Copper transport mediated by nanocarrier systems in a blood-brain barrier in vitro model

Copper (Cu) is a cofactor of various metalloenzymes and has a role in neurodegenerative diseases with disturbed Cu homeostasis, for example, in Alzheimers disease (AD) and Menkes disease. To address Cu imbalances, we synthesized two different dendritic nanoparticles (NP) for the transport of Cu(II) ions across the blood-brain barrier (BBB). The synthesized NPs show low toxicity and high water solubility and can stabilize high amounts of Cu(II). The Cu(II)-laden NPs crossed cellular membranes and increased the cellular Cu level. A human brain microvascular endothelial cell (HBMEC) model was established to investigate the permeability of the NPs through the BBB. By comparing the permeability × surface area product (PS_e) of reference substances with those of NPs, we observed that NPs crossed the BBB model two times more effectively than ¹⁴C-sucrose and sodium fluorescein (NaFl) and up to 60× better than Evans Blue labeled albumin (EBA). Our results clearly indicate that NPs cross the BBB model effectively. Furthermore, Cu was shielded by the NPs, which decreased the Cu toxicity. The novel design of the core-shell NP enabled the complexation of Cu(II) in the outer shell and therefore facilitated the pH-dependent release of Cu in contrast to core-multishell NPs, where the Cu(II) ions are encapsulated in the core. This allows a release of Cu into the cytoplasm. In addition, by using a cellular detection system based on a metal response element with green fluorescent protein (MRE-GFP), we demonstrated that Cu could also be released intracellularly from NPs and is accessible for biological processes. Our results indicate that NPs are potential candidates to rebalance metal-ion homeostasis in disease conditions affecting brain and neuronal systems.