Plant extracellular vesicles (pEVs) derived from numerous edible sources gain a lot of attention in recent years, mainly due to the potential to efficiently carry bioactive molecules into mammalian cells. In the present study, we focus on isolation of PDNVs (plant-derived nanovesicles) and pEVs from callus culture and from BY-2 culture of Nicotiana tabacum (tobacco). Tobacco was selected as a source of plant vesicles, as it is commonly used by human, moreover it is a model organism with established techniques for cultivation of explant cultures in vitro. Explant cultures are suitable for the isolation of pEVs in large quantities, due to their fast growth in sterile conditions. As the efficiency of isolation methods varies, we were comparing two methods of isolation. We evaluated biophysical and biochemical properties of plant vesicles, as well as differences between isolates. We encountered difficulties in the form of vesicles aggregation, which is often described in publications focused on mammalian nanovesicles. In an effort to prevent vesicle aggregation, we used trehalose in different stages of isolation. We show tobacco-derived vesicles successfully enter tobacco and mesenchymal cell lines. We observed that tobacco-nanovesicles isolated by different methods incorporated fluorescent dye with different efficiency. The results of our study show tobacco-derived vesicles isolated by various isolation methods are able to enter plant, as well as mammalian cells.

Extracellular vesicles are small membrane particles (30-1000 nm) released by Bacteria, Eukaryotes and Archaea. They have been shown to play an important role in intracellular and intercellular communication, within and between kingdoms via transport of bioactive molecules. Thus, they can be involved in altering gene expression and regulation of physiological and pathological processes of the recipient. Their unique properties make extracellular vesicles a perfect candidate vector for targeted drug delivery or a biomarker. For a long time, animal and mainly mammal extracellular vesicles have been used in research. But for plants, there had been speculations about the existence of nanovesicles due to the presence of a cell wall. Today, awareness of plant extracellular vesicles is on the rise and their research has proved they have various functions, such as protein secretion, transport of bioactive molecules or defense against pathogens. Further potential of plant extracellular vesicles is stressed in this review.

The slug Arion vulgaris has attracted major attention as one of the worst invasive herbivore pests in Europe and is renowned for the stiff mucus it secretes for locomotion. In this study we focused on the isolation and characterisation of extracellular vesicles, specifically exosomes and exosome-like vesicles, from Arion secretions. We developed a method for slug mucus collection and subsequent vesicle isolation by ultracentrifugation. The isolated vesicles with an average diameter of ~ 100 nm carry abundant proteins and short RNAs, as well as adhesion molecules similar to mammalian galectins. We demonstrated that the slug extracellular vesicles are internalised by plant cells and human cancer cells in in vitro assays and are loadable by bioactive compounds, which makes them an interesting tool for utilisation in biotechnology.