## 3 Results and Discussion
### Photophysical Properties  
One of the most significant advances to be improved in PDT is the progression to the treatment of pathologies originating from deeper tissues, which can barely be treated with current porphyrin-basedphotosensitizers [61]. Thus, these light-sensitive compounds should possess photophysical properties such as high absorption capacity within the so-called \"phototherapeutic window\" (650-850 nm) and great singlet oxygen quantum yield. The photosensitizers that absorb in this electromagnetic spectrum region are the ones that can be the most easily excited in deeper tissues, since our body's constituents, such as proteins and other biomolecules, do not absorb radiation at this spectral range [62; 63]. Once the photodynamic activity of a photosensitizer may be due to two reaction types, type I or type II reactions [64], the singlet oxygen quantum yield measurement is relevant for this study since this value reflects the ability of new photosensitizing molecules to produce this highly cytotoxic ROS [55] and allows us to infer about the nature of the main reaction type to be produced in biological media [8]. All the prepared dyes exhibited sharp and intense absorption in the Vis/NIR region, matching the tissue transparency spectral region (Figure 1; Table 1). In DMSO, the four-membered central ring unsubstituted squaraine dye **10** displayed absorption at longer wavelengths than the aminosquaraine ones (**12** and **13**), in which the insertion of amine groups was found to produce a slight hypsochromic effect [4] (Figure 1. Compared to the non-iodinated analog dyes reported in our previous study [25], there is a bathochromic shift of at least 10 nm, so the introduction of this heavy atom influenced this photophysical property. When dissolved in Dulbecco's Modified Eagle Medium (DMEM), the dyes exhibited broader absorption bands, behavior possibly explained by the formation of aggregates in this aqueous medium [65] (Figure 1). The acquisition of these spectra in this culture medium is due to the need to construct a light source that emits radiation close to the maximum absorption wavelength of the dyes in the solvent where cytotoxicity assays were carried out so that the squaraines perform the maximum activity. \(O\)-methyl ether dye **11** was not entered in this photodynamic study, and it was not shown the cell culture media spectra since it easily hydrolyzes in aqueous medium yielding its unsubstituted precursor **10**. To evaluate the photosensitizing ability of the synthesized dyes, the singlet oxygen formation quantum yields were measured in chloroform, and phenazine was used as a standard for its \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Dye**} & \multicolumn{2}{c}{**DMSO**} & \multicolumn{2}{c}{**DMEM**} & \\ \cline{2-5}  & \(\lambda_{\text{max}}\) (nm) & \(\log\epsilon\) (M\({}^{-1}\).cm\({}^{-1}\)) & \(\lambda_{\text{max}}\) (nm) & \\ \hline
**10** & 726 & 5.26 & 640 & 0.03 \\
**11** & 677 & 5.25 & – & – \\
**12** & 705 & 5.17 & 619 & 0.03 \\
**13** & 715 & 5.18 & 637 & 0.05 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Relevant Vis/NIR spectral data and singlet oxygen quantum yields (\(\Phi_{\Delta}\)) of squaraine cyanine dyes **10–13**. Figure 1: Vis/NIR absorption spectra obtained in dimethyl sulfoxide (DMSO) and in Dulbecco’s Modified Eagle Medium (DMEM) dye solutions. Absorbance was normalized and is presented as arbitrary units (a.u.). determination, as already described [55] (Table 1). In general, all iodinated dyes displayed quantum yields of less than 0.1, which is considered very low. Thus, compared to the non-iodinated dyes that we reported [25] and contrary to what is described in the literature [9], the heavy atom introduction into the heterocyclic quinoline moiety does not produced an increase in the singlet oxygen production ability of these dyes.