Cyanine Lipids Promote the Shedding of Extracellular Vesicles from Cell Membranes

Extracellular vesicles (EV) have garnered significant attention in the fields of drug delivery, imaging, and immunotherapy. There is a need in methods to enhance release of EVs from cells. We found that at high labeling concentrations (100µM), indocarbocyanine lipids DiD and DiR that are commonly used for labeling cells, nanoparticles and EVs, promoted shedding of cell membrane tetraspanins with concomitant release of EVs in the medium. To further investigate this phenomenon, we screened a library of lipids and liposomal formulations for the release of membrane marker CD63 from THP-1 cells, and membrane red nanolantern (RNL) from 4T1 cells. We found a strong dependency of the EV release on lipid structure. In general, lipids that had a cyanine headgroup were more efficient than PEGylated phospholipids, neutral and cationic liposomes, with some lipids enhancing the release of CD63 up to 4-fold, and of RNL up to 8-fold, over vehicle treated control. A side-by-side comparison of cyanine lipid derivatives and corresponding precursor lipids confirmed that the cyanine headgroup significantly promoted shedding of RNL. Mutation of an exosome biogenesis regulator UNC13D did not hinder the release. Lipid-released EV could be modified with anti-interleukin 13 receptor alpha 2 antibody and targeted to glioma cells, suggesting potential utility in drug delivery. Furthermore, the impact of extraneously added lipids on cell membrane integrity should be carefully considered in cell labeling and drug delivery applications.