Improved cardiac-specific delivery of RAGE siRNA within small extracellular vesicles engineered to express intense cardiac targeting peptide attenuates myocarditis

Figures

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  Figure 1. Characterization and cardiac-specific cellular uptake of C-sEVs without cytotoxicity in vitro (A) Schematic representation of sEVs targeting cardiac cells. (B) Schematic representation of targeting via CTP-expressing sEVs. (C–E) TEM images (scale bar, 100 nm) (C), size distribution (D), and zeta potential (E) of sEVs. (F) Western blot analysis of FLAG and GAPDH from stable cell lines as well as FLAG, LAMP2, Alix, and CD81 in sEVs. CTP expression levels were indirectly quantified via the detection of FLAG expression. (G) Cell viability was measured through an MTS assay. (H) Expression of FLAG-LAMP2b in H9C2 cells. (I) Fluorescence microscopy images of PKH26-labeled (red) sEVs in H9C2 cells. Values are expressed as mean ± SEM. ∗p < 0.05 (compared to N-sEVs).

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  Figure 2. Characterization and effects of siRAGE-loaded C-sEVs in a cellular model of inflammation (A) Schematic representation of siRNA-loaded sEVs. (B) TEM images of siRAGE-loaded sEVs. H9C2 cells were treated with siRNA-loaded sEVs for 48 h. (C) Measurement of H9C2 cell viability via MTS assays after treatment with siRAGE-loaded sEVs. (D) Fluorescence microscopy images of NC-siRNA-loaded (green) and PKH26-labeled (red) sEVs in H9C2 cells, with cell nuclei (blue). (E) Western blot and (F) qRT-PCR analyses of H9C2 cells treated with siRAGE-loaded sEVs. H9C2 cells were treated with siRAGE-loaded sEVs for 48 h before inducing inflammation via TNF-α treatment for 24 h. (G) Inflammation was monitored through detection of IL-6 levels in culture medium. TF, transfection. Values are expressed as mean ± SEM. ∗p < 0.05 (compared to control); †p < 0.05 (compared to N-sEVs). See also Figure S2.

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  Figure 3. In vivo cardiac targeting of C-sEV U without cytotoxicity (A) H&E staining of the heart, liver, lung, kidney, and spleen 2 days after sEV treatment. (B) Representative near-infrared fluorescence (NIRF) images (overlaid on photograph) of hearts from rats administered PBS or PKH26-labeled sEVs. (C) Quantification of fluorescence intensity in the heart. (D) Representative NIRF images (overlaid on photograph) of rat organs. (E) Quantification of fluorescence intensity in the liver, lung, kidney, and spleen. (F and G) Western blot analysis (F) and qRT-PCR detection (G) of RAGE in heart tissue from rats treated with siRAGE-loaded sEVs. (H) qRT-PCR of RAGE mRNA in the liver, lung, kidney, and spleen. Values are expressed as the mean ± SEM. ∗p < 0.05 (compared to PBS); †p < 0.05 (compared to N-sEVs). See also Figure S3.

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  Figure 4. Effects of siRAGE-loaded C-sEV U in a rat EAM model To induce myocarditis, rats were immunized with purified cardiac myosin derived from porcine ventricular muscle. On days 0 and 7, each rat was subcutaneously injected with an emulsion (0.2 mL) containing cardiac myosin (1.0 mg) in the footpads. On day 7, siRAGE-loaded N-sEVs, C-sEV U, or PBS (control group) was administered. (A) Representative H&E-stained sections of hearts from all four groups. (B) Representative M-mode echocardiograms and comparison of LVEF, LVEDD, and LVESD. (C) Western blot analysis of RAGE, IL-6, TNF- α, COX2, HMGB1, p-p65, p65, and GAPDH. Values are expressed as mean ± SEM. ∗p < 0.05 (compared to normal); †p < 0.05 (compared to N-sEVs).