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Systematic characterization of extracellular vesicles sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Voshol, Johannes and Trojer, Dominic and Corso, Guilia and Heusermann, Wolf and Görgens, Andre and Steib, Emmanuelle and Graff, Alexandra and Genoud, Christel and Lee, Yi and Hean, Justin and Nordin, Joel and Wiklander, Oskar and El Andaloussi, Samir and Meisner-Kober, Nicole (2019) Systematic characterization of extracellular vesicles sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging. Journal of Extracellular Vesicles, 8 (1). ISSN 2001-3078

Abstract

Extracellular vesicles (EV) efficiently convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected twelve EV related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV producing cells and both parent cells as well as the recovered vesicles were characterised biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP tagged molecules per vesicle. The co-expression of each target protein with CD63 was further quantified by confocal imaging of single vesicles after double transfection of parent cells. We observed highly different loading efficiencies and specificities for the different proteins into EVs. Even for the most abundant Tetraspanins, the molecular levels in the vesicles did not exceed ca 30-40 fluorescent proteins per vesicle despite overexpression in the cells. Some of the GFP tagged EV reporters were either non-vesicular, despite co-purification with EVs, showed quenched fluorescence or comprised a significant fraction of truncated GFP. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs, and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterisation of fluorescent EVs to link single vesicle analysis with single molecule quantification.

Item Type: Article
Date Deposited: 02 Oct 2019 00:45
Last Modified: 02 Oct 2019 00:45
URI: https://oak.novartis.com/id/eprint/39423

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