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Author
Prous Conde, Anna
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Abstract
Cells release several biomolecules into the extracellular environment as cell-to-cell commu-nication fucntion. Recently, it was described that most of them are released inside extracellu-lar vesicles (EVs), defined as nanosized membranous vesicles which are released from most kinds of cells. Their content mainly comprises proteins, sugars, lipids, and an extensive variety of genetic materials, such as DNA, mRNA, and non-coding RNA. The natural role of these vesicles is to deliver these molecules into recipient cells to effectively modulate their biological response, which evidences their potential to be used as carriers for therapeutic purposes, as delivery systems.
Until now, several methods have been designed to load different cargos into EVs. However, some challenges need to be overcome, amongst which the limited uptake by recipient cells of mRNA exogenously loaded into EVs can be emphasized. The present study pretends to optimize the loading of small extracellular vesicles with mRNA for their use as vaccines for non-small cell lung cancer (NSCLC) therapy. The methodology consists of fluorescent label-ing both the mRNA and the exosomes to track their uptake by an immortalized human tumoral cell culture. Incubation has been used as a passive method to load EVs with nucleic acids. Afterward, further analysis such as fluorescence microscopy or flow cytometry have been used to evaluate the fluorescence expression, which demands further optimization. Addition-ally, flow cytometry has also been conducted to understand how mRNA encoding for GFP is internalized and expressed by cells, to better adapt the following experiments. Finally, cyto-toxicity assays have also been performed to confirm if crystal violet is adequate for charac-terization as well as non-toxic to cells when used as a staining method for extracellular vesi-cles. Crystal violet has proved to be suitable to characterize EVs, as seen by confocal micros-copy, but not when it needs to be internalized into cells, as MTS assay revealed.
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