Quantification of extracellular vesicles with unaltered surface membranes using an internalized oligonucleotide tracer and droplet digital PCR applied to modeling ofin vivokinetics

Extracellular vesicles (EVs) continue to attract interest for their potential role in targeted therapeutics and as biomarkers of disease and drug response. In order to achieve clinical utility, it is important to determine the pharmacokinetic parameters of candidate EVs during preclinical development using in vivo animal models. To date, no methods exist for studying EV kinetics without modification to surface ligands that may affect normal behavior. Here we introduce an accessible method for labeling and quantifying EVs administered to conscious animals, without disrupting endogenous ligands. Our method relies upon established laboratory techniques and can be tailored to a variety of biological questions. Digital PCR is leveraged to detect a non-homologous oligonucleotide tracer introduced into the vesicles, allowing for quantification over a wide dynamic range. Using an application of this method, we found differences in the in vivo kinetics of EVs from three different cell types using non-linear mixed effects modeling. We propose that this method will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.