Christe, L.; Haessler, A.; Gier, S.; Schmeck, B.; Jung, N.; Windbergs, M.

Bacterial membrane vesicles (BMVs) have attracted significant attention as highly efficient transport vehicles for molecules crossing biological barriers and as key mediators in infection processes. As interest in BMVs increases, the need for standardized isolation protocols and comprehensive analytical approaches becomes apparent. This study introduces Raman spectroscopy as a novel, chemically selective monitoring approach for the analysis of subtle biochemical changes in BMVs across different bacterial growth phases. BMVs derived from Pseudomonas aeruginosa, a Gram-negative human pathogen responsible for severe nosocomial infections, were isolated at six different time points and analyzed via established physicochemical and functional assays, as well as Raman spectroscopy. While established analytics revealed growth phase-dependent variations in protein content, surface charge, and immunogenic effects on human immune cells, Raman spectroscopy enabled the comprehensive analysis of molecular-level changes between isolation time points. Significant shifts in protein-to-lipid ratios, higher lipid saturation, and changes in protein secondary structure were detected in BMVs isolated from later growth phases. Further, the absence of spectral markers for nucleic acids enabled the identification of BMVs as outer membrane vesicles. These findings emphasize the critical influence of the isolation time point on BMV properties and highlight Raman spectroscopy as a powerful tool for semi-quantitative chemical profiling, revealing minuscule yet biologically significant changes in BMVs depending on isolation time points.