## 3 Results and Discussion
### Analysis of DOX-NPs (7)-Induced Genotoxic Effect in Cancer Cells  
Two mechanisms of action have been proposed by which doxorubicin acts in cancer cells: (i) intercalation in DNA and disruption of DNA repair; and (ii) generation of free radicals with subsequent damage in cell membranes, DNA, and proteins [34]. However, the confirmation of a genotoxic effect of these DOX-NPs (7) nanoformulations loaded with DOX in cancer cells confirms the in situ cytoplasmic release of the drug from the nanoparticle, and confirms that it reaches the nucleus; thus, the efficacy of the pH release strategy is corroborated. The genotoxic effect of the DOX conjugated to NPs (DOX-NPs (7)) was evaluated in the cell models studied, focusing on the damage caused in the DNA through detection of the phosphorylated form of the variant histone H2 AX (\(\gamma\)-H2 AX). The cell responds to DNA damage through the phosphorylation of thousands of H2 AX molecules flanking the damaged site. This highly amplified response can be visualized as a \(\gamma\)-H2 AX focus in the chromatin that can be detected in situ with the appropriate antibody (antiphospho-H2A.X.) using confocal microscopy analysis [28]. For this purpose, DNA damage in MDA-M-231, H520, and A549 cancer cells was determined by immunostaining of phospho-H2A.X foci (Figure 4). Untreated cells  
Figure 3: Effect of DOX-NPs (7) on cell viability. (**a**) Dose–response curves (percentage of cell viability versus concentration) of the treatment of A549, H520, and MDA-MB-231 cancer cells with DOX-NPs (7) represented in nM. The IC50 value was determined using the logarithm (inhibitor) versus normalized response: variable slope using the GraphPad software. (**b**) Bar graph showing cell viability of these three cell lines treated with DOX-NPs (7) and NAKED-NPs (1). (**c**) Comparison of therapeutic effect of DOX-NPs (7) compared to corresponding concentration of free doxorubicin (40,000 NPs/cell, 500 nM). Statistical significance was determined by Student’s \(t\)-test (* \(p\)-value \(<0.0001\)). The viability data represent the mean \(\pm\) SEM of the results of three independent experiments with six points each.  
(UNT) and cells treated with NAKED-NPs (**1**) were used as negative controls for this experiment. As expected, DNA damage was not observed when cells were not treated or treated with nanoparticles without the drug being loaded (Figure 4a,b). Conversely, cells treated with DOX-NPs (**7**) but without primary staining with \(\gamma\)-H2 Ax were analyzed to confirm the specificity of this assay. Noteworthy nanoparticles were detected (red dots) but no signal coming from unspecific immunostaining was observed (Figure 4c). In addition, the obtained results of specific immunostaining with \(\gamma\)-H2 Ax following the treatment of the cells with DOX-NPs (**7**) show that DNA damage can be clearly observed following staining with a secondary antibody for the green channel (green foci, Figure 4d). This result corroborated the efficiency of the pH-sensitive release strategy of the drug from the nanoparticles, since the drug must be released cytoplasmically so that it is possible to enter the nucleus where DOX-NP (**7**)-induced DNA damage occurs.