The completely dried lipid film was then hydrated with 2 ml of aqueous medium (7.5 mg/ml of carboxyfluorescein solution in PBS, pH 7.4). The sample and the extruder (Avanti Mini Extruder) were heated to 69 °C for 10 minutes and the sample was then vortexed to form polydisperse multilamellar liposomes. To increase the uniformity, the sample was extruded at least 21 times through a membrane with a pore size of 200 nm (at 69 °C).