*3.1. Characterization of NS and NF*

Spermidine and fenretinide nanomicelles were obtained by dispersion in PBS of the semisolid mixtures made by phospholipids, spermidine or fenretinide, glyceryl tributyrate, and 2-hydroxypropyl beta cyclodextrin. The spontaneous self-assembling, in the aqueous phase, of the mixture components triggered nanomicelles formation and inclusion of spermidine (Figure 2) or fenretinide [21,22] in the amphiphilic nanomicelle matrix.

Particle size, polydispersity, and zeta potential were measured on the nanomicelle suspensions starting from 50 mg/mL and progressively diluting with PBS containing 10% (*v*/*v*) HS to simulate nanoparticle dilution in vivo after injection.

The mean diameter of the nanomicelles resulted optimally sized for tumor accumulation by the Enhanced Permeability and Retention (EPR) effect [42–46], being 148.4 ± 3.4 nm for NS and 154.1 ± 10.3 nm for NF. The polydispersity was always lower than 0.3, indicating good dimensional homogeneity (Table 1).

**Figure 2.** Schematic representation of the supramolecular organization of Nanospermidine main constituents: spermidine, phospholipids, 2-hydroxypropyl beta cyclodextrin.

**Table 1.** Physicochemical Characteristics of Nanospermidine (NS), Nanofenretinide (NF), and empty nanomicelles (No) in PBS at 50 mg/mL.


As is known, the EPR effect depends on the discontinuity of the tumor capillaries and the impaired lymphatic drainage in the tumor environment.

Capillary discontinuities, generally between 200 nm and 1.2 µm in diameter, have been shown to allow injected nanoparticles, smaller than 500 nm, to access tumor tissues by extravasation and accumulate due to reduced lymphatic drainage [47–50].

The surface of the nanomicelles was always negatively charged, as indicated by the zeta potential values (Table 1). Spermidine nanomicelles were characterized by the lowest absolute value of zeta potential in accordance with the alkaline character of spermidine which induces a decrease in the negative charge on the nanomicelle surface.

Dilution slightly increased the nanomicelle size (Figure 3A). The size increase was very low in the 50 to 1 mg/mL dilution range and higher between 0.5 and 0.05 mg/mL. The maximum size was obtained at 0.05 mg/mL, being 221.4 ± 23.7 nm for NF and

270.5 ± 16.1 nm for NS. This corresponded to diameter expansions of 43.67% for NF and 82.27% for NS. Size expansion at high dilutions could represent a favorable feature for nanomedicines because it could prevent retro-diffusion towards the venous circulation of the extravasated nanoparticles when their size increases due to the high dilutions provided by the tumor matrix.

**Figure 3.** (**A**) Dimensional stability of the Nanomicelles to dilution in PBS containing 10% HS. (**B**) Stability of the Nanomicelles to leakage of their Spermidine (NS) or Fenretinide (NF) content in PBS containing 10% HS.

Spermidine and fenretinide release from NS and NF at 24 h was 18.83% ± 5.13 and 11.82% ± 2.33, respectively (Figure 3B), indicating stability towards drug leakage in circulation for time frames longer than those required for nanoparticle accumulation in solid tumors by extravasation in accordance with the EPR effect.
