## 3 Results and Discussion
Interaction of GO-AuNP Hybrids with Neuroblastoma Cells: Cytotoxicity, Mitochondrial Dysfunction and ROS Generation  
GO nanosheets at high concentrations (>80 ug/mL) have a cytotoxic effect in human cancer neuroblastoma cells (SH-SYSY line) after long times of incubation (96 h) [6; 52]. In the same cell line, we previously reported that 12 nm AuNPs at the concentrations of 5 and 10 nM did not show any cytotoxicity after 24 h of treatment [53]. In order to compare the cytotoxic effect of the hybrid GO-AuNP on undifferentiated neuroblastoma SH-SYSY cell line with the bare AuNPs and GO sheets, cells were incubated for 24 h with the samples at different concentrations: 5, 10, 15 nM of AuNPs; 5, 10, 20 ug/mL of GO into the hybrids and 5, 10, 20 ug/mL of GO alone. Figure 5, in agreement with previously obtained data, shows that the synthetized gold nanosystems and GO alone, do not exhibit significant dose-dependent cytotoxicity at the tested condition. Differently, the hybrids GO-AuNP show a clearly dose-depended cytotoxicity on neuroblastoma cell line with a significant cell viability reduction after the treatment with 10 and 20 ug/mL of GO into the nanocomposites. Figure 4: AC-mode atomic force microscopy (AFM) height images in air for (pellet 2) samples of: (**a**) AuNPs, (**b**) GO, (**c**) GO-Au-NP; \(z\) scale = 10 nm. The panels below each micrograph show the section analysis plots corresponding to the drawn line. Figure 5: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzoolium bromide (MTT) results of dose-response experiment on neuroblastoma SH-SYSY cell line. Cells were incubated for 24 h with AuNPs, GO-AuNP, and free GO nanosheets, at different concentrations: 5, 10, 15 nM of AuNPs; 5, 10, 20 ug/mL of GO into the hybrids; 5, 10, 20 ug/mL of GO alone. Results are presented as mean \(\pm\) SEM from experiments in triplicate and normalized with respect to the control untreated cells. Asterisk (*) represents the correlation significant at the \(p\leq 0.05\) level with respect to the control. These results suggest that the cytotoxic effect in response to the treatment with GO-AuNP hybrids is due to a common action of both GO nanosheets and AuNPs in a single platform. Probably, the hybrids pass through the cell membrane more easily than GO alone or/and directly move into the cell organelles thus inducing cytotoxicity. Physicochemical reactivity of nanomaterials leads to the formation of reactive oxygen species (ROS) and results in oxidative stress through the activation of oxidative enzymatic pathways [54]. Some studies demonstrate the cytotoxicity of graphene in neural pheochromocytoma-derived PC12, lung carcinoma A549 and breast cancer MCF-7 cells through the generation of ROS [55; 56; 57]. To analyse the molecular mechanisms involved in the toxicity of GO-AuNP we monitored oxidative stress by measuring the mitochondrial dysfunction and ROS levels through MitoSOX and DCF assays, respectively. Neuroblastoma cells were treated for 24 h in dose-dependent manner with AuNPs, GO-AuNP and GO. The results in Figure 6 show an increase of the MitoSOX emission signal in a dose-dependent manner after the treatment both with GO and with GO-AuNP. On the other hand, the cells incubation with AuNPs does not show any significant difference between treated and control cells. Noteworthy, the exposition to the hybrids GO-AuNP more significantly increases ROS production respect to GO alone. These data confirm our theory that the presence of gold nanosystems supports the entrance of GO sheets into the cancer cells and strongly influence mitochondria. The link between ROS overproduction and mitochondrial damage is well known [58]. Therefore, we analysed the levels of the overall ROS by DCF assay on neuroblastoma cells after the treatment with GO, GO-AuNP, and AuNPs for 24 h. As shown in Figure 7, the exposure to AuNPs slightly favours ROS generation (38-40 \(\pm\) 16-18%). Total in cell ROS production after the incubation with GO shows a trend similar to that shown by the mitochondrial O\({}^{\bullet-}\) level.