*2.2. Preparation of Spermidine Nanomicelles (NS) and Fenretinide Nanomicelles (NF)*

Spermidine nanomicelles were prepared by mixing soy phosphatidylcholine (4 mmoles), glyceryl tributyrate (2 mmoles), spermidine (2 mmoles), 2-hydroxypropyl beta cyclodextrin (0.4 mmoles), and KOH 10 N (400 µL, 4 mmoles) to obtain a semisolid phase. Mixing to homogeneity was carried out by pressure and friction in a mortar grinder (RM 200 Retsch Verder, Italy) at 37 ◦C for 30 min at a 100 min−<sup>1</sup> rate. The resultant semisolid phase was dispersed in PBS pH 7.4 to 50 mg/mL, filtered through 0.2 µm cellulose acetate filters (Fisher Scientific, Pittsburgh, PA, USA), and dialyzed for 72 h (dialysis membrane Mw cutoff 10 KD, Fisher Scientific) against PBS pH 7.4. The dialyzed phase was lyophilized. The dry residue was reconstituted with water and filtered again through 0.2 µm filters to obtain 50 mg/mL NS, representing the final formulative dispersion that was stored at −22 ◦C until use.

Fenretinide nanomicelles were prepared according to a method previously reported [21–24] by mixing soy phosphatidylcholine (4 mmoles), glyceryl tributyrate (2 mmoles), spermidine (2 mmoles), 2-hydroxypropyl beta cyclodextrin (0.4 mmoles), and KOH 10 N (400 µL, 4 mmoles) to obtain a semisolid phase. Mixing to homogeneity was carried out by pressure

and friction in a mortar grinder at 37 ◦C for 30 min at a 100 min−<sup>1</sup> rate. Fenretinide (1.2 mmoles) was dissolved in ethanol (300 µL) and KOH 10 N (120 µL, 1.2 mmole) and was subsequently added to the mixture. Homogenization was carried out in a mortar grinder for 30 min at a 100 min−<sup>1</sup> rate. The resultant semisolid phase was dispersed in PBS pH 7.4 to 50 mg/mL, filtered through 0.2 µm filters, and dialyzed for 72 h (dialysis membrane Mw cutoff 10 KD) against PBS pH 7.4. The dialyzed phase was lyophilized. The dry residue was reconstituted with water and filtered again through 0.2 µm filters to obtain 50 mg/mL NF, representing the formulative dispersion that was stored at −22 ◦C until use.
