Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking

We present a method that takes advantage of the fluorophore loading dependence of fluorescence nanoparticle tracking (fNTA) to determine the content of specific miRNA targets in extracellular vesicles (EVs) and their stoichiometry across the entire EV population. The method is based on an assay for detecting EV miRNA by hybridization to fluorescently labeled, miRNA-specific molecular beacons encapsulated in cationic lipoplex nanoparticles that fuse non-specifically with negatively charged EVs. To demonstrate the method, we carry out a stoichiometric analysis of miR-21 in EVs released from A549 lung cancer cells. We find approximately 2.3% of the A549 EVs have an average copy number of ~44 miR-21/A549 EV and contain at least a threshold number of 33 miR-21 copies/A549 EV required for fluorescence tracking. Potential applications of sizing, enumerating, and phenotyping EVs using this method include specifying dosages for therapeutic applications and identifying specific EV subpopulations in patient samples for diagnostic applications.