## 3 Results and Discussion
### Assessment of CTAB and DDAB-Decorated SLNs Cytotoxicity
* Indicates statistical significance (\(p<0.05\)) between control and different concentrations, for each condition (CTAB-SLNs and DDAB-SLNs). Statistical significance (\(p<0.05\)) between the effect of DDAB-SLNs and CTAB-SLNs, at same concentration, was found to all values (not indicated). \begin{table}
\begin{tabular}{c c c c} \hline \multirow{2}{*}{**Cell Line**} & \multirow{2}{*}{**Time**} & \multicolumn{3}{c}{**IC50 (\(\upmu\)g/mL) *} \\ \cline{3-4}  & & **CTAB-SLNs** & **DDAB-SLNs** \\ \hline \multirow{2}{*}{**Caco-2**} & 24 h & 8.91 \(\pm\) 0.020 & 983.99 \(\pm\) 97.01 \\  & 48 h & 8.72 \(\pm\) 0.119 & 845.20 \(\pm\) 54.98 \\ \hline \multirow{2}{*}{**HepG2**} & 24 h & 9.70 \(\pm\) 0.158 & 510.25 \(\pm\) 18.10 \\  & 48 h & 9.04 \(\pm\) 0.026 & 343.53 \(\pm\) 10.56 \\ \hline \multirow{2}{*}{**MCF-7**} & 24 h & 8.91 \(\pm\) 0.099 & 1121.14 \(\pm\) 104.28 \\  & 48 h & 9.11 \(\pm\) 0.099 & 869.88 \(\pm\) 62.45 \\ \hline \multirow{2}{*}{**SV-80**} & 24 h & 9.31 \(\pm\) 0.079 & 594.06 \(\pm\) 16.43 \\  & 48 h & 8.91 \(\pm\) 0.152 & 284.06 \(\pm\) 17.01 \\ \hline \multirow{2}{*}{**Y-79**} & 24 h & 8.72 \(\pm\) 0.059 & 1015.28 \(\pm\) 39.50 \\  & 48 h & 9.24 \(\pm\) 0.059 & 540.21 \(\pm\) 10.49 \\ \hline \end{tabular}  
* Values given in \(\upmu\)g/mL of CTAB or DDAB in formulation.  
\end{table} 
Table 3: IC\({}_{50}\) values calculated for each cell line exposed to CTAB-SLNs and DDAB-SLNs for 24 or 48 h (as indicated). Values represent the mean \(\pm\) S.D. (see methods for details). the toxicity was due to both the CTAB molecules surfacing Au-NRs and to those that were left in the suspended solution. CTAB molecules permeate cell membranes, enter the cell and damage the mitochondria resulting in depolarization, increased reactive oxygen species (ROS), and apoptosis. High and dose-dependent intracellular concentration of ROS was observed in HepG2 cells, either exposed to SLNs (containing CTAB as surfactant) or to CTAB alone, triggering oxidative stress and resulting in activation of intracellular antioxidant enzymes system [19]. The cytotoxicity of CTAB in human keratinocytes (HaCaT) and osteoblast-like (SAOS-2) cell lines, in the presence or absence of serum (FBS), was shown to be CTAB dose-dependent and was affected by the presence of FBS which clearly showed cell protection [15]. In the absence of FBS (the same conditions in which our incubations were performed), 200 uM (72.88 ug/mL) of surfactant reduced the metabolic activity of cells (SAOS-2 to ~20% and HaCaT to less than 10%), although in live/dead assays, at 200 uM only a slight decrease in SAOS-2 cells viability was observed [15]. Although the metabolic activity is low, the percentage of dead cells is still low, suggesting that, after 24 h incubation, cells still trigger the apoptotic events. On the other hand, DDAB-SLNs produced very low toxicity when compared to CTAB-SLNs (Figure 1 and Table 3), the cell viability being higher than 90% of control (for all tested cell lines), at 165 ug/mL DDAB (or, 261.51 uM; see Table 2), and higher than 50% for twice this concentration (330 ug/mL; also for all cell lines). On average, cell viability was still above 50% for Caco-2 (Figure 1A), MCF-7 (Figure 1C), and Y-79 (Figure 1E) cells exposed to 660 ug/mL DDAB-SLNs. While all cell lines are highly and equally affected by CTAB-SLNs (IC\({}_{50}\) \(<\) 10 ug/mL; identical to all cell lines), the effect of DDAB-SLNs is cell line-dependent, and shows an order of potency (at 24 h exposure): HepG2 \(>\) SV-80 \(>\) Caco-2 \(>\) Y-79 \(>\) MCF-7, the IC\({}_{50}\) obtained for HepG2 and MCF-7 exposure to DDAB-NPs being ~50-fold and ~130-fold, respectively, higher than for CTAB-NPs exposure (Table 3). These results translate a higher safety profile of DDAB when compared to CTAB.