Coincident Fluorescence‐Burst Analysis of the Loading Yields of Exosome‐Mimetic Nanovesicles with Fluorescently‐Labeled Cargo Molecules

Abstract The possible targeting functionality and low immunogenicity of exosomes and exosome‐like nanovesicles make them promising as drug‐delivery carriers. To tap into this potential, accurate non‐destructive methods to load them and characterize their contents are of utmost importance. However, the small size, polydispersity, and aggregation of nanovesicles in solution make quantitative characterizations of their loading particularly challenging. Here, an ad‐hoc methodology is developed based on burst analysis of dual‐color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome‐like nanovesicles and of their fluorescently‐labeled loading. It is applied to study exosome‐mimetic nanovesicles derived from animal extracellular‐vesicles and human red blood cell detergent‐resistant membranes, loaded with fluorescently‐tagged dUTP cargo molecules. For both classes of nanovesicles, successful loading is proved and by dual‐color coincident fluorescence burst analysis, size statistics and loading yields are retrieved and quantified. The procedure affords single‐vesicle characterizations well‐suited for the investigation of a variety of cargo molecules and biological nanovesicle combinations besides the proof‐of‐principle demonstrations of this study. The results highlight a powerful characterization tool essential for optimizing the loading process and for advanced engineering of biomimetic nanovesicles for therapeutic drug delivery.