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

Abstract

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.

Investigation of Transfection Barriers Involved in Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines

“…Cell experiments were performed at both 37 °C and 4 °C in order to distinguish cellular uptake from adsorption of lipoplexes to the outside of the cell membrane or their fusion with the cell membrane. It has been described that no energy dependent internalization process can take place at 4 °C [18]. As the size of the DC-30 ® lipoplexes was determined to be in the range of 300 and 500 nm [5], we assumed internalization to be an active process.…”

Targeting of Drugs to ICAM for Treatment of Acute Lung Injury

“…The inducible expression of selectins has been used as a means to enable intracellular delivery to activated EC. For instance, anti-E-selectin targeted liposomes or conjugates have been found to internalize via clathrin-coated pits within EC, to enable intracellular delivery of anti-inflammatory dmes [176,177].…”

“…Moreover, sitespecific delivery vehicles targeted to endothelium through Eselectin (i.e. anti-E-selectin liposomes and conjugates) traffic rapidly to multivesicular bodies and other acidic compartments [176,177], which could considerably reduce the half-life of potential cargoes. In epithelial cells, IgA is internalized by clathrin-mediated endocytosis and subsequently transcytosed firom the basolateral to the apical plasma membrane [241].…”

Inhibition of pulmonary metastasis in a human MT3 breast cancer xenograft model by dual liposomes preventing intravasal fibrin clot formation

“…Efforts to prevent tumor cells from adhering to endothelial cells has mostly focussed on a competitive blockade of the E-selectin receptor on vascular endothelial cells [21,22]. In recent studies, we used liposomes bearing Sialyl-Lewis Xligands; these prevented the binding of different tumor cell lines to endothelial cells in vitro, but failed in vivo [23].…”