2.4.3. Determination of Phospholipid Content of Lipid Vesicles

The method of Rouser et al. was used to determine the amount of phosphatidylcholine (PC) incorporated into the different vesicles (*n* = 3) [53]. Briefly, 100 µl of the liposomal aqueous samples was heated at 270 ◦C until complete liquid evaporation, followed by addition of 450 µL of HClO<sup>4</sup> (70% *v/v*). Next, the mixture was heated to 250 ◦C for 30 min. After cooling down, 3.5 mL of water, 500 µL of ammonium molybdate (2.5% *w/v*), and 500 µL of ascorbic acid (10% *w/v*) were added. The mixture was vortexed and incubated at 100 ◦C for 7 min. After the tubes were cooled down, the absorbance was measured at 820 nm (spectrophotometer HITACHI U-2900, Milan, Italy).
