ATR-FTIR analysis of temperature-dependent changes in extracellular vesicles and associated released components

Recent advances in vibrational spectroscopy techniques have proven to be reliable, fast and operator-independent tools for extracellular vesicles (EVs) study. In an attempt to further reveal the wide potential that the vibrational spectroscopy can achieve in EV characterization, in the present study we analysed the EVs isolated from various cell lines employing Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy. The acquired spectra of the EVs were noisy and affected by moisture after sample defrosting at 4 °C following storage at -80 °C, however a blow-drying pre-processing step could effectively remove water hence its influence on spectra, and EV distinctive signals were clearly identified. The analysis of specific band range (1800-650 cm^-1) from the ATR-FTIR spectra led to clear discrimination between the epithelial cells (EPI) EVs on one hand, and the hepatocellular cancer cells (HEP-G2) EVs and the embryotic kidney cells (HEK293) EVs on the other hand. We were able to detect signals corresponding to EVs after separating them from PBS and subjecting them to blow-drying without heat, followed by examination of alterations upon heat treatment. The fingerprint of compounds released by EVs after disruption at 60 °C was not detected under blow-drying conditions. However, when the filtrate was freeze-dried, the signal after disruption through heating at 60 °C was preserved, suggesting the importance of freeze-drying for maintaining compounds integrity. The comparative ATR-FTIR analysis of EVs and their released compounds revealed distinct molecular compositions, with EVs enriched in phospholipids and proteins, while the filtrates consisted of degraded fractions containing more homogeneous molecules.