VIVID: a Source-Agnostic DNA-Tag Method for Sensitive and Quantitative Tracking of Extracellular Vesicle Cargo Delivery In Vivo

Mesenchymal stem cell therapies are increasingly understood to act through paracrine mechanisms, with extracellular vesicles (EVs) and their nucleic acid cargos now recognized as major drivers of therapeutic effects. As EVs gain momentum as naturally optimized RNA and DNA delivery systems, progress is limited by the absence of a method capable of directly and sensitively tracking nucleic acid cargo in vivo after systemic administration. Optical tracers lack accuracy, reporter systems are constrained by low sensitivity or genetic requirements, and endogenous nucleic acid markers are too scarce or model-dependent to be generalized. To overcome these barriers, we developed VIVID (Vesicle In Vivo Identification using DNA), a DNA tag-based platform that enables quantitative tracking of EV-delivered nucleic acids using a defined PCR-amplifiable sequence loaded under standard engineering conditions. As a proof-of-concept, we intravenously injected red blood cell-derived EVs endowed with DNA through polyethyleneimine (PEI)-mediated loading in C57BL/6 mice and obtained complete biodistribution profiles across major organs at 1, 3, 6, and 24 hours. VIVID achieved up to 37,000-fold higher sensitivity in liver and 1,000-fold in lungs compared to fluorescence tracing, revealing low-dose delivery events and organ-specific kinetics undetectable with optical methods. The platform captured rapid hepatic clearance within the first hour, stable pulmonary retention over time, and delayed renal accumulation consistent with DNA tag excretion. VIVID also identified substantial formulation-dependent shifts in cargo delivery, including strong increases in liver, spleen, and lung accumulation when PEI was retained on EVs, illustrating how engineering reshapes in vivo fate. By selectively reporting on nucleic-acid-bearing EVs, VIVID provides a biologically meaningful map of functional cargo delivery. Its ability to generate sensitive, time-resolved biodistribution profiles positions VIVID as a powerful new approach methodology for advancing EV-based RNA and DNA therapeutics from any cell source, including stem-cell-derived EVs.