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 int erleukin-1 activated human endothelial cells has been analysed by several s pectroscopical 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 physiolog ical conditions, we demonstrate that E-Selectin-directed immunoliposomes ar e able to bind their target cells under the simulated shear force condition s of capillary blood flow cumulatively for up to 18 It. In order to consequ ently follow the fate of liposomes after target binding, we analysed the ro ute and degree of liposome internalization of the cells concentrating on ce ll activation state or various liposomal parameters, e.g., sterical stabili zation, type of antibody or antibody coupling strategy. The use of NBD-labe lled liposomes and subsequent fluorescence quenching outside the cells with dithionite show that circa 25% of the targeted immunoliposomes were intern alized. According to inhibition experiments with agents that interfered wit h the endocytotic pathway, we found out that the major mechanism of liposom e entry is endocytic. The entry involves, at least in part, receptor-mediat ed endocytosis via E-Selectin, a liposome accumulation in the endosomes and their acidification was proved by pyranine spectroscopic results. Furtherm ore, microscopical investigations demonstrate that also a fusion of liposom es 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 de livery at inflammatory sites. (C) 2001 Elsevier Science B.V. All rights res erved.