Extended Data Fig. 4: Cytotoxicity of EV preparations and preparation and characterization of EV-liposome hybrids.
(a) Effect of purified EVs on cellular metabolic activity. EVs were titrated onto Vero E6 cells and incubated with mock virus (medium only). Medium was replaced after 2 h and 2 days later, intracellular ATP levels were determined via CellTiter-Glo luminescent viability assay. Values normalized to untreated cells. n = 3 biological replicates, mean ± SD. (b) Evaluation of direct virucidal effects of semen EVs or liposomes. Virus stocks were treated with vesicles (1 × 1012 particles/ml) or 0.1% Triton X-100, TX, as a positive control for 2 h before determining residual infectious titer by TCID50 endpoint titration on Vero E6 and evaluation of CPE 7 days post-infection. One-way ANOVA with Bonferroni’s post-test, all without indication p > 0.05 = ns compared to PBS treated. n = 3 (liposomes), n = 5 (PBS), n = 6 (Triton X-100) and n = 9 (semen EVs) independently treated samples. Mean ± SEM. (c) Workflow for preparation of hybrid EV-liposome vesicles. (d) Representative NTA histograms showing size distribution detected in scatter (grey) or 640 nm fluorescence mode (red), n = 3 replicate acquisitions, shaded area show SD. (E) Bead-assisted flow cytometric analysis of EV surface proteins by TSPN staining or detecting Cy5 with PC liposomes as control. n = 3 replicates (f) Antiviral activity (ZIKV MR766 MOI 0.2 on Vero E6) of unlabeled semen EVs, labeled semen EVs and liposomes, as well as labeling-liposomes. n = 3 biological replicates, means ± SD. (g) Attachment of virions (left) or vesicles (right) after addition of labeling-liposomes and ZIKV MR766 (MOI 35) to Vero E6 cells. Quantification of 3 separate image per condition (n = 3), means ± SD. (h) Representative confocal microscopy images as used for quantification shown in (F) including images for or DOPC liposomes mixed with labeling-liposomes (shown in Fig. 4c). Scale bars = 20 μm.
