Melatonin Engineered Adipose-Derived Biomimetic Nanovesicles Regulate Mitochondrial Functions and Promote Myocardial Repair in Myocardial Infarction

Figures

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  Figure 1. Characterization of ADSCs and ADSC-derived biomimetic nanovesicles. (A) The morphology of ADSCs by using a microscope. Scale bar = 200 μm. (B–D) Osteogenic, adipogenic, and chondrogenic differentiation of ADSCs. Scale bar = 200 μm. (E) Analysis of flow cytometry indicating surface markers of ADSCs. (F) The characteristic image of ADSC-derived biomimetic nanovesicles under TEM. Scale bar = 100 nm. (G) Western blot analysis of nanovesicles' positive markers including CD9, CD63, CD81, Hsp70, and TSG101. Calnexin was used as the negative control. (H,I) Size distribution and zeta potential of nanovesicles.

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  Figure 2. Preparation and characterization of Mel@NVs. (A) The characteristic images of NVs and Mel@NVs via TEM. Scale bar = 100 nm. (B,C) Size distribution and zeta potential of Mel@NVs. (D) HPLC analysis of loading efficiency. A different amount of melatonin was added into NVs (10 μg). The loading was sonicated when 40 μg melatonin was added. The results represented the mean ± standard deviation (n = 3).

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  Figure 3. Cellular uptake and cardioprotective effects of Mel@NVs in H9C2 cells. (A) Representative images of NVs and Mel@NVs taken up by H9C2 cells in control and H/SD conditions. (B) The apoptotic rate of annexin-V/PI staining in different conditions by using flow cytometric detection. (C) The analysis of the quantity of apoptotic and necrotic cells by flow cytometry (**p < 0.01). (D) Detection of apoptosis-related proteins using Western blotting test. (E) The amount of anti-inflammatory and pro-inflammatory cytokines using ELISA in H9C2 cells with different treatments (*p < 0.05; **p < 0.01; ns, not significant. n = 3).

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  Figure 4. The effect of Mel@NVs on reactive oxygen species levels in H/SD condition. (A–D) The ROS levels in cells by using DHE probe and the images were obtained using fluorescence microscopy. Scale bar = 200 μm. (E) The quantification of the DHE fluorescence intensity is shown in a bar graph (*p < 0.05 v.s. control; #p < 0.05 v.s. H/SD treatment; &p < 0.05 v.s. NVs treatment). (F–I) The oxygen species levels in mitochondria were observed by using a Mito-SOX probe and the images were obtained using confocal laser scanning microscopy. Scale bar = 25 μm. (J) The quantification of Mito-SOX fluorescence intensity is detected in different assays (*p < 0.05 v.s. control; #p < 0.05 v.s. H/SD treatment; &p < 0.05 v.s. NVs treatment). The results represented the mean ± standard deviation (n = 3).

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  Figure 5. Apoptotic effect and cardiac function at 4 weeks after MI treatment in mice. (A) Representative fluorescence images of TUNEL-stained myocardial sections. The images showed TUNEL-positive cells (green color), myocardium stained with monoclonal antibody cTnI (red color), and total nuclei stained with DAPI (blue color). Scale bar = 20 μm. (B) M-mode echocardiography for heart function evaluation. (C) The quantification of representative TUNEL-positive apoptotic cells (**p < 0.01). The results represented the mean ± standard deviation (n = 5). (D) The ejection fraction rate (EF%) standing for heart function is presented as a bar graph (**p < 0.01). The results expressed the mean ± standard deviation (n = 5). (E) The fractional shortening (FS%) standing for heart function is represented as a bar graph (*p < 0.05; **p < 0.01). The results represented the mean ± standard deviation (n = 5).

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  Figure 6. Histological evaluation of the effect of Mel@NVs in MI model at 4 weeks after MI in mice. (A) H&E staining in different groups. (B) Masson's trichrome staining in different groups. (C) Representative immunohistochemical dyed for CD31-positive cells in different groups (red color), and DAPI (blue color). (D) Quantitative analysis of micro-vessels in different groups (**p < 0.01). The results represented the mean ± standard deviation (n = 5).