From labeling, dosage, size to biodistribution and function of extracellular vesicles – a bioimaging perspective

Lai Charles^1,2,3*

¹Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
²Chemical Biology and Molecular Biophysics Program, TIGP, Academia Sinica, Taipei, Taiwan
³Genome and Systems Biology Degree Program, National Taiwan Univeristy, Academia Sinica, Taipei, Taiwan

* Presenter:Lai Charles, email:laicharles@sinica.edu.tw

Extracellular vesicles (EVs) are varying sized bionanoparticles released by cells to mediate intercellular communication under (patho)physiological conditions. While a majority of EV-related studies focuses on small EVs (sEVs; e.g., exosomes; <100 - 200 nm), less research examines medium and large EVs (collectively termed big EVs [bEVs]; e.g., microvesicles; >200 nm) and their functions. Meanwhile, a high dose of EV is commonly applied to exert a phenotype in vivo, often as an attempt to detect dye-labeled EVs which yields a low signal-to-noise signal. We thus investigated key parameters of EV in vivo study such as labeling methods, EV sizes, as well as their subsequent biodistribution and biological function. We compared biophysical and in vivo property of EVs labeled with lipophilic dyes and our latest bioluminescence resonance energy-based EV reporter, PalmGRET. DiR significantly alters the size and biodistribution of the labeled EVs when compared to PalmGRET-labeled counterparts. Notably, a high dose of DiR-EV (50 – 100 μg per mouse) is required to attain an appreciable signal when administered intravenously, and is often lethal. By contrast, PalmGRET-EVs can be readily detected for sensitive biodistribution analysis at a non-lethal and physiological dose (5 – 10 μg). Importantly, sEV and bEVs comprise of varying sized EV populations. Using PalmGRET-EVs at the physiological dosage, we observed a differential biodistribution between sEV and bEV derived from 4T1 breast cancer, and further demonstrated their protumorigenic potential in breast cancer-bearing mice. To the best of our knowledge, this is the first study which empirically optimized the labeling method and dosage for EV in vivo study, and subsequently revealed distinct biodistribution profile and protumorigenic function of both sEV and bEVs.

Keywords: BRET, bioluminescence, fluorescence, exosomes, pharmacokinetics