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

## TL;DR

The study explores how extracellular vesicles from red blood cells interact with lipoproteins in different environments, revealing complex and dynamic binding patterns.

## Contribution

The work introduces a novel methodological framework using multiple analytical techniques to study extracellular vesicle-lipoprotein interactions at various scales.

## Key findings

- Lipoproteins bind to extracellular vesicles with affinities ranging from 10 nM to 1 μM.
- Up to 100% of extracellular vesicles interact with high-density lipoproteins in plasma conditions.
- The study reveals class-specific and context-dependent associations between extracellular vesicles and lipoproteins.

## Abstract

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.