*3.4. ROS Increase in Treated Cells*

Treatment with nanospermidine elicited ROS increases in both tumor cell lines. ROS increase was proportional to nanospermidine concentration (Figure 13). The highest ROS levels were obtained in NLF at 200 µM nanospermidine.

The presence of fenretinide further increased ROS at each nanospermidine concentration analyzed. At 200 µM nanospermidine, fenretinide provided comparable ROS levels in both cell lines.

**Figure 13.** Effect of Nanospermidine (NS) and Nanospermidine in combination with Nanofenretinide (NS + NF) on ROS increase. ROS production was measured by H2DClFDA fluorescence in BR6 and NLF cells treated 4 h with Nanospermidine at concentrations corresponding to 50, 100, 150, and 200 µM spermidine or with Nanospermidine in combination with Nanofenretinide at 0.05 mg/mL corresponding to 10 µM fenretinide. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\*\* *p* < 0.01, \*\*\* *p* < 0.001).
