*2.3. Characterization of the Nanomicelles*

Spermidine loading was evaluated in 50 mg/mL NS dispersions diluted (1:3, *v*/*v*) with an ethanol:water (1:1, *v*/*v*) mixture and analyzed by a fluorimetric method in comparison with the empty nanomicelles. The concentration obtained represented spermidine, both encapsulated in the nanomicelles and free in the aqueous phase. Therefore, to obtain the concentration of free spermidine, the nanomicelle dispersion was centrifuged in a 3.5 mL Ultra 5 KDa filter (Merck Millipore, Burlington, MA, USA) at 4000× *g* for 30 min and the ultrafiltrate was analyzed for spermidine content after dilution 1:3 with an ethanol:water (1:1, *v*/*v*) mixture. The difference between spermidine concentration in the nanomicelle dispersion and in the ultrafiltrate provided the concentration of the encapsulated spermidine. The nanomicelle loading was obtained as the ratio between the concentration (*w*/*v*) of the encapsulated spermidine and the concentration (*w*/*v*) of the nanomicelle dispersion.

The fluorimetric evaluation of spermidine in the nanomicelle dispersion and in the ultrafiltrate was carried out by a polyamine assay kit (Biovision, Milan, Italy) containing enzymes for the generation of hydrogen peroxide from spermidine and subsequent reaction of hydrogen peroxide with a fluorometric probe (Ex/Em = 535/587 nm) to yield a signal proportional to the amount of the polyamine present. The analysis was carried out according to the manufacturer's instructions.

Fenretinide loading was evaluated in 50 mg/mL NF dispersions diluted (1:3 *v*/*v*) with an ethanol:water (1:1, *v*/*v*) mixture and analyzed by UV spectroscopy (Shimadzu UV-1601) at 360 nm in comparison with the empty nanomicelles. The concentration obtained represented fenretinide, both encapsulated in the nanomicelles and free in the aqueous phase. To obtain the concentration of the free drug, the nanomicelle dispersion was centrifuged in a 3.5 mL Ultra 5 KDa filter at 4000× *g* for 30 min, and the ultrafiltrate was spectrophotometrically analyzed for drug content after dilution 1:3 with an ethanol:water (1:1, *v*/*v*) mixture. The difference between the drug concentration in the nanomicelle suspension and in the ultrafiltrate provided the concentration of the encapsulated drug. The nanomicelle loading was obtained as the ratio between the concentration (*w*/*v*) of the encapsulated drug and the concentration (*w*/*v*) of the nanomicelle dispersion.

Particle size, polydispersity, and zeta potential were measured at 37 ◦C (Malvern Nano-ZS Spectrometer, Malvern, UK) on the nanomicelle suspensions prepared in PBS and on the nanomicelle suspensions progressively diluted up to 1:100 (*v*/*v*) starting from 50 mg/mL concentration. Dilutions were made with PBS containing 10% (*v*/*v*) Human Serum (HS) to simulate the in-vivo dilution of the nanomicelles injected into the bloodstream. A minimum of 12 measurements per sample were made. Results were the combination of 3 10-min runs for a total accumulation correlation function time of 30 min. The results were volume-weighted.

Leakage of spermidine and fenretinide from NS and NF, respectively, was measured by dialysis at 37 ◦C, as previously described [21,22], with some minor changes. Briefly, the nanomicelles suspensions, prepared in NaCl 0.9% (*w*/*v*) at 50 mg/mL, were diluted to 10 mg/mL with pH 7.4 PBS containing 10% HS, and 1 mL of the diluted suspension was placed in a releasing chamber separated from a receiving compartment by a dialysis membrane (Mw cutoff 5KD, Fisher Scientific). The receiving compartment was filled with 10 mL pH 7.4 PBS containing 10% HS.

Leakage from the nanomicelles was determined by evaluating spermidine or fenretinide concentrations in the receiving phase at increasing time intervals. Spermidine concentration was obtained by the polyamine assay kit previously described, and fenretinide was evaluated spectrophotometrically by its maximum absorption wavelength (360 nm). Sink conditions were monitored throughout the experiment.
