# The Comparison between Different Extracellular Vesicle Isolation Methods by AFM-IR Nanospectroscopy

## TL;DR

This paper compares methods for isolating extracellular vesicles using a nanospectroscopy technique to understand how each method affects their molecular and structural properties.

## Contribution

The study introduces AFM-IR nanospectroscopy as a novel method to analyze EVs at the nanoscale and compare isolation techniques.

## Key findings

- AFM-IR reveals that SEC depletes biomolecules above 1150 cm–1, including α-helix and fatty acids.
- TEIR preserves protein conformational diversity but damages nucleic acids and glycoproteins.
- Ultracentrifugation preserves the most biomolecules but causes membrane structural damage.

## Abstract

Extracellular vesicles
(EVs) contain cell-type-specific
signatures
and have been proposed as biomarkers in various diseases. However,
due to their small dimensions, quantification and size distribution,
the biophysical characterization of these particles is challenging
and still controversial. The diffraction-limiting effect limits acquiring
spectral data on EV samples in the mid-IR range, making it impossible
to capture images and spectra at the nanoscale by conventional vibrational
spectroscopy methods. Here, we employed atomic force microscopy–infrared
nanospectroscopy (AFM-IR) technique to elucidate the molecular signatures
of EVs isolated from blood (serum and plasma), as well as the direct
correlation of their topography and biochemical signature with respect
to the isolation method. EVs were extracted from serum or plasma from
C57BL/6 male mice by Total Exosome Isolation Reagent (TEIR), ultracentrifugation,
and size-exclusion chromatography (SEC), and evaluated by microreflectance
in FTIR and AFM-IR analysis. The multivariate analysis by PCA and
PLS-DA showed that phosphate groups could be significantly altered
depending on the EV isolation/purification method, as well as glycosidic
linkages. The SEC processing strongly depletes the amount of biomolecules
whose bands fall in the region above 1150 cm–1,
which includes α-helix and fatty acid content of EVs, while
the β-sheet and random coil/turn/loop conformational content
of proteins, nucleic acids, phospholipids, carbohydrates, and glycoproteins
is almost preserved for SEC. The TEIR method can sustain the diverse
fractions of conformational content of proteins at the cost of damaging
nucleic acids, phospholipids, carbohydrates, and glycoproteins. Still,
the UC processing was able to preserve the largest amount of biomolecules
present in EVs. However, structural damage on membranes was observed,
e.g., smearing out of phospholipids related to membrane-packing bands
(1720–1800 cm–1 region) and an overall decrease
in protein bands decreasing.