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

Scheme 1. Mitochondria-targeted nanomedicine VB@MOF/TA: Preparation and therapeutic mechanism in MI/RI. (A) The synthesis process of VB@MOF/TA. (B) Schematic of targeted therapy after circulatory administration. (C) Therapeutic mechanisms of mitochondria-targeted nanomedicine VB@MOF/TA for myocardial injury. The underlying mechanisms involve: (I) Mitochondrial ROS was reduced; (II) The opening of the mitochondrial permeability transition pore (mPTP) was inhibited, which restores mitochondrial membrane potential (ΔΨm), reduces the release of cytochrome c (Cyt c), and consequently attenuates cardiomyocyte apoptosis; (III) Restoration of mitochondrial bioenergetics, as ROS reduction alleviates suppression of the mitochondrial electron transport chain (ETC), thereby enhancing ATP production and maintaining energy supply to cardiomyocytes during reperfusion; (IV) Attenuation of nuclear DNA damage, whereby decreased cytoplasmic ROS levels reduce oxidative DNA damage in the nucleus, leading to downregulation of phospho-histone 2AX (γ-H2AX) protein expression; (V) Suppression of inflammatory signaling, as intracellular ROS depletion diminishes nuclear inflammatory complex formation and…

Fig. 1. Synthesis and characterization of VB@MOF/TA. (A) Schematic illustration of the synthesis for VB@MOF/TA. (B) TEM image of VB@MOF, scale bar: 200 nm. (C) TEM image of VB@MOF/TA, scale bar: 200 nm. (D) Zeta potentials of MOF, VB, VB@MOF, TA, and VB@MOF/TA measured by PALS, n ≥ 3. (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 1 s spectra of VB@MOF and VB@MOF/TA. (J) UV-Vis spectra of VB@MOF/TA, VB@MOF, MOF, and VB

Fig. 2. Multienzyme-mimicking antioxidant properties and free radical scavenging capability of VB@MOF/TA. (A) Schematic of the mechanism for ABTS•+ scavenging by VB@MOF/TA. (B) UV-Vis spectral changes during the scavenging of ABTS•+ by VB@MOF/TA. (C) Reaction kinetic curve of VB@MOF/TA scavenging ABTS•+. (D) Schematic of the mechanism for DPPH• scavenging by VB@MOF/TA. (E) UV-Vis spectral changes during the scavenging of DPPH• by VB@MOF/TA. (F) Reaction kinetic curve of VB@MOF/TA scavenging DPPH•. (G) Concentration-dependent SOD-like activities of VB@MOF/TA, VB@MOF, VB, and MOF. (H) CAT-like activities of VB@MOF/TA, VB@MOF, VB, and MOF. (I) POD-like activities of VB@MOF/TA, VB@MOF, VB, and MOF. (J) UV-Vis spectra showing the radical elimination activities of VB@MOF/TA for •O2−. (K) The ability to resist •O2− of VB@MOF/TA at different concentrations. (L) UV-Vis spectra showing the radical elimination 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. Data are represented as the mean ± standard deviation

Fig. 3. The representative confocal fluorescence images of mitochondria-targeting and restoration of mitochondrial function by antioxidant ability. (A) The representative fluorescence images of mitochondria-targeting of RhB@MOF. (B) The representative fluorescence images of mitochondria-targeting of RhB@MOF/TA. (C) Quantification analysis of the colocalization of the RhB@MOF and RhB@MOF/TA with mitochondria stained with Mito Tracker Green (n = 3, **p = 0.0080). (D) Quantification analysis of the colocalization of the RhB@MOF and RhB@MOF/TA with lysosome stained with Lyso Tracker Green (n = 3, ***p < 0.001). (E) Schematic diagram of the cell experiment. (F) Intracellular ROS detected by flow cytometry. (G) The level of cellular CAT (n = 3, **p (OGD/R, VB) = 0.0014, nsp (VB, VB@MOF) = 0.6357, *p (VB@MOF, VB@MOF/TA) = 0.0157, ***p (OGD/R, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0034). (H) The levels of cellular SOD (n = 3, ***p (OGD/R, VB) < 0.001, *p (VB, VB@MOF) = 0.0236, ***p (VB@MOF, VB@MOF/TA) < 0.001, ***p (OGD/R, VB@MOF/TA) < 0.001, ***p (VB, VB@MOF/TA) < 0.001). (I) Representative fluorescent images and quantification of JC-1 in different groups (n = 6, ***p (Control, OGD/R) <…

Fig. 4. Cytoprotective effect of VB and VB@MOF/TA. (A) LDH level (n = 6, ***p (Control, OGD/R) < 0.001, **p (OGD/R, VB) = 0.0064, ***p (OGD/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0223). (B) CK-MB level (n = 6, ***p (Control, OGD/R) < 0.001, **p (OGD/R, VB) = 0.0027, ***p (OGD/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0363). (C) The levels of cellular MDA (n = 6, ***p (Control, OGD/R) < 0.001, ***p (OGD/R, VB) < 0.001, ***p (OGD/R, VB@MOF/TA) < 0.001, ***p (VB, VB@MOF/TA) < 0.001). (D) Quantification of positive cells detected by the TUNEL cell apoptosis kit (n = 3, ***p (Control, OGD/R) < 0.001, ***p (OGD/R, VB) < 0.001, ***p (OGD/R, VB@MOF/TA) < 0.001, ***p (VB, VB@MOF/TA) < 0.001). (E) Representative fluorescent images of apoptotic cells accessed by the TUNEL assay. (F) Representative flow cytometry detection of cell apoptosis through Annexin V/PI assay and relevant quantification of apoptotic cells (n = 3, ***p (Control, OGD/R) < 0.001, ***p (OGD/R, VB) < 0.001, ***p (OGD/R, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.009). (G) Representative images of western blot and quantification of Bax, C-casp 3, Bcl-2 (n = 6, for Bax, ***p (Control, OGD/R) < 0.001, *p (OGD/R, VB) =…

Fig. 5. The inflammatory response was alleviated by VB and VB@MOF/TA through their modulation of the M1/M2 macrophage equilibrium. (A) Schematic illustration of the experiment. (B) Heatmap illustrating the gene expression levels of TNF-α, IL-6, IL-1β, and IL-10 in RAW 264.7 macrophages assessed by qPCR assay (n = 3). (C) Quantification of TNF-α, IL-6, IL-1β and IL-10 in RAW 264.7 macrophages assessed by ELISA after different treatments (n = 3, for TNF- α, ***p (Control, LPS) < 0.001, ***p (LPS, VB) < 0.001, ***p (LPS, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0012; for IL-6, ***p (Control, LPS) < 0.001, *p (LPS, VB) = 0.0142, ***p (LPS, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0050; for IL-1β, ***p (Control, LPS) < 0.001, **p (LPS, VB) = 0.0043, ***p (LPS, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0052; for IL-10, nsp (Control, LPS) > 0.9999, ***p (LPS, VB) = 0.0043, ***p (LPS, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0319). (D) 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

Fig. 6. Transcriptomic analysis of VB@MOF/TA anti-inflammatory mechanisms. (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) Gene set enrichment analysis (GSEA) for gene sets

Fig. 7. Therapeutic efficacy of VB and VB@MOF/TA on myocardial I/R injury. (A) Schematic illustration of the animal experiment. (B) Serum level of myocardial enzyme spectrum LDH (n = 6, ***p (Control, I/R) < 0.001, *p (I/R, VB) = 0.0268, ***p (I/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0408). (C) Serum level of myocardial enzyme spectrum CK-MB (n = 6, ***p (Control, I/R) < 0.001, *p (I/R, VB) = 0.0230, ***p (I/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0249). (D) Representative photographs of TTC staining (n = 3). (E) Representative images of transthoracic echocardiography. (F) quantitative analysis of cardiac function (n ≥ 3, comparison between I/R and I/R + VB, EF: ***p (1d) < 0.001, ***p (7d) < 0.001, **p (14d) = 0.0011, ***p (28d) < 0.001, FS: ***p (1d) < 0.001, *p (7d) = 0.0209, *p (14d) = 0.0217, **p (28d) = 0.0019; comparison between I/R + VB and I/R + VB@MOF/TA, EF: ###p (1d) < 0.001, nsp (7d) = 0.3084, ##p (14d) = 0.0068, #p (28d) < 0.0490, FS: ###p (1d) < 0.001, #p (7d) = 0.0251, #p (14d) = 0.0408, #p (28d) = 0.0181). (G) Representative images of western blot and quantification of Bax, C-casp 3, Bcl-2 (n = 6, for Bax, ***p (Control, I/R) < 0.001, *p (I/R, VB) = 0.0370,…

Fig. 8. Inflammatory regulation and angiogenesis of VB@MOF/TA in vivo. (A) Serum levels of pro-inflammatory cytokine TNF- α, IL-1 β, IL-6, and anti-inflammatory cytokine IL-10 detected by ELISA kit (n = 6, for TNF-α and IL-1 β, ***p (Control, I/R) < 0.001, ***p (I/R, VB) < 0.001, ***p (I/R, VB@MOF/TA) < 0.001, ***p (VB, VB@MOF/TA) < 0.001; for IL-6, ***p (Control, I/R) < 0.001, **p (I/R, VB) = 0.0029, ***p (I/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0433; for IL-10, nsp (Control, I/R) = 0.0567, ***p (I/R, VB) < 0.001, ***p (I/R, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0017). (B) Quantification analysis for CD86 (n = 3, CD86, ***p (Control, I/R) < 0.001, ***p (I/R, VB) < 0.001, ***p (I/R, VB@MOF/TA) < 0.001, *p (VB, VB@MOF/TA) = 0.0107). (C) Representative images of CD86 and CD206, scale bar: 50 μm. (D) Quantification analysis for CD206 (n = 3, CD206, *p (Control, I/R) = 0.0430, ***p (I/R, VB) < 0.001, ***p (I/R, VB@MOF/TA) < 0.001, **p (VB, VB@MOF/TA) = 0.0013). (E) Representative images and quantification analysis for CD31 (n = 4, ***p (Control, I/R) < 0.001, **p (I/R, VB) = 0.0015, ***p (I/R, VB@MOF/TA) < 0.001, ***p (VB, VB@MOF/TA) < 0.001), scale bar: 50 μm. (F)…