Nanomaterials Covered with Cell Membranes for Intracellular Delivery Without Lysosomal Degradation and Innate...

Cellular uptake of nanomaterials is based on endocytosis with their endosomal-lysosomal entrapment resulting in enzymatic hydrolysis. Besides biodegradation, the antigen presentation induces innate and adaptive immunity. Our goal was isolation of extracellular particles to study their structures, penetration into cells, stability, intracellular distribution, and interferon (IFN) production. Extracellular nanomaterials were isolated from conditioned culture media of human embryonic and cancer cells by two-stage differential centrifugation. Cellular uptake of Cy5-labeled particles was evaluated using spectrofluorimetry and confocal fluorescent microscopy. IFN gene expression was analyzed by reverse transcription with real-time PCR and ELISA. Vesicles of 10-200 nm were isolated by centrifugation at 20,800× g at +4 °C for 30 min. The fluorescent vesicles were gradually accumulated inside cells for seven days. Intracellular distribution patterns of the Cy5-labeled vesicles differed from lysosomes stained with LysoRed tracker. IFNs α, β and γ were not detected after treatment with the vesicles. IFN λ was found in cells in the presence of allogenic but not autologous particles. The gradual cellular uptake occurred without significant differences between autologous and heterologous vesicles. Different localization of the extracellular vesicles (EV) and lysosomes along with weak innate immune response (if any) suggested membrane fusion.