4.1. Synthesis and Characterization of PDA NPs

PDA NPs were synthesized by the oxidation and self-polymerization of dopamine in a basic aqueous medium. Briefly, deionized H₂O (H₂O(d), 90 mL) was mixed with pure ethanol (40 mL) and NH₄OH (25–28%, 4.2 mL), and the resulting mixture was kept under vigorous stirring for 30 min. Then, dopamine hydrochloride (0.5 g) was dissolved in H₂O(d) (10 mL) and this solution was added to the previous mixture. The polymerization reaction was allowed to proceed for 24 h and after that time, PDA NPs obtained were isolated by centrifugation [13,17,18]. Next, four centrifugation–redispersion cycles in H₂O(d) were performed to eliminate any residue, and washed PDA NPs were finally resuspended in H₂O(d) in a final concentration of 2 mg/mL.

To characterize them, TEM images were taken (Tecnai Spirit Twin, Fei Company, Hillsboro, OR, USA) with a voltage acceleration of 120 kV. PDA NPs were resuspended in H₂O(d) in a concentration inferior to 0.01% WT, and drops of this dispersion were deposited on copper grids with a collodium membrane. They were allowed to dry for 24 h, TEM images were taken and size-range histograms were obtained after determining the size of at least 300 different PDA NPs (ImageJ software, NIH, Bethesda, MD, USA). In addition, PDA NP hydrodynamic diameter was also determined by DLS (Zetasizer Nano ZS90, Malvern Instruments Inc., Royston, Hertfordshire, UK) on the basis of their intensity-average size distribution. On this occasion, PDA NPs were dispersed in Trizma base solution (pH 10.0), also in a concentration lower than 0.01% WT.