Figure 1.

Physical characterization of semen exosomes (SE) from different clinical groups: (A) Exosomes were isolated from semen specimens obtained from donors in different clinical groups—HIV−Drug−, HIV−Drug+, HIV+Drug−, and HIV+Drug+. Seminal plasma from different participants were pooled, and the exosomes were purified by size exclusion chromatography (SEC). Six pools of clarified seminal plasma (n = 2, 100 µL/donor) from each of the four clinical groups were purified by size exclusion chromatography (SEC), and fractions were collected. UV–Vis was used to monitor absorbance at 280 nm and turbidity at 600 nm, indicative of the presence of proteins and lipid-containing vesicles, respectively. The dotted curve and filled curve represent absorbance profiles at 280 nm (protein) and 600 nm (lipid), respectively. The gray vertical rectangle highlights exosome fraction. (B) Purified SE fractions were pooled, and total protein concentration was determined by Bradford assay. Nano Tracking Analysis (NTA) measurements of the different SE physical properties: (C) size distribution profile, (D) NTA-based particle mean size, (E) mean particle concentration, and (F) Mean zeta potential (ζ-potential, mV). (G) Negative-stain TEM images of purified SE from the four clinical groups: The insets correspond to zoomed areas indicated by the arrow. All scale bars = 100 nm. (H) TEM-based mean particle size from Figure 1G determined with Image J. Assessment of HIV proteins: (I) HIV reverse transcriptase (RT) and (J) HIV p24. A total of 30 µg purified SE (~6 × 10⁹ particles) from HIV+ groups (6 pools of 2 donors each) were tested in triplicate. An equivalent volume of PBS was used as the negative control. (K) Cocaine metabolite ELISA. A total of 30 µg purified SE (~6 × 10⁹ particles) from Drug+ groups (6 pools of 2 donors each) were tested in triplicate. Equivalent volume of Phosphate Buffered Saline (PBS) was used as a negative control. The numbers in the graphs of Figure 1B,D–F,H indicate mean values. Error bars indicate SEM of 6 biological replicates. For a two-group comparison, unpaired t-test with Welch’s correction was used to determine the differences between the groups. For a four-group comparison, ordinary one-way ANOVA test (Dunnett’s correction) was used to determine the differences between the SE groups as compared to HIV−Drug−. * p < 0.05, ** p < 0.01, and ns, nonsignificant.