## 2 Materials and Methods
### Cell Culture and Viability Assay  
**Cell culture and maintenance**: Caco-2 (human epithelial colorectal adenocarcinoma), HepG2 (human liver hepatocellular carcinoma), and SV-80 (human lung fibroblast) cells were maintained in DMEM (Dulbecco's Modified Eagle Medium), MCF-7 (human breast adenocarcinoma) in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), and Y-79 (human retina retinoblastoma) in RPMI-1640 (Roswell Park Memorial Institute (RPMI) 1640 medium), supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, and antibiotics (100 U/mL penicillin and 100 \(\upmu\)g/mL of streptomycin) in an atmosphere of 5% CO\({}_{2}\) in air, at 37 \({}^{\circ}\)C with controlled humidity. Cell lines: Caco-2, SV-80, and Y-79 were acquired from Cell Lines Service (CLS, Eppelheim, Germany), HepG2 and MCF-7 were acquired from ATCC (ATCC, Rockville, MD, USA). All cell culture media and supplements were from Gibco (Alfagene, Lisbon, Portugal). **Cell preparation prior to cytotoxicity assays**: Confluent cells were subjected to trypsin treatment (except Y-79, which grows in suspension), as described [26]. Briefly, after trypsinization, cells were counted, diluted in culture media to achieve a density of \(5\times 10^{4}\) cells/mL and then were plated in 96-well plates (100 \(\upmu\)L/well). Cells were further cultured, for at least 24 h, to allow cell adherence and reestablishment of growth. Y-79 cells were aspirated from growing flasks using a serological pipette, placed in 15-mL sterile centrifuge tube and then centrifuged (to eliminate the supernatant). The pellet containing the cells was re-suspended in fresh culture media; as these cells form clusters. The cell cluster disaggregation was performed by applying gentle aspirations with a Pasteur pipette. After re-suspension and disaggregation, cells were counted, diluted to a density of \(1\times 10^{5}\) cells/mL, in culture media, and then were seeded in 96-well plates pre-coated with poly-\(L\)-lysine (100 \(\upmu\)L/well, i.e., \(1\times 10^{4}\) cells/well). Cells were left in culture for at least 24 h to allow cell adherence. **Cytotoxicity/viability assay**: Two different LNs formulations: CTAB-SLNs and DDAB-SLNs were assayed for cytotoxicity, at four concentrations, calculated as surfactant (CTAB or DDAB) concentration in the formulation, in \(\upmu\)g mL\({}^{-1}\): 66.0, 165.0, 330.0, and 660.0. Test solutions were prepared just prior to cell application, by dispersing the required volume of particles in FBS-free culture media to achieve the desired final concentration. Test solutions (100 \(\upmu\)L/well; in quadruplicates) were added to the cells, after removing the growing culture media, and cells were exposed for 24 h or 48 h to test solutions before the cell viability assessment using the Alamar Blue (Invitrogen; Alfagene, Lisbon, Portugal), as previously reported [27; 28]. Briefly, 24 h or 48 h after cell exposure, the incubating test solutions were removed and replaced by Alamar Blue solution (10% (v/v) in FBS-free culture media; 100 \(\upmu\)L/well). The absorbances, at 570 and 620 nm (reduced and oxidative form or resazurin, respectively [22]) were read \(\sim\)5 h after the addition of Alamar Blue solution. Data were analyzed by calculating the percentage of Alamar blue reduction (using the equations and procedures as previously reported [4; 26; 27]) and results were expressed as percentage of control (untreated cells). **Determination of IC\({}_{\mathbf{50}}\)**: The IC\({}_{\mathbf{50}}\) (half maximal inhibitory concentration or concentration that inhibits 50% of cell viability/proliferation) was calculated as reported by [PERSON] et al. [25], from each individual dose-response experiment using Excel or GraphPad Prism tolls (\(n=3\) independent experiments; each experiment in quadruplicate).