Injectable hydrogel with miR-222-engineered extracellular vesicles ameliorates myocardial ischemic reperfusion injury via mechanotransduction

Abstract

Cardiac ischemic reperfusion injury (IRI) significantly exacerbates cardiac dysfunction and heart failure, causing high mortality. Despite the severity of IRI, effective therapeutic strategies remain elusive. Acellular cardiac patches have shown considerable efficacy in delivering therapeutics directly to cardiac tissues. Herein, we develop injectable GelMA (GEL) hydrogels with controlled mechanical properties. Targeting miR-222-engineered extracellular vesicles (TeEVs), tailored with cardiac-ischemia-targeting peptides (CTPs), are developed as ischemic TeEV therapeutics. These TeEVs are encapsulated within mechanical hydrogels to create injectable TeEV-loaded cardiac patches, enabling minimal invasiveness to attenuate IRI. The injectable patches facilitate the precise targeting of TeEVs for the efficient rescue of damaged cells. Persistent delivery of TeEVs into the infarcted region alleviates acute IRI and mitigated remodeling post IRI. This is linked to focal adhesion activation, cytoskeleton force enhancement, and nuclear force-sensing preservation. These findings may pave the way for force-sensing approaches to cardiac therapy using bioengineered therapeutic patches.

Keywords: IRI remodeling; force-sensing mechanotransduction; injectable hydrogel patch; miR-222-engineered EV; targeting cardiac peptide.

Graphical abstract

Figure 1

Generation and characterization of targeting miR-222-engineered EVs, mechanical hydrogels (GEL), and TeEV-loaded hydrogel patches (GEL-TeEV) (A) Representative TEM images of EVs modified by miR-222-engineered and cardiac-ischemia targeting peptides (CTsP) as miR-222-engineered EVs (eEVs) and targeting miR-222-engineered EVs (TeEVs) in both Mus musculus (mmu) and Rattus norvegicus (rno). (B) Nanoparticle size distribution of engineered EVs by NTA analysis. (C) Force-distance curves by AFM measurement. (D) Young’s modulus of GEL and GEL-TeEV. (E) Adhesive force of GEL and GEL-TeEV. (F) TeEV release rate in GEL at different time points. (G) Absorption ratio of mechanical hydrogels. (H) Degradability evaluated by weight loss of hydrogels. (I) Cell viability by PBS, GEL, TeEV, and GEL-TeEV treatment. Data present mean ± SD; n = 3 (F) or n = 6 per group. Unpaired Student’s t test (D–H) and one-way ANOVA test followed by Bonferroni test (I) were used for statistical analysis. ∗∗∗p < 0.001. See also Figure S1.

Figure 2

Targeting cell internalization and anti-apoptosis ability of GEL-TeEVs in OGDR-stressed NRCMs (A) Representative fluorescence images of PBS, miR-222-engineered EV (eEV), and targeting miR-222-engineered EV (TeEV) with or without OGDR-stressed overload. α-actinin: green; DiD: red; nuclei: blue. Scale bar: 50 μm. (B) Targeting uptake ability calculated by normalized fluorescence intensity. (C) Flow cytometry evaluation of PBS, eEV, and TeEV with or without OGDR-stressed overload. (D) Normalized fluorescence quantification by flow cytometry analysis. (E) Representative fluorescence images of Tunel staining in OGDR-stressed NRCMs. Tunel: green; α-actinin: red; nuclei: blue. The white arrows point to Tunel-positive cells. Scale bar: 100 μm. (F) Percentage of Tunel-positive cardiomyocytes. (G) WB estimation for apoptosis-related marker analysis of Bax, Bcl2, cleaved caspase-3, and caspase-3 in OGDR-stressed NRCMs. (H) Bax/Bcl2 and cleaved caspase-3/caspase-3 ratios from WB analysis in OGDR-stressed NRCMs. Data present mean ± SD; n = 6 per group. Two-way ANOVA test followed by Tukey post hoc test (B, D) and one-way ANOVA test followed by Bonferroni test (F, H) were used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2.

Figure 3

Myocardial targeting delivery and retention of TeEVs via pericardial hydrogel injection in vivo (A) Illustration of minimally invasive injection in pericardial cavity as a natural mold to form cardiac hydrogel patches in situ. (B) Fluorescent images of CTP-targeting engineered EVs (TeEVs) in DiD-labeled hearts. (C) Total radiant efficiency of DiD-labeled fluorescence in hearts. (D) Fluorescent images of the cross-sections of IRI hearts from base and mid to apex in vitro. (E) Representative fluorescence images of DiD-labeled eEVs and TeEVs with or without hydrogel patches to detect targeting delivery and retention ability of therapeutics in cardiac tissues. DiD: red; α-actinin: green; nuclei: blue. Scale bar: 50 μm. (F) Normalized IF efficiency of eEVs and TeEVs to detect internalization efficacy in vivo. (G) Representative in vivo fluorescence images of DiD-labeled TeEVs with or without hydrogel patches in mice at 24 h, 3 days, 7 days, 14 days, and 21 days (H) Total radiant efficiency of quantitative fluorescence intensity in TeEVs and GEL-TeEVs. (I) Normalized area under the radiant curves of total radiant efficiency. Data present mean ± SD; n = 6 (C–F) or n = 5 (H and I) per group. One-way ANOVA test followed by Bonferroni test (C, F) and unpaired Student’s t test (H, I) were used for statistical analysis. ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3.

Figure 4

GEL-TeEV patches alleviated myocardial acute IRI (A) Establishment of AIRI model after 30-min ischemia and then protected by GEL-TeEV patches followed by 24-h reperfusion in vivo. (B) Cardiac 2,3,5-triphenyl tetrazolium chloride (TTC) staining. (C) Detection of the infarction area/area at risk (INF/AAR) ratio and the area at risk/left ventricle weight (AAR/LV) ratio after AIRI. (D) Representative fluorescence images of Tunel staining in myocardial tissues. Tunel: green; α-actinin: red; nuclei: blue. The white arrows point to Tunel-positive cells. Scale bar: 100 μm. (E) Percentage of Tunel-positive cells. (F) WB estimation for apoptosis-related marker analysis of Bax, Bcl2, cleaved caspase-3, and caspase-3. (G) Bax/Bcl2 and cleaved caspase-3/caspase-3 ratios from WB analysis. (H) Lactate dehydrogenase (LDH) expression level. (I) Relative expression levels of inflammatory factors, including IL-1β, TNF-α, and IL-6. Data present mean ± SD; n = 6 per group. One-way ANOVA test followed by Bonferroni test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S4.

Figure 5

GEL-TeEV patches ameliorated cardiac dysfunction in cardiac IRI remodeling (A) Establishment of IRI remodeling model after 30-min ischemia and then protected by GEL-TeEV patches followed by 3-week reperfusion in vivo. (B) Representative echocardiography images of sham, IRI+PBS, IRI+GEL, IRI+TeEV, and IRI+GEL-TeEV groups. (C) Left ventricle ejection fraction (EF). (D) Fractional shortening (FS). (E) Masson staining. Scale bar: 100 μm. (F) Cardiac fibrosis detected by Masson staining. (G) WB analysis of fibrosis-related protein (α-SMA). (H) Quantitative protein expression level of α-SMA. (I) Col1a1 gene expression level quantified by qPCR analysis. (J) Col3a1 gene expression level quantified by qPCR analysis. (K) Relative expression level of pathological cardiac genes, including ANP, BNP, and β-MHC. Data present mean ± SD; n = 6 per group. One-way ANOVA test followed by Bonferroni test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S5.

Figure 6

Activation of GEL-TeEV patches on intracellular adhesion-cytoskeleton-nucleus mechanotransduction of myocardial cells in vivo (A) Representative fluorescence images of integrin (green). α-actinin: red; nuclei: blue. Scale bar: 100 μm. (B) WB analysis of vinculin to detect focal adhesion formation. (C) Quantitative vinculin expression level by WB analysis. (D) Representative fluorescence images of talin-1 (green). Scale bar: 100 μm. Magnified talin-1 images were shown in the second row. Scale bar: 50 μm. (E) Normalized myosin immunofluorescence (IF) intensity per cell. (F) Normalized Piezo1 IF intensity per cell. (G) Young’s modulus of cardiac tissues measured by AFM. (H) Adhesion force of cardiac tissues. (I) WB analysis of YAP. (J) Quantitative YAP expression level. (K) WB analysis of laminA/C to detect nuclear skeleton structures. (L) Quantitative laminA/C expression level. Data present mean ± SD; n = 6 per group. One-way ANOVA test followed by Bonferroni test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S6.

Figure 7

Enhanced nanomechanics by GEL-TeEV patches in NRCMs to induce cell focal adhesion formation and well-organized cytoskeleton structures for force-activated uptake of TeEVs (A) Representative fluorescence images of vinculin (green). Scale bar: 100 μm. (B) Normalized vinculin IF intensity per cell. (C) Normalized myosin IF intensity per cell. (D) Normalized Piezo1 IF intensity per cell. (E) Young’s modulus of NRCMs measured by AFM. (F) Adhesion force of NRCMs. (G) Representative fluorescence images of YAP (green). Nuclei: blue. Scale bar: 100 μm. (H) YAP nucleus/cytoplasm ratio of NRCMs. (I) Mechanical hydrogel patches were used to induce FA-coupling receptor formation, cytoskeletal nanomechanics, and nuclear mechanotransduction for mechanical signaling activation and to promote TeEV uptake for IRI repair. Data present mean ± SD; n = 6 per group. One-way ANOVA test followed by Bonferroni test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S7.