanning microscopy (CLSM) was used to assess the intracellular uptake of RhB@MOF/TA at various time points (0, 1, 4, 8, and 12 h). Fluorescence imaging revealed that RhB@MOF/TA (red fluorescence) was extensively internalized by H9C2 cells after just 1 h of co-incubation. Quantitative fluorescence intensity analysis revealed no statistically significant differences across incubation periods (1 h vs. 4/8/12 h) (Figure S10). Lysosomes, the primary digestive organelles within cells, play a crucial role in cellular defense by mediating the degradation of exogenous materials internalized through endocytosis. These membrane-bound compartments form a critical biological barrier to mitochondrial delivery of antioxidant nanoparticles[ 28 ]. We further investigated whether TA-modified nanozymes could evade lysosomal entrapment to reach the mitochondria. H9C2 cells were incubated with RhB@MOF and RhB@MOF/TA for 1 h, followed by co-staining with Mito Tracker Green (mitochondria) and Lyso Tracker Green (lysosomes). Colocalization analysis of red (nanoparticles) and green (organelle markers) fluorescence signals revealed distinct distribution patterns. CLSM images showed that RhB@MOF predominantly localized within lysosomes (Fig. 3 A), while RhB@MOF/TA efficiently escaped lysosomal compartments and colocalized with mitochondria (Fig. 3 B). Additionally, mitochondrial colocalization analysis revealed a significantly higher Pearson coefficient for VB@MOF/TA (0.68) compared to VB@MOF (0.30) (Figure S11a). In contrast, lysosomal colocalization showed that VB@MOF/TA exhibited a lower Pearson coefficient (0.30) than VB@MOF (0.60) (Figure S11b). These findings demonstrated that TA modification facilitates lysosomal escape and promotes mitochondrial targeting of VB@MOF. The antioxidant and mitochondrial function recovery after oxygen-glucose deprivation/reoxygenation (OGD/R) injury During the initial phase of reperfusion, the sudden restoration of oxygen supply to cardiomyocytes leads to massive generation and accumulation of ROS. As the primary source of ROS, mitochondria are the most susceptible to oxidative damage, which further exacerbates the explosive release of ROS, forming a vicious cycle of “ROS-induced mitochondrial damage”. This process can trigger mitochondrial damage, impaired ATP synthesis, cardiomyocyte apoptosis, and ultimately lead to deterioration of cardiac function[ 29 , 30 ]. The studies, as mentioned above, have confirmed that VB@MOF/TA possessed multienzyme-mimicking activities and could efficiently scavenge ROS. VB@MOF/TA demonstrated good cytocompatibility, and in H9C2 cells, we have verified that VB@MOF/TA can be extensively internalized by cells and specifically targeted to mitochondria after 1 h of co-culturing. We further evaluated the potential applications of VB and VB@MOF/TA in protecting cells against OGD/R injury. The OGD/R protocol[ 26 ] was employed as an in vitro model to simulate MI/RI injury (Fig. 3 C). Both VB and VB@MOF/TA at 25 µg/mL effectively quenched the majority of intracellular ROS. Both VB and VB@MOF/TA significantly attenuated intracellular ROS accumulation (Figs. 3 D, S12, and S13), detecting by the 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, while restoring physiological SOD and CAT activity levels, thereby demonstrating potent antioxidant efficacy under OGD/R conditions (Figs. 3 E and 3 F). Based on quantitative analysis of ROS levels, VB@MOF/TA demonstrated more significant antioxidant activity compared to free VB. Mitochondria serve as the primary site of ROS generation. They are particularly vulnerable to ROS-induced damage, typically manifesting as decreased mitochondrial membrane potential, reduced ATP production, and altered mitochondrial structure[ 31 , 32 ]. Our evaluation of mitochondrial transmembrane potential demonstrated that both VB and VB@MOF/TA could suppress ROS, thereby restore mitochondrial membrane potential (Figs. 3 G and 3 I) and enhancing ATP synthesis (Figure S14). Furthermore, TEM analysis of ultrastructural changes in H9C2 cell mitochondria revealed distinct morphological alterations. As shown in Figure S15, mitochondria in the OGD/R group exhibited significant swelling and rounding, accompanied by disrupted cristae. After treatment with the VB, the mitochondrial swelling was attenuated, but the cristae structure remained unclear. In contrast, the VB@MOF/TA group displayed markedly reduced swelling, with better-preserved cristae morphology. H2A histone family member X (H2AX), undergoes rapid phosphorylation at Ser139 within minutes following DNA double-strand breaks, forming γ-H2AX[ 33 ]. This phosphorylation event plays a crucial role in the DNA damage response and repair, serving as a sensitive biomarker for DNA damage[ 34 , 35 ]. Given that ROS can induce irreversible oxidative DNA damage, we subsequently assessed γ-H2AX levels. Both immunofluorescence analysis (Figs. 3 H and 3 J) and Western blot results (Figs. 3 K and 3 L) confirmed that VB@MOF/TA effectively mitigated the OGD/R-induced γ-H2AX elevation. Notably, VB@MOF/TA demonstrated superior DNA damage protection compared to free VB, attributable to its targeted mitochondrial delivery and potent ROS scavenging capacity. Anti-apoptotic effect of VB and VB@MOF/TA in OGD/R injury Our study using the OGD/R cellular model confirmed the antioxidative protective effects of VB@MOF/TA. Following cardiomyocyte injury, lactate dehydrogenase (LDH) and creatine kinase-myocardial band (CK-MB) are released, while intracellular malondialdehyde (MDA) concentration serves as a positive correlate of cellular membrane damage[ 36 , 37 ]. We found that treatment with both VB and VB@MOF/TA not only reduced the release of cardiac injury markers LDH and CK-MB (Figs. 4 A and 4 B) but also decreased intracellular MDA levels (Fig. 4 C), with VB@MOF/TA demonstrating more pronounced cardioprotective effects. Considering the established link between oxidative stress and apoptosis, we further investigated the anti-apoptotic properties of VB and VB@MOF/TA. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining revealed that OGD/R induced substantial apoptosis, which was significantly attenuated by both treatments; however, VB@MOF/TA demonstrated superior anti-apoptotic efficacy compared to VB alone (Figs. 4 D and 4 F). Flow cytometric analysis further confirmed that OGD/R markedly increased apoptosis, whereas both treatment groups exhibited reduced apoptotic rates, with VB@MOF/TA showing a significantly greater reduction (Figs. 4 E and 4 G). Western blot analysis of apoptosis-related proteins revealed that OGD/R upregulated pro-apoptotic proteins (C-casp 3 and Bax), while downregulating the anti-apoptotic Bcl-2 expression. Notably, VB@MOF/TA treatment effectively reversed these changes, suppressing pro-apoptotic proteins while enhancing Bcl-2 expression (Figs. 4 H-K). Consistent with these findings, live/dead cell staining confirmed that VB@MOF/TA more effectively inhibited OGD/R induced cell death compared to VB monotherapy (Figure S16). Collectively, these results demonstrate that both VB and VB@MOF/TA effectively scavenge ROS and inhibit cardiomyocyte apoptosis. Since the apoptotic process stimulates the release of inflammatory cytokines and subsequent inflammatory cascades[ 3 ], we quantified pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokine levels in H9C2 cells. While OGD/R promoted the release of pro-inflammatory cytokines and decreased the release of IL-10, both VB and VB@MOF/TA significantly inhibited inflammation, with VB@MOF/TA showing greater efficacy (Figure S17). These findings established that VB and VB@MOF/TA not only provided potent ROS scavenging and anti-apoptotic effects but also disrupted the ROS-inflammation cycle by modulating cytokine expression and restoring inflammatory homeostasis. The regulatory effect of VB@MOF/TA on the inflammatory microenvironment Macrophages play a pivotal role in myocardial tissue repair following MI/RI. During the reparative phase of MI/RI, M2 macrophages become predominant, suppressing M1-mediated inflammatory responses to maintain a balanced fibrosis and facilitate favorable ventricular remodeling[ 38 ]. Conversely, excessive activation of pro-inflammatory macrophages not only exacerbates oxidative stress and myocardial damage but also promotes pathological fibrosis, leading to adverse ventricular remodeling[ 39 , 40 ]. These findings underscore the therapeutic importance of modulating macrophage phenotype and function to optimize the inflammatory microenvironment. To investigate whether macrophages can effectively internalize VB@MOF/TA to exert intracellular effects, we first conducted cellular uptake studies in RAW 264.7 macrophages. Using RhB@MOF/TA as a fluorescent tracer, we employed CLSM to evaluate intracellular uptake at various time points (0, 1, 4, 8, and 12 h). The results paralleled our observations in H9C2 cells; fluorescence imaging revealed that RhB@MOF/TA (red fluorescence) was extensively internalized by RAW 264.7 macrophages within just 1h of co-incubation. Quantitative analysis of fluorescence intensity showed no statistically significant differences between the 1, 4, 8, and 12 h time points (Figure S18). To investigate the regulatory effects of VB and VB@MOF/TA, RAW 264.7 cells were cultured for 12 h and subsequently treated with phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS + VB, or LPS + VB@MOF/TA for an additional 24 h (Fig. 5 A). LPS stimulation of RAW 264.7 cells induce inflammation-associated oxidative stress, and the DCFH-DA fluorescent probe was used to detect ROS. The ROS-scavenging efficacy of VB and VB@MOF/TA in macrophages was assessed via fluorescence microscopy. Distinct green fluorescence was observed in LPS-stimulated macrophages, indicating substantial intracellular ROS accumulation resulting from the LPS-induced inflammatory response. Following treatment with either free VB or VB@MOF/TA, both groups exhibited markedly reduced green fluorescence. Notably, the VB@MOF/TA group demonstrated significantly greater attenuation of green fluorescence intensity compared to the VB group, confirming superior efficacy of VB@MOF/TA in suppressing LPS-induced oxidative stress (Figure S19). We further examined the expression of inflammatory genes in cells and the levels of inflammatory cytokines released. Quantitative real-time polymerase chain reaction (qPCR) results (Fig. 5 B) demonstrated that LPS stimulation significantly upregulated the expression of pro-inflammatory genes ( TNF-α , IL-6 , and IL-1β ) compared to the Control group. In contrast, the anti-inflammatory gene ( IL-10 ) showed no statistically significant change. Inflammatory cytokine levels in cell culture supernatant (Figs. 5 C-F) aligned with qPCR findings. Both VB and VB@MOF/TA treatments effectively suppressed LPS-induced overexpression of pro-inflammatory genes (Fig. 5 B) and reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Figs. 5 C-E). Concurrently, the expression of the anti-inflammatory gene (IL-10) (Fig. 5 B) and the level of IL-10 in cell supernatant were significantly enhanced (Fig. 5 F). To further investigate the regulatory effects of VB and VB@MOF/TA on macrophage polarization, we assessed the expression of M1 markers (CD86) and M2 markers (CD206) using immunofluorescence staining and flow cytometry. Compared to the LPS group, both VB and VB@MOF/TA groups exhibited weaker fluorescence signals for the M1 marker (CD86) and stronger signals for the M2 marker (CD206) (Figs. 5 G, S20, and S21). Additionally, immunofluorescence staining and Western blot analysis confirmed that VB and VB@MOF/TA treatment resulted in decreased expression of iNOS (M1 marker) and increased expression of Arg-1 (M2 marker) (Figures S22 and S23). These findings indicated that VB and VB@MOF/TA effectively counteract LPS-induced oxidative stress and modulate the inflammatory microenvironment by regulating macrophage polarization. Notably, VB@MOF/TA demonstrated superior efficacy compared to VB alone in scavenging intracellular ROS under inflammatory conditions and promoting M2 macrophage polarization. Transcriptomic Analysis of Molecular Mechanism Excessive ROS not only directly damages cardiomyocytes but also promotes macrophage recruitment to injured areas, establishing a self-perpetuating “ROS-inflammation” feedback loop that exacerbates myocardial injury[ 41 , 42 ]. The intensity of macrophage-mediated inflammatory responses critically influences fibrotic scar formation during cardiac repair, ultimately determining ventricular remodeling and long-term cardiac function[ 43 ]. To elucidate the molecular mechanisms of the immunomodulatory effects of VB@MOF/TA, we performed transcriptomic analysis of LPS-induced RAW 264.7 cells treated with either PBS or VB@MOF/TA (Fig. 6 A). Principal component analysis (PCA) revealed differentially expressed genes (DEGs) between the VB@MOF/TA group and the control group. DEGs analysis identified 2,786 significantly altered genes, including 1,370 upregulated genes and 1,416 downregulated genes (Figs. 6 B and 6 C). Gene Ontology (GO) enrichment demonstrated these genes were predominantly involved in immune-related biological processes (Fig. 6 D). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that VB@MOF/TA significantly modulated inflammatory signaling pathways, including the TNF pathway and the NF-κB pathway (Figs. 6 E and 6 F). Notably, the Gene Set Enrichment Analysis (GSEA) revealed profound alterations in metabolic pathways—specifically glycolysis (enrichment score − 1.30) and tryptophan metabolism (enrichment score 1.79) (Figs. 6 G and 6 H), providing a new perspective for future research on inflammatory response and mitochondrial metabolism. The findings above demonstrated that VB@MOF/TA likely exerts its immunomodulatory effects through the regulation of inflammatory signaling pathways and metabolic reprogramming, with the tryptophan metabolism pathway emerging as a potential novel therapeutic target for macrophage-associated inflammatory diseases. Collectively, these results provide crucial evidence for understanding the potential of VB@MOF/TA to modulate macrophage function, suppress inflammatory cascades, and consequently promote tissue repair. Therapeutic effects of VB and VB@MOF/TA in mouse MI/RI model Building upon VB@MOF/TA’s demonstrated capacity for targeted ROS scavenging, cryoprotection, and inflammation modulation in vitro , we further investigated its therapeutic potential in myocardial ischemia/reperfusion (I/R) injury using an established murine model. MI/RI was applied with transient left anterior descending (LAD) occlusion for 45 min, followed by reperfusion for specified durations (Fig. 7 A)[ 26 ]. VB and VB@MOF/TA were administered via tail vein injection immediately before reperfusion. At 24 h post-reperfusion, serum levels of myocardial injury markers LDH and CK-MB in the I/R group were significantly elevated (Figs. 7 B and 7 C), indicating substantial cardiomyocyte damage. Following treatment with either VB or VB@MOF/TA, serum LDH and CK-MB levels were significantly reduced (Figs. 7 B and 7 C). 2,3,5-Triphenyltetrazolium chloride (TTC) staining revealed that MI/RI mice treated with VB or VB@MOF/TA exhibited significantly reduced infarct areas compared to the I/R group (Fig. 7 D, Tables S1 and S2). Cardiac function was assessed by measuring ejection fraction (EF) and fractional shortening (FS) via transthoracic echocardiography from preoperative to endpoint observations (Figs. 7 E-G). On postoperative day 1, the I/R group showed markedly decreased EF and FS values, while both the VB and VB@MOF/TA groups demonstrated improved cardiac parameters. Throughout the observation period, all groups showed progressive functional recovery. By postoperative day 28, the EF and FS of the VB@MOF/TA group increased by 23.30% ± 3.19% and 13.66% ± 1.76% respectively, compared to the I/R group. Further analysis revealed that these parameters were also 7.72% ± 3.71% (EF) and 5.10% ± 2.32% (FS) greater than with VB monotherapy. These findings were corroborated by left ventricular short-axis echocardiographic analysis (Figure S24, Tables S1 and S2). To investigate whether VB and VB@MOF/TA protect cells from damage through potential anti-apoptotic effects, we subsequently conducted experiments related to apoptosis. During I/R injury, the levels of pro-apoptotic proteins C-casp 3 and Bax were upregulated while Bcl-2 was downregulated (Figs. 7 H-K). The administration of both VB and VB@MOF/TA downregulated the expression of cleaved-caspase 3 (C-casp 3) and Bax, while upregulating Bcl-2 levels. TUNEL staining revealed that VB and VB@MOF/TA treatment alleviated I/R injury-induced cardiomyocyte apoptosis, with the VB@MOF/TA group showing a 2.59-fold greater reduction in apoptosis rate compared to the VB group (Figs. 7 L and 7 M). Quantitative analysis of both Western blot and TUNEL assay results demonstrated that VB@MOF/TA exhibited stronger anti-apoptotic capabilities than VB following I/R injury, confirming its superior advantages in reducing cardiac damage. VB@MOF /TA regulates the inflammatory microenvironment to promote cardiac repair Following acute myocardial ischemia and hypoxia, Ly6C high monocytes rapidly accumulate at the injury site, reaching peak abundance and constituting the dominant immune cell population responding to cardiomyocyte death[ 44 ]. These infiltrating Ly6C high monocytes differentiate into M1 macrophages, which secrete pro-inflammatory cytokines, thereby driving the initial inflammatory response. Upon reperfusion therapy, cardiomyocytes subjected to ROS attack release multiple inflammatory factors that trigger inflammatory cascades. Subsequently, Ly6C high monocytes are recruited to the injured area and differentiate into M2 macrophages, which mitigate inflammation through the secretion of the anti-inflammatory cytokine IL-10 while promoting extracellular matrix remodeling and angiogenesis[ 45 ]. The persistent inflammatory response during early reperfusion promotes myocardial fibrosis and impairs cardiac tissue recovery post-injury[ 46 ]. Precise modulation of this inflammatory response represents a critical therapeutic target for optimizing tissue repair following myocardial reperfusion injury. VB, a bioactive constituent of the medicinal plant Plantago asiatica L. , exhibits well-documented anti-inflammatory pharmacological properties. To investigate whether VB could enhance cardiac tissue repair through modulation of inflammation, we examined the effects of VB and VB@MOF/TA on myocardial tissue. In our murine model, inflammatory responses peaked 24 h post-I/R[ 47 ], prompting an analysis of serum cytokine levels and macrophage marker expression in myocardial tissue at 24 h post-reperfusion. The I/R group exhibited significantly elevated serum levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), without a concurrent increase in the anti-inflammatory cytokine IL-10. Both VB and VB@MOF/TA treatments reversed this profile, reducing pro-inflammatory cytokines while elevating IL-10 levels to 3.8-fold (VB) and 5.1-fold (VB@MOF/TA) of the I/R group, respectively (Figs. 8 A-D). Immunofluorescence staining of cardiac specimens (Figs. 8 E-G) demonstrated decreased CD86 and increased CD206 expression in treatment groups, with VB@MOF/TA showing more pronounced effects on macrophage subtype distribution. These findings confirm that both compounds promote M2 macrophage polarization, with VB@MOF/TA demonstrating superior efficacy to VB monotherapy in restoring inflammatory homeostasis. At day 28 post-reperfusion therapy, we histologically evaluated myocardial tissue recovery. Immunofluorescence staining for CD31 and α-smooth muscle actin (α-SMA) revealed that CD31 + capillary density in the VB@MOF/TA and VB groups increased to 1.60-fold and 2.41-fold of the I/R group, respectively, and α-SMA + arteriole density reached 1.73-fold and 2.58-fold of the I/R group, respectively (Figs. 8 H-K). Wheat germ agglutinin (WGA) fluorescent staining demonstrated that both VB and VB@MOF/TA treatments significantly attenuated cardiomyocyte hypertrophy following I/R injury (Fig. 8 L). Collectively, these findings indicated that the modulated inflammatory microenvironment promotes both angiogenesis and vascular preservation post-MI/RI, thereby enhancing myocardial tissue repair. In vivo biosafety assessment We also conducted a series of biocompatibility evaluation of VB@MOF/TA. Our preliminary in vitro studies demonstrated favorable biocompatibility of VB@MOF/TA. Critical in vivo investigations, conducted through histological examination of major organs, revealed no detectable pathological alterations (Figure S25), indicating that neither VB nor VB@MOF/TA induced pulmonary, hepatic, splenic, or renal significant damage. Complementary hematological analysis of hepatic and renal function markers showed no statistically significant differences across all parameters (Figure S26), providing additional confirmation of its biosafety. This integrated dataset from multiple experimental approaches provides conclusive validation of the good biocompatibility and favorable safety characteristics of VB@MOF/TA for potential therapeutic applications. Conclusion To address the dual challenges of cardiomyocyte injury and pathological remodeling in acute myocardial infarction reperfusion therapy, we developed VB@MOF/TA, a mitochondria-targeted nanoformulation that simultaneously scavenges ROS and modulates the inflammatory microenvironment. This work demonstrates three key innovations: 1. A new targeting strategy. The TA-mediated mitochondrial targeting mechanism represents a breakthrough in organelle-specific drug delivery, enabling precise accumulation of VB@MOF/TA within cardiomyocyte mitochondria. 2. An advanced antioxidant/anti-inflammatory platform based on VB. Our pioneering MOF-based VB delivery system overcomes critical clinical limitations of free VB while enhancing antioxidant and anti-inflammatory efficacy. In vivo validation demonstrated that VB@MOF/TA improved the left ventricular ejection fraction by 23.30% ± 3.19% compared to the I/R group and by 7.72% ± 3.71% compared to VB monotherapy. 3. A potential anti-inflammatory pathway. Mechanistic studies revealed that VB@MOF/TA significantly modulates tryptophan metabolism, identifying this pathway as a potential new therapeutic target for regulating inflammation. Certainly, this study also has many limitations: the long-term biosafety, metabolism, and precise mechanisms underlying the anti-inflammatory effects of VB@MOF/TA require further investigation. Given that this system possesses the triple functions of combating oxidative stress, regulating mitochondrial quality, and modulating inflammatory responses, it holds considerable promise for treating oxidative stress-related diseases, such as diabetes-related complications, atherosclerosis, and Alzheimer's disease. Abbreviations AMI MI/RI TA MOF VB ROS mPTP ETC γ-H2AX 2-MI Zn(NO 3 ) 2 ·6H 2 O DPPH ABTS LDH CK-MB ALT AST BUN CRE MDA ELISA DAPI DCFH-DA FBS ATP SEM XRD FTIR UV-Vis TMB •OH •O 2 − OGD/R Calcein-AM/PI qPCR SDS-PAGE PVDF TBST ECL BSA WGA TTC Acute myocardial infarction myocardial ischemia/reperfusion injury tannic acid metal-organic framework verbascoside reactive oxygen species mitochondrial permeability transition pore electron transport chain phospho-histone 2AX 2-methylimidazole Zinc nitrate hexahydrate 2,2-Diphenyl-1-picrylhydrazyl 2,2¢-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) lactate dehydrogenase creatine kinase-myocardial band alanine aminotransferase aspartate aminotransferase blood urea nitrogen creatinine Malondialdehyde enzyme-linked immunosorbent assay 4,6-diamidino-2-phenylindole 2¢,7¢-dichlorofluorescin diacetate Fetal bovine serum Adenosine triphosphate scanning electron microscopy X-ray diffraction Fourier-transform infrared spectroscopy Ultraviolet-visible 3,3¢,5,5¢-Tetramethylbenzidine Hydroxyl radical superoxide radical oxygen–glucose deprivation and reperfusion calcein acetoxymethyl ester/propidium iodide Quantitative real-time polymerase chain reaction sodium dodecyl sulfate-polyacrylamide gel electrophoresis Polyvinylidene fluoride Tris-buffered saline/Tween 20 enhanced chemiluminescence bovine serum albumin Wheat germ agglutinin 2,3,5-Triphenyltetrazolium chloride Declarations Ethics approval and consent to participate Animal experiments were permitted by the animal ethics committee of Nanchang University (Nanchang, China, NCULAE-20250120001). Consent for publication Not applicable. Availability of data and materials All relevant data are within the manuscript and its Supporting Information files. Competing interests All authors declared that no conflict of interest existed. Funding This work was funded by The National Natural Science Foundation of China (82360105 to Yanhua Tang), The Natural Science Foundation of Jiangxi Province (20232ACB206001 to Yanhua Tang), The Key Research and Development Program of Jiangxi Province (20223BBG71010 to Yanhua Tang), The Clinical Trial Research Projects (2021efyA02 to Yanhua Tang), The Key Research and Development Program of Jiangxi Province (20212BBG73004 to Xiaolei Wang), The Jiangxi Province Key Laboratory of Bioengineering Drugs (No.2024SSY07061 to Xiaolei Wang) and The Interdiscipline Innovation Fund Project of Nanchang University (PYJX20230001 to Xiaolei Wang). Authors' contributions X. Wang and Y. Tang guided the project. X. Wang, Y. Tang and C. Li conceived the idea and conceptualized the manuscript. C. Li, Z. Zhang, C. Luo, W. Lan, C. Liu, W. Liu, J. Yang, H. Xiang, Y. Tang and X. Wang participated in the design of experimental methods and sample analysis. C. Li completed the processing of experimental data and wrote the manuscript. X. Wang and Y. Tang reviewed and edited the manuscript. All authors reviewed the manuscript. Acknowledgements Not applicable. References G.A. Roth, G.A. Mensah, C.O. Johnson, G. Addolorato, E. Ammirati, L.M. Baddour, N.C. Barengo, A.Z. Beaton, E.J. Benjamin, C.P. Benziger, A. Bonny, M. Brauer, M. Brodmann, T.J. Cahill, J. Carapetis, A.L. Catapano, S.S. Chugh, L.T. Cooper, J. Coresh, M. Criqui, N. DeCleene, K.A. Eagle, S. Emmons-Bell, V.L. Feigin, J. Fernández-Solà, G. Fowkes, E. Gakidou, S.M. Grundy, F.J. He, G. Howard, F. Hu, L. Inker, G. Karthikeyan, N. Kassebaum, W. Koroshetz, C. Lavie, D. Lloyd-Jones, H.S. Lu, A. Mirijello, A.M. Temesgen, A. Mokdad, A.E. Moran, P. Muntner, J. Narula, B. 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Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingMaterials.docx Graphicalabstract.docx Scheme1.docx Cite Share Download PDF Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 16 Sep, 2025 Reviews received at journal 15 Sep, 2025 Reviews received at journal 14 Sep, 2025 Reviews received at journal 11 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 25 Aug, 2025 Submission checks completed at journal 25 Aug, 2025 First submitted to journal 24 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7445253","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513683411,"identity":"83b4e718-1d28-470e-9540-94240c30cd50","order_by":0,"name":"Congcong Li","email":"","orcid":"","institution":"Department of Cardiovascular Surgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Congcong","middleName":"","lastName":"Li","suffix":""},{"id":513683412,"identity":"8eb0ca78-2c24-43e8-9503-72eabdc9c02a","order_by":1,"name":"Zhengfeng 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University","correspondingAuthor":false,"prefix":"","firstName":"Wanqi","middleName":"","lastName":"Lan","suffix":""},{"id":513683415,"identity":"a844a71e-cb3d-4b76-8fa3-9183f8692c35","order_by":4,"name":"Chen Liu","email":"","orcid":"","institution":"Department of Ultrasound, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Liu","suffix":""},{"id":513683416,"identity":"bf9a228d-5119-47dc-adfa-2bd74f61ed6d","order_by":5,"name":"Wu Liu","email":"","orcid":"","institution":"Department of Cardiovascular Surgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"Liu","suffix":""},{"id":513683417,"identity":"35e06088-5c9c-486d-bf12-eef6385556e4","order_by":6,"name":"Haiyan Xiang","email":"","orcid":"","institution":"Department of Cardiovascular Surgery, The Second 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09:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7445253/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7445253/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03810-3","type":"published","date":"2025-11-05T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91157732,"identity":"9fd2ab9f-de1a-41aa-bd82-eccec8c90295","added_by":"auto","created_at":"2025-09-12 08:23:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1084301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of VB@MOF/TA.\u003c/strong\u003e (A) Schematic illustration of the synthesis for VB@MOF/TA. (B-C) TEM images of VB@MOF and VB@MOF/TA, scale bar: 200 nm. (D) Zeta potentials of MOF, VB@MOF, and VB@MOF/TA measured by DLS. (E) XRD patterns of MOF, VB@MOF, and VB@MOF/TA. (F) FTIR spectra of VB@MOF/TA, VB@MOF, MOF, and VB. (G) XPS survey spectra of MOF, VB@MOF, and VB@MOF/TA. (H-I) XPS C 1s spectra of VB@MOF and VB@MOF/TA. (J) UV-Vis spectra of VB@MOF/TA, VB@MOF, MOF, and VB.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/7553ce0f33e80f05e6ebfba2.png"},{"id":91157748,"identity":"7e218659-f4b8-4ddb-8f13-b25bebd6dd14","added_by":"auto","created_at":"2025-09-12 08:23:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":893325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultienzyme-mimicking antioxidant properties and free radical scavenging capability of VB@MOF/TA. \u003c/strong\u003e(A-C) Schematic diagram of the mechanism of VB@MOF/TA scavenging ABTS•\u003csup\u003e+\u003c/sup\u003e (A), UV spectral changes of the reaction solution (B), and reaction kinetic curve (C). (D-F) Schematic diagram of the mechanism of VB@MOF/TA scavenging DPPH• (D), UV spectral changes of the reaction solution (E), and reaction kinetic curve (F).\u003cstrong\u003e \u003c/strong\u003e(G-I) Concentration-dependent SOD-like (G), CAT-like (H), POD-like activities (I) of VB@MOF/TA, VB@MOF, and MOF. (J) UV-Vis spectra showing the radicals eliminating activities of VB@MOF/TA for •O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e. (K) The ability to resist •O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e of VB@MOF/TA at different concentrations. (L) UV-Vis spectra showing the radicals eliminating activities of VB@MOF/TA for •OH. (M) The ability to resist •OH of VB@MOF/TA at different concentrations. Experiments were repeated three times with similar results.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/15a17c462b677c9a85e79601.png"},{"id":91158453,"identity":"05bb59d3-c110-470a-9080-b8786a7db21d","added_by":"auto","created_at":"2025-09-12 08:31:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2325469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe representative confocal fluorescence images of mitochondria-targeting and maintaining mitochondrial function by antioxidant ability.\u003c/strong\u003e (A, B) The representative fluorescence images of mitochondria-targeting of RhB@MOF (A) and RhB@MOF/TA (B). (C) Schematic diagram of the cell experiment. (D) Intracellular ROS detected by flow cytometry. (E) The level of cellular CAT (n = 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0028, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0067). (F) The levels of cellular SOD (n = 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0030). (G) Representative fluorescent images of JC-1. (H) Immunofluorescence staining of phosphorylated γ-H2AX. (I) Quantification analysis of JC-1 (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0288). (J) The proportion of γ-H2AX positive nuclei (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001). (K) Representative images of Western blot of γ-H2AX. (L) Quantification analysis of γ-H2AX (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0141, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0477). Data are analyzed using One-way ANOVA and represented as the mean ± standard deviation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/9ad6ad7a88a907fa692e78ce.png"},{"id":91157781,"identity":"84441a63-78e1-4145-8fc0-b427f50d3b2b","added_by":"auto","created_at":"2025-09-12 08:23:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":974574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytoprotective effect of VB and VB@MOF/TA.\u003c/strong\u003e (A) LDH level (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0064, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0223). (B) CK-MB level (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0027, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0363). (C) The levels of cellular MDA (n = 6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001). (D) Quantification of positive cells detected by the TUNEL Cell Apoptosis Kit (n = 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001). (E) Relevant quantification of apoptotic cells (n = 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.009). (F) Representative fluorescent images of apoptotic cells accessed by the TUNEL assay. (G) Representative flow cytometry detection of cell apoptosis through Annexin V/PI assay. (H-K) Representative images of western blot (H) and quantification of Bax (I), C-casp 3 (J), Bcl-2 (K) (n = 6, for Bax, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0269, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0439; C-casp 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e = 0.0062, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0086; Bcl-2, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, OGD/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(OGD/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0048). Data are analyzed using One-way ANOVA and represented as the mean ± standard deviation. FC, fold-change.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/4128f3cd485d2d2da0d1b9f6.png"},{"id":91157735,"identity":"0ba2e366-e341-49ab-bac3-65ce1bb28501","added_by":"auto","created_at":"2025-09-12 08:23:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1511149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe inflammatory response was alleviated by VB and VB@MOF/TA through their modulation of the M1/M2 macrophage equilibrium.\u003c/strong\u003e (A) Schematic illustration of experiment. (B) Heatmap illustrating the gene expression levels of \u003cem\u003eTNF-a\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-1b\u003c/em\u003e and \u003cem\u003eIL-10\u003c/em\u003e in RAW 264.7 macrophages assessed by qPCR assay (n = 3). (C-F) Quantification of TNF-a (C), IL-6 (D), IL-1b (E) and IL-10 (F) in RAW 264.7 macrophages assessed by ELISA after different treatments (n = 3, for TNF-𝛼, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, LPS)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0012; for IL-6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, LPS)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep \u003c/em\u003e\u003csub\u003e(LPS, VB)\u003c/sub\u003e = 0.0142, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0050; for IL-1b, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, LPS)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB)\u003c/sub\u003e = 0.0043, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0052; for IL-10, \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, LPS)\u003c/sub\u003e \u0026gt; 0.9999, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB)\u003c/sub\u003e = 0.0043, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(LPS, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0319). (G) Representative immunofluorescence images of M1 marker (CD86) and M2 marker (CD206) expression in RAW 264.7 cells after different treatments. Data are analyzed using One-way ANOVA and represented as the mean ± standard deviation. ns, no significance.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/d282593989e9740113ae42e3.png"},{"id":91158459,"identity":"fb852e1a-32e3-4add-9146-c89cfcf70709","added_by":"auto","created_at":"2025-09-12 08:31:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1702403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of VB@MOF/TA anti-inflammatory mechanisms.\u003c/strong\u003e (A) Principal component analysis (PCA) of the sequence results in RAW 264.7 macrophages treated with LPS + PBS and LPS + VB@MOF/TA. (B) Heatmap between PBS and VB@MOF/TA group. (C) Volcano plot of DEGs between PBS and VB@MOF/TA group. (D) GO enrichment analysis for DEGs. (E) KEGG pathway enrichment analysis. (F) Chord diagram illustrating KEGG enrichment terms for DEGs. (G, H) Gene set enrichment analysis (GSEA) for gene sets.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/a870d5b7875fde5696c0a0e1.png"},{"id":91158449,"identity":"fd0856bf-b3a9-4739-80b0-25a77b8c67d5","added_by":"auto","created_at":"2025-09-12 08:31:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2232166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic efficacy of VB and VB@MOF/TA on myocardial I/R injury.\u003c/strong\u003e (A) Schematic illustration of the animal experiment. (B, C) Serum level of myocardial enzyme spectrum LDH (B) and CK-MB (C) (for LDH, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0268, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0408; for CK-MB, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0230, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0249). (D) Representative photographs of TTC staining (n = 3). (E-G) Representative images of transthoracic echocardiography and quantitative analysis of cardiac function (comparison between I/R and I/R + VB, EF: ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(1d)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(7d)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(14d)\u003c/sub\u003e = 0.0011, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(28d)\u003c/sub\u003e \u0026lt; 0.001, FS: ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(1d)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(7d)\u003c/sub\u003e = 0.0209, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(14d)\u003c/sub\u003e = 0.0217, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(28d)\u003c/sub\u003e = 0.0019; comparison between I/R + VB and I/R + VB@MOF/TA, EF: \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(1d)\u003c/sub\u003e \u0026lt; 0.001, \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u003csub\u003e(7d)\u003c/sub\u003e = 0.3084, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(14d)\u003c/sub\u003e = 0.0068, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(28d)\u003c/sub\u003e \u0026lt; 0.0490, FS: \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(1d)\u003c/sub\u003e \u0026lt; 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(7d)\u003c/sub\u003e = 0.0251, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(14d)\u003c/sub\u003e = 0.0408, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(28d)\u003c/sub\u003e = 0.0181). (H-K) Representative images of western blot (H) and quantification of Bax (I), C-casp 3 (J), Bcl-2 (K) (n = 6, for Bax, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0370, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0124; C-casp 3, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0026, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0317; Bcl-2, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0288, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0060). (L, M) Representative image (L) and quantification (M) of apoptotic cells evaluated by TUNEL assay (scale bar: 50 µm). Data are analyzed using One-way or Two-way ANOVA and represented as the mean ± standard deviation. FC, fold-change.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/b07bbe5fdfd693f92302af78.png"},{"id":91158452,"identity":"058650f2-7eab-44fc-a40d-0b6dcb890604","added_by":"auto","created_at":"2025-09-12 08:31:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":320896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammatory regulation and angiogenesis of VB@MOF/TA \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A-D) Serum levels of pro-inflammatory cytokine TNF-𝛼 (A), IL-1𝛽 (B), IL-6 (C), and anti-inflammatory cytokine IL-10 (D) detected by ELISA kit (n = 6, for TNF-𝛼 and IL-1𝛽, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001; for IL-6, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0029, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0433; for IL-10, \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e = 0.0567, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0017). (E-G) Representative images (F) and quantification analysis for CD86 (E) and CD206 (G) in different groups (n = 3, CD86, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0107; CD206, *\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e = 0.0430, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e = 0.0013), scale bar: 50 µm. (H, I) Representative images (H) and quantification analysis (I) for CD31 (n = 4, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0015, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001), scale bar: 50 µm. (J, K) Representative images (J) and quantification analysis (K) for a-SMA (n = 4, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(Control, I/R)\u003c/sub\u003e \u0026lt; 0.001, **\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB)\u003c/sub\u003e = 0.0018, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(I/R, VB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001, ***\u003cem\u003ep\u003c/em\u003e \u003csub\u003e(VB,\u003c/sub\u003e \u003csub\u003eVB@MOF/TA)\u003c/sub\u003e \u0026lt; 0.001), scale bar: 50 µm. (L) Representative WGA staining of cardiomyocytes in the I/R area (n = 4), scale bar: 50 µm. Data are analyzed using One-way ANOVA and represented as the mean ± standard deviation. ns, no significance.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/e2efd6a59bc4d6ec13821c48.png"},{"id":95564139,"identity":"31d279e0-e38f-4bb9-bfed-422b665c2e82","added_by":"auto","created_at":"2025-11-10 16:08:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11711497,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/988a117b-7b62-4100-be2b-4a791eead17b.pdf"},{"id":91157793,"identity":"fc10d2f9-3af8-4978-bf26-701df8d2e82e","added_by":"auto","created_at":"2025-09-12 08:23:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":58386066,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/72ca09c7bedc15cc147336da.docx"},{"id":91157853,"identity":"11ab131f-43e5-47fe-be2d-ca4e717d3bcb","added_by":"auto","created_at":"2025-09-12 08:23:56","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1947997,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/f813dac8f4b6d7d12a54e832.docx"},{"id":91157788,"identity":"8d1a55b4-bb63-47df-928d-81aaa2a1ffa5","added_by":"auto","created_at":"2025-09-12 08:23:52","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6831554,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7445253/v1/c7c23f6b74e8f47386f424cd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A mitochondria-targeted nanozyme for myocardial ischemia/reperfusion injury with synergistic antioxidant and anti-inflammatory properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myocardial infarction (AMI) remains the leading cause of mortality worldwide, posing a significant threat to global public health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathological process involves cardiomyocyte necrosis secondary to acute coronary occlusion, with timely reperfusion therapy serving as the standard clinical strategy to salvage ischemic myocardium[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, reperfusion exhibits a \u0026ldquo;double-edged sword\u0026rdquo; effect while restoring blood flow, it paradoxically aggravates myocardial damage through ischemia/reperfusion injury (MI/RI)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite therapeutic advances, current MI/RI management faces several critical limitations, including poor drug target specificity, safety concerns with combination therapies, and inadequate long-term prevention of myocardial fibrosis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Effective MI/RI mitigation is crucial for minimizing cardiomyocyte death, preserving post-reperfusion cardiac function, and preventing adverse ventricular remodeling[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mitochondrial reactive oxygen species (ROS) overload and the ensuing inflammatory cascade are now regarded as the core drivers of MI/RI[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This positions mitochondria as a rational therapeutic target to fine-tune ROS output, safeguard organelle quality, rebalance metabolism, and curb maladaptive inflammation.\u003c/p\u003e\u003cp\u003eAddressing these challenges, this study presents the first construction of VB@MOF/TA, a mitochondria-targeting nanozyme, with potent antioxidant and anti-inflammatory activities for the prevention and treatment of MI/RI. Leveraging the exceptional porosity and tunable pore distribution of metal-organic framework (MOF)-based nanozymes[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], high-efficiency encapsulation of verbascoside (VB) was achieved, yielding the VB@MOF nanozyme (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). VB, a phenylethanoid glycoside with established antioxidant and anti-inflammatory properties, is a bioactive constituent derived from \u003cem\u003ePlantago asiatica L.\u003c/em\u003e, a traditional Chinese herb native to central China[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The MOF carrier system was specifically engineered to overcome clinical limitations of VB[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]: (1) Chemical instability due to its polyhydroxy structure, (2) Poor lipophilicity, (3) Pronounced first-pass effect (oral bioavailability as low as 0.12%)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To enable mitochondrial targeting, we functionalized the VB@MOF nanoparticles with tannic acid (TA), which facilitates charge-independent mitochondrial accumulation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, this surface engineering approach yielded the mitochondria-targeting nanozyme VB@MOF/TA, which was then applied to alleviate MI/RI (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The nanozyme selectively accumulates in mitochondria \u003cem\u003evia\u003c/em\u003e TA-mediated tropism, where it efficiently scavenges ROS, restores mitochondrial membrane potential (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-I), and enhances adenosine triphosphate (ATP) production (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-II), thereby precisely regulating mitochondrial quality. Consequently, deoxyribonucleic acid (DNA) damage induced by ROS (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-III) and inflammatory cytokine release (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-IV) are significantly reduced, enabling intracellular targeted antioxidant protection against reperfusion injury. Furthermore, the dual antioxidant and anti-inflammatory action of the nanozyme promotes M2 macrophage polarization while suppressing pro-inflammatory M1 polarization, leading to the downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the upregulation of the anti-inflammatory cytokine (IL-10), thereby ameliorating the inflammatory microenvironment (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Ultimately, this approach attenuates adverse cardiac remodeling (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In summary, this multifunctional nanoplatform, which integrates mitochondrial targeting, antioxidant, and anti-inflammatory properties, represents a promising translatable strategy for MI/RI therapy. Subsequent experiments were conducted to validate the feasibility of this system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Experimental Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e\n\u003cp\u003e2-methylimidazole (2-MI) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Zinc nitrate hexahydrate (Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), tannic acid (TA), and verbascoside (VB) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and the 2,2\u0026prime;-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Phygene Life Sciences Co., Ltd. (Fuzhou, China). Inhibition and produce superoxide anion assay kit, hydroxyl free radical assay kit, hydrogen peroxide assay kit, lactate dehydrogenase (LDH) assay kit, creatine kinase-myocardial band (CK-MB) isoenzyme assay kit, alanine aminotransferase (ALT) assay kit, aspartate aminotransferase (AST) assay kit, blood urea nitrogen (BUN) assay kit, and creatinine (CRE) assay kit were purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China). Malondialdehyde (MDA) assay kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kits of mouse TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-10 were purchased from BOSTER Biological Technology Co., Ltd. (Wuhan, China). 4,6-diamidino-2-phenylindole (DAPI) solution, 2\u0026prime;,7\u0026prime;-dichlorofluorescin diacetate (DCFH-DA), mitochondrial membrane potential assay kit with JC-1, catalase activity assay kit, superoxide dismutase activity assay kit, and CCK-8 cell proliferation and cytotoxicity assay kit were purchased from Solarbio Technology Co., Ltd. (Beijing, China). YF\u0026reg;488 TUNEL cell apoptosis assay kit was purchased from UElandy (Suzhou, China). Annexin V-FITC/PI double-stained cell apoptosis detection kit and Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) were purchased from Key GEN BioTECH (Nanjing, China). Fetal bovine serum (FBS) was purchased from Abcell (Beijing, China). The Calcein-AM/PI live/dead cell double staining kit was purchased from BIOPRIMACY (Wuhan, China). Adenosine triphosphate (ATP) assay kit, Mtio-Tracker Green, and Lyso-Tracker Green were purchased from Beyotime Biotechnology (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eSynthesis of VB@MOF/TA\u003c/p\u003e\n\u003cp\u003eFirstly, 2-MI (6.568 g, 80 mmol) and Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (2.9749 g, 10 mmol) were fully dissolved in methanol solution[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The reaction solution was stirred for 3 h at room temperature and then allowed to stand for 12 h. The samples in the centrifuge tube were washed 3 times with methanol, and the metal organic framework (MOF) nanoparticles were obtained after centrifuging at 1000 rpm for 10 min. The sample was dried and then stored at room temperature. Afterward, the MOF methanol solution, followed by ultrasonication, was mixed with 20 mg of VB, the mixture was stirred for 24 h, and the VB@MOF nanomedicine was collected by centrifugation. Subsequently, the VB@MOF methanol solution was added to the TA solution (12 mM, 5 mL, pH 7.5), and the mixture was further stirred for 5 min. Then, VB@MOF/TA was washed 3 times with methanol and collected by centrifugation. Finally, the VB@MOF/TA nanomedicines were obtained by vacuum drying.\u003c/p\u003e\n\u003cp\u003eCharacterization of VB@MOF/TA\u003c/p\u003e\n\u003cp\u003eThe morphology was observed using scanning electron microscopy (SEM, ZEISS Sigma 300, Germany) and transmission electron microscopy (HT7800, Hitachi Ltd., Japan). A particle size and \u0026zeta; potential analyzer (NanoBrook, Brookhaven, USA) was used to measure size distribution and \u0026zeta; potential. X-ray diffraction (XRD, Bruker, D8ADVANCE, Germany) was employed to confirm the crystalline structure. The chemical structure and composition were characterized using Fourier-transform infrared spectroscopy (FTIR) (Thermo Nicolet, Nicolet 5700, USA) and X-ray photoelectron spectroscopy (XPS, ESCALAB25OXI, Thermo Fisher, USA), respectively. The spectrum of VB aqueous solution was obtained using a Ultraviolet-visible (UV-Vis) spectrophotometer (UV-2600, SHIMADZU, Japan) and plotted as a standard curve. The VB content in VB@MOF and VB@MOF/TA was quantified by UV-Vis spectrophotometry at 334 nm, with concentrations determined using the pre-established VB standard curve.\u003c/p\u003e\n\u003cp\u003eSOD-like activity\u003c/p\u003e\n\u003cp\u003eThe SOD-like activity was measured with SOD activity assay kit (Solarbio). Different concentrations of samples (20 \u0026micro;L) were incubated with the working solution in a 37 ℃ water bath for 30 min, following the manufacturer\u0026rsquo;s instructions. Absorbance changes were monitored \u003cem\u003evia\u003c/em\u003e Multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 450 nm.\u003c/p\u003e\n\u003cp\u003eCAT-like activity\u003c/p\u003e\n\u003cp\u003eThe CAT-like activity was measured with the CAT activity assay kit (Solarbio). Different concentrations of samples (10 \u0026micro;L) were mixed with the working solution (190 \u0026micro;L) prepared according to the manufacturer\u0026rsquo;s instructions. Absorbance changes were monitored immediately \u003cem\u003evia\u003c/em\u003e Multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 240 nm.\u003c/p\u003e\n\u003cp\u003ePOD-like activity\u003c/p\u003e\n\u003cp\u003eThe POD-like activity was performed according to the mechanism in which colorless 3,3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine (TMB) is oxidized in the presence of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to produce blue oxidized TMB (oxTMB) and H\u003csub\u003e2\u003c/sub\u003eO. The HAc/NaAc buffer (0.1 M, pH 4.5) was mixed with TMB (Aladdin, \u0026ge; 99%) (1.0 mM), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1.0 mM), and different concentrations of MOF, VB@MOF, and VB@MOF/TA. The reaction was performed using TMB (Aladdin, \u0026ge; 99%) (1.0 mM), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1.0 mM), and varying concentrations of MOF, VB@MOF, and VB@MOF/TA, all at room temperature. Absorbance at 652 nm was monitored by a UV-Vis spectrophotometer (UV-2600, SHIMADZU, Japan).\u003c/p\u003e\n\u003cp\u003eDPPH free radical scavenging activity\u003c/p\u003e\n\u003cp\u003eThe mixed solution of different concentrations of VB@MOF/TA (50 \u0026micro;L), PBS (150 \u0026micro;L), and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) ethanol solution (0.3 mM, 200 \u0026micro;L) was incubated in the dark. The UV-Vis absorbance was monitored every 2 min for 30 min at 517 nm, and UV-Vis spectra were recorded between 400 nm and 700 nm at 30 min. The standard curve of DPPH\u0026bull; was calculated using UV-Vis absorbance at 517 nm to estimate DPPH scavenging capacity.\u003c/p\u003e\n\u003cp\u003eABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e free radical scavenging activity\u003c/p\u003e\n\u003cp\u003eThe ABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e working solution was prepared 24 h in advance. Briefly, an equal volume of ABTS (7.4 mM) and K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e (2.6 mM) was mixed in the PBS (pH 7.4) in the dark. The ABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e working solution was obtained by diluting the concentrated solution with PBS (pH 7.4) to an absorbance of 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm. Then, different concentrations of VB@MOF/TA (20 \u0026micro;L) and PBS buffer (80 \u0026micro;L) were added to the ABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e working solution (400 \u0026micro;L). The UV-Vis absorbance was monitored every 2 min for 30 min at 734 nm, and UV-Vis spectra of 500\u0026ndash;900 nm were detected at 30 min. The standard curve of ABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e was calculated using UV-Vis absorbance at 734 nm to estimate ABTS\u0026bull;\u003csup\u003e+\u003c/sup\u003e scavenging capacity.\u003c/p\u003e\n\u003cp\u003eHydroxyl radical (\u0026bull;OH) and superoxide radical (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) scavenging activity\u003c/p\u003e\n\u003cp\u003eThe hydroxyl radical (\u0026bull;OH) and superoxide Radical (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) scavenging activity of VB@MOF/TA was measured by using the hydroxyl free radical assay kit and the inhibition and produce superoxide anion assay kit, respectively, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eHemolysis\u003c/p\u003e\n\u003cp\u003eThe centrifuged erythrocytes were obtained from fresh blood samples, and the erythrocytes (1.00 mL) were diluted with saline (3.67 mL). The diluted erythrocytes (100 \u0026micro;L) were added to physiological saline solution (1 mL) containing different concentrations of MOF, VB@MOF, and VB@MOF/TA. The mixture was incubated at 37 ℃ for 3 h, and then centrifuged at 12000 rpm for 15 min. The absorbance of supernatants was monitored by a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA) at 540 nm. The rate of hemolysis was measured, with ddH\u003csub\u003e2\u003c/sub\u003eO serving as a positive control and saline as a negative control.\u003c/p\u003e\n\u003cp\u003eCell culture\u003c/p\u003e\n\u003cp\u003eRAW 264.7 macrophages, H9C2 cells, and HUVECs were cultured in DMEM with 1% (V/V) penicillin/streptomycin (Key GEN BioTECH) and 10% (V/V) FBS (Abcell) in a 5% (V/V) carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) incubator at 37 ℃ (standard conditions).\u003c/p\u003e\n\u003cp\u003eCytocompatibility assay\u003c/p\u003e\n\u003cp\u003eH9C2 cells, RAW 264.7 macrophages, and HUVECs were seeded into 96-well plates (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well) and incubated for 24 h, allowing the cells to adhere fully to the wall. The cells were washed with PBS and replaced with fresh medium (100 \u0026micro;L/well) containing various concentrations of different materials. The cells were then incubated for 24, 48, and 72 h. Subsequently, the cells were rinsed twice with PBS, and then the CCK-8 assay (Solarbio) was performed according to the manufacturer\u0026rsquo;s instructions. The absorbance was monitored at 450 nm using a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA). The formula calculated cell viability:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eA is the absorbance of the cells treated with materials, A \u003csub\u003econtrol\u003c/sub\u003e is the absorbance of the cells treated with PBS, and A \u003csub\u003eblank\u003c/sub\u003e is the absorbance of the medium containing CCK-8 without cells.\u003c/p\u003e\n\u003cp\u003eCellular Uptake and mitochondria-targeting ability\u003c/p\u003e\n\u003cp\u003eTo evaluate whether VB@MOF/TA can be taken up by macrophages and cardiomyocytes, rhodamine B (RhB), a fluorescence probe, was used instead of VB in RhB@MOF/TA to track the uptake process. RAW 264.7 cells and H9C2 cells were cultured in a confocal culture dish at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells and incubated for 24 h. After the cells were adherent, fresh medium containing RhB@MOF/TA was added. The cells and RhB@MOF/TA were co-cultured for 0 h, 1 h, 4 h, 8 h, and 12 h in CO\u003csub\u003e2\u003c/sub\u003e incubator at 37 ℃, RhB@MOF/TA solution was sucked away and washed thrice with PBS. Subsequently, the nuclei were stained withDAPI (Solarbio) for 5 min. A laser scanning confocal microscope (LSM980, ZEISS, Germany) was used to visualize, followed by three washes.\u003c/p\u003e\n\u003cp\u003eTo evaluate whether the VB@MOF/TA can effectively escape from lysosomes and target mitochondria. The H9C2 cells were cultured in a confocal dish (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) for 24 h, and then treated with RhB@MOF (red) and RhB@MOF/TA (red) in cell culture medium for 1 h, respectively. The cells were washed three times with PBS and stained with Mito-Tracker Green (Beyotime) and Lyso-Tracker Green (Beyotime) for 30 min in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37 ℃. After being washed thrice with PBS, the cells were photographed using a laser scanning confocal microscope (LSM980, ZEISS, Germany).\u003c/p\u003e\n\u003cp\u003eExtracorporeal oxygen\u0026ndash;glucose deprivation and reperfusion (OGD/R) process\u003c/p\u003e\n\u003cp\u003eMyocardial cell hypoxia/reperfusion injury was simulated using an in vitro oxygen-glucose deprivation (OGD/R) model constructed according to reported protocols[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. H9C2 cells were seeded in 6-well plate at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well, when the density of the cells reached 70\u0026ndash;80%, the H9C2 cells were exposed to 0.1% oxygen in serum-free no glucose DMEM at 37 ℃ to achieve OGD treatment for 10 h, followed by culturing under standard DMEM added VB@MOF/TA (25 \u0026micro;g/mL), or VB (25 \u0026micro;g/mL), or same dose of PBS for recovery oxygenation 12 h. The cells treated with the OGD/R process were used to detect ROS, assess mitochondrial function, evaluate cell apoptosis, and investigate inflammation.\u003c/p\u003e\n\u003cp\u003eLive/dead cell stain\u003c/p\u003e\n\u003cp\u003eAfter the H9C2 cells were treated with OGD/R process, the cells were stained with calcein acetoxymethyl ester/propidium iodide (Calcein-AM/PI) using a Calcein-AM/PI live/dead cell double staining kit (BIOPRIMACY, China), and visualized by fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany) after incubating for 15 min in the dark.\u003c/p\u003e\n\u003cp\u003eMitochondrial membrane potential\u003c/p\u003e\n\u003cp\u003eH9C2 cells were seeded in a 6-well plate at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. The mitochondrial membrane potential of cells after the OGD/R model was measured using the mitochondrial membrane potential assay kit with JC-1 (Solarbio) under the protocol. Fluorescently labeled cells were photographed using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany). The ratio between the monomer (green) and aggregate (red) mean fluorescence intensities was calculated using ImageJ software as a measure of mitochondrial membrane potential.\u003c/p\u003e\n\u003cp\u003eIntracellular MDA and ATP\u003c/p\u003e\n\u003cp\u003eThe levels of intracellular MDA and ATP were detected with the MDA assay kit (Shanghai Enzyme-linked Biotechnology) and the ATP assay kit (Beyotime), respectively. Cells that had undergone the OGD/R procedure were collected and processed following the manufacturer\u0026rsquo;s protocol. For the determination of MDA, absorbance at 532 nm and 600 nm was detected by a multimode plate reader (VICTOR Nivo 3S, PerkinElmer, USA), and protein concentration was tested by BCA protein assay kit (Beyotime). The level of ATP was detected by a luminometer (LB960, Berthold Technologies, Germany).\u003c/p\u003e\n\u003cp\u003ePolarization regulation of RAW 264.7 macrophages\u003c/p\u003e\n\u003cp\u003eRAW 264.7 macrophages were seeded into 6-well plate (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) and incubated for 12 h. To mimic inflammatory microenvironments, RAW 264.7 macrophages of experiment group were treated with LPS (300 ng/mL), the cells in the control group were treated with same dose of PBS, the cells of VB group were treated with LPS (300 ng/mL)\u0026thinsp;+\u0026thinsp;VB (25 \u0026micro;g/mL), the cells of VB@MOF/TA group were treated with LPS (300 ng/mL)\u0026thinsp;+\u0026thinsp;VB@MOF/TA (25 \u0026micro;g/mL), all cells were co-cultured for another 24 h. The impact of VB and VB@MOF/TA on regulating macrophage phenotype and inflammation was demonstrated through immunofluorescence staining, Western blot, qPCR, flow cytometry, and ELISA.\u003c/p\u003e\n\u003cp\u003eIntracellular ROS scavenging\u003c/p\u003e\n\u003cp\u003eThe intracellular levels of total ROS were assessed using DCFH-DA. DCFH-DA (10 \u0026micro;M) was incubated with OGD/R-treated H9C2 cells and LPS-induced RAW 264.7 macrophages for 30 min. The cells were then observed using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany).\u003c/p\u003e\n\u003cp\u003eMoreover, flow cytometry was also used to collect quantitative data about the intracellular ROS level of H9C2 cells. The cells were subjected to the OGD/R process and then incubated with DCFH-DA (10 \u0026micro;M) for 30 min. After trypsinization, the cells were harvested, and the fluorescence intensity was measured using flow cytometry (ID7000, Sony Group Corporation, Japan). Data was analyzed using Flow Jov_10.9.0. The gating strategy was presented in Figure S27a.\u003c/p\u003e\n\u003cp\u003eQuantitative real-time polymerase chain reaction(qPCR)\u003c/p\u003e\n\u003cp\u003eThe qPCR was used to detect the relative mRNA expression levels of pro-inflammatory cytokine (\u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e) and anti-inflammatory cytokine (\u003cem\u003eIL-10\u003c/em\u003e). The total RNA of macrophages was extracted by SteadyPure Quick RNA Extraction Kit (Accurate Biology, China) according to the manufacturer\u0026rsquo;s instructions, and reverse transcription of mRNA was performed utilizing the Hifair\u003csup\u003e\u0026reg;\u003c/sup\u003e AdvanceFast 1st Strand cDNA Synthesis SuperMix for qPCR (DNA digester plus) (YEASEN, China). QPCR was conducted using a TB Green Premix Ex Taq II on a ViiA 7 Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA). The relative gene expression levels in all samples were normalized using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method, with GAPDH as the internal control for comparison.\u003c/p\u003e\n\u003cp\u003eFlow cytometry of CD86 and CD206\u003c/p\u003e\n\u003cp\u003eRAW 264.7 macrophages were resuspended with PBS (100 \u0026micro;L) after being collected and placed in flow cytometry tubes from different groups. PE Anti-Mouse CD86 Antibody (Elabscience) or APC Anti-Mouse CD206/MMR Antibody (Elabscience) was used for staining, and the cells were detected by flow cytometry.\u003c/p\u003e\n\u003cp\u003eMyocardial infarction injury model in mouse\u003c/p\u003e\n\u003cp\u003eC57BL/6C mice (6\u0026ndash;8 weeks, male) were obtained from Charles River Laboratories (Beijing, China) and were bred in a specific pathogen-free (SPF) environment. All animals were handled in accordance with protocols approved by the Nanchang University Laboratory Animal Center and its internal ethics committee (NCULAE-20250120001). The mice were housed in a 12-h photoperiod with standard temperature and humidity, and given free access to water and food. The mice were acclimated for 1 week before the operations. Mice were anesthetized and sustained with 1.5% isoflurane, the left open thoracotomy was performed to visualize the heart, the left anterior descending artery (LAD) of the coronary artery was temporarily ligated with a 7\u0026ndash;0 suture for 45 min. The saline, VB, and VB@MOF/TA were treated (dose: 1 mg/kg) via tail vein injection at 40 min, followed by suture removal for reperfusion after 5 min. The mice of the control group underwent the same surgical process, and the LAD was passed with a suture without ligation; an equal amount of saline was injected after 40 min. Surgical wounds of all mice were sutured using sterile 4\u0026ndash;0 sutures.\u003c/p\u003e\n\u003cp\u003eTransthoracic echocardiography\u003c/p\u003e\n\u003cp\u003eTransthoracic echocardiography was performed using an Animal ultrasound imaging system (VINNO6 LAB, VINNO, China), in a blinded manner. Mice were anesthetized and sustained with isoflurane after depilation of the chest area skin. Hearts were visualized from short-axis and long-axis views. The images of B-mode and M-mode were captured. The EF and FS were calculated automatically by the software in the Animal Ultrasound Imaging System.\u003c/p\u003e\n\u003cp\u003eMyocardial enzyme spectrum\u003c/p\u003e\n\u003cp\u003eMouse blood was collected at 24 h post-surgery and centrifuged to obtain serum. The culture medium from H9C2 cells after OGD/R was collected and centrifuged to obtain the supernatant in different groups. The myocardial enzyme spectrum in serum and cell supernatant was detected using the LDH assay kit and the CK-MB isoenzyme assay kit, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eDetection of cytokines\u003c/p\u003e\n\u003cp\u003eThe ELISA kits were used to detect the secretion of pro-inflammatory and anti-inflammatory cytokines. For H9C2 cells and RAW 264.7 macrophages, the medium was collected after various treatments and centrifuged to obtain the supernatants. The serum of mice was obtained at 24 h after surgery. The concentrations of pro-inflammatory cytokines (IL-1\u0026beta;, IL-6, TNF-\u0026alpha;) and anti-inflammatory cytokines (IL-10) in serum and cell supernatant were measured according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e2,3,5-Triphenyltetrazolium chloride (TTC) staining\u003c/p\u003e\n\u003cp\u003eThe hearts of mice were obtained after perfusing with PBS at 24 h post-surgery and stored in \u0026minus;\u0026thinsp;80 ℃. The 1-mm-thick cross sections of the heart were incubated with 2% TTC (Solarbio, China) solution at 37 ℃ for 15 min in the dark. After that, the slices were fixed with 4% paraformaldehyde, and photographs of the slices were taken.\u003c/p\u003e\n\u003cp\u003eWestern blot\u003c/p\u003e\n\u003cp\u003eThe radioimmunoprecipitation assay lysis buffer (RIPA, Solarbio, China), which contained proteinase and phosphatase inhibitors (Salorbio, China), was used to extract protein from cells and heart tissue. Protein concentration was measured by the BCA Protein Assay Kit (Beyotime, China). Proteins were separated by 6\u0026ndash;12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to 0.2\u0026ndash;0.45 \u0026micro;m Polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk dissolved in Tris-buffered saline/Tween 20 (TBST) for 1\u0026ndash;2 h. Primary antibodies were incubated overnight at 4 ℃. After being washed with TBST, secondary antibodies were incubated for 2 h at room temperature. The membranes were immersed in enhanced chemiluminescence (ECL) and analyzed using an automated gel imaging system (Tanon 5200, Shanghai, China). Protein levels were quantified with the ImageJ software. The following primary antibodies were used: \u0026beta;-actin (1:5000, Proteintech), Bax (1:3334, Proteintech), C-casp 3 (1:1000, Abmart), Bcl-2 (1:1000, Abmart), iNOS (1:1000, Abmart), Arg-1 (1:1000, Servicebio), \u0026gamma;-H2AX (1:1000, Abmart). The secondary antibodies used were HRP-conjugated IgG antibody (Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) antibody, 1:10000, Proteintech, or anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) antibody, 1:10000, Proteintech).\u003c/p\u003e\n\u003cp\u003eImmunofluorescence analysis\u003c/p\u003e\n\u003cp\u003eFor cell samples, cells were fixed with 4% paraformaldehyde and permeabilized with a 0.5% Triton X-100 solution in PBS. 5% bovine serum albumin (BSA, Solarbio) was used to block, and then cells were incubated with primary antibodies (\u0026gamma;-H2AX, 1:200, Abmart; CD86, 1:400, Proteintech; CD206, 1:500, Proteintech; iNOS, 1:200, Abmart; Arg-1, 1:200, Servicebio) overnight at 4 ℃. After washing with PBS, cells were incubated with fluorescently coupled secondary antibodies (CoraLite\u003csup\u003e\u0026reg;\u003c/sup\u003e488-Conjugated Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), 1:1000, Proteintech; Multi-rAb\u003csup\u003e\u0026trade;\u003c/sup\u003e CoraLite\u003csup\u003e\u0026reg;\u003c/sup\u003e Plus 555-Goat Anti-Rabbit Recombinant Secondary Antibody (H\u0026thinsp;+\u0026thinsp;L), 1:1000, Proteintech). 4,6-diamidino-2-phenylindole (DAPI, Solarbio) was used to visualize nuclei, followed by washing with PBS. The cells were visualized using a laser scanning confocal microscope (LSM980, ZEISS, Germany).\u003c/p\u003e\n\u003cp\u003eCardiac tissue samples were cut into 7-\u0026micro;m tissue sections, and fixation and permeabilization blocking were carried out. The tissues were then washed and incubated overnight at 4 ℃ with primary antibodies (CD86, 1:400, Proteintech; CD206, 1:500, Proteintech; CD31, 1:200, Protentech; \u0026alpha;-SMA, 1:500, Proteintech). The tissues were incubated with fluorescently coupled secondary antibodies for 1 h at room temperature after rinsing, and then stained with DAPI. The slides were visualized using a laser scanning confocal microscope (LSM980, ZEISS, Germany).\u003c/p\u003e\n\u003cp\u003eApoptosis assays\u003c/p\u003e\n\u003cp\u003eFor Annexin V/PI staining of cells, an Annexin V-FITC/PI double-stained cell apoptosis detection kit (Key GEN) was used. Cells with OGD/R disposal were digested with EDTA-free trypsin (Solarbio) to form a single-cell suspension (500 \u0026micro;L). 5 \u0026micro;L of Annexin V-FITC and 5 \u0026micro;L of Propidium Iodide were gently mixed with the suspension, and the mixture was incubated for 10 min at room temperature in the dark. Samples were assessed using a flow cytometer (ID7000, Sony Group Corporation, Japan), and the data were analyzed using FlowJo 10.9.0. The gating strategy was presented in Figure S27b.\u003c/p\u003e\n\u003cp\u003eFor TUNEL staining of cells and tissue, cells and cardiac tissue sections were fixed with 4% paraformaldehyde and permeabilized with a 0.5% Triton X-100 solution. They were then stained using a TUNEL kit (Uelandy) and visualized using a fluorescence microscope (DM i8, Leica Microsystems CMS GmbH, Germany).\u003c/p\u003e\n\u003cp\u003eWheat germ agglutinin (WGA) staining\u003c/p\u003e\n\u003cp\u003eCardiomyocyte area was presented with WGA staining. Cardiac tissue sections were washed and then fixed with 4% paraformaldehyde. WGA (50 \u0026micro;g/mL) was applied to the tissue and incubated for 30 min at room temperature. Short-axis imaging of the heart was taken.\u003c/p\u003e\n\u003cp\u003eIn vivo safety evaluation\u003c/p\u003e\n\u003cp\u003eFor the detection of hepatic and renal function, blood was collected 28 days post-I/R injury, centrifuged at 3000 rpm for 10 min at 4 ℃ to obtain serum. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured by the ALT assay kit, the AST assay kit, the BUN assay kit, and the CRE assay kit.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll the data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons of two groups were performed using a t-test. One-way ANOVA or Two-way ANOVA was used for the comparisons of more than two groups. The significance level was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Calculations were performed using GraphPad Prism 9.0 (GraphPad Prism Software, USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eSynthesis and characterization of VB@MOF/TA\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, VB@MOF/TA nanoparticles were synthesized \u003cem\u003evia\u003c/em\u003e a three-step fabrication process. First, MOF nanoparticles were prepared through a self-assembly reaction using 2-methylimidazole (2-MI) and zinc nitrate hexahydrate (Zn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) as precursors. Subsequently, VB was encapsulated within the MOF nanoparticles \u003cem\u003evia\u003c/em\u003e physical embedding, yielding the VB@MOF composite. Finally, TA was conjugated onto the surface of VB@MOF to obtain the VB@MOF/TA nanoparticles. Scanning electron microscopy (SEM) analysis (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and transmission electron microscopy (TEM) imaging (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, and S2) demonstrated that pristine MOF nanoparticles maintained a well-defined dodecahedral morphology, with structural integrity preserved even after VB encapsulation and TA surface modification. Dynamic light scattering (DLS) measurements (Figure S3) yielded hydrodynamic diameters of approximately 90 nm, 250 nm, and 350 nm for MOF, VB@MOF, and VB@MOF/TA, respectively, which is consistent with the electron microscopy observations. Zeta potential (ζ) measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) showed that TA modification induced a significant negative shift in surface charge, with VB@MOF/TA exhibiting a ζ potential of \u0026minus;\u0026thinsp;29.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 mV, compared to 28.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mV for MOF and \u0026minus;\u0026thinsp;11.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 mV for VB@MOF. This negative surface charge has been demonstrated to concurrently enhance both the colloidal stability and biocompatibility of the material[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. X-ray diffraction (XRD) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) confirmed that both VB@MOF and VB@MOF/TA retained the characteristic diffraction peaks of the MOF, indicating that neither VB encapsulation nor TA conjugation disrupted the crystalline structure. Fourier-transform infrared (FTIR) spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) further validated the chemical composition of VB@MOF/TA. The key absorption bands of MOF, including the C\u0026thinsp;=\u0026thinsp;N stretching vibration at 1460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Zn\u0026thinsp;\u0026minus;\u0026thinsp;O coordination bond at 780 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and Zn\u0026thinsp;\u0026minus;\u0026thinsp;N coordination at 695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed in VB@MOF and VB@MOF/TA[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The FTIR spectrum of VB@MOF/TA exhibited absorption bands of C\u0026thinsp;\u0026minus;\u0026thinsp;O\u0026minus;C stretching vibrations of ester/ether bonds at 1110 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1270 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating VB encapsulation. The FTIR spectrum of VB@MOF/TA exhibited a significantly weakened bonding absorption peak between 3200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was attributed to the O\u0026thinsp;\u0026minus;\u0026thinsp;H stretching vibrations of TA, confirming the successful surface modification of VB@MOF with TA. X-ray photoelectron spectroscopy (XPS) provided additional evidence of successful synthesis, with the Zn 2p\u003csup\u003e3\u003c/sup\u003e peak persisting in both VB@MOF and VB@MOF/TA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The C 1s spectra (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, and S4) revealed a distinct C\u0026thinsp;=\u0026thinsp;O peak at 288 eV in VB@MOF and VB@MOF/TA, absent in pristine MOF, directly confirming VB encapsulation. UV-Vis spectrophotometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ) demonstrated a redshift in the absorption peak of VB from 332 nm to 372 nm post-encapsulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMulti-enzyme mimicking VB@MOF/TA for broad-spectrum free radical scavenging\u003c/p\u003e\u003cp\u003eThis study systematically evaluated the multienzyme-mimicking properties and free radical scavenging efficacy of VB@MOF/TA \u003cem\u003ein vitro\u003c/em\u003e. Firstly, we evaluated the free radical scavenging capacity of VB@MOF/TA against various free radical. Initially, standard radical reagents, specifically 2,2\u0026prime;-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS\u0026bull;⁺) and 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH\u0026bull;), were incubated with VB@MOF/TA at different concentrations (6.25, 12.5, 25, 50, 100, and 200 \u0026micro;g/mL) for 30 min to assess their scavenging capability. The ABTS\u0026bull;⁺ solution exhibited a blue coloration with a characteristic UV-Vis absorption peak at 734 nm[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The characteristic absorption peak at 734 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated that VB@MOF/TA scavenged ABTS\u0026bull;⁺ in a concentration-dependent manner, with the blue ABTS\u0026bull;⁺ solution decolorizing to colorless (Figure S5a). The radical scavenging kinetics and rate also displayed concentration-dependent and time-dependent characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At 30 min post-reaction, VB@MOF/TA (200 \u0026micro;g/mL) achieved an ABTS\u0026bull;⁺ scavenging rate of about 79.84%. Similarly, in the DPPH\u0026bull; scavenging assay, the DPPH\u0026bull; ethanol solution was observed to be deep purple (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) with a characteristic UV-Vis absorption peak at 517 nm[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The absorption peak intensity decreased proportionally with increasing VB@MOF/TA concentration in the DPPH\u0026bull; solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), accompanied by a color transition from purple to yellow (Figure S5b). The DPPH\u0026bull; scavenging efficiency of VB@MOF/TA exhibited both concentration-dependence and time-dependence, reaching about 66.12% at 200 \u0026micro;g/mL after 30 min of reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eSuperoxide dismutase (SOD) activity was measured using the water-soluble tetrazolium salt-1 (WST-1) method, which is based on SOD-catalyzed dismutation of superoxide anions (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The results showed that both VB@MOF and VB@MOF/TA exhibited comparably high \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e inhibition percentages, significantly outperforming the MOF alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), This confirmed VB@MOF/TA\u0026rsquo;s superior SOD-like activity, primarily attributed to VB loading, while TA surface modification preserved the enzymatic performance without interference. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) displays a characteristic absorption peak at 240 nm. Catalase (CAT)-like activity was assessed by monitoring the time-dependent decrease in absorbance at 240 nm, reflecting H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition. The results demonstrated that VB@MOF/TA exhibited excellent CAT-like activity, with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging efficiency showing a positive correlation with nanoparticle concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Furthermore, the peroxidase (POD)-like activity of VB@MOF/TA was evaluated using the 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB)-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e chromogenic system. In this reaction, colorless TMB is oxidized by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate blue oxidized TMB (oxTMB) and H\u003csub\u003e2\u003c/sub\u003eO, with POD-like activity quantified by monitoring the characteristic oxTMB absorption at 652 nm[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The VB@MOF/TA-treated solution showed significantly reduced absorbance at 652 nm, indicating that VB@MOF/TA displayed superior POD-like catalytic activity compared to MOF alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). The catalytic performance showed a concentration-dependent enhancement, with higher concentrations yielding greater activity (Figure S6). Finally, we evaluated the scavenging efficacy of VB@MOF/TA against \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and hydroxyl radicals (\u0026bull;OH). Using the superoxide anion radical inhibition and generation assay kit, we observed a dose-dependent decrease in characteristic absorbance at 550 nm with increasing concentrations of VB@MOF/TA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Quantitative analysis revealed that at a concentration of 200 \u0026micro;g/mL, the \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e scavenging capacity reached about 82.90 U/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). For evaluation of \u0026bull;OH scavenging capacity using the hydroxyl radical detection kit, the experimental results showed a concentration-dependent attenuation of the 550 nm absorbance signal (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and S7), correlating with a visible transition from pink to colorless solution. At the concentration of 200 \u0026micro;g/mL, the \u0026bull;OH scavenging efficiency achieved about 31.88 U/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Comprehensive analysis revealed that the VB@MOF/TA nanocomposite possessed multiple enzyme-mimetic activities (SOD, CAT, and POD). A quantitative comparison showed that its activity intensity was similar to VB@MOF but superior to MOF, confirming that the incorporation of VB enhanced the antioxidant properties, while TA surface modification did not affect the multi-enzyme activities.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBiocompatibility of VB@MOF/TA\u003c/p\u003e\u003cp\u003eHemolysis assays were performed to assess the blood compatibility of the nanomaterials. Fresh red blood cells were incubated with varying concentrations of MOF, VB@MOF, and VB@MOF/TA. All experimental groups showed hemolysis rates below 5%, confirming excellent hemocompatibility of the three nanomaterials (Figure S8). Subsequently, the cytocompatibility of MOF, VB@MOF, and VB@MOF/TA was evaluated in rat cardiomyocyte cells (H9C2 cells), mouse macrophages (RAW 264.7 macrophages), and human umbilical vein endothelial cells (HUVECs) using the cell counting kit-8 (CCK-8) assay. The results demonstrated that cell viability remained above 80% at concentrations\u0026thinsp;\u0026le;\u0026thinsp;25 \u0026micro;g/mL, establishing this as the biocompatible threshold (Figure S9).\u003c/p\u003e\u003cp\u003eCellular uptake and mitochondrial targeting of VB@MOF/TA\u003c/p\u003e\u003cp\u003eEffective cellular internalization is critical for VB@MOF/TA to exert its intracellular functions. We investigated both the uptake capability and temporal dynamics of VB@MOF/TA in H9C2 cells. For visual tracking of cellular uptake, the fluorescent probe Rhodamine B (RhB) was encapsulated in RhB@MOF/TA as a substitute for VB. Confocal laser scanning microscopy (CLSM) was used to assess the intracellular uptake of RhB@MOF/TA at various time points (0, 1, 4, 8, and 12 h). Fluorescence imaging revealed that RhB@MOF/TA (red fluorescence) was extensively internalized by H9C2 cells after just 1 h of co-incubation. Quantitative fluorescence intensity analysis revealed no statistically significant differences across incubation periods (1 h vs. 4/8/12 h) (Figure S10). Lysosomes, the primary digestive organelles within cells, play a crucial role in cellular defense by mediating the degradation of exogenous materials internalized through endocytosis. These membrane-bound compartments form a critical biological barrier to mitochondrial delivery of antioxidant nanoparticles[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We further investigated whether TA-modified nanozymes could evade lysosomal entrapment to reach the mitochondria. H9C2 cells were incubated with RhB@MOF and RhB@MOF/TA for 1 h, followed by co-staining with Mito Tracker Green (mitochondria) and Lyso Tracker Green (lysosomes). Colocalization analysis of red (nanoparticles) and green (organelle markers) fluorescence signals revealed distinct distribution patterns. CLSM images showed that RhB@MOF predominantly localized within lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while RhB@MOF/TA efficiently escaped lysosomal compartments and colocalized with mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, mitochondrial colocalization analysis revealed a significantly higher Pearson coefficient for VB@MOF/TA (0.68) compared to VB@MOF (0.30) (Figure S11a). In contrast, lysosomal colocalization showed that VB@MOF/TA exhibited a lower Pearson coefficient (0.30) than VB@MOF (0.60) (Figure S11b). These findings demonstrated that TA modification facilitates lysosomal escape and promotes mitochondrial targeting of VB@MOF.\u003c/p\u003e\u003cp\u003eThe antioxidant and mitochondrial function recovery after oxygen-glucose deprivation/reoxygenation (OGD/R) injury\u003c/p\u003e\u003cp\u003eDuring the initial phase of reperfusion, the sudden restoration of oxygen supply to cardiomyocytes leads to massive generation and accumulation of ROS. As the primary source of ROS, mitochondria are the most susceptible to oxidative damage, which further exacerbates the explosive release of ROS, forming a vicious cycle of \u0026ldquo;ROS-induced mitochondrial damage\u0026rdquo;. This process can trigger mitochondrial damage, impaired ATP synthesis, cardiomyocyte apoptosis, and ultimately lead to deterioration of cardiac function[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The studies, as mentioned above, have confirmed that VB@MOF/TA possessed multienzyme-mimicking activities and could efficiently scavenge ROS. VB@MOF/TA demonstrated good cytocompatibility, and in H9C2 cells, we have verified that VB@MOF/TA can be extensively internalized by cells and specifically targeted to mitochondria after 1 h of co-culturing. We further evaluated the potential applications of VB and VB@MOF/TA in protecting cells against OGD/R injury. The OGD/R protocol[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] was employed as an \u003cem\u003ein vitro\u003c/em\u003e model to simulate MI/RI injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Both VB and VB@MOF/TA at 25 \u0026micro;g/mL effectively quenched the majority of intracellular ROS. Both VB and VB@MOF/TA significantly attenuated intracellular ROS accumulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, S12, and S13), detecting by the 2\u0026prime;,7\u0026prime;-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, while restoring physiological SOD and CAT activity levels, thereby demonstrating potent antioxidant efficacy under OGD/R conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Based on quantitative analysis of ROS levels, VB@MOF/TA demonstrated more significant antioxidant activity compared to free VB.\u003c/p\u003e\u003cp\u003eMitochondria serve as the primary site of ROS generation. They are particularly vulnerable to ROS-induced damage, typically manifesting as decreased mitochondrial membrane potential, reduced ATP production, and altered mitochondrial structure[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our evaluation of mitochondrial transmembrane potential demonstrated that both VB and VB@MOF/TA could suppress ROS, thereby restore mitochondrial membrane potential (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and enhancing ATP synthesis (Figure S14). Furthermore, TEM analysis of ultrastructural changes in H9C2 cell mitochondria revealed distinct morphological alterations. As shown in Figure S15, mitochondria in the OGD/R group exhibited significant swelling and rounding, accompanied by disrupted cristae. After treatment with the VB, the mitochondrial swelling was attenuated, but the cristae structure remained unclear. In contrast, the VB@MOF/TA group displayed markedly reduced swelling, with better-preserved cristae morphology. H2A histone family member X (H2AX), undergoes rapid phosphorylation at Ser139 within minutes following DNA double-strand breaks, forming γ-H2AX[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This phosphorylation event plays a crucial role in the DNA damage response and repair, serving as a sensitive biomarker for DNA damage[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Given that ROS can induce irreversible oxidative DNA damage, we subsequently assessed γ-H2AX levels. Both immunofluorescence analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) and Western blot results (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL) confirmed that VB@MOF/TA effectively mitigated the OGD/R-induced γ-H2AX elevation. Notably, VB@MOF/TA demonstrated superior DNA damage protection compared to free VB, attributable to its targeted mitochondrial delivery and potent ROS scavenging capacity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnti-apoptotic effect of VB and VB@MOF/TA in OGD/R injury\u003c/p\u003e\u003cp\u003eOur study using the OGD/R cellular model confirmed the antioxidative protective effects of VB@MOF/TA. Following cardiomyocyte injury, lactate dehydrogenase (LDH) and creatine kinase-myocardial band (CK-MB) are released, while intracellular malondialdehyde (MDA) concentration serves as a positive correlate of cellular membrane damage[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We found that treatment with both VB and VB@MOF/TA not only reduced the release of cardiac injury markers LDH and CK-MB (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) but also decreased intracellular MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with VB@MOF/TA demonstrating more pronounced cardioprotective effects. Considering the established link between oxidative stress and apoptosis, we further investigated the anti-apoptotic properties of VB and VB@MOF/TA. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining revealed that OGD/R induced substantial apoptosis, which was significantly attenuated by both treatments; however, VB@MOF/TA demonstrated superior anti-apoptotic efficacy compared to VB alone (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Flow cytometric analysis further confirmed that OGD/R markedly increased apoptosis, whereas both treatment groups exhibited reduced apoptotic rates, with VB@MOF/TA showing a significantly greater reduction (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Western blot analysis of apoptosis-related proteins revealed that OGD/R upregulated pro-apoptotic proteins (C-casp 3 and Bax), while downregulating the anti-apoptotic Bcl-2 expression. Notably, VB@MOF/TA treatment effectively reversed these changes, suppressing pro-apoptotic proteins while enhancing Bcl-2 expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-K). Consistent with these findings, live/dead cell staining confirmed that VB@MOF/TA more effectively inhibited OGD/R induced cell death compared to VB monotherapy (Figure S16). Collectively, these results demonstrate that both VB and VB@MOF/TA effectively scavenge ROS and inhibit cardiomyocyte apoptosis. Since the apoptotic process stimulates the release of inflammatory cytokines and subsequent inflammatory cascades[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], we quantified pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokine levels in H9C2 cells. While OGD/R promoted the release of pro-inflammatory cytokines and decreased the release of IL-10, both VB and VB@MOF/TA significantly inhibited inflammation, with VB@MOF/TA showing greater efficacy (Figure S17). These findings established that VB and VB@MOF/TA not only provided potent ROS scavenging and anti-apoptotic effects but also disrupted the ROS-inflammation cycle by modulating cytokine expression and restoring inflammatory homeostasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe regulatory effect of VB@MOF/TA on the inflammatory microenvironment\u003c/p\u003e\u003cp\u003eMacrophages play a pivotal role in myocardial tissue repair following MI/RI. During the reparative phase of MI/RI, M2 macrophages become predominant, suppressing M1-mediated inflammatory responses to maintain a balanced fibrosis and facilitate favorable ventricular remodeling[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Conversely, excessive activation of pro-inflammatory macrophages not only exacerbates oxidative stress and myocardial damage but also promotes pathological fibrosis, leading to adverse ventricular remodeling[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These findings underscore the therapeutic importance of modulating macrophage phenotype and function to optimize the inflammatory microenvironment. To investigate whether macrophages can effectively internalize VB@MOF/TA to exert intracellular effects, we first conducted cellular uptake studies in RAW 264.7 macrophages. Using RhB@MOF/TA as a fluorescent tracer, we employed CLSM to evaluate intracellular uptake at various time points (0, 1, 4, 8, and 12 h). The results paralleled our observations in H9C2 cells; fluorescence imaging revealed that RhB@MOF/TA (red fluorescence) was extensively internalized by RAW 264.7 macrophages within just 1h of co-incubation. Quantitative analysis of fluorescence intensity showed no statistically significant differences between the 1, 4, 8, and 12 h time points (Figure S18).\u003c/p\u003e\u003cp\u003eTo investigate the regulatory effects of VB and VB@MOF/TA, RAW 264.7 cells were cultured for 12 h and subsequently treated with phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS\u0026thinsp;+\u0026thinsp;VB, or LPS\u0026thinsp;+\u0026thinsp;VB@MOF/TA for an additional 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). LPS stimulation of RAW 264.7 cells induce inflammation-associated oxidative stress, and the DCFH-DA fluorescent probe was used to detect ROS. The ROS-scavenging efficacy of VB and VB@MOF/TA in macrophages was assessed \u003cem\u003evia\u003c/em\u003e fluorescence microscopy. Distinct green fluorescence was observed in LPS-stimulated macrophages, indicating substantial intracellular ROS accumulation resulting from the LPS-induced inflammatory response. Following treatment with either free VB or VB@MOF/TA, both groups exhibited markedly reduced green fluorescence. Notably, the VB@MOF/TA group demonstrated significantly greater attenuation of green fluorescence intensity compared to the VB group, confirming superior efficacy of VB@MOF/TA in suppressing LPS-induced oxidative stress (Figure S19).\u003c/p\u003e\u003cp\u003eWe further examined the expression of inflammatory genes in cells and the levels of inflammatory cytokines released. Quantitative real-time polymerase chain reaction (qPCR) results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) demonstrated that LPS stimulation significantly upregulated the expression of pro-inflammatory genes (\u003cem\u003eTNF-α\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eIL-1β\u003c/em\u003e) compared to the Control group. In contrast, the anti-inflammatory gene (\u003cem\u003eIL-10\u003c/em\u003e) showed no statistically significant change. Inflammatory cytokine levels in cell culture supernatant (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F) aligned with qPCR findings. Both VB and VB@MOF/TA treatments effectively suppressed LPS-induced overexpression of pro-inflammatory genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E). Concurrently, the expression of the anti-inflammatory gene (IL-10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and the level of IL-10 in cell supernatant were significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). To further investigate the regulatory effects of VB and VB@MOF/TA on macrophage polarization, we assessed the expression of M1 markers (CD86) and M2 markers (CD206) using immunofluorescence staining and flow cytometry. Compared to the LPS group, both VB and VB@MOF/TA groups exhibited weaker fluorescence signals for the M1 marker (CD86) and stronger signals for the M2 marker (CD206) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, S20, and S21). Additionally, immunofluorescence staining and Western blot analysis confirmed that VB and VB@MOF/TA treatment resulted in decreased expression of iNOS (M1 marker) and increased expression of Arg-1 (M2 marker) (Figures S22 and S23). These findings indicated that VB and VB@MOF/TA effectively counteract LPS-induced oxidative stress and modulate the inflammatory microenvironment by regulating macrophage polarization. Notably, VB@MOF/TA demonstrated superior efficacy compared to VB alone in scavenging intracellular ROS under inflammatory conditions and promoting M2 macrophage polarization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranscriptomic Analysis of Molecular Mechanism\u003c/p\u003e\u003cp\u003eExcessive ROS not only directly damages cardiomyocytes but also promotes macrophage recruitment to injured areas, establishing a self-perpetuating \u0026ldquo;ROS-inflammation\u0026rdquo; feedback loop that exacerbates myocardial injury[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The intensity of macrophage-mediated inflammatory responses critically influences fibrotic scar formation during cardiac repair, ultimately determining ventricular remodeling and long-term cardiac function[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To elucidate the molecular mechanisms of the immunomodulatory effects of VB@MOF/TA, we performed transcriptomic analysis of LPS-induced RAW 264.7 cells treated with either PBS or VB@MOF/TA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Principal component analysis (PCA) revealed differentially expressed genes (DEGs) between the VB@MOF/TA group and the control group. DEGs analysis identified 2,786 significantly altered genes, including 1,370 upregulated genes and 1,416 downregulated genes (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Gene Ontology (GO) enrichment demonstrated these genes were predominantly involved in immune-related biological processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that VB@MOF/TA significantly modulated inflammatory signaling pathways, including the TNF pathway and the NF-κB pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Notably, the Gene Set Enrichment Analysis (GSEA) revealed profound alterations in metabolic pathways\u0026mdash;specifically glycolysis (enrichment score \u0026minus;\u0026thinsp;1.30) and tryptophan metabolism (enrichment score 1.79) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), providing a new perspective for future research on inflammatory response and mitochondrial metabolism. The findings above demonstrated that VB@MOF/TA likely exerts its immunomodulatory effects through the regulation of inflammatory signaling pathways and metabolic reprogramming, with the tryptophan metabolism pathway emerging as a potential novel therapeutic target for macrophage-associated inflammatory diseases. Collectively, these results provide crucial evidence for understanding the potential of VB@MOF/TA to modulate macrophage function, suppress inflammatory cascades, and consequently promote tissue repair.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTherapeutic effects of VB and VB@MOF/TA in mouse MI/RI model\u003c/p\u003e\u003cp\u003eBuilding upon VB@MOF/TA\u0026rsquo;s demonstrated capacity for targeted ROS scavenging, cryoprotection, and inflammation modulation \u003cem\u003ein vitro\u003c/em\u003e, we further investigated its therapeutic potential in myocardial ischemia/reperfusion (I/R) injury using an established murine model. MI/RI was applied with transient left anterior descending (LAD) occlusion for 45 min, followed by reperfusion for specified durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. VB and VB@MOF/TA were administered \u003cem\u003evia\u003c/em\u003e tail vein injection immediately before reperfusion. At 24 h post-reperfusion, serum levels of myocardial injury markers LDH and CK-MB in the I/R group were significantly elevated (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), indicating substantial cardiomyocyte damage. Following treatment with either VB or VB@MOF/TA, serum LDH and CK-MB levels were significantly reduced (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). 2,3,5-Triphenyltetrazolium chloride (TTC) staining revealed that MI/RI mice treated with VB or VB@MOF/TA exhibited significantly reduced infarct areas compared to the I/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, Tables S1 and S2). Cardiac function was assessed by measuring ejection fraction (EF) and fractional shortening (FS) \u003cem\u003evia\u003c/em\u003e transthoracic echocardiography from preoperative to endpoint observations (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). On postoperative day 1, the I/R group showed markedly decreased EF and FS values, while both the VB and VB@MOF/TA groups demonstrated improved cardiac parameters. Throughout the observation period, all groups showed progressive functional recovery. By postoperative day 28, the EF and FS of the VB@MOF/TA group increased by 23.30% \u0026plusmn; 3.19% and 13.66% \u0026plusmn; 1.76% respectively, compared to the I/R group. Further analysis revealed that these parameters were also 7.72% \u0026plusmn; 3.71% (EF) and 5.10% \u0026plusmn; 2.32% (FS) greater than with VB monotherapy. These findings were corroborated by left ventricular short-axis echocardiographic analysis (Figure S24, Tables S1 and S2). To investigate whether VB and VB@MOF/TA protect cells from damage through potential anti-apoptotic effects, we subsequently conducted experiments related to apoptosis. During I/R injury, the levels of pro-apoptotic proteins C-casp 3 and Bax were upregulated while Bcl-2 was downregulated (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-K). The administration of both VB and VB@MOF/TA downregulated the expression of cleaved-caspase 3 (C-casp 3) and Bax, while upregulating Bcl-2 levels. TUNEL staining revealed that VB and VB@MOF/TA treatment alleviated I/R injury-induced cardiomyocyte apoptosis, with the VB@MOF/TA group showing a 2.59-fold greater reduction in apoptosis rate compared to the VB group (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM). Quantitative analysis of both Western blot and TUNEL assay results demonstrated that VB@MOF/TA exhibited stronger anti-apoptotic capabilities than VB following I/R injury, confirming its superior advantages in reducing cardiac damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eVB@MOF /TA regulates the inflammatory microenvironment to promote cardiac repair\u003c/p\u003e\u003cp\u003eFollowing acute myocardial ischemia and hypoxia, Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes rapidly accumulate at the injury site, reaching peak abundance and constituting the dominant immune cell population responding to cardiomyocyte death[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These infiltrating Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes differentiate into M1 macrophages, which secrete pro-inflammatory cytokines, thereby driving the initial inflammatory response. Upon reperfusion therapy, cardiomyocytes subjected to ROS attack release multiple inflammatory factors that trigger inflammatory cascades. Subsequently, Ly6C\u003csup\u003ehigh\u003c/sup\u003e monocytes are recruited to the injured area and differentiate into M2 macrophages, which mitigate inflammation through the secretion of the anti-inflammatory cytokine IL-10 while promoting extracellular matrix remodeling and angiogenesis[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The persistent inflammatory response during early reperfusion promotes myocardial fibrosis and impairs cardiac tissue recovery post-injury[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Precise modulation of this inflammatory response represents a critical therapeutic target for optimizing tissue repair following myocardial reperfusion injury.\u003c/p\u003e\u003cp\u003eVB, a bioactive constituent of the medicinal plant \u003cem\u003ePlantago asiatica L.\u003c/em\u003e, exhibits well-documented anti-inflammatory pharmacological properties. To investigate whether VB could enhance cardiac tissue repair through modulation of inflammation, we examined the effects of VB and VB@MOF/TA on myocardial tissue. In our murine model, inflammatory responses peaked 24 h post-I/R[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], prompting an analysis of serum cytokine levels and macrophage marker expression in myocardial tissue at 24 h post-reperfusion. The I/R group exhibited significantly elevated serum levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), without a concurrent increase in the anti-inflammatory cytokine IL-10. Both VB and VB@MOF/TA treatments reversed this profile, reducing pro-inflammatory cytokines while elevating IL-10 levels to 3.8-fold (VB) and 5.1-fold (VB@MOF/TA) of the I/R group, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D). Immunofluorescence staining of cardiac specimens (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-G) demonstrated decreased CD86 and increased CD206 expression in treatment groups, with VB@MOF/TA showing more pronounced effects on macrophage subtype distribution. These findings confirm that both compounds promote M2 macrophage polarization, with VB@MOF/TA demonstrating superior efficacy to VB monotherapy in restoring inflammatory homeostasis.\u003c/p\u003e\u003cp\u003eAt day 28 post-reperfusion therapy, we histologically evaluated myocardial tissue recovery. Immunofluorescence staining for CD31 and α-smooth muscle actin (α-SMA) revealed that CD31\u003csup\u003e+\u003c/sup\u003e capillary density in the VB@MOF/TA and VB groups increased to 1.60-fold and 2.41-fold of the I/R group, respectively, and α-SMA\u003csup\u003e+\u003c/sup\u003e arteriole density reached 1.73-fold and 2.58-fold of the I/R group, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH-K). Wheat germ agglutinin (WGA) fluorescent staining demonstrated that both VB and VB@MOF/TA treatments significantly attenuated cardiomyocyte hypertrophy following I/R injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). Collectively, these findings indicated that the modulated inflammatory microenvironment promotes both angiogenesis and vascular preservation post-MI/RI, thereby enhancing myocardial tissue repair.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn vivo biosafety assessment\u003c/p\u003e\u003cp\u003eWe also conducted a series of biocompatibility evaluation of VB@MOF/TA. Our preliminary \u003cem\u003ein vitro\u003c/em\u003e studies demonstrated favorable biocompatibility of VB@MOF/TA. Critical \u003cem\u003ein vivo\u003c/em\u003e investigations, conducted through histological examination of major organs, revealed no detectable pathological alterations (Figure S25), indicating that neither VB nor VB@MOF/TA induced pulmonary, hepatic, splenic, or renal significant damage. Complementary hematological analysis of hepatic and renal function markers showed no statistically significant differences across all parameters (Figure S26), providing additional confirmation of its biosafety. This integrated dataset from multiple experimental approaches provides conclusive validation of the good biocompatibility and favorable safety characteristics of VB@MOF/TA for potential therapeutic applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo address the dual challenges of cardiomyocyte injury and pathological remodeling in acute myocardial infarction reperfusion therapy, we developed VB@MOF/TA, a mitochondria-targeted nanoformulation that simultaneously scavenges ROS and modulates the inflammatory microenvironment. This work demonstrates three key innovations: 1. A new targeting strategy. The TA-mediated mitochondrial targeting mechanism represents a breakthrough in organelle-specific drug delivery, enabling precise accumulation of VB@MOF/TA within cardiomyocyte mitochondria. 2. An advanced antioxidant/anti-inflammatory platform based on VB. Our pioneering MOF-based VB delivery system overcomes critical clinical limitations of free VB while enhancing antioxidant and anti-inflammatory efficacy. \u003cem\u003eIn vivo\u003c/em\u003e validation demonstrated that VB@MOF/TA improved the left ventricular ejection fraction by 23.30% \u0026plusmn; 3.19% compared to the I/R group and by 7.72% \u0026plusmn; 3.71% compared to VB monotherapy. 3. A potential anti-inflammatory pathway. Mechanistic studies revealed that VB@MOF/TA significantly modulates tryptophan metabolism, identifying this pathway as a potential new therapeutic target for regulating inflammation. Certainly, this study also has many limitations: the long-term biosafety, metabolism, and precise mechanisms underlying the anti-inflammatory effects of VB@MOF/TA require further investigation. Given that this system possesses the triple functions of combating oxidative stress, regulating mitochondrial quality, and modulating inflammatory responses, it holds considerable promise for treating oxidative stress-related diseases, such as diabetes-related complications, atherosclerosis, and Alzheimer's disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAMI\u003c/p\u003e\n \u003cp\u003eMI/RI\u003c/p\u003e\n \u003cp\u003eTA\u003c/p\u003e\n \u003cp\u003eMOF\u003c/p\u003e\n \u003cp\u003eVB\u003c/p\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003cp\u003emPTP\u003c/p\u003e\n \u003cp\u003eETC\u003c/p\u003e\n \u003cp\u003eγ-H2AX\u003c/p\u003e\n \u003cp\u003e2-MI\u003c/p\u003e\n \u003cp\u003eZn(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e·6H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003cp\u003eDPPH\u003c/p\u003e\n \u003cp\u003eABTS\u003c/p\u003e\n \u003cp\u003eLDH\u003c/p\u003e\n \u003cp\u003eCK-MB\u003c/p\u003e\n \u003cp\u003eALT\u003c/p\u003e\n \u003cp\u003eAST\u003c/p\u003e\n \u003cp\u003eBUN\u003c/p\u003e\n \u003cp\u003eCRE\u003c/p\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003cp\u003eDCFH-DA\u003c/p\u003e\n \u003cp\u003eFBS\u003c/p\u003e\n \u003cp\u003eATP\u003c/p\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003cp\u003eXRD\u003c/p\u003e\n \u003cp\u003eFTIR\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eUV-Vis\u003c/p\u003e\n \u003cp\u003eTMB\u003c/p\u003e\n \u003cp\u003e•OH\u003c/p\u003e\n \u003cp\u003e•O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eOGD/R\u003c/p\u003e\n \u003cp\u003eCalcein-AM/PI\u003c/p\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003cp\u003eSDS-PAGE\u003c/p\u003e\n \u003cp\u003ePVDF\u003c/p\u003e\n \u003cp\u003eTBST\u003c/p\u003e\n \u003cp\u003eECL\u003c/p\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003cp\u003eWGA\u003c/p\u003e\n \u003cp\u003eTTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcute myocardial infarction\u003c/p\u003e\n \u003cp\u003emyocardial ischemia/reperfusion injury\u003c/p\u003e\n \u003cp\u003etannic acid\u003c/p\u003e\n \u003cp\u003emetal-organic framework\u003c/p\u003e\n \u003cp\u003everbascoside\u003c/p\u003e\n \u003cp\u003ereactive oxygen species\u003c/p\u003e\n \u003cp\u003emitochondrial permeability transition pore\u003c/p\u003e\n \u003cp\u003eelectron transport chain\u003c/p\u003e\n \u003cp\u003ephospho-histone 2AX\u003c/p\u003e\n \u003cp\u003e2-methylimidazole\u003c/p\u003e\n \u003cp\u003eZinc nitrate hexahydrate\u003c/p\u003e\n \u003cp\u003e2,2-Diphenyl-1-picrylhydrazyl\u003c/p\u003e\n \u003cp\u003e2,2¢-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)\u003c/p\u003e\n \u003cp\u003elactate dehydrogenase\u003c/p\u003e\n \u003cp\u003ecreatine kinase-myocardial band\u003c/p\u003e\n \u003cp\u003ealanine aminotransferase\u003c/p\u003e\n \u003cp\u003easpartate aminotransferase\u003c/p\u003e\n \u003cp\u003eblood urea nitrogen\u003c/p\u003e\n \u003cp\u003ecreatinine\u003c/p\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e\n \u003cp\u003e4,6-diamidino-2-phenylindole\u003c/p\u003e\n \u003cp\u003e2¢,7¢-dichlorofluorescin diacetate\u003c/p\u003e\n \u003cp\u003eFetal bovine serum\u003c/p\u003e\n \u003cp\u003eAdenosine triphosphate\u003c/p\u003e\n \u003cp\u003escanning electron microscopy\u003c/p\u003e\n \u003cp\u003eX-ray diffraction\u003c/p\u003e\n \u003cp\u003eFourier-transform infrared spectroscopy\u003c/p\u003e\n \u003cp\u003eUltraviolet-visible\u003c/p\u003e\n \u003cp\u003e3,3¢,5,5¢-Tetramethylbenzidine\u003c/p\u003e\n \u003cp\u003eHydroxyl radical\u003c/p\u003e\n \u003cp\u003esuperoxide radical\u003c/p\u003e\n \u003cp\u003eoxygen–glucose deprivation and reperfusion\u003c/p\u003e\n \u003cp\u003ecalcein acetoxymethyl ester/propidium iodide\u003c/p\u003e\n \u003cp\u003eQuantitative real-time polymerase chain reaction\u003c/p\u003e\n \u003cp\u003esodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e\n \u003cp\u003ePolyvinylidene fluoride\u003c/p\u003e\n \u003cp\u003eTris-buffered saline/Tween 20\u003c/p\u003e\n \u003cp\u003eenhanced chemiluminescence\u003c/p\u003e\n \u003cp\u003ebovine serum albumin\u003c/p\u003e\n \u003cp\u003eWheat germ agglutinin\u003c/p\u003e\n \u003cp\u003e2,3,5-Triphenyltetrazolium chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were permitted by the animal ethics committee of Nanchang University (Nanchang, China, NCULAE-20250120001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are within the manuscript and its Supporting Information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declared that no conflict of interest existed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by The National Natural Science Foundation of China (82360105 to Yanhua Tang), The Natural Science Foundation of Jiangxi Province (20232ACB206001 to Yanhua Tang), The Key Research and Development Program of Jiangxi Province (20223BBG71010 to Yanhua Tang), The Clinical Trial Research Projects (2021efyA02 to Yanhua Tang), The Key Research and Development Program of Jiangxi Province (20212BBG73004 to Xiaolei Wang), The Jiangxi Province Key Laboratory of Bioengineering Drugs (No.2024SSY07061 to Xiaolei Wang) and The Interdiscipline Innovation Fund Project of Nanchang University (PYJX20230001 to Xiaolei Wang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX. Wang and Y. Tang guided the project. X. Wang, Y. Tang and C. Li conceived the idea and conceptualized the manuscript. C. Li, Z. Zhang, C. Luo, W. Lan, C. Liu, W. Liu, J. Yang, H. Xiang, Y. Tang and X. Wang participated in the design of experimental methods and sample analysis. C. Li completed the processing of experimental data and wrote the manuscript. X. Wang and Y. Tang reviewed and edited the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eG.A. Roth, G.A. Mensah, C.O. Johnson, G. Addolorato, E. Ammirati, L.M. Baddour, N.C. Barengo, A.Z. Beaton, E.J. Benjamin, C.P. Benziger, A. Bonny, M. Brauer, M. Brodmann, T.J. Cahill, J. Carapetis, A.L. Catapano, S.S. Chugh, L.T. Cooper, J. Coresh, M. Criqui, N. DeCleene, K.A. Eagle, S. Emmons-Bell, V.L. Feigin, J. 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Hu, Modulation of NLRP3 Inflammasome: Advantages of Chinese Herbal Medicine in Treating Myocardial Ischemia/Reperfusion Injury, Am J Chin Med 53(3) (2025) 737-769.\u003c/li\u003e\n \u003cli\u003eC. Peet, A. Ivetic, D.I. Bromage, A.M. Shah, Cardiac monocytes and macrophages after myocardial infarction, Cardiovasc Res 116(6) (2020) 1101-1112.\u003c/li\u003e\n \u003cli\u003eN.G. Frangogiannis, M.L. Entman, Chemokines in myocardial ischemia, Trends Cardiovasc Med 15(5) (2005) 163-9.\u003c/li\u003e\n \u003cli\u003eF.C. Sim\u0026otilde;es, P.R. Riley, Immune cells in cardiac repair and regeneration, Development 149(8) (2022).\u003c/li\u003e\n \u003cli\u003eF. Shahid, G.Y.H. Lip, E. Shantsila, Role of Monocytes in Heart Failure and Atrial Fibrillation, J Am Heart Assoc 7(3) (2018).\u003c/li\u003e\n \u003cli\u003eA.J. Mouton, K.Y. DeLeon-Pennell, O.J. Rivera Gonzalez, E.R. Flynn, T.C. Freeman, J.J. Saucerman, M.R. Garrett, Y. Ma, R. Harmancey, M.L. Lindsey, Mapping macrophage polarization over the myocardial infarction time continuum, Basic Res Cardiol 113(4) (2018) 26.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"myocardial ischemia/reperfusion injury, mitochondria-targeted nanozymes, reactive oxygen species, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-7445253/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7445253/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute myocardial infarction (AMI) remains the leading cause of mortality worldwide, posing a significant threat to global public health. Although revascularization strategies such as percutaneous coronary intervention represent the standard treatment for AMI, myocardial cell death caused by myocardial ischemia/reperfusion injury (MI/RI)significantly compromises clinical efficacy. The clinical application of anti-inflammatory and antioxidant therapeutic strategies for MI/RI is confronted with critical limitations due to poor targeting and low bioavailability. This study successfully constructed a new mitochondria-targeted nanozyme, VB@MOF/TA, in which tannic acid (TA) mediates specific mitochondrial targeting, and the metal-organic framework (MOF) serves as a carrier to synergistically enhance the antioxidant and anti-inflammatory effects of verbascoside (VB). Cellular experiments demonstrate that VB@MOF/TA co-localizes with mitochondria, exerts potent antioxidant effects, significantly suppresses oxygen-glucose deprivation/reoxygenation-induced cardiomyocyte apoptosis, and effectively modulates macrophage polarization. \u003cem\u003eIn vivo\u003c/em\u003estudies confirm that, compared with VB monotherapy, the VB@MOF/TA group exhibits a 2.59-fold reduction in apoptosis rate, a 7.72% ± 3.71% improvement in left ventricular ejection fraction, and a 2.50-fold increase in vascular density. These findings indicate that VB@MOF/TA significantly mitigates MI/RI and promotes myocardial tissue remodeling through its targeted antioxidant and synergistic anti-inflammatory mechanisms, highlighting its substantial clinical translational potential.\u003c/p\u003e","manuscriptTitle":"A mitochondria-targeted nanozyme for myocardial ischemia/reperfusion injury with synergistic antioxidant and anti-inflammatory properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 08:23:31","doi":"10.21203/rs.3.rs-7445253/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-16T09:42:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T01:04:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T03:57:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-11T13:10:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27020369422526550074230459113988111761","date":"2025-09-10T10:28:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305188631851368051492408407009274436221","date":"2025-09-09T01:48:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160434634858311627443884375574378159969","date":"2025-09-08T08:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125501440624053510686460838060577114212","date":"2025-09-08T03:58:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197015044999475828281132949939716290371","date":"2025-09-07T05:55:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201492233470600651278608505324787068707","date":"2025-09-07T00:50:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T12:52:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-25T04:56:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-25T04:56:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-08-24T09:17:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c445acf-09c2-4fc0-b8f7-65a6c550955f","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:04:30+00:00","versionOfRecord":{"articleIdentity":"rs-7445253","link":"https://doi.org/10.1186/s12951-025-03810-3","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-11-05 15:57:34","publishedOnDateReadable":"November 5th, 2025"},"versionCreatedAt":"2025-09-12 08:23:31","video":"","vorDoi":"10.1186/s12951-025-03810-3","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03810-3","workflowStages":[]},"version":"v1","identity":"rs-7445253","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7445253","identity":"rs-7445253","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}