TY - JOUR
T1 - Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking
AU - Baldwin, Scott
AU - Deighan, Clayton
AU - Bandeira, Elga
AU - Kwak, Kwang J.
AU - Rahman, Mohammad
AU - Nana-Sinkam, Patrick
AU - Lee, L. James
AU - Paulaitis, Michael E.
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health (1UH2TR000914-01) and the NSF (EEC-0425626 and EEC-0914790).
Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2017/2/1
Y1 - 2017/2/1
N2 - 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.
AB - 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.
KW - Cationic lipoplex nanoparticles
KW - Extracellular vesicles
KW - Fluorescence detection
KW - Nanoparticle tracking analysis
KW - miRNA
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U2 - 10.1016/j.nano.2016.10.013
DO - 10.1016/j.nano.2016.10.013
M3 - Article
C2 - 27989853
AN - SCOPUS:85010408611
SN - 1549-9634
VL - 13
SP - 765
EP - 770
JO - Nanomedicine: Nanotechnology, Biology, and Medicine
JF - Nanomedicine: Nanotechnology, Biology, and Medicine
IS - 2
ER -