Recipient Cell Type-Dependent Association of Extracellular Vesicles from Different Cellular Origins

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors influencing their interactions with recipient cells remain incompletely understood. This study investigated recipient-cell-specific EV association using mNG-CD81-labelled EVs derived from HEK293T, SKBR-3, and Yummer cells. EV-associated fluorescence was assessed by flow cytometry following incubation with human epithelial, immune, and murine melanoma cell lines at different doses and time points. Marked differences were observed across recipient-cell types. Liver-derived Huh7 and HepG2 cells exhibited the highest percentages of fluorescence-positive cells among human epithelial cells, whereas parental HEK293T cells showed the lowest. THP-1 cells exhibited greater EV-associated fluorescence than Jurkat cells, while Yummer melanoma cells showed higher fluorescence than B16F10 cells following exposure to HEK293T-derived EVs. Similar recipient-cell patterns were observed with SKBR-3- and Yummer-derived EVs. Notably, Yummer cells exhibited relatively high association with their own EVs, demonstrating that low parental-cell association is not universal. EV-associated fluorescence generally increased with dose and incubation time. Although these findings highlight recipient-cell specificity, the relative contributions of EV donor origin and recipient-cell characteristics remain unresolved. Moreover, flow cytometry cannot distinguish surface-associated from internalized EVs. Together, these findings identify recipient-cell specificity as an important determinant of EV-associated fluorescence, providing a foundation for understanding EV–cell interactions and informing the development of targeted EV-based delivery strategies.