2.2.1. PLGA Nanoparticles Preparation

Polymeric nanoparticles were prepared by the nanoprecipitation method [20]. For blank or empty NPs, 50 mg of biodegradable polymer (PLGA) was dissolved in 5 mL of THF, with 2 h magnetic stirring, and slowly added dropwise over a 2% w/v aqueous solution of an emulsion stabilizer, in this case polyvinyl alcohol (PVA). For drug-loaded NPs, the PLGA solution was mixed with 5 or 10 mg of the drug dissolved in 2.5 and 5 mL of THF/MeOH (4:1), respectively, prior to addition to the aqueous solution. For the solvent evaporation, the resulting emulsion was stirred for 2–3 h at room temperature (RT). NPs were collected by centrifugation (Sorvall RC-5C Centrifuge, SS-34 Rotor, Hampton, NY, USA) at 15,000 rpm for 10 min and washed out three times with deionized water. Finally, NPs were frozen at −80 °C in 50 mL Falcon^® tubes and lyophilized using a fast-freeze flask (Labconco Corp. 750 mL cat ref #10033692) at −45 °C and 3.2 × 10⁻² mbar pressure.