Intelligent responsive PVA hydrogel loaded with composite MXene nanozyme enables efficient myocardial infarction repair by disrupting the oxidative stress–inflammation cascade

Scheme 1. Schematic diagram of synthesis and action mechanism of 4-OI@MXene@Gel.

Scheme 2. Preparation process of the composition hydrogel.

Fig. 1. Physicochemical characterization of MXene and 4-OI-loaded MXene nanosheets (A) Representative AFM image and 3D surface morphology of MXene nanosheets. (B) Thickness measurement of MXene based on AFM analysis. Scale bar = 400 μm. (C) Representative TEM images and elemental mapping (C, O, Ti) of MXene and 4-OI@MXene. Scale bar = 200 μm. (D–E) Raman spectra of MXene and 4-OI@MXene. (F) FTIR spectra of MXene and 4-OI@MXene. (G) XPS survey spectra of 4-OI@MXene. (H–I) High-resolution XPS spectra of C 1s and O 1s for MXene and 4-OI@MXene.

Fig. 2. Physicochemical characterization of 4-OI@MXene@Gel composite hydrogel (A) Schematic illustration of the preparation process of the composite hydrogel. (B–D) Representative SEM images of Gel, MXene@Gel, and 4-OI@MXene@Gel. Scale bar = 20 μm. (E) Rheological analysis of the composite hydrogels. (F) Photographs demonstrating the appearance and injectability of the hydrogel. (G) Evaluation of the stretchability and adhesiveness of the hydrogel. (H) DPPH radical scavenging ability of 4-OI@MXene@Gel at different concentrations (n = 5). (I) ABTS radical scavenging ability of 4-OI@MXene@Gel at different concentrations (n = 5).

Fig. 3. In vitro evaluation of the biocompatibility, antioxidant capacity, and anti-apoptotic effects of the composite hydrogel (A) Relative viability of H9C2 cells after 24 h and 48 h of treatment with different concentrations of 4-OI@MXene@Gel. (B) Protective effects of various concentrations of 4-OI@MXene@Gel on H9C2 cells under H2O2-induced oxidative stress. (C) Live/dead staining of H9C2 cells treated with Control, Gel, MXene@Gel, and 4-OI@MXene@Gel at different time points (24 h, 48 h, and 72 h). Scale bar = 250 μm. (D) Phalloidin staining of the cytoskeleton to assess morphological changes in cells under different treatments. Scale bar = 100 μm. (E) Detection of intracellular ROS levels using DCFH-DA staining. Scale bar = 100 μm. (F) JC-1 staining to evaluate mitochondrial membrane potential. Scale bar = 150 μm. All treatment groups were conducted under H2O2 exposure (n = 3). All statistical comparisons were made versus the H2O2 group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 4. In vitro anti-inflammatory effects and immunomodulatory mechanism of 4-OI@MXene@Gel composite hydrogel (A) ELISA analysis of pro-inflammatory and anti-inflammatory cytokine levels in the culture supernatant of each treatment group. (B) Western blot detection of representative pro-inflammatory and anti-inflammatory protein expression. (C) Immunofluorescence staining of macrophage surface markers CD86 and CD206 to assess polarization status. Scale bar = 100 μm. (D) Western blot analysis of proteins related to oxidative stress signaling pathways. All treatment groups were conducted under H2O2 exposure (n = 3). All statistical comparisons were made versus the H2O2 group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 5. 4-OI@MXene@Gel improves cardiac function, reduces oxidative stress and apoptosis, and promotes myocardial repair (A) Schematic timeline of the experimental procedure. (B) Masson and H&E staining of heart tissue on day 28 to assess infarct size. (C) Echocardiographic evaluation of cardiac function parameters in each group. (D) DHE fluorescence staining to detect ROS levels in myocardial tissue. Scale bar = 100 μm. (E) Caspase-3 immunofluorescence staining to evaluate cardiomyocyte apoptosis. Scale bar = 50 μm. All statistical comparisons were made versus the Saline group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 6. Transcriptomic analysis and GO/KEGG functional enrichment results (A) Volcano plot showing differentially expressed genes between the 4-OI@MXene@Gel group and the saline group. (B) Heatmap displaying hierarchical clustering of gene expression profiles in both groups. (C) GO enrichment analysis indicating regulation of multiple biological processes. (D) KEGG pathway enrichment analysis revealing modulation of several key signaling pathways. (E–F) Western blot validation of representative proteins involved in relevant signaling pathways. All statistical comparisons were made versus the Saline group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 7. 4-OI@MXene@Gel modulates the post-infarction immune microenvironment (A) ELISA analysis of pro-inflammatory and anti-inflammatory cytokine levels in myocardial tissue from each group. (B) CD68/CD206 dual immunofluorescence staining showing macrophage infiltration and polarization phenotype. Scale bar = 100 μm, Zoom panle sacle bar = 30 μm (C) Western blot quantification of macrophage phenotype-related protein expression. All statistical comparisons were made versus the Saline group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 8. In vivo biosafety evaluation of 4-OI@MXene@Gel (A) Serum biochemical analysis of liver and kidney function markers in rats from each group. (B) H&E staining of major organs including lung, liver, spleen, and kidney, showing no significant histopathological abnormalities. Scale bar = 400 μm ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.