Abstract
Microglia, the resident macrophages of the brain, become activated during glioblastoma multiforme (GMB) progression and release proinflammatory factors that drive tumor progression. Effective treatment of brain tumors is severely hindered by the blood‒brain barrier (BBB), which is characterized by tightly closed endothelial cells that prevent therapeutic agents from reaching the brain. Nevertheless, chitosan nanoparticles (CsNPs) are known to cross the BBB and confer neuroprotective and anti-inflammatory effects. Therefore, this study aimed to develop CsNPs linked to a lipophilic alkyl chain and assess their cellular uptake for brain drug delivery purposes. Chitosan was chemically modified with butyl glycidyl ether and labeled with fluorescein isothiocyanate (FITC) before generating NPs by ionic gelation. Cellular uptake studies of the NPs were performed on a murine SIM-A9 microglial cell line and an in vitro model of the BBB generated from hCMEC/D3 brain endothelial cells. In particular, microglia were treated with lipopolysaccharide (LPS) to simulate an inflammatory environment typical of brain tumors. Moreover, the intracellular pathways activated by NPs in microglia were investigated by treating cells with sucrose, an inhibitor of clathrin-mediated endocytosis (CME), and by labeling microglia with CD68, which is expressed by lysosomes. Finally, the trypan blue (TB) technique was used to quench the extracellular fluorescence associated with FITC. The results showed that butyl-C NPs were larger (456 ± 5 nm) than CsNPs were (170 ± 10 nm) but were still effectively taken up by the in vitro human BBB model and SIM-A9 cells, even under LPS-activated conditions. NPs did not undergo degradation, while treatment with sucrose did not prevent the uptake of NPs, suggesting that the NPs were engulfed by micropinocytosis. Finally, the TB technique showed that the NPs were effectively localized inside the cytoplasm of the microglia. These data suggested that the butyl chain increased the hydrophobicity, facilitating NP engulfment regardless of size. Due to their lipophilic component, butyl-CsNPs may encapsulate lipophilic drugs to reduce the inflammation associated with activated microglia.