Purification, profiling and bioengineering of extracellular vesicles

Abstract: The nano-sized membrane enclosed extracellular vesicles (EVs) transfer macromolecular information in the form of proteins, nucleic acids and lipids across cells. They are important mediators of cell-to-cell communication, but their relatively small size makes EV isolation and down-stream analysis challenging. In this thesis, we ventured through some of the current challenges in the EV field touching upon purification, small RNA and protein content profiling and ultimately characterization of engineered vesicles at the molecular level. The isolation of EVs from the complex fluid they are surrounded by, represents the first hindrance in studying these vesicles. Ideally, the purification method should preserve the integrity and natural properties of the EVs and simultaneously deplete the vesicular portion from unwanted components. In Paper I, we describe a novel liquid chromatography technique for EV purification, that combines size separation with bind elution (BE-SEC) entrapping molecules smaller than 700 kDa within the matrix core. The BE-SEC isolation method yields high particles recovery in a reproducible and time-efficient way, without neither affecting the EVs natural surface protein signature nor their physicochemical properties. By adding a prior tangential flow filtration step, the BE-SEC could be scaled-up and the EV preparation further depleted from unwanted non-vesicular proteins and RNAs. Secondly, therapeutically engineered EVs are promising delivery vehicles and linking the administered vesicular dose to the molecular cargo concentration is of extreme relevance to achieve a desired response. Therefore, analytical methods focused on single vesicles quantification rather than ‘bulk’ analysis and improved bioengineered vesicles are of utmost importance for therapeutic applications. In Paper II, we extensively characterize a set of fluorescently labelled EV-associated proteins, employing several qualitative and quantitative methods. Using Fluorescence Correlation Spectroscopy, we quantify the number of fluorescent molecules per single loaded vesicle. Different loading efficiencies were observed for the tested proteins, with the tetraspanins (CD63, CD9 and CD81) showing the highest loading efficiency with an average of 40-60 fluorescent molecules per vesicle. To summarize, we provide a reference for selecting EV sorting domains that best fit the desired outcome, as well as an array of quantitative and qualitative methodologies to support EV engineering. In Paper III, we investigate the native RNA and protein content of EVs, with a focus on small RNAs and RNA binding proteins respectively. Across different mouse and human cell-derived EVs, a deficiency of miRNA sequences and relative depletion of ‘miRNA-related’ proteins were observed. The majority of the RNA sequences detected in EVs was represented by rRNA-, coding- and tRNA fragments, reflecting the observations in the respective protein portion, where ribosomal and translational proteins were predominantly identified. In conclusion, this thesis explores and advances some of the challenges encountered in the EV field by ameliorating the EV isolation workflow in terms of time and scalability, linking the vesicular transcriptome and proteome of EVs derived from various cell lines, and systematically comparing and quantifying the sorting efficiency of different proteins into EVs.

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