## 3 Results and Discussion
### Assessment of CTAB and DDAB-Decorated SLNs Cytotoxicity
The effect of different sets of SLNs containing different concentrations of DDAB was tested in colorectal carcinoma (HCT-116) and bronchial epithelial (16-HBE) human cell lines [7], showing that at the highest tested SLN concentration (500 ug/mL, ~40 ug/mL of DDAB), the loss of cell viability was ~20% for both cell lines. These concentrations are significantly lower than those tested in our work (Figure 1), showing that toxicity might also be dependent on the other components of the formulation (Precirol ATO 5 and the surfactants Brij 76 [7]) and/or of the cell line. The IC\({}_{50}\) values reported by [PERSON] et al. [7], together with the observed low toxicity, is in line with our results. The effect of DDAB in solution was assessed in 29 human and non-human cell lines, showing a cell-dependent toxicity, the Neuro2a (mouse neuroblastoma; EC\({}_{50}\) = 0.64 uM; 0.40 ug/mL) being the most affected with U-937 (Human leukemia; EC\({}_{50}\) = 2.12 uM; 1.33 ug/mL) and, the MDCK (dog kidney; EC\({}_{50}\) = 21.4 uM; 13.50 ug/mL) the least affected [31]. In this set of cells, A-172 (human glioblastoma; EC\({}_{50}\) = 15 uM; 9.46 ug/mL), Caco-2, HepG2, and MCF-7 showed EC\({}_{50}\)'s of 11.4 \(\pm\) 1.14, 13.4 \(\pm\) 0.80, and 9.63 \(\pm\) 4.11, respectively [31], indicating that nanoparticles are less toxic than the individual counterparts. [PERSON] and [PERSON] [31] attributed cytotoxicity of DDAB to the formation of pores in cell membrane leading to apoptotic events, either intrinsic or extrinsic. Other important and distinctive characteristic of these two surfactants is the critical micellar concentration (CMC; defined as the minimal concentration of surfactants that induces the spontaneous formation of micelles) [32; 33], which is ~1 mM to CTAB [34] and ~14 mM to DDAB [35]. It has been reported that cell viability decrease and cell lysis only occur at or near the CMC of the surfactant [36], therefore the lowest tested CTAB and DDAB concentrations were ~5-fold and 134-fold lower than the respective CMC. For this reason, we have observed a higher toxicity for CTAB as the used concentration is closer to the CMC. In order to counteract the surfactant-induced toxicity, a preventive measure is to decrease the concentration of free/unbound surfactant in nanoparticles suspension by, e.g., centrifugation as described by [PERSON] et al. [29]. However, this method does not prevent the CTAB-induced damage, as CTAB may eventually unbind from the nanoparticles [29; 37]. Other strategies include the application of a coat to the surfactant layer, creating a barrier between the nanoparticle and the cell membrane, thereby preventing the toxic interaction. This coating can be made of several components, such as proteins (e.g., BSA), polyelectrolytes (e.g., polystyrene sulfonate), or silica [37]. With the results here discussed, one should always have in mind that the components of SLNs might be as toxic as the encapsulated drugs. Thus, if not previously assessed adequately, the non-specific damage caused by SLNs components might be higher than the desired therapeutic effect. Thus, not only the cytotoxicity of the drug-loaded SLNs should be assessed, but also the blank (non-loaded) SLNs as their surface properties play a decisive role both in vitro and in vivo.