Isolation of Extracellular Vesicles Derived from Dried Leaves of Kinkéliba (Combretum micranthum G. Don) Using Tangential Flow Filtration

Extracellular vesicles (EVs) are particles released from all cells across kingdoms of life, delimited by a lipid bilayer, which cannot replicate on their own. They have high biological relevance, influencing various physiological and pathological processes. However, knowledge about their isolation and characterization remains a key challenge for certain sources, particularly in the plant kingdom, despite their emerging biomedical application potential. In addition, the studied plant is recognized for its traditional medicinal use and its potential therapeutic properties, which further supports the relevance of investigating its extracellular vesicles. The objective of this study was to isolate and characterize EV-enriched preparations from the dried leaves of Combretum micranthum G. Don using tangential flow filtration, optimizing different isolation protocols to increase the yield and purity of the obtained EVs. After isolation, the concentration and the size distribution of particles in the final sample were measured by NTA; FPLC-SEC was used to analyze the presence of additional particle subpopulations and non-EV-associated proteins, while TEM was used to evaluate the morphology of vesicle-like structures. The total lipid content was determined by the Sulfo-Phospho-Vanillin Assay (SPVA), and the total protein content was determined by means of the BCA assay. This approach allowed to isolate 2–4 × 1011 vesicles per gram of plant material with relatively high reproducibility in particle yields and lipid and protein content. In conclusion, these results suggest that TFF is a suitable approach for the isolation of plant-derived EVs from dried leaves, while providing a scalable and cost-effective production method for scientific and industrial applications.