# Theranostic Nanoplatforms of Thiolated Reduced Graphene Oxide Nanosheets and Gold Nanoparticles
## 3 Results and Discussions
### Biological Characterization of GO and GO@AuNP Nanocomposite Platforms  
The theranostic capabilities of the fabricated GO@AuNP were tested in terms of cytotoxicity on human neuroblastoma cells (SH-SYSY line).  
The cell viability results from MTT assays demonstrated that the cells' treatment with bwGO and rGOSH at the tested conditions did not induce cytotoxic effects (Figure 8). Such results are in agreement with literature data, which evidence only a slight cytotoxicity on SH-SYSY cells for treatments with GO at concentrations lower than 80 \(\mu\)g/mL and a decrease (about 20% than control untreated) in the cell viability after 96-h treatment with 100 \(\mu\)g/mL GO [24; 103].  
As to the cellular treatments with the nanoparticles, after 24 h of cells' incubation with AuNS or hybrid GO@AuNS, still no significant toxicity was detected (Figure 8a). On the contrary, the cellular treatments with AuNR or GO@AuNR induced a viability decrease of about 80% with respect to the control untreated cells (Figure 8b).  
To note, the size, shape, and surface functional groups are pivotal to drive bioavailability, uptake, subcellular distribution, metabolism, and degradation of the nanoparticle. In particular, according to many reports, gold nanoparticles in the size range of 4-18 nm, upon capping with agents like citrate, cysteine, glucose, and biotin, are nontoxic in several cell lines, including leukemia cells (nanoparticle concentration up to 200 micromolar (\(\mu\)M), neuroblastoma and brain glioblastoma (nanoparticle concentration lower than 10 nanomolar [57; 58]), and human immune system cells (nanoparticle concentration up to 100 micromolar [104]).  
On the other hand, the synthesis of rod-like gold nanoparticles used in the present work required a seed-mediated approach based on the use of CTAB cationic surfactant [105], which formed a bilayer at the nanoparticle surface, which can be toxic to cells even at sub-micromolar dose [106]. Moreover, CTAB drives the growth and the formation of the rod [107] and, simultaneously, inferring also a positive charge to the nanoparticle [108]. However, the bilayer structure of CTAB on the AuNRs has been demonstrated being able to generate defects on the cell membrane, inducing cell death [109]. Moreover, as a cationic surfactant, CTAB itself can easily pass through the cell membrane and target mitochondria, thus causing apoptosis [110]. It has been demonstrated that the cellular toxicity of CTAB-capped gold nanoparticles is significantly reduced when the nanoparticles are centrifuged and washed with water, to remove unbound CTAB [111].  
Although all the dispersions with rod-like gold nanoparticles used in this study were purified by centrifugation and water-washing (repeated a limited number of times for avoiding excessive aggregation) to remove the CTAB in excess (see Materials and Methods), the AFM and FTIR results discussed above rule out the presence of a significant residual amount of unbound CTAB. Hence, even if the concentrations of GO samples, nanorods, and related hybrid nanocomposites were scaled down for the cellular treatments, in comparison with the corresponding cellular experiments with AuNS, the cellular treatments either with bwGO@AuNR or rGOSH@AuNR nanocomposites resulted as still highly cytotoxic against the tumor cell line used in the present experiments.