Kinesis of a fluorescently labelled gold nanoparticle through the blood brain barrier of mice, in vivo, and in the murine glioblastoma cell line, GL261

Abstract

Glioblastoma multiforme (GBM) is a cancer originated from the uncontrolled proliferation of astrocyte cells. These cells are the most abundant cell type in the human brain. Several functions are attributed to astrocyte cells, such as: i. A constituent of the blood brain barrier (BBB); ii. Nourish neighboring cells, such neurons; iii. Keep the extracellular ions balance; and iv. Help in the repair and cicatrization of central nervous system (CNS) after injury. The glioblastoma multiforme is the most common primary brain tumor, accounting for 82% of those tumors and for 69% of all gliomas. It is one of the most devastating and lethal human cancer since it can kill those affected in a short time (by 15 months in average), even those submitted to the usual treatment. It is known that inflammation plays a role for the full development of glioblastoma multiforme. However, even we demonstrated gold nanoparticles (AuNPs) with 20 nm diameter, in average, reduces inflammation and oxidative stress, we did not observe reduction in the tumor volume in mice-carrying glioblastoma chronically treated with those nanoparticles (data not published). Then, in this study we aim to find out if AuNPs cross the blood brain barrier (BBB) of mice, in vivo, and the plasma membrane of the murine glioblastoma cell lineage, GL261. To answer the first question, C57Bl/6 mice 8 to 12-week-old will be used. The fluorescent marker DY-495 was covalently conjugated to the AuNPs (AuNP-DY), (final average diameter is 35.6 ± 2.1 nanometers and zeta potential, -7,7 ± 0,6 mV), and the resulting nanoparticles will be intravenously (IV) injected (1.9x10 to the power of 11 nanoparticles) and fluorescence measured, in vivo, by fluorescence intravital microscopy after performing craniotomy for pial vessels exposition. Fluorescence inside and outside blood vessels will be measured the following time points after AuNPs-DY injection: 10 minutes, 30 minutes and two hours. The same protocol will be used after albumine-fluorescein (albumine-FITC 150,000 MW) injection (at 10 mg/mL), which will be used as control (for the effect of craniotomy on the BBB). In order to clarify if AuNPs-DY can get into the glioblastoma GL261 cells and the mechanism of entrance, GL261 cells will be plated for 1 hour in contact with cell culture media containing 1% AuNP-DY, in presence or not of 8 mM methyl-²-cyclodextrin, 1 mM cytochalasin D, 250 ouabain, 0,5 M saccharose, 10 ug/mL filipin, or 5 mM amiloride, at 37oC or 4oC. Fluorescence will be visualized using fluorescence microscopy.