GOLD NANOPARTICLES (AuNPS) IN VIVO AND IN VITRO NEURONAL INTERACTION

Drugs delivery into the brain across the blood brain barrier has always remained a challenging task for neuroscientists. Nanotechnology is now providing new prospective in the treatment of neurological cancer, neurovascular disorders and neurodegenerative diseases. This study aims to explore whether gold nanoparticles (AuNPs) are able to cross the blood brain barrier (BBB) and more importantly whether they have cytotoxic effects after in vivo and in vitro uptake. We selected AuNPs for their stability, size controlled synthesis, relatively easy surface modification with amine and thiol groups and capability to be studied at ultrastructural level with electron microscopy. Alkanethiol pyrazol was used to stabilize AuNPs. Thiol ligands strongly bind gold molecules due to soft character of both Au and S. Nanoparticles with a mean particle size < 2 nm were obtained. These nanoparticles had an absorption spectrum at 350 nm and showed a broad emission ranging from 400 to 600 nm depending on their quantum size since their emission shift from green to red when they form clusters of ~200 nm of diameter. In order to explore in vivo uptake of gold nanoparticles, 0.2 ml of saline suspension containing AuNPs (1x105) or physiological solution (as control) were injected into the carotid artery respectively of n=3 anesthetized mice. After 90 min from administration, the animals were sacrificed and the hindbrain slices were studied with an epifluorescence microscope. A strong particleassociated florescence was observed in the Caudato Putamen region (CPu) for AuNPs treated and not for untreated control. The fluorescence shows a more evenly spread distribution in the neuronal but not in glial cells, as shown by GFAP, Iba1 and NeuN immunoistochemistry. In order to explore in vitro cellular uptake the human neuroblastoma cells SH-SY5Y were incubated with 1 ml of saline suspension containing AuNPs (1x105) at 20 min, 1, 2 and 4 hrs. AuNPs internalization was observed through Leica microscope workstation B6000 equipped for in vivo time lapse analysis and, furthermore, confirmed by transmission electron microscopy observations. The effect of AuNPs was investigated both in vivo and in vitro using caspase-3 immunoreactivity and TUNEL test which showed apoptosis. In vivo many CPu neurons loaded with AuNPs were both caspase- 3 and TUNEL labelled as in vitro neuroblastoma cells whereas glial cells were AuNPs unloaded and caspase-3 or TUNEL negatives. Our data suggest that AuNPs cross the BBB and have particular imaging properties since they cluster into the cells changing the emission spectrum from green to red. Noteworthy AuNPs are cytotoxic so they are an attractive platform for neurological diseases if specifically addressed toward target cells.