Coincident Fluorescence Burst Analysis of dUTP-Loaded Exosome-Mimetic Nanovesicles

AbstractThe targeting functionality and low immunogenicity of exosomes and exosome-mimetic 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, their small size, polydispersity and aggregation in solution make quantitative characterizations of their loading particularly challenging. Here we develop an ad-hoc methodology based on a burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their loading. We apply it to study bioengineered nanovesicles, loaded with dUTP cargo molecules, synthetized from detergent-resistant membranes of animal extracellular vesicles and human red blood cells. For both classes of bioengineered nanovesicles we prove, by means of dual-color fluorescence cross-correlation spectroscopy (FCCS), successful loading. Furthermore, by a dual-color coincident fluorescence burst (DC-CFB) analysis of the experimental data, we retrieve size and loading statistics for both types of nanovesicles. The procedure affords single-vesicle characterizations, which are essential for reliable quantitative studies of loading processes in exosomes and exosome-mimetic nanovesicles, especially in light of the typically high heterogeneity of their populations. Moreover, the method implementation can be easily adapted to the investigation of a variety of combinations of different cargo molecules and biological nanovesicles besides the proof-of-principle demonstrations considered in this study. The results provide a powerful characterization tool, well-suited for the optimization of loading processes of biomimetic nanovesicles and their advanced engineering for therapeutic drug delivery.