Andreia M. Silva, 1 Elisa Lázaro‐Ibáñez, 1 , 2 Anders Gunnarsson, 3 Aditya Dhande, 4 George Daaboul, 4 Ben Peacock, 5 Xabier Osteikoetxea, 6 Nikki Salmond, 6 Kristina Pagh Friis, 2 Olga Shatnyeva,corresponding author 1 and Niek Dekkercorresponding author


1 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg Sweden,
2 Advanced Drug Delivery, Pharmaceutical Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg Sweden,
3 Structure and Biophysics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg Sweden,
4 NanoView Biosciences, Boston Massachusetts, USA,
5 NanoFCM INC., Nottingham UK,
6 Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Alderley Park UK,
Olga Shatnyeva, Email: [email protected]


Medical Research, Molecular Biology


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 PDGFRβ 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.



Samples were imaged at multiple dilutions using a 60x oil immersion objective (NA = 1.49) mounted on a Ti Eclipse inverted microscope (Nikon), equipped with a FITC filter cube for GFP fluorescence imaging, CoolLED pE4000 illumination system (490 nm illumination) and an Orca Flash 4.0 CMOS camera (Hamamatsu).

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The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores

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Journal of Extracellular Vesicles

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