Injured Cardiac Tissue-Targeted Delivery of TGFβ1 siRNA by FAP Aptamer-Functionalized Extracellular Vesicles Promotes Cardiac Repair

Figures

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  Figure 1. Expression of FAP in injured cardiac tissues. (A) qRT-PCR analysis of FAP levels in human cardiac tissues; n = 6 per group. Data are normalized to GAPDH. (B and C) Representative blots and quantified data showing FAP levels in the indicated groups. GAPDH served as a loading control. Uncropped blots are shown in Figure S7. (D and E) Representative immunofluorescence images of FAP (red)‐ and DAPI (blue)‐stained cardiac tissue sections and quantified data showing FAP-positive areas (%). (F) qRT-PCR analysis of FAP levels in cardiac tissues of untreated (n = 4) and Ang II-treated mice (n = 8). Data are normalized to GAPDH. (G and H) Representative immunofluorescence images of FAP (red)‐ and DAPI (blue)‐stained cardiac tissue sections and quantified data showing FAP-positive areas (%). Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 2. Characterization of hEV@FAP and its toxicity in vitro. (A) Schematic illustration of hEV@FAP preparation by embedding a cholesterol-conjugated FAP aptamer onto the hEV. Cy5.5-labeled FAP aptamer was conjugated to visualize the surface modification of hEV@FAP. (B) Representative flow cytometric images of the modification efficiency of Cy5.5-labeled FAP aptamer with different concentrations on the hEV. (C) Transmission electron micrograph of hEV, hEV@Scr, and hEV@FAP. Scale bar = 100 nm. (D) Size distribution of hEV, hEV@Scr, and hEV@FAP. (E) Representative blots of TSG101, CD81, and GRP94 in three types of EVs. Uncropped blots are shown in Figure S7. (F) Zeta potential analysis of hEV, hEV@Scr, and hEV@FAP. (G and H) The analysis of cell viability and LDH release in HEK293 cells treated with hEV, hEV@Scr, or hEV@FAP.

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  Figure 3. In vitro and in vivo targeting selectivity of hEV@FAP. (A and B) Representative flow cytometric images and quantified data showing cellular uptake of Cy5.5 (red)-labeled hEV@Scr and hEV@FAP by HEK293 cells transfected with GFP-tagged NC vector or GFP-tagged FAP expression vector. (C and D) Representative immunofluorescence images and quantified data showing NC- or FAP-transfected HEK293 cells (green). Nuclei were stained with Hoechst 33342 (blue). Red fluorescence indicates Cy5.5-labeled aptamer-conjugated hEV. Scale bar = 50 μm. (E and F) Representative IVIS images and quantified data showing the fluorescence intensity in the cardiac tissues of Ang II-treated mice at 24 h after intravenous injection of PBS, hEV@Scr, or hEV@FAP; n = 5 per group. (G) Representative immunofluorescence images of PBS, hEV@Scr, and hEV@FAP accumulated in FAP-positive areas. Nuclei were stained with DAPI (blue); n = 5 per group. Scale bar = 20 μm. *P < 0.05, **P < 0.01.

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  Figure 4. Preparation and characterization of hEV@FAP-siTGFβ1. (A) Schematic illustration showing generation of siTGFβ1-loaded hEV@FAP. (B and C) Transmission electron micrograph (B) and size distribution (C) of hEV@FAP-siCtrl, hEV@Scr-siTGFβ1, and hEV@FAP-siTGFβ1. Scale bar = 100 nm. (D) Representative blots of TSG101, CD81, and GRP94 in three types of EVs. Uncropped blots are shown in Figure S7. (E) Zeta potential analysis of hEV@FAP-siCtrl, hEV@Scr-siTGFβ1, and hEV@FAP-siTGFβ1. (F) qRT-PCR analysis of TGFβ1 levels in the indicated groups. Data are normalized to GAPDH. (G and H) Representative IVIS images and quantified data showing the fluorescence intensity in the cardiac tissues of Ang II-treated mice at 24 h after intravenous injection of PBS, hEV@FAP-siCtrl, hEV@Scr-siTGFβ1, or hEV@FAP-siTGFβ1; n = 5 per group. *P < 0.05, **P < 0.01, ****P < 0.0001. FAP TF = FAP-transfected HEK293 cells.

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  Figure 5. Therapeutic effects of hEV@FAP-siTGFβ1 in vivo. (A) Schematic illustration of in vivo experimental procedure. (B) qRT-PCR analysis of TGFβ1 levels in the cardiac tissues of mice; n = 5 per group. Data are normalized to GAPDH. (C and D) Representative images of MT-stained cardiac sections at low (× 12.5) to high (× 200) magnification and quantified data showing fibrotic area (%); n = 5 per group. Scale bar = 1 mm (top row) and 50 μm (bottom row). (E and F) Representative immunofluorescence images of WGA-stained cardiac sections and quantified data showing CSA; n = 5 per group. Scale bar = 20 μm. (G and H) Representative M-mode images (G) and quantified data showing LVEF, LVFS, LVID;d, and LVID;s (H) in the indicated groups; n = 5 per group. (I and J) qRT-PCR analysis of collagen I, collagen III, ANP, and BNP levels in the indicated groups; n = 5 per group. Data are normalized to GAPDH. *indicates comparison with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #indicates comparison with the Ang II-treated group, #P < 0.05, ##P < 0.01, ####P < 0.0001.

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  Figure 6. Biosafety of hEV@FAP-siTGFβ1 in vivo. (A) Representative images of H&E-stained different organs (heart, liver, spleen, lung, and kidneys) at 24 h after intravenous injection of PBS, hEV@Scr, hEV@FAP, hEV@FAP-siCtrl, hEV@Scr-siTGFβ1, or hEV@FAP-siTGFβ1; n = 3 per group. Scale bar = 50 μm. (B–G) The levels of ALT, AST, ALB, ALP, BUN, and CRE in serum of mice at 24 h after intravenous injection of each EV sample; n = 3 per group.