In cancer radiotherapy, gold nanoparticles (AuNPs) have emerged as promising radiosensitizers, which, when accumulated in the tumor, increase the effectiveness of external beam radiotherapy by local production of reactive oxygen species (ROS) and secondary electrons upon irradiation. At UNamur, 5 nm gold nanoparticles coated with an organic shell of polyallylamine are produced by plasma vapor deposition (AuNPs@PPAA) (1). Optionally, the AuNPs@PPAA can be conjugated to anti-EGFR antibodies (Cetuximab) (AuNPs@PPAA-Ctxb) which actively target EGFR-overexpressing cancer cells in vitro and in vivo (2-3) and for which in vivo biodistribution studies have demonstrated a significant accumulation in the liver and the spleen. Therefore, the cytotoxicity profile of the gold nanoparticles should be properly investigated in healthy cell types prior to use the AuNPs@PPAA-Ctxb in clinical applications.
Human kidney (HK-2) cells and telomerase-immortalized microvascular endothelial (TIME) cells were studied as first examples of healthy cells. We performed MTS cytotoxicity assays after 3 hours and 24 hours of incubation and live cell imaging with apoptotic markers Annexin V and caspase 3/7 during 72 hours of incubation with AuNPs@PPAA(-Ctxb). Transmission electron microscopy (TEM) was performed to visualize nanoparticle uptake after 30 minutes, 3 hours and 24 hours in the presence of nanoparticles with concentrations ranging from 0.001 mg/ml to 0.05 mg/ml of gold.
TEM imaging demonstrated gold nanoparticle uptake in the cytoplasm already after 30 minutes of incubation with AuNPs@PPAA-Ctxb and a further increase after 3 hours and 24 hours of incubation. The MTS cytotoxicity assay showed a significant concentration-dependent reduction in cell viability of TIME cells after 3 hours and 24 hours of incubation with AuNPs@PPAA(-Ctxb), and of HK-2 cells after 24 hours of incubation. For the latter time point, TIME cells showed a lower EC50 value than that of HK-2 cells.
Using live cell imaging, we observed a significant increase in Annexin V labelling and caspase 3/7 activation in HK2 cells and TIME cells after 8 and 10 hours respectively in a concentration- and time-dependent manner, indicating apoptosis as the process of cell death. Co-exposure of cells with gold nanoparticles and a potent antioxidant significantly reduced or delayed apoptosis indicating that AuNPs@PPAA(-Ctxb) induced an oxidative stress.
In the future, we will quantify the cellular AuNPs@PPAA-Ctxb uptake by means of ICP-MS. Furthermore, ELISA and western blot will give more details on damages induced by the oxidative stress. Finally, the toxicity evaluation will be completed using a liver cell line.