Orthogonal investigation at single-particle and ensemble levels uncovers lipoprotein-extracellular vesicle binding

Mesoscale interactions—such as biomolecular coronas, transient associations, aggregation, and fusion—are increasingly recognized for their biological significance and, under certain conditions, are essential for extracellular nanoparticles to fulfill their functions. Among these interactions, the binding between extracellular vesicles and lipoproteins has recently gained attention for its potential impact on extracellular vesicle function and fate in vivo. These interactions must be understood to clarify and possibly engineer and exploit the biological modes of action of EVs. A bottom-up simplified system consisting of red blood cell-derived extracellular vesicles and purified human lipoproteins was used to investigate extracellular vesicles-lipoprotein binding in saline buffer and in human plasma. A customized toolbox of orthogonal analytical techniques was developed to characterize these interactions at multiple scales, also using label-free materials, while preserving their natural binding states. This toolbox includes fluorescence cross-correlation spectroscopy, super-resolution microscopy, flow cytometry, and Single Molecule Array assay. Our findings reveal that lipoproteins bind to red blood cell EVs with affinities ranging from 10 nM to 1 µM. The percentage of individual extracellular vesicles interacting with lipoproteins is dependent on the specific lipoprotein class and the incubation conditions, and is always considerable, with up to 100% EV interacting with High Density Lipoproteins in the presence of plasma proteins. Such binding is stable, proving resistant also to several washing steps. Our data depict the EV – lipoprotein interaction as a generalizable phenomenon, which is shared among all the lipoprotein classes to different degrees. This implies that in physiological conditions, EVs may be constantly associated with a certain number of lipoproteins in the bloodstream, with possible impacts on EV surface identity and, therefore, function. This finding advances our understanding of extracellular nanoparticle interactome, and provides a step forward in deciphering the physicochemical foundations of biodistribution and clearance mechanisms of natural, synthetic, and hybrid nanoparticles.