*2.2. PVA-PLGA Nanoparticle Production and Characterization*

NPs containing either synthetic Esc(1-21) or its diastereomer, Esc(1-21)-1c, at a theoretical loading of 2% (2 mg of peptide *per* 100 mg of NPs) were prepared by emulsion/solvent diffusion, as previously reported [27]. Briefly, 100 μL of an aqueous solution of each Esc peptide was added to a solution of uncapped PLGA 50:50 (Resomer® RG 502H, Mw 7000–17,000 Da, inherent viscosity 0.16–0.24 dL/g, Evonik, Germany) in methylene chloride (1 mL, 10 mg/mL) under vortex mixing (Reax top, Heidolph, Germany). The resulting water-in-oil emulsion was poured into 12.5 mL of ethanol to induce the production of NPs. Then, the NP dispersion was diluted with 12.5 mL of aqueous 0.1% (*w/v*) PVA (Mowiol® 40–88, average Mw ∼205 000 Da, 87–89% hydrolyzed, Merck, Italy) and kept under magnetic stirring for 10 min at room temperature. The residual organic solvent was evaporated under vacuum at 30 ◦C (Rotavapor®, Heidolph VV 2000, Germany). NP colloidal dispersion was collected, adjusted to a final volume of 5 mL, and centrifuged at 7000× *g* for 20 min at 4 ◦C (Hettich Zentrifugen, Universal 16R, Hettich, Germany) to isolate NPs. The resulting NP pellet was diluted in ultrapure water up to the desired final concentration. When needed, Esc-peptide-loaded NPs were freeze-dried, adding trehalose as a cryoprotectant, with a NP/trehalose ratio of 1:25 *w*/*w*, as previously reported [27]. Control bare PVA-PLGA NPs were prepared in the absence of Esc peptides.
