43 - Investigating NPS-induced electro-exocytosis of extracellular vesicles

Background: Electro-exocytosis is a cellular process characterized by the voltage-dependent release of intracellular components, usually extracellular vesicles (EVs) or small organelles. It is mostly mediated by Ca2+ flux and observable uniquely in excitable cells, such as neurons. Nano-Pulse Stimulation (NPS) is based on the application of ultrashort (nanosecond) electric fields to cells or tissues. While high field intensities may elicit cell ablation and death, lower doses are able to stimulate proliferation and increase metabolism. Hypothesis: We hypothesized that NPS could be employed to stimulate cells in releasing biologically active EVs. Methods: We performed initial NPS dose-response studies to define optimal parameters for the release of EVs in Jurkat Cells (1 pulse of 20kV to 10 pulses of 40kV). EVs were then isolated and purified from media of Jurkat cells and quantified by Nanotracking analysis (NTA, Malvern ns300). EVs were also collected, lysed and protein extracts analyzed by Western Blotting. Results: We observed a biphasic effects on NPS-mediated increased EVs production at 1p20kV and at 3-5 p 40kV. This increase in EVs concentration was associated with a population of EVs between 80nm and 150nm observed in size/distribution analysis. WB analysis displayed increased levels of CD63 expression uniquely in EVs produced during 3p, 50ns at 40kV, compared to sham-treated controls. Conclusion: Our preliminary data suggest that NPS technology could be exploited to increase the production of extracellular vesicles. NPS may elicit the electro-exocytosis of EVs from the MVB and the external plasma membrane, depending on field intensity. Funding: The current project is funded by a Frank Reidy Research Center for Bioelectrics ODU SEED Grant.