期刊
JOURNAL OF EXTRACELLULAR VESICLES
卷 10, 期 10, 页码 -出版社
WILEY
DOI: 10.1002/jev2.12130
关键词
EV cargo sorting; exosomes; ExoView; extracellular vesicles; nanoflow cytometry; protein delivery vehicle; single-molecule localization microscopy
类别
This study introduced a workflow for accurately quantifying the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. By using a combination of techniques, TSPAN14, CD63, and CD63/CD81 fused to the PDGFR beta transmembrane domain were identified as the most efficient EV-sorting proteins, accumulating an average of 50-170 single GFP molecules per vesicle.
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63(+), CD81(+), or CD9(+) EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFR beta transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.
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