## 3 Results and Discussion
### Photocytotoxicity Studies  
An efficient PDT photosensitizer needs to possess relevant photocytotoxicity and low dark toxicity [30]. The evaluation of the toxicity level of novel compounds using cell lines as in vitro models is recurrent, as they have prolonged life expectancy, phenotypic stability and easy handling [72]. HepG2 is a human hepatoma nontumorigenic cell line with high growth and proliferation rates that allows the hepatotoxicity and drug metabolism research [72]. The photocytotoxicity study in the human colorectal carcinoma cell line Caco-2 is of particular interest as these cells have morphological and biochemical characteristics similar to those of the intestinal epithelium, as well as being an ideal in vitro model of the third most common malignant pathology and second cancer-related cause of death [73; 74]. Thus, to determine the concentration range at which a in vitro phototherapeutic effect is produced, Caco-2 and HepG2 cells were exposed to increasing concentrations (0.1, 1.0, 5.0 and 10.0 \(\upmu\)M) of squaraine cyanine dyes **10**, **12** and **13**. Irradiation was executed by a self-made LED system centered at \(630.8\pm 0.8\) nm with a power of \(4.3\pm 0.5\) mW for periods of 7 or 14 min, and a non-irradiated control was also performed. Cell viability was evaluated at 1 h or 24 h after irradiation by using Alamar Blue, a reliable and straightforward colorimetric method, which has several advantages compared to standard techniques such as 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolzolium bromide (MTT) reduction assay, due to its high sensitivity, stability and minimal cell toxicity that allows continuous viability monitoring [75]. Dark and light cytotoxicity of culture medium containing the maximum DMSO concentration used were also assessed, and data confirmed that these did not influence the cell viability percentage (data not shown). All the squaraine cyanine dyes induced a decrease of Caco-2 cell proliferation and dose-dependent cytotoxicity was observed. Interestingly, the unsubstituted squaraine dye **10** showed the most significant phototherapeutic potential in the Caco-2 cell line compared to aminosquaraine dyes, since that at 5.0 and 10.0 \(\upmu\)M the presence of radiation was necessary to reduce cell viability in more than 30% (threshold level for cytotoxicity according to the Generally Recognised As Safe (GRAS) standards [76]) (Figure 4). The exposure times of Caco-2 cells to synthesized squaraine dyes showed to have an impact on dye cytotoxicity. Proof of this is the abrupt decrease in cell viability occurred at the concentration of 1.0 \(\upmu\)M, 23 h after 1 h exposure reading with the irradiated dye, as well as at 10.0 \(\upmu\)M without irradiation, regardless of the dye evaluated. The substitution of hydrogen atom by the methyl group did not potentiate the photodynamic activity of dye **13** since both aminosquaraines (**12** and **13**) showed very similar cytotoxicity results for the colorectal adenocarcinoma cells. The latter produced a good phototherapeutic effect at 5.0 \(\upmu\)M when cells were exposed for 1 h to irradiated dye and at 1.0 and 5.0 \(\upmu\)M when cells were analysed 24 h after exposure. Thus, the incubation of the cells over a more extended time period made it possible to increase the cytotoxic effect of these aminosquaraine dyes produced in vitro as photodynamic effect. Note that at the 1.0 \(\upmu\)M concentration and an incubation time of 24 h, aminosquaraines can only abruptly reduce cell viability at 14 min of irradiation, so that the amount of energy absorbed may be strictly linked to the photosensitizer candidates' biological action. The dyes showed different degrees of cytotoxicity depending on the cell lines under study.