Orthogonal Investigation at Single-Particle and Ensemble Levels Uncovers Lipoprotein-Extracellular Vesicle Binding

Mesoscale interactions critically shape the biological identity of extracellular nanoparticles, including extracellular vesicles. These interactions encompass biomolecular coronas, transient aggregation, and fusion events. Among them, the interaction between extracellular vesicles and lipoproteins has recently garnered significant attention due to their potential impact on functionality and in vivo fate of extracellular vesicles. In this work, we present a first investigation of the binding between human red blood cell-derived extracellular vesicles and lipoproteins across multiple scales, in both buffer and plasma. Red blood cell-derived extracellular vesicles were selected as a model system for their physicochemical homogeneity, potential in personalized medicine, and production scalability. To achieve this, we employed an ad hoc suite of orthogonal analytical techniques: fluorescence cross-correlation spectroscopy (FCCS), super-resolution microscopy, flow cytometry, and Single Molecule Array assays (Simoa). Our results reveal class-specific and context-dependent extracellular vesicle-lipoprotein associations. Notably, lipoproteins bind to extracellular vesicles with affinities ranging from 10 nM to 1 μM and with up to 100% extracellular vesicles interacting with high-density lipoproteins in the presence of plasma proteins. These findings uncover a complex and dynamic interactome of red blood cell-derived extracellular vesicles across lipoprotein classes. This work establishes a robust methodological framework for studying mesoscale interactions of extracellular nanoparticles under physiologically relevant conditions. Its versatility allows for its application to diverse interaction scenarios, supporting systematic investigation of context-dependent effects on EV-LP binding.