Ameliorating post-infarction myocardial fibrosis and cardiac function via ROS-responsive hydrogel-mediated IL-11 antibody delivery

Figures

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  Fig. 1. Schematic of the formation and therapeutic mechanism of the ROS-degradable PVA-TSPBA@MAB hydrogel for treating myocardial infarction (MI).

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  Fig. 2. The expression of interleukin-11 (IL-11) is increased in myocardial fibrosis in vitro and in vivo. A. Western blot analysis of IL-11 and vimentin expression in angiotensin II (AngII, 100 nM) or TGF-β (10 ng/mL)-treated cardiac fibroblasts. B. Quantification of (A). C. TUNEL staining and (D) its quantitative analysis in treated cells. E. CCK-8 viability assays (n = 3). F. Circulating IL-11 levels in mouse blood measured by ELISA (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Fig. 3. Characterization of reactive oxygen species (ROS) sensitive hydrogels. A. Schematic of the ROS-responsive Gel@VHH structure. B. A 9 % PVA – 3 % TSPBA mixture exhibits rapid sol–gel transition, forming PVA-TSPBA hydrogel. C. Injectability and macroscopic morphology of PVA-TSPBA@MAB hydrogel.D. Time-dependent swelling profile (n = 3). E. Compressive stress–strain behavior. F. Rheological frequency sweeps showing storage (G′) and loss (G″) moduli. G. Degradation of hydrogels in PBS versus 100 μM H2O2. H. Live/dead staining of AC16 cells after 1, 3, and 7 days of culture with P-T@MAB. Scale bar = 100 μm. I. Quantitative viability based on (H). J. ROS concentration-dependent gel disassembly. (K) Degradation kinetics in PBS and H2O2 (n = 3).

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  Fig. 4. Assessment of intrapericardial MAB delivery and P-T@MAB combination therapy in mice. A. Experimental timeline of animal studies. B. In vivo cardiac IVIS imaging following intrapericardial injection of free MAB or P-T@MAB at indicated time points (0 h, 24 h, 2 d, 4 d; n = 3). C. Quantification of cardiac MAB fluorescence. D–I. Serum levels of IL-11, TIMP-1, MMP-2, MMP-9, CCL8, and CXCL2 measured by ELISA (n = 3). n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Fig. 5. Western blot and immunofluorescence analysis of myocardial fibrosis markers in myocardial infarction (MI) mice. A. Western blot analysis for interleukin-11 (IL-11) and vimentin expression in heart tissue of MI mice after treatment with phosphate buffered saline, Gel, MAB, and P-T@MAB and sham group. B-C. Statistical evaluation of IL-11 and vimentin expression. D. Western blot of phosphorylated extracellular signal-regulated kinase (ERK) in heart tissues from MI mice. E.Quantification of (D) (n = 3). F–G. Immunofluorescence staining of α-SMA, collagen Ⅰ, and collagen Ⅲ in scar tissue (H) with corresponding quantitative analysis. Scale bar: 100 μm (n = 5). n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Fig. 6. P-T@MAB injection promotes angiomyogenesis. Immunostaining images show TUNEL at 3 days post-injection (A), Ki67 (C), vWF (E), and CD31 (F) expression in heart sections at 4 weeks post-injection. Quantitative analysis of TUNEL (B), Ki67 (D), vWF (G), and CD31 (G) is presented (n = 5). Scale bars: 100 μm. n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Fig. 7. Functional benefits of P-T@MAB therapy in myocardial infarction (MI) mice. A. Representative Masson's trichrome-stained myocardial sections 4 weeks after treatment. B. Quantification of viable myocardium. C. Echocardiographic left ventricular end-diastolic diameter. D. LV ejection fraction (LVEF) and (E) fractional shortening (LVFS) after 4 weeks of treatment (n = 5). (n = 5, Scale bars, 100 μm). ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Fig. 8. RNA-seq analysis explored the mechanism of MAB delivered via hydrogel. A. Heatmap of gene expression across conditions (three biological replicates per group. B–C. Volcano plots of differentially expressed genes (DEGs) between P-T@MAB vs.PBS and MAB vs.PBS groups. D–E. KEGG and Gene Ontology analysis of DEGs in P-T@MAB vs. Gel. F–K. Gene Set Enrichment Analysis showing pathways including Immune Response, Extracellular Region, Immune System Process, Collagen-Containing ECM, PPAR, and AMPK Signaling in P-T@MAB vs. Gel.