title: Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking.
abstract: 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.
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA; Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, Ohio State University, Columbus, OH, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Baldwin
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA; Malvern Instruments, Westborough, MA, USA.
authors.forename: Clayton
authors.initials: C
authors.lastname: Deighan
authors.affiliation: Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Elga
authors.initials: E
authors.lastname: Bandeira
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA; Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, Ohio State University, Columbus, OH, USA.
authors.forename: Kwang J
authors.initials: KJ
authors.lastname: Kwak
authors.affiliation: Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Wexner Medical Center, Ohio State University, Columbus, OH, USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rahman
authors.affiliation: Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Wexner Medical Center, Ohio State University, Columbus, OH, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Nana-Sinkam
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA; Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, Ohio State University, Columbus, OH, USA.
authors.forename: L James
authors.initials: LJ
authors.lastname: Lee
authors.affiliation: William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, USA; Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, Ohio State University, Columbus, OH, USA; Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: michaelp@jhmi.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Paulaitis
mesh_terms: D000067128:Extracellular Vesicles; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D008175:Lung Neoplasms; D035683:MicroRNAs / Q000493:pharmacokinetics*; D053758:Nanoparticles; D010641:Phenotype; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D035683:MicroRNAs
keywords: Cationic lipoplex nanoparticles; Extracellular vesicles; Fluorescence detection; Nanoparticle tracking analysis; miRNA