Cellular Output and\nPhysicochemical Properties of\nthe Membrane-Derived Vesicles Depend on Chemical Stimulants

Synthetic liposomes are widely used as drug delivery\nvehicles in\nbiomedical treatments, such as for mRNA-based antiviral vaccines like\nthose recently developed against SARS-CoV-2. Extracellular vesicles\n(EVs), which are naturally produced by cells, have emerged as a next-generation\ndelivery system. However, key questions regarding their origin within\ncells remain unresolved. In this regard, plasma membrane vesicles\n(PMVs), which are essentially produced from the cellular plasma membrane\n(PM), present a promising alternative. Unfortunately, their properties\nrelevant to biomedical applications have not be extensively studied.\nTherefore, we conducted a thorough investigation of the methods used\nin the production of PMVs. By leveraging advanced fluorescence techniques\nin microscopy and flow cytometry, we demonstrated a strong dependence\nof the physicochemical attributes of PMVs on the chemicals used during\ntheir production. Following established protocols employing chemicals\nsuch as paraformaldehyde (PFA), <i>N-</i>ethylmaleimide\n(NEM) or dl-dithiothreitol (DTT) and by developing a modified\nNEM-based method that involved a hypotonic shock step, we generated\nPMVs from THP-1 CD1d cells. We systematically compared key parameters\nsuch as vesicle output, their size distribution, vesicular content\nanalysis, vesicular membrane lipid organization and the mobility of\na transmembrane protein. Our results revealed distinct trends: PMVs\nisolated using NEM-based protocols closely resembled natural vesicles,\nwhereas PFA induced significant molecular cross-linking, leading to\nnotable changes in the biophysical properties of the vesicles. Furthermore,\nour novel NEM protocol enhanced the efficiency of PMV production.\nIn conclusion, our study highlights the unique characteristics of\nchemically produced PMVs and offers insights into their potentially\ndiverse yet valuable biological functions.