2.2. Preparation of PC- and PS-based Liposomes and NCs

The NCs were obtained from nano-sized liposomes (LPs), which were prepared according to the film hydration method [19]. The liposomes were formulated as follows: the required amounts of phospholipids (60 mg) and cholesterol (20 mg) were dissolved in a dichloromethane/methanol mixture (20 mL of a mixture, 3:2 v/v). The obtained organic solution was evaporated under vacuum and the lipid film was hydrated by the addition of PBS (10 mL) using a mechanical stirrer (RW20 digital, IKA, Staufen im Breisgau, Germany) for 30 min in a water bath at a constant temperature of 37 °C for PC and 60 °C for PS. The resulting formulations were optimised by ultrasonication (3 min, two cycles of 90 s) in an ice bath to prevent lipid degradation. Subsequently, a gentle centrifugation (1205× g, 1 min) was performed to remove possible metallic particles released during the ultrasonication. The NCs were prepared from the nanoliposomes according to the trapping method, described by Asprea et al. [20]. Briefly, a 0.1 M solution of CaCl₂ was added drop-by-drop to the liposomal suspension under magnetic stirring (150 rpm, room temperature) until the formulation appeared cloudy, indicating the formation of NCs. The molar ratio between PC and CaCl₂ was 1:1, while the molar ratio between PS and CaCl₂ was 1:4.