2.2. Preparation of Non-Targeted Flavonoid-Loaded Nanoemulsions

A previously optimized method for the preparation of nanoemulsions was employed [14]. The nanoemulsions formula contains the organic phase consisting of SPC (9.8 mM), Mal-PEG-DSPE (0.2 mM) and 0.5% v/v soybean oil, reconstituted in an aqueous phase containing glycerin, as surfactant. Lipid nanoemulsions were prepared by the ultrasonication method. Briefly, the protocol was carried out as follows: the organic phase, and a fixed volume of ethanol (60 µL) containing or not the flavonoid, was evaporated on a rotary evaporator (Laborota 4000, Heidolph) at 40 °C, in vacuum. The residual was resuspended in the aqueous phase, containing 1 mL of water and 10% glycerin. The coarse emulsion was sonicated for 10 min using a UPH200H probe-type sonicator from Hielscher. The nanoemulsions was further centrifuged using a 100 kDa cut-off Amicon centrifugal column to remove non-encapsulated flavonoid and traces of organic solvents. Light exposure was minimized to prevent the photodegradation of flavonoids. The lipid nanoemulsions were characterized by determining the size and zeta potential using a ZetaSizer NanoZS instrument (Malvern Instruments, Malvern, UK).

To fluorescently label the lipid nanoemulsions, 1.5% (mole/mole) Rhodamine-PE was added as an ethanol solution subsequent to LN preparation and incubated 30 min at room temperature in the dark.