VIVID: A qPCR‐Based Platform for Sensitive and Quantitative In Vivo Tracking of Extracellular Vesicles

ABSTRACT Extracellular vesicles (EVs) represent promising carriers for nucleic acid therapeutics, offering advantages over synthetic nanoparticles such as biocompatibility and the ability to cross biological barriers. However, in vivo tracking methods most often rely on fluorescent labeling of the EV membrane or surface proteins, which does not accurately reflect the biodistribution of the nucleic acid cargo. This study introduces VIVID (Vesicle In Vivo Identification using DNA), a qPCR‐based platform that tracks PCR‐amplifiable DNA tags loaded into EVs instead of therapeutic nucleic acids. Following systemic injection, the biodistribution of intact tags is quantified by qPCR in tissue lysates. VIVID demonstrates far superior sensitivity compared to fluorescence and reveals cargo distribution profiles that differ substantially from those inferred from membrane labeling. Whereas fluorescence indicates prolonged circulation and redistribution, VIVID shows faster clearance of nucleic acid cargo, detects signals in fluorescence‐negative tissues, and reveals distinct liver‐to‐spleen distribution ratios. VIVID further enables evaluation of EV engineering strategies, revealing that retention of polyethyleneimine improves circulation time and markedly increases delivery to tissues, including the lungs. By enabling direct, sensitive, and quantitative tracking of EV‐delivered nucleic acids in vivo, VIVID addresses a key challenge in the development of EV‐based therapeutics.