## 2 Materials and Methods
### In Vitro Photobiological Evaluation  
The human colon adenocarcinoma and hepatocellular carcinoma cell lines, Caco-2 (Cell Lines Service, Eppelheim, Germany) and HepG2 (American Type Culture Collection, Rockville, USA) respectively, were grown as a monolayer culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (_v/v_) heat-inactivated fetal bovine serum (FBS), 1 mM l-glutamine, 100 U/mL penicillin and 100 ug/mL streptomycin, at 37 \({}^{\circ}\)C in a humidified atmosphere of 5% CO\({}_{2}\). The medium of these two cell lines was changed every two days, and cells were related before reaching confluence. All reagents and consumables for cell culture were obtained from Gibco (Alfagene, Lisbon, Portugal). The general methods applied for handling these cell lines are already described in the literature [51; 52].  
At confluence, cells were trypsinized, counted, and resuspended in culture media at a density of 5 \(\times\) 10\({}^{4}\) cells/mL, and then 100 \(\upmu\)l/well of cell suspension was added to a 96-well culture plate for seeding and incubated at 37 \({}^{\circ}\)C under 5% CO\({}_{2}\). After 24 h of cell growth, the medium was removed, and the cells incubated with serum-free culture medium solution of differing squaraine dyes' concentrations (0.1, 1, 1.5 and 5 \(\upmu\)M). The maximum DMSO concentration used in cell viability assays did not produce significant effects on cell proliferation (data not shown). A negative control was performed exposing the cells only to the cell culture medium.  
After 24 h of squaraine dyes incubation, cells were exposed to three different irradiation conditions: (i) 0 min of irradiation (without irradiation; cells were protected from ambient light), (ii) 7 min of irradiation and (iii) 14 min of irradiation. The irradiation was performed with a self-designed and constructed apparatus consisting of an arrangement of LEDs of required wavelength. The device was built using aluminum gallium indium phosphide LEDs (B5B-445-TL, Roithner, Vienna, Austria) with a spectral emission peak at \(\uplambda=630.8\pm 0.8\) nm with \(P=4.3\pm 0.5\) mW, operating at 20 mA. The apparatus was placed over the 96-well plates, with the LEDs fronting the cells, and each LED irradiated a single well at approximately 1.5 cm away from the wellbore, as previously described [25]. After light-treatment, cells were incubated for 1 h or 24 h with irradiated dyes. Later the media containing the dye solutions was removed, and the cells were washed with phosphate-buffered saline solution (PBS; 137.0 mM NaCl, 2.7 mM KCl, 1.75 mM KH\({}_{2}\)PO\({}_{4}\), 10.0 mM Na\({}_{2}\)HPO\({}_{4}\), pH 7.4).  
Finally, in order to assess the photodynamic effects of squaraine dyes **10**, **12** and **13**, the culture medium containing the irradiated dyes was replaced by a 10% (\(\uplambda\)/\(\uppi\)) Alamar Blue solution diluted in a serum-free culture medium. After about 4 to 5 h of Alamar Blue incubation, absorbance at 570 (reduced form) and 620 nm (oxidized form) was read in a Multiskan EX microplate reader (MTX Labsystems, Vantaa, Finland). The data were analyzed by calculating the percentage of Alamar Blue reduction and expressed as a percentage of control (untreated cells), as previously described [51].  
The photobiological assays were performed in quadruplicate, and its data are expressed as mean value \(\pm\) standard deviation (mean \(\pm\) SD). Statistical significance was determined using a Student's \(t\)-test of the data of dye treatments and control, and differences were considered statistically significant for a \(p\)-value lower than 0.05. The IC\({}_{50}\) values determination was done from the concentration-response curves by sigmoidal fitting analysis considering a 95% confidence interval and with the bottom of the curves constrained to greater than 0% of relative cell proliferation. All data shown are representative of at least three independent experiments.