Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis

Extracellular vesicles (EVs) were isolated from Pectobacterium zantedeschiae culturing media using direct ultracentrifugation (UC), iodixanol cushion ultracentrifugation (ICUC), and iodixanol density gradient ultracentrifugation (IDGUC) techniques. The isolates were characterized with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A satisfactory correlation (R-2 > 0.94) between quantitative results obtained with BCA, NTA and CE was achieved only for isolates obtained with the IDGUC. The correlation between protein content and CE was proved to be related to the isolates' purity. The NTA was found unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC, due to the co-isolated particulate impurities. Moreover, the work reports polysaccharides, used as culturing media components, as a potential source of bias of quantitation with total protein content assay and NTA. The study demonstrates the advantageous selectivity of CE in quality control of EVs and its ability to differentiate subpopulations of EVs of Pectobacterium.

Automated summary

This study isolated EVs from Pectobacterium zantedeschiae culture media using different ultracentrifugation techniques and characterized them using BCA, NTA, and CE. The results showed that there was a satisfactory correlation between quantitative results obtained with BCA, NTA, and CE only for isolates obtained with the IDGUC. Moreover, NTA was found to be unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC.