Platelet-Membrane-Encapsulated Carvedilol with Improved Targeting Ability for Relieving Myocardial Ischemia–Reperfusion Injury

Figures

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  Figure 1. Schematic diagram of PMVs@Carvedilol fabrication and its targeted therapy towards myocardial ischemia–reperfusion injury.

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  Figure 2. Characterization of PLT and PMVs in vitro. (A) SEM image of PLT. a—Scale bar, 1.0 μm; b—Scale bar, 100 nm. (B) A transmission electron micrograph of platelet membrane vesicles (PMVs). Scale bar, 100 nm. (C) a,b—Size distribution of PLT and PMVs by nanoparticle tracking analysis. (D) Western blot analysis revealed the expression of platelet-specific markers including CD41 in platelets, PMVs. (E) Quantitative analysis of the membrane protein CD41. (F) CD62P and GAPDH expression levels of PLT, PLT (active), and PMVs (PLTs were activated with 1% CaCl2 for 2 h). (G) Quantitative analysis of CD62P and GAPDH. * p < 0.05; ** p < 0.01; *** p < 0.001; ns > 0.05.

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  Figure 3. Verification of PMV targeting in vivo. (A) A representative in vitro fluorescence image showing intravenous Dil-labeled PMV binding in hearts with or without MIRI. (B,C) Representative fluorescent image showing the targeting of Dil-labeled PMVs (red) to the MIRI area and quantitative fluorescence analysis. (D,E) Retention time and fluorescence quantification of fluorescent PMVs in the MIRI rat heart. * p < 0.05; ** p < 0.01; *** p < 0.001; ns > 0.05.

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  Figure 4. Determination of optimal drug encapsulation and loading rate for PMVs@Carvedilol and validation of toxicity in vivo. (A) Comparison of the maximum absorption of Carvedilol at 285 nm, 319 nm, 331 nm. (B) Comparison of the encapsulation rate at different concentrations and volume ratios. (C) Comparison of the drug loading rate at different concentrations and volume ratios. (D) Changes in the encapsulation concentration of PMVs@Carvedilol at different time points (0 h, 2 h, 6 h, 8 h, 12 h, 24 h). (n = 3) (E) Analysis of various blood parameters of normal rats at different time periods after tail vein injection of PMVs@Carvedilol. * p < 0.05; ** p < 0.01; **** p < 0.0001.

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  Figure 5. PMVs encapsulation reduces infarction area and boosts therapeutic outcomes in rats with MIRI. (A) A schematic showing the animal study design. (B) Representative TTC-stained myocardial sections 24 h after treatment (red fluorescence, scar tissue; red, viable myocardium). Scale bar, 2 mm. (C) Quantitative analyses of viable myocardium and scar size from the TTC staining images. (i.p. means intraperitoneal injection; i.v. means intravenous injection.) * p < 0.05; ** p < 0.01; ns > 0.05.

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  Figure 6. Detection of ultrasound cardiogram in rats from different groups. (A) Echocardiography in SD rats 24 h and 1 week after MIRI. (B) Quantitative analyses of 24 h heart LVEF% and LVFS%. (C) Quantitative analyses of 1 week heart LVEF% and LVFS%. (D) Quantitative analyses of 24 h heart LVDd and LVDs. (E) Quantitative analyses of 1w heart LVDd and LVDs. (i.p. means intraperitoneal injection; i.v. means intravenous injection.) * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 7. PMVs@Carvedilol therapy reduces myocyte cell apoptosis. (A) Expression of antiapoptotic molecule Bcl2 under immunofluorescence. Scale bar, 600 μm; 20× Scale bar, 150 μm. (B) Quantitative analyses of antiapoptotic molecule Bcl2. (C) The expression of apoptotic molecules Bax under immunofluorescence. Scale bar, 600 μm; 20× Scale bar,150 μm. (D) Quantitative analyses of apoptotic molecule Bax. (i.p. means intraperitoneal injection; i.v. means intravenous injection.) ** p < 0.01; *** p < 0.001; **** p < 0.0001.