# Monitoring extracellular vesicle surface glyco-properties using fluorescent lectins and nanoparticle tracking analysis

## TL;DR

This study compares methods to isolate extracellular vesicles and finds that certain techniques change their surface sugar patterns, which could impact their function.

## Contribution

A new method using fluorescent lectins and nanoparticle tracking analysis is introduced to monitor extracellular vesicle surface glycosylation during isolation.

## Key findings

- Lectin binding to extracellular vesicles changes with isolation methods like size exclusion chromatography and microfiltration.
- Lectin-positive extracellular vesicles larger than 200 nm increase in samples isolated with size exclusion chromatography or microfiltration.
- Ultracentrifugation alone preserves original surface glycosylation patterns better than combined methods.

## Abstract

Extracellular vesicles are small particles released by all cell types. Different extracellular vesicle isolation methods are widely used, yet none achieve an optimal balance between yield, purity and structural integrity. This study aimed to establish a comparative approach for evaluating different extracellular vesicle preparations using nanoparticle tracking analysis. A simple one-step assay relying on fluorescence-based nanoparticle tracking analysis was used to evaluate lectin binding to extracellular vesicles as a measure of possible changes in their surface glycosylation during various isolation methods. Seminal extracellular vesicles were isolated from normozoospermic men using ultracentrifugation alone—UC-sEVs—or combined with size exclusion chromatography—UC-SEC-sEVs—or microfiltration—UC-MF-sEVs. They were analysed based on their size and lectin-binding properties using wheat germ agglutinin and Ricinus communis agglutinin I. While total seminal extracellular vesicles and tetraspanin-positive seminal extracellular vesicles maintained similar size distributions across all isolates, lectin-positive seminal extracellular vesicles displayed a shift towards larger than 200 nm seminal extracellular vesicles in UC-SEC-sEVs and UC-MF-sEVs, as compared to UC-sEVs. The ratio of larger (>200 nm) to smaller (30–200 nm) lectin-positive sEVs was increased, particularly for wheat germ agglutinin in UC-MF-sEVs and Ricinus communis agglutinin I in UC-SEC-sEVs. These findings demonstrate that size exclusion chromatography and microfiltration combined with ultracentrifugation influence seminal extracellular vesicle surface glycosylation and alter lectin binding across extracellular vesicles of different sizes.