Lipidomics and biodistribution of extracellular vesicles‐secreted by hepatocytes from Zucker lean and fatty rats

Figures

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  FIGURE 1. Comparative Raman Tweezers Micrsospectroscopy characterization of EVs from ZL versus ZF hepatocytes, for three different EV preparations. Individual EVs from ZL hepatocytes (left panels, blue bars) were optically trapped and analysed in total 112 times, while EVs from ZF hepatocytes (right panels, red bars) were analysed 129 times. Raman spectra recorded in each trapping event were classified by an educated observer into one of four biomolecular groups: P = dominant proteins; P+L = proteins and lipids in various non‐negligible proportions; L = dominant unsaturated lipids, negligible proteins; SL+P = saturated lipids and proteins in various proportions. Error bars correspond to the uncertainty of spectra attribution to a particular biomolecular group.

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  FIGURE 2. Bodipy staining of lipids from ZL and ZF primary hepatocytes. (a) Confocal representative images were taken 4 h and 36 h after cell seeding. (b) Quantification of the area of Bodipy signal from ZL and ZF hepatocytes 4 h and 36 h after cell seeding. The p‐values were denoted as follows: 0.01‐0.05 = *,0.01‐0.001 = **, 0.001‐0.0001 = *** n = 4.

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  FIGURE 3. Direct Lipid content analysis in intact Zucker rat hepatocyte‐derived EVs. The content of lipids in EV samples was analysed by flow cytometry of Bodipy stained EV‐samples. (a) Overlay histograms of triplicates show the differences between ZL (blue) and ZF (red) EVs with (b) both the percentage of positive particles as well as the intensity means were increased in EVs derived from ZF hepatocytes, when compared with ZL EVs. Each biological replicate (n = 3) was calculated using 30,000 events.

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  FIGURE 4. Lipidomic analysis. (a) Score scatter plot (left) and Loadings scatter plot (right) of the OPLS‐DA model of hepatocytes obtained from lean and obese rats. Model diagnostics (A = 3; R2X = 0.778; Q2X = 0.268). (b) Score plot (left) and loadings plot (right) of the OPLS‐DA model of EVs obtained from hepatocytes of ZL and ZF rats. Model diagnostics (A = 5; R2X = 0.904; Q2X = 0.976). Cer: Ceramides; Cho: Cholesterol; ChoE: Cholesteryl Esters; HexCer: Monohexosylceramides; DAG: Diacylglycerols; PC: Phosphatidylcholines; PE: Phosphatidylethanolamines; PI: Phosphoinositols; SM: Sphingolipids; TAG: Triacylglycerols. n = 4 per condition.

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  FIGURE 5. Biodistribution of Zucker rat hepatocyte‐derived EVs. (a) In vivo biodistribution of radiolabelled EVs from ZL and ZF rats, at t = 1, 3 and 6 h after intravenous administration, as determined by PET imaging. Left: representative PET images (maximum intensity projections, cor‐registered with rendered CT images; coronal view) for the different EVs at different time points; right; quantification values for the different organs. Values are expressed as mean ± standard deviation, n = 4 per EV and time point. (b) Concentration (%) of radioactivity in the different ex vivo organs at t = 6 h after intravenous administration of ZL and ZF EVs, as determined by dissection and gamma counting. Values are expressed as mean ± standard deviation, n = 4 per EV. NA: not applicable.