*2.5. MTT Assay*

The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell proliferation and availability. Briefly, this assay is based on the reduction of MTT to the insoluble formazan salt by the cellular dehydrogenase. For this reason, the amount of formazan produced is considered a good indicator of the number of viable cells in the sample [38,39]. To perform the MTT assay, the cells were seeded at <sup>10</sup> <sup>×</sup> <sup>10</sup><sup>3</sup> cell/cm<sup>2</sup> in 96 multiwell plates, and, after 24 h, they were treated with free spermidine at concentrations varying from 10 µM to 2000 µM and with NS at nanoparticles concentrations ranging from 0.05 to 0.2 mg/mL corresponding to spermidine concentrations between 50 µM and 200 µM. Cells were also treated with fenretinide nanomicelles in combination with both free spermidine and nanospermidine. These studies were performed at constant fenretinide concentration (0.05 mg/mL nanoparticles corresponding to 10 µM fenretinide) and increasing concentrations of free spermidine (10 µM–2000 µM) or NS (nanoparticle concentrations 0.05–0.2 mg/mL corresponding to 50 µM–200 µM spermidine). After 24, 48, or 72 h, 10 µL of a 5 mg/mL MTT solution were added to each well to a final concentration of 0.5 mg/mL. After 4 h at 37 ◦C in the dark, 100 µL of a solution containing 10% (*w*/*v*) sodium dodecylsulfate (SDS) and HCl 0.01 mM were added to each well to dissolve the insoluble purple formazan crystals and left overnight on a shaker. The absorbance of each well was read on a TECAN plate reader (Männedorf, Switzerland) at 570 nm. To avoid the turbidity interference of biological samples, the read absorbance was normalized by a second reading at 690 nM.
