In recent years, there has been a growing interest in plant extracellular vesicles (pEVs) due to their immense potential for medical applications, particularly as carriers for drug delivery. To use the benefits of pEVs in the future, it is necessary to identify methods that facilitate their production in sufficient quantities while maintaining high quality. In this study, a comparative analysis of yields of tobacco pEV derived from apoplastic fluid, sterile calli, and suspension cultures, was performed to identify the most suitable plant material for vesicle isolation. Subsequent experiments focused on assessing the efficiency of small interfering RNA (siRNA) loading into callus-derived vesicles, employing various methods such as sonication, incubation, incubation supplemented with saponin, lipofection, and electroporation. Differences in loading efficiency among vesicles derived from apoplastic fluid, calli, and suspension cultures were observed. Moreover, our investigation extended to the presence of tobacco secondary metabolites, specifically anabasine and nicotine, within vesicles originating from three distinct tobacco sources. The outcomes of our study highlight variations not only in vesicle yields based on their source but also in their loadability and the presence of nicotine and anabasine. These findings contribute valuable insights into optimizing the production and application of pEVs for future medicinal purposes.

Centre for Nanomaterials and Biotechnologies Faculty of Science Jan Evangelista Purkyně University in Ústí Nad Labem Ústí Nad Labem Czech Republic

Department of Environmental Chemistry and Technology Faculty of Environment Jan Evangelista Purkyně University in Ústí Nad Labem Ústí Nad Labem Czech Republic

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