Isolation and Properties of Extracellular Vesicles

For the prolonged intracellular action of pharmaceutical drugs, penetration through cell membranes and escape from endosomal-lysosomal entrapment are necessary. Inorganic and organic nanomaterials enter eukaryotic cells mainly through endocytosis with biodegradation with lysosomal enzymes; antigen presentation to the immune system; and the induction of innate, trained, and adaptive immunity. The fusion of extracellular vesicle membranes and cell membranes permit to escape from lysosomes, enzymatic hydrolysis, and cytokine gene expression. The study aims to isolate extracellular vesicles and analyze their physical and chemical properties. Vesicles with a diameter of 10–200 nm surrounded by membranes with a thickness of 7–10 nm were isolated from the conditioned culture media of embryonic and transformed human cells, as well from apple juice, using differential centrifugation at 20 800 g. They consisted of lipids and proteins, were stable during isolation, fluorescent labeling, and storage at +4°C for 5 months. Loaded extracellular vesicles were fabricated by hydration of a thin layer of dried cell membranes. Fluorescent vesicles were nontoxic to autologous and heterologous human cells and gradually accumulated in cells over 7 days of observation.