Browsing by Subject "nanoparticle-mediated drug delivery"

Blood-brain barrier (BBB) is a physical barrier between the blood and the brain. BBB restricts drugs transport from blood stream to the brain, which sets challenges in drug delivery to the brain. Nanoparticles can be utilized in drug delivery to the central nervous system (CNS). Nanoparticles are internalized via endocytosis. However it remains unknown which endocytic pathways are active in brain endothelial cells. The characterization of BBB cells would help light on the exact mechanism of nanoparticle delivery into the brain, which would enable the design of targeted nanoparticles to deliver drugs to the CNS. In present study we characterized human brain endothelial cells, hCMEC/D3, which are widely utilized as BBB in vitro model. As brain endothelial cells are polarized in vivo, the aim of the study was to demonstrate the cell polarization of hCMEC/D3 cells and to study the activity and functionality of different endocytic pathways as a function of cell polarization. The level of cell polarization in cells grown on transwell permeable supports was characterized at multiple timepoints with four different methods: transepithelial electrical resistance (TEER) measurement, lucifer yellow permeability assay, alkaline phosphatase expression and ZO-1 expression. To characterize hCMEC/D3 cells for the presence of specific endocytic pathways, proteins involved into each pathway were selected. Expression of these proteins at mRNA level was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). For clathrin-mediated endocytosis, mRNA level of CHC protein was further correlated with the protein level of this protein, and the activity of clathrin-mediated endocytosis was analyzed by fluorescence activated cell sorting (FACS). Our results showed that hCMEC/D3 cells are best polarized after growing on transwell permeable support for 7 days. At the later timepoints, the cell polarization started to decrease, probably due to multilayer formation. We concluded that measuring TEER alone is not a reliable method to determine polarization status of the cells. mRNA levels of endocytosis-related proteins did not change remarkably as a function of cell polarization. In case of clathrin-mediated endocytosis, there was lack of correlation between CHC mRNA and protein level, but good correlation between mRNA level and activity of the pathway.