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Contains fulltext : 206962.pdf (Publisher’s version ) (Open Access) The knowledge of in vitro and in vivo stability of polymeric nanoparticles is vital for the development of clinical formulations for drug delivery and cell labeling applications. Forster resonance energy transfer (FRET)-based fluorescence labeling approaches are promising tools to study nanoparticle stability under different physiological conditions. Here, we present the FRET-based stability assessment of poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating BODIPY-FL12 and Nile Red as the donor and acceptor, respectively. The stability of PLGA nanoparticles is studied via monitoring the variations of fluorescence emission characteristics along with colloidal characterization. Accordingly, PLGA nanoparticles are colloidally stable for more than 2 weeks when incubated in aqueous buffers in situ, whereas in vitro particle degradation starts in between 24 and 48 h, reaching a complete loss of FRET at 72 h as shown with fluorescence microscopy imaging and flow cytometry analysis. PLGA nanoparticles systemically administered to mice predominantly accumulate in the liver, in which FRET no longer takes place at time points as early as 24 h postadministration as determined by ex vivo organ imaging and flow cytometry analysis. The results of this study expand our knowledge on drug release and degradation behavior of PLGA nanoparticles under different physiological conditions, which will prove useful for the rational design of PLGA-based formulations for various applications that can be translated into clinical practice.