Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed.

Current analysis of exosomes focuses primarily on bulk analysis, where exosome‐to‐exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell‐line‐averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell‐line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non‐cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ – potential measurements have shown a decreased ζ – potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.