*3.2. Effect of Free Spermidine and Nanospermidine on Cell Viability*

Treatment with free spermidine in the concentration range of 10 µM–2000 µM did not elicit any detrimental effects up to 1200 µM (Figure 4). The higher concentration of 1500 µM induced a slowdown of cell proliferation in NLF and a complete inhibition in BR6. At 2000 µM, free spermidine significantly reduced the 72 h cell viability in both cell lines.

**Figure 4.** Relative viability of BR6 and NLF cells treated with increasing concentrations of Free Spermidine for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\*\* *p* < 0.01, \*\*\* *p* < 0.001).

Nanospermidine had a tenfold higher effect than free spermidine in both tumor cell lines, as cell viability was significantly decreased at a nanospermidine concentration of 0.15 mg/mL corresponding to 150 µM spermidine (Figure 5).

**Figure 5.** Relative viability of BR6 and NLF cells treated with increasing concentrations of Nanospermidine (NS) for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\* *p* < 0.05, \*\*\* *p* < 0.001).

We had previously demonstrated the antitumor activity of nanofenretinide in these cell lines [21,22]; therefore, we evaluated the combination of nanospermidine with nanofenretinide to assess if the contribution of fenretinide could improve the cytotoxic effect of nanospermidine.

In combination with nanofenretinide, a slightly increased overall activity was obtained with nanospermidine (Figure 6) at the same concentrations that triggered cytotoxicity in single administrations: 0.15 mg/mL and 0.20 mg/mL nanospermidine, corresponding to 150 µM and 200 µM spermidine, respectively.

**Figure 6.** Relative viability of BR6 and NLF cells treated with increasing concentrations of Nanospermidine (NS) in the presence of Nanofenretinide (NF, 10 µM) for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\*\*\* *p* < 0.001) and versus NF (## *p* < 0.01, ### *p* < 0.001).

As a comparison, we evaluated the combination of free spermidine with nanofenretinide and, also, in this case, we observed increased activity at the concentrations that triggered cytotoxicity in single administrations: 1500 µM and 2000 µM spermidine (Figure 7).

**Figure 7.** Relative viability of BR6 and NLF cells treated with increasing concentrations of Free Spermidine in the presence of Nanofenretinide (NF, 10 µM) for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\*\*\* *p* < 0.001) and versus NF (## *p* < 0.01, ### *p* < 0.001).

To exclude any contribution of the nanomicelles to the improved cytotoxicity of nanospermidine, we evaluated the effect of empty nanomicelles on cell viability. No decrease in cell vitality was observed up to 0.2 mg/mL nanomicelles, corresponding to the maximum concentration used in this study. However, higher concentrations induced a slight but significant decrease in cell viability in both cell lines (Figure 8).

**Figure 8.** Relative viability of BR6 and NLF cells treated with Empty Nanomicelles at increasing concentrations for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\* *p* < 0.05, \*\* *p* < 0.01).

Finally, the effect of nanospermidine was evaluated in normal WS1 fibroblasts at the same concentrations used in the tumor cells (Figure 9). No decrease in viability was obtained up to 150 µM spermidine, corresponding to the cytotoxic concentration in tumor cells. At 200 µM, a 28.36% viability decrease was observed after 72 h.

**Figure 9.** Relative viability of WS1 fibroblasts treated with increasing concentrations of Nanospermidine (NS) for 24, 48, and 72 h assessed by MTT assay. Data are presented as percentage versus control cells (100%) (mean ± SD, *n* = 6) (\*\*\* *p* < 0.001).
