Exploring FTIR Spectroscopy as an Orthogonal Approach for Detecting Processing-Induced Changes in Extracellular Vesicle Preparations

Extracellular vesicles (EVs) require complementary methods for their comprehensive characterization. This study investigates the potential of attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy to provide orthogonal information on alterations in EV preparations subjected to different processing conditions. EVs were isolated using a previously established nanobody-based immunoaffinity approach, and further subjected to lyophilization, repeated freeze–thawing, or heat treatment followed by sonication. The EV preparations were characterized using nanoparticle tracking analysis, transmission electron microscopy, biochemical assays, flow cytometry and ATR-FTIR spectroscopy. Although the overall FTIR spectral profiles remained broadly similar, multivariate and targeted spectral analyses revealed differences in the heat-and-sonicated preparation, which also showed the most pronounced changes observed by complementary characterization methods. The findings provide proof-of-concept evidence that ATR-FTIR can provide orthogonal information for the assessment of processing-induced changes in EV preparations.