Precision targeting of FDX1-mediated cuproptosis by a ROS-responsive hydrogel for myocardial ischemia-reperfusion injury treatment

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  Scheme 1. Injectable hydrogel OD@G4CAsi-FDX1 releases CA (ROS scavenger) and si-FDX1 (cuproptosis inhibitor) upon ROS-triggered degradation, synergistically repairing MIRI.

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  Figure 1. Copper dysregulation and FDX1-driven mitochondrial damage in MIRI. A) DEGs in the IRI model compared to the sham operation group were shown by volcano plot (Red dots: upregulated genes, green dots: downregulated genes). B) GSEA demonstrating enrichment of the “response to copper ions” gene set in the ranked list of DEGs from (A). C) Total Cu concentration in serum and in the heart tissue from the sham operation and IRI groups (n = 30). D) Representative Cu2+ (red) staining of NRCMs from different groups (scale bar: 200 µm). Flow cytometric profile and quantitative results (n = 6). E) Representative Cu+ (green) staining of NRCMs from different groups (scale bar: 40 µm). Flow cytometric profile and quantitative results (n ≥ 6). F, G) Protein levels of FDX1 (14 kD), DLAT oligomers (250 kD) and DLAT (70 kD) in NRCMs after different treatments. The left panels are WB bands and the right panels are quantitative data (n = 6). H) Activity of α-KGDH and PDH in NRCMs after different treatments. I) Concentrations of α-KG and PA in NRCMs after different treatments. J) Cell viability was measured with the CCK-8 assay from different groups. K) Concentration of cTNT in NRCMs was detected from…

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  Figure 2. Synthesis and characterization of ROS-responsive hydrogel. A) Schematic design of OD@G4CAsi-FDX1 hydrogel. Integration of ROS-cleavable Schiff base bonds (enabling ROS-triggered degradation) and G4CAsi-FDX1 NPs (providing dual drug/gene delivery). B) UV/Vis spectra validating component integration. C) Agarose gel electrophoresis of si-FDX1 complexation efficiency (G4CA:si-FDX1 w:w ratios, from 0.2:1 to 5:1). D) Hydrodynamic size distribution of G4CAsi-FDX1 NPs. E) TEM images of G4CAsi-FDX1 NPs (scale bar: 100 nm and 50 nm). F) Confocal images showing intracellular trafficking of G4CAsi-FDX1 NPs in NRCMs after 1, 4, 12, 24 h of incubation (scale bar: 40 μm and 20 μm) and co-localization quantification via Pearson's correlation coefficient (n = 3). Late endosomes and lysosomes were stained with LysoTracker (green), while nuclei were stained with DAPI (blue). G) Cell viability of NRCMs treated by G4CAsi-FDX1 NPs with different concentration. H) Injectability of the hydrogel. I) Representative SEM image of the freeze-dried hydrogel (scale bar: 20 μm). J) Self-healing property of the hydrogel. K) Macroscopic observation of the ROS responsiveness of the hydrogel. L) Kinetic degradation…

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  Figure 3. Antioxidant and anti-inflammatory effects of G4CAsi-FDX1 NPs. A) Intracellular ROS levels in NRCMs assessed by ROS probe (green) staining (scale bar: 100 μm and 200 μm) and ROS level quantification in NRCMs with different treatments (n = 6). B) ABTS radical scavenging activity of the various concentration of G4CAsi-FDX1 NPs (n = 3). C) JC-10-based mitochondrial membrane potential assay (scale bar: 100 μm and 300 μm). Red and green fluorescence indicate J-aggregates (polarized) and monomers (depolarized), respectively. Red/green ratio quantification (right, n = 6). qRT-PCR analysis of D) Nfe2l2 (NRF2), Sod1 (SOD1), and Hmox1 (HO1) mRNA levels, and E) Tnf (TNF-α) and Il1b (IL1β) mRNA levels in NRCMs under the indicated treatments as labeled on the figure (n = 3). F) Illustration of cuproptosis mechanism and G4CAsi-FDX1 NPs' targeting action on FDX1 inhibition (via siRNA-mediated silencing) and ROS scavenging (via CA).

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  Figure 4. Hydrogel's Release Characteristics and Molecular Effects in MIRI Model. A) Schematic of the mouse I/R model operation flowchart. B) In vivo retention of the ICG-labeled hydrogel in mouse myocardium at 0, 1, 3, 24 and 72 h post injection (left). Quantification of hydrogel retention (n = 3, fluorescence intensity normalized to 0 h, right). C) Release kinetics of Cy5-labeled NPs at 0, 0.5, 3, 24 and 72 h post administration (left). Relative drug release profile (n = 3, right). D) Protein levels of FDX1 in heart tissue after different treatments. The left panels are WB bands and the right panels are quantitative data (n = 3). E) qRT-PCR analysis of Fdx1 (FDX1) mRNA level expression (n = 9). F) Immunohistochemistry of FDX1 protein expression in heart sections (scale bar: 100 μm and 50 μm). Quantitative analysis of Average Optical Density (AOD, right, n = 9). AOD was defined as the total optical density divided by the positive staining area. For quantitative data (D-F), data are mean ± s.e.m. Statistical significance is indicated by p < 0.05. P values were determined using one-way ANOVA with Tukey's post hoc analysis.

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  Figure 5. Therapeutic Efficacy of ROS-responsive Hydrogel in a Mouse MIRI Model. A, B) Echocardiography and measured EF%, FS%, CO%, SV% (n = 12). C) The left panels are representative longitudinal 2D B-mode images and strain traces across different groups. The right panels are quantitative result of GLS (n = 6). D) Representative images of heart sections stained with TTC. E) Heart weight to tibia length ratio (HW/TL). F) Serum levels of LDH and CK-MB in mice at day 1 postinjection (n = 8). G, H) Representative images of HE staining of the heart tissues from mice at day 1 postinjection (scale bar: 100 μm and 50 μm) and quantitative analysis of inflammatory cell infiltration (n = 3). I, J) Representative images of Masson's staining (scale bar: 1mm) and measured fibrosis area% (n = 3).