Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells

In the present study the cellular uptake of targeted immunoliposomes by interleukin-1 activated human endothelial cells has been analysed by several spectroscopical and microscopical fluorescence techniques. Previous in vitro experiments demonstrated that the targeting of immunoliposomes to vascular selectins is a potential way for a selective drug delivery at inflammatory sites. In attempts to further adapt the targeting experiments to physiological conditions, we demonstrate that E-Selectin-directed immunoliposomes are able to bind their target cells under the simulated shear force conditions of capillary blood flow cumulatively for up to 18 h. In order to consequently follow the fate of liposomes after target binding, we analysed the route and degree of liposome internalization of the cells concentrating on cell activation state or various liposomal parameters, e.g., sterical stabilization, type of antibody or antibody coupling strategy. The use of NBD-labelled liposomes and subsequent fluorescence quenching outside the cells with dithionite show that circa 25% of the targeted immunoliposomes were internalized. According to inhibition experiments with agents that interfered with the endocytotic pathway, we found out that the major mechanism of liposome entry is endocytic. The entry involves, at least in part, receptor-mediated endocytosis via E-Selectin, a liposome accumulation in the endosomes and their acidification was proved by pyranine spectroscopic results. Furthermore, microscopical investigations demonstrate that also a fusion of liposomes with the cell membrane occurs followed by a release of entrapped calcein into the cytoplasm. These observations gain insight into the behaviour of E-Selectin-targeted immunoliposomes and indicate that these immunoliposomes have great potential to be used as drug carriers for intracellular drug delivery at inflammatory sites.