Injection of ROS-Responsive Hydrogel Loaded with IL-1β-targeted nanobody for ameliorating myocardial infarction

Fig. 1. Schematic illustration of the formation and mechanism of the reactive oxygen species (ROS) degradable PVA-TSPBA@VHH hydrogel for efficient myocardial infarction (MI) treatment.

Fig. 2. Characterization of ROS sensitive hydrogels. (A) The schematic illustrates the composition of the ROS-Responsive Gel@VHH; (B) 9 % (w/v) polyvinyl alcohol (PVA) −3% (w/v) TSPBA complexes shows a rapid sol–gel transition, generating PVA-TSPBA hydrogels; (C) The FTIR characterization of PVA, TSPBA, and P-T hydrogel; (D) The morphology of hydrogel; (E)Rheological frequency sweep of storage (G′) and loss (G″) moduli of the hydrogels; (F) Compression stress strain curves of the hydrogels; (G) Swelling ratio of the hydrogels (n = 3, mean ± SD).(H) ROS-triggered gel disassembly. PVA-TSPBA gel were incubated with ROS at different concentrations; (I) Degradation pattern of the ROS-sensitive hydrogels when immersing in PBS and 100 μM H2O2 solution (n = 3, mean ± SD); (J)The release profile of VHH from hydrogels under pH 7.4 PBS and H2O2 solution.

Fig. 3. Intrapericardial injection of Gel-VHH and cardiac retention. (A) Timeline of animal studies; (B) Ex vivo IVIS imaging of hearts after intrapericardial injection of VHH alone or Gel-VHH at baseline, 2 d and 4 d, n = 3; (C) Quantification of fluorescence intensities of VHH in the hearts; (D) Confocal fluorescence microscopy images showing released VHH into the myocardiμm. Scale bar, 50 μm. White arrows indicate the approximity of released VHH to cardiomyocytes. ∗∗∗∗ indicates p < 0.0001.

Fig. 4. Gel-VHH injection promotes angiomyogenesis. Immunostaining of (A)TUNEL on day7 (n = 5), (B) Ki67, and (C) CD31 staining of heart sections 4 weeks after injection. Scale bars, 100 μm. (D–F) Quantitative data corresponding to TUNEL, ki67, CD31 staining (n = 5). ∗∗∗ and ∗∗∗∗ indicates p < 0.005 and p < 0.0001, respectively.

Fig. 5. Functional benefits of Gel- VHH therapy in MI mice. (A) Representative Masson's stained myocardial sections 4 weeks after treatment; (B) Quantitative analyses of viable myocardiμm from the Masson's images; (C) Left ventricular ejection fractions (LVEFs) and (F) fractional shortening (LVFS) of mouse 4 weeks after treatment (n = 5); (E) Co-immunostaining of pan-macrophage cell surface marker CD68 (red), and the M2 marker CD206 (green) were merged with nuclear staining Hoechst(blue). Scale bars, 100 μm ∗∗ and ∗∗∗∗ indicate p < 0.01 and p < 0.0001, respectively.

Fig. 6. Gel-VHH injection promotes angiomyogenesis. Immunostaining of (A)TUNEL on day7(n = 5), (B) Ki67, and (C) CD31 staining of heart sections 4 weeks after injection. Scale bars, 100 μm. (D–F) Quantitative data corresponding to TUNEL, ki67, CD31 staining (n = 5). ∗∗, ∗∗∗ and ∗∗∗∗ indicates p < 0.001, p < 0.005 and p < 0.0001, respectively.

Fig. 7. Functional benefits of Gel- VHH therapy in MI rats. (A) Representative Masson's trichrome-stained myocardial sections 4 weeks after treatment. (B) Quantitative analyses of viable myocardiμm from the Masson's trichrome images. (C) Left ventricular ejection fractions (LVEFs) and (D) fractional shortening (LVFS) of rat 4 weeks after treatment (n = 5). (E–F) Co-immunostaining of pan-macrophage cell surface marker CD68 (red), and the M2 marker CD206 (green) were merged with nuclear staining Hoechst(blue). Scale bars, 100 μm ∗, ∗∗ and ∗∗∗∗ indicate p > 0.05, p < 0.01 and p < 0.0001, respectively.

Fig. 8. Transcriptome sequencing analysis of VHH treated with TSPBA-PVA gel. (A) Volcano plot analyses of total differentially expressed genes (DEGs) between the Gel treatment group and the Gel-VHH group. Green and orange dots represent down-regulated and up-regulated genes. Grey dots represent the genes not statistically different. (B) KEGG analysis of DEGs. (C) GO analysis of DEGs. (D) Bubble plot of KEGG analyses in the “MAPK signaling” pathway. (E) Bubble plot of GO term. Clustering heat map of genes associated with (F)MAPK signaling pathway, (G) Cardiac muscle contraction and (F) Insulin resistance. h) Clustering heat map of genes associated with ABC transporters. (I) KEGG chord diagram in the “MAPK signaling” and “Cardiac muscle contraction” pathway. (J) Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) of some important genes.