Comparison of two isolation methods of tobacco-derived extracellular vesicles, their characterization and uptake by plant and rat cells

Abstract

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.

Plant-Derived Extracellular Vesicles: A New Revolutionization of Modern Healthy Diets and Biomedical Applications

“…165 Similarly, tobacco-derived EVs isolated from BY-2 suspension cultures and callus homogenates and exosome-like nanoparticles isolated from blueberries using PEG precipitation methods could be taken up by plant or mammalian cells. 113,168 Unexpectedly, despite polymerase or protein contamination of EVs isolated from mammalian cells or plants using this method, the biochemical composition of PEG-precipitated PDEVs was found to be comparable to that of ultracentrifugation-isolated PDEVs, which had similar biological activities. 165,168 Moreover, PEG precipitation allows the enabling surface modification of the EVs to improve their stability and enhance their cellular uptake.…”

“…113,168 Unexpectedly, despite polymerase or protein contamination of EVs isolated from mammalian cells or plants using this method, the biochemical composition of PEG-precipitated PDEVs was found to be comparable to that of ultracentrifugation-isolated PDEVs, which had similar biological activities. 165,168 Moreover, PEG precipitation allows the enabling surface modification of the EVs to improve their stability and enhance their cellular uptake. 146,169 isolation.…”

“…Suresh et al further improved this method and significantly enhanced its yield of ginger-derived EVs via acidification during PEG precipitation . Similarly, tobacco-derived EVs isolated from BY-2 suspension cultures and callus homogenates and exosome-like nanoparticles isolated from blueberries using PEG precipitation methods could be taken up by plant or mammalian cells. , Unexpectedly, despite polymerase or protein contamination of EVs isolated from mammalian cells or plants using this method, the biochemical composition of PEG-precipitated PDEVs was found to be comparable to that of ultracentrifugation-isolated PDEVs, which had similar biological activities. , Moreover, PEG precipitation allows the enabling surface modification of the EVs to improve their stability and enhance their cellular uptake. , These reports demonstrate that the simple and scalable PEG precipitation procedure represents a new possibility for large-scale PDEV isolation. However, this approach has not yet been widely applied in isolating PDEVs.…”

Plant-derived extracellular vesicles (PDEVs) in nanomedicine for human disease and therapeutic modalities

“…Although efficacious, most of these processes depend on specialized instruments or lack standard protocols, consequently representing a limitation for their clinical applications [ 106 ]. The method using polyethylene glycol (PEG) could effectively prevent Nicotiana tabacum -derived vesicles from forming the aggregates [ 122 ]. In the future, there is an urgent need to develop the standardization of qualitative and quantitative procedures to better achieve the successful commercialization of PDEVs in the field of translational medicine.…”

Establishing the Callus-Based Isolation of Extracellular Vesicles from Cissus quadrangularis and Elucidating Their Role in Osteogenic Differentiation

“…There was a long-standing belief that due to the presence of the cell wall, plants were incapable of producing extracellular vesicles (EVs). This notion was proven wrong in recent years, and it is now widely accepted that EVs can be released from any cell, including those of humans, animals, and even plants [2,3]. Although mammalian extracellular vesicles (MEVs) have shown promise in broad applications within the field of tissue engineering and regenerative medicine [4], their usage is surmounted by 2 of 17 numerous challenges such as tissue specificity, toxicity, difficulty in isolation from biological fluids, and large-scale production costs [5,6].…”