An improvised one-step OptiPrep cushion ultracentrifugation method for outer membrane vesicles isolation of Klebsiella pneumoniae

Abstract Background Extracellular vesicles (EVs) play a crucial role in intraspecies and interspecies communication, significantly influencing physiological and pathological processes. Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria are rich in components from the parent cells and are important for bacterial communication, immune evasion, and pathogenic mechanisms. However, the extraction and purification of OMVs face numerous challenges due to their small size and heterogeneity. Results This study proposes an innovative strategy that combines traditional differential centrifugation (DC) with one-step ultracentrifugation (ODG) to develop a dual differential gradient centrifugation (DDGC) method for extracting outer membrane vesicles from Klebsiella pneumoniae. By comparing the DC and DDGC extraction methods, we found that OMVs extracted by DDGC exhibited more typical morphology, clearer backgrounds, and more uniform particle size distribution. The lipid polysaccharide (LPS) content in OMVs extracted by DDGC was significantly higher than that obtained by DC, and the outer membrane protein content was also greater, demonstrating enhanced biological activity. Biological activity assays indicated that OMVs extracted by DDGC showed stronger cytotoxicity to A549 lung epithelial cells, a significant decrease in cell viability, and higher levels of inflammatory factor expression(IL-6, TNF-α, IL-1β, and IL-8). Conclusion Our study demonstrates the advantages of the DDGC method in extracting K. pneumoniae OMVs, showing improvements in morphology, particle size distribution, protein content, and biological activity. This provides a solid foundation for further exploration of the biological functions of OMVs and their potential applications in the biomedical field.