## 3 Results and Discussion
### Photocytotoxicity Studies
These differences are due to the fact that each cell line is a system with its unique biological characteristics, nevertheless of whether they originate from the same tissue, species or histological tumor type, given the individual properties of the biological material from which the cell lines have been established and heterogeneity of the tumor cells [77; 78]. Among the three dyes, the one that stood out most in the HepG2 cell line was the unsubstituted dye **10** (Figure 5). This dye produces, compared to the Caco-2 cell line, a more evident photodynamic effect at 5 and 10 \(\upmu\)M concentrations, 1 h after irradiation. This effect is explained by the total absence of hepatotoxicityoxicity in the absence of radiation, which despite showing less than 30% decrease in cell viability, was statistically significant at the 10 \(\upmu\)M concentration for the colorectal carcinoma cell line. Figure 4: In vitro phototherapeutic effects of squaraine cyanine dyes **10**, **12** and **13** on Caco-2 cells. Cells were incubated for 24 h with the dyes at several concentrations (0.1, 1.0, 5.0 and 10.0 \(\upmu\)M) and then exposed to the radiation of a LED system centered at 630.8 + 0.8 nm and with the power of 4.3 \(\pm\) 0.5 mW for 0, 7 and 14 min. Irradiated dyes were in contact with the cells for 1 h or for 24 h. Data are expressed as a percentage of control (untreated cells) and presented as mean \(\pm\) standard deviation of three independent assays performed in quadruplicate. Data points without error bars showed no differences between the independent tests performed. Experiments with \(t\)-Student’s statistical significance compared to the negative control are signed by an asterisk (*) of the color relative to the irradiation time. In contrast to the Caco-2 cell line, in which aminosquaraine **12** showed therapeutic properties, this dye evidenced weak photodynamic activity for HepG2 cells, since the conditions in which light produces reduced cell viability also decreased in the absence of irradiation. Finally, the compound bearing methylamino group **13** shows a slight photodynamic activity at a concentration of 1 \(\upmu\)M under both irradiation conditions and times after irradiation. Similar effects of these dyes were produced in Caco-2 cells, but at a concentration of 5 \(\upmu\)M, so the compound showed higher potency for the hepatocyte cell line. Furthermore, the half-inhibitory concentration (IC\({}_{50}\)) of the evaluated squaraines was determined for each incubation and irradiation times of both studied cell lines by sigmoidal fitting analysis, and these values confirm the relevance and phototherapeutic ability of unsubstituted dye **10** (Table 3). Figure 5: In vitro phototherapeutic effects of squaraine cyanine dyes **10**, **12** and **13** on HepG2 cells. Cells were incubated for 24 h with the dyes at several concentrations (0.1, 1.0, 5.0 and 10.0 \(\upmu\)M) and then exposed to the radiation of a LED system centered at 630.8 \(\pm\) 0.8 nm and with the power of 4.3 \(\pm\) 0.5 mW for 0, 7 and 14 min. Irradiated dyes were in contact with the cells for 1 h or for 24 h. Data are expressed as a percentage of control (untreated cells) and presented as mean \(\pm\) standard deviation of three independent assays performed in quadruplicate. Data points without error bars showed no differences between the independent tests performed. Experiments with \(t\)-Student’s statistical significance compared to the negative control are signed by an asterisk (*) of the color relative to the irradiation time. Of the evaluated compounds, dye **10** has the highest IC\({}_{50}\) values in the absence of light, regardless of cell line and incubation time, indicating low dark toxicity, and clearly lower IC\({}_{50}\) values when irradiated, specifying good phototherapeutic response. The sensitivity of the hepatocyte cell line to aminosquaraine dyes **12** and **13** is evidenced by the significantly lower IC\({}_{50}\) values under most conditions compared to the colorectal cell line (Table 3).