Effect of the development of a cell barrier on nanoparticle uptake in endothelial cells

In order to improve the current success of nanomedicine, a better understanding of how nano-sized materials interact with and are processed by cells is required. Typical in vitro nanoparticle-cell interaction studies often make use of cells cultured at different cell densities. However, in vivo, for their successful delivery to the target tissue, nanomedicines need to overcome several barriers, such as endothelial and epithelial cell barriers. Unlike sub-confluent or confluent cell cultures, cell barriers are tight cell monolayers, expressing a series of specialized tight junction proteins between adjacent cells to limit paracellular transport and ensure close cell-to-cell interactions. A clear understanding on how the development of cells into a cell barrier may affect the uptake of nano-sized drug carriers is still missing. To this aim, here, human primary umbilical vein endothelial cells (HUVEC) are used as a model cell line to form endothelial cell barriers. Then, nanoparticle uptake is assessed in the developed endothelial barriers and compared to the uptake in sub-confluent or confluent HUVEC cultures. The results clearly show that the organization of cells into a cell barrier leads to a differential gene expression of endocytic markers, and - interestingly - this is accompanied by reduced nanoparticle uptake levels. Transport inhibitors are used to characterise the mechanisms involved in the uptake. However, we show that some of them can strongly compromise barrier integrity, thus impairing the interpretation of the outcomes, and overall, only a partial inhibition of nanoparticle uptake could be obtained.