[66]. These authors hypothesised that AuNP-induced oxidative stress in the HL7702 human liver cell line is related to the binding of these NPs to endogenous antioxidants (GSH), leading to complete depletion

after 48 h. The increase in surface area associated with the decrease in size allows for more GSH binding and thus depletion. They also reported that the extent of oxidative stress depends on NP access to cytosolic GSH or mitochondrial GSH reserves. Hence, increased oxidative stress may occur with smaller NPs. This notion would explain the different levels of ROS production observed in this study, in particular the higher ROS levels elicited by Au[(Gly-Tyr-TrCys)2B] (the AuNPs present in the smallest hydrodynamic size, as shown by DLS). GF120918 Evidence of dark assemblies in Hep G2 cells exposed to AuNP Au[(Gly-Tyr-TrCys)2B] would suggest cellular interaction/internalisation; however, further studies are needed. Cells undergoing autophagy have clearly visible autophagosomes, which form around degraded cellular components. The dark assemblages present in Hep G2 cells after exposure to Au[(Gly-Tyr-TrCys)2B] resemble these autophagosomes. Li et al. [67] proposed a cell survival mechanism of autophagy upon exposure to AuNPs. This mechanism has been studied further by Ma et al. [68], who showed that AuNPs that are taken up and accumulate in lysosomes p38 MAPK assay induce autophagosome Fludarabine clinical trial accumulation through the blockage of the autophagy

flux. This observation supports the findings in this study for Au[(Gly-Tyr-TrCys)2B]. In this case, despite the high levels of ROS produced, the cells did not succumb to the same loss in viability as that these registered for the other NPs at 48 h of exposure. This phenomenon was observed only for cells exposed to the AuNP Au[(Gly-Tyr-TrCys)2B], thus suggesting that the unique state of

this NP in the culture medium influences the NP-cell interaction. In fact, AuNPs eliciting the lowest increase in ROS levels after 24 h also showed the greatest loss in viability after 48 h of incubation: exposure to Au[(Gly-Trp-Met)2B], Au[(Gly-Tyr-Met)2B] and Au[(Met)2B] reduced viability to 69%, 71% and 68%, respectively. These AuNPs all formed large agglomerates and had Met groups in their PBH-capping agents. Several considerations need to be made when studying NP toxicity. One must be aware that NPs may interact unfavourably with assay components. The AuNPs described herein absorb at the same wavelength as those used for the MTT cytotoxicity assay (570 nm) and NRU assay (550 nm). NP interferences with commonly used toxicity assays, such as NRU and MTT, have been reported previously [69, 70]. In addition, AuNP interference was also observed when carrying out the GSH/GSSG ratio assay. Care should be taken when interpreting results in order to avoid false positive results. One should also consider that the physico-chemical state of the NP under distinct assay conditions may also lead to differences in levels of interference.