Background: Extracellular vesicles (EVs) are important mediators of intercellular communication. In COPD, EVs spread cellular senescence by transferring selected microRNAs to neighbouring healthy cells. Thus, EVs participate in the accumulation of senescent cells in tissues which may account for disease progression. However, the mechanism of EV uptake by recipient cells is unknown.

Aim: To compare the effect of endocytosis inhibitors on the internalisation of EVs in recipient epithelial cells.

Methods: EVs were isolated from BEAS2B cell media by ultracentrifugation and stained with the lipophilic membrane dye, pkh67. Recipient BEAS2B cells were cultured with and without drugs that inhibit different mechanisms of endocytosis (pitstop, cytochalasin b, cytochalasin d, 30?M dynasore, nocodazole and ?-cyclodextrin) and pkh67-labelled EVs for up to 12h. Internalisation of EVs in recipient cells was analysed by flow cytometry.

Results: Internalisation of EVs was active and time dependent as the proportion of recipient BEAS2B cells positive for EVs increased from 0.99 ± 0.22% to 36.97 ± 10.29% after 3h and to 61.60 ± 11.40% after 12h (n=4, p=0.01). Pitstop, cytochalasin b and d, nocodazole and ?-cyclodextrin had no effect on EVs uptake by BEAS2B (n=4). Treatment with dynasore, a dynamin inhibitor, blocked the internalisation of EVs by BEAS2B cells after 3 and 12h (after 3h, untreated: 36.97 ± 10.29% vs dynasore: 5.47 ± 2.37%, n=4, p=0.001).

Conclusion: These data suggest that EVs are internalised in epithelial cells in a dynamin-dependent mechanism. Further exploration of this mechanism will enable the development of strategies to inhibit the interaction between EVs and recipient cells.