Expanding the Analytical Toolbox for Extracellular Vesicle Biochemical Profiling: A Multiplatform Spectroscopic and Chromatographic Strategy.

The molecular characterization of extracellular vesicles (EVs) remains a major analytical challenge, particularly regarding lipid components that govern vesicle stability, biogenesis, and biological activity. Here we present an integrated workflow combining high-performance liquid chromatography with diode array detection (HPLC-DAD), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and gas chromatography-mass spectrometry (GC-MS) to profile EV lipids from both mammalian (human embryonic kidney HEK293T human cell line) and microalgal (Tetraselmis chuii) cell-derived EVs. Quantitative phospholipid analysis by HPLC-DAD and fatty acid profiling by GC-MS revealed phosphatidylserine as a conserved and selectively enriched lipid signature in EVs across phylogenetically distant species, along with system-specific differences in acyl chain saturation. FTIR spectroscopy was extended beyond conventional biochemical fingerprinting to derive spectroscopic indicators (including protein-to-lipid ratio, saturated/unsaturated balance, acyl chain length, and lateral packing) that are effectively related to EV sources. This multitechnique approach provides comprehensive structural readouts that translate complex molecular diversity into reproducible descriptors of extracellular vesicle identity and function. Our findings also identify distinctive lipid and spectroscopic signatures in microalgal EVs, reinforcing their potential as sustainable nanobiotechnological platforms.