## 3 Results and Discussion
### Evaluation of the Efficiency of Cellular Uptake of DOX-NP (7)  
To investigate the efficiency of nanofection (cellular uptake and internalization) of DOX-NPs (7),) three cancer cell lines were chosen: two from lung cancer (A549 and H520) and one from triple negative breast cancer (MDA MB 231). The selection of these cell lines was based on the fact that doxorubicin is routinely used as a first-line chemotherapeutic agent in the treatment of these types of cancer [29; 30; 31]. For this purpose, the number of nanoparticles per cell needed to cause 50% of the cells to be nanofected (MNF50 index) was calculated in order to quantify the nanofection capacity of DOX-NPs (7)) following a protocol previously reported by us [26]. Nanofection efficiency of the DOX-NPs (7) in these three cell lines was quantitatively determined by flow cytometry based on the intrinsic red fluorescence of DOX. To this end, different concentrations of these nanoconjugates were incubated for 24 h, in an increasing gradient of concentration with between 50 and 10,000 NPs added per cell. After the incubation time, the cells were washed, trypsinized, and analyzed by flow cytometry. The results obtained on the cellular nanofection of DOX-NPs (7) revealed that they are internalized efficiently by the cancer cells studied. As shown in Figure 2a, a concentration-dependent effect on uptake efficiency was observed in the three cell lines. The analysis of the data showed that these three cell lines demonstrated a very similar MNF50 index, with values of \(1720\pm 69.2\) in the A549, \(1328\pm 69\) in the H520, and \(1385\pm 68.4\) for the MDA MB 231 cells (Figure 2b). However, the analysis of the increase in the median fluorescence (AMFI) indicated that, although the MNF50 was similar in the three cell lines and the internalization capacity gradually increased as the number of DOX-NPs (7) increased, starting at 2500 NPs/cell, the behavior of the lung cancer lines became different from that of the breast cancer cell line; the fluorescence intensity in the MDA MB 231 cells doubled by increasing the concentration range until reaching the saturation level. While the internalization of DOX-NPs (7) in the lung cells lines demonstrated a similar behavior, it was not the same, since the cellular entry was higher in the A549 without reaching the saturation point in both cases (Figure 2c). The cellular uptake of DOX-NPs (7) was also verified by confocal fluorescence microscopy. Figure 2d shows that cell internalization occurred efficiently in the three cell lines tested, confirming the cytoplasmic localization of DOX-NPs (7). Furthermore, to corroborate the intracellular location of DOX-NPs (7) and to discard any adsorption of these NPs onto the cell surface, a confocal microscopy analysis was carried out. Confocal microscopy image of A549 cells treated with DOX-NPs (7) showed that the location of these nanoparticles was intracellular (Figure 2e). Figure 2: Evaluation of the cellular nanofection of DOX-NPs (**7**) in the cell lines A549, H520, and MDA MB 231 by flow cytometry. (**a**) Dot plots representative of flow cytometry obtained by incubating different concentrations of NPs for 24 h in the cell lines. (**b**) Percentage of cells containing the different concentrations of DOX-NPs (**7**). The data (mean \(\pm\) SEM) are represented with a hyperbola equation model showing the different degrees of cellular internalization according to the number of DOX-NPs (**7**). (**c**) Representation of bars to compare \(\Delta\)MFI between cell lines: A549 (blue), H520 (red), and MDA MB 231 (green). The arrow indicates the MNF50. The experiments were carried out in triplicate and the results are expressed with the values of the mean \(\pm\) SEM. The statistical significance was determined by the analysis of the variance of a factor (ANOVA) using the Bonferroni multiple comparison (* \(p\)-value \(<\) 0.05, **\(p\)**-value \(<\) 0.001, ****\(p\)**-value \(<\) 0.0001). MNF50: Multiplicity of nanofection 50.