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

作者：Giulia Corso, Wolf Heusermann, Dominic Trojer, André Görgens, Emmanuelle Steib, Johannes Voshol, Alexandra Graff, Christel Genoud, Yi Lee, Justin Hean, Joel Z. Nordin, Oscar P. B. Wiklander, Samir EL Andaloussi, Nicole C. Meisner-Kober · 发表于：Journal of Extracellular Vesicles · 年份：2019 · DOI：10.1080/20013078.2019.1663043 · 被引用次数：184 · 研究领域：Extracellular vesicles in disease、Cell Adhesion Molecules Research、RNA Interference and Gene Delivery

Extracellular vesicles (EV) 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 the 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 12 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; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40-60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification wi...