2.3. Characterization of Drug-Loaded Liposomes

The size and surface charge of liposome formulations were measured using the dynamic light scattering (DLS, Zeta sizer, Malvern Panalytical, Malvern, UK) instrument after diluting liposomes in DW. Encapsulation efficiency (EE) is the ratio of the amount of drug encapsulated in liposomes to the amount of drug injected. After centrifugation at 3000 rpm for 15 min, the supernatant was collected to separate Lipo + EVL from free drug, and 0.9 mL of methanol was mixed with 0.1 mL of the supernatant and sonicated for 15 min [34]. The quantification of Lipo + EVL was determined using a high-performance liquid chromatography (HPLC, LC 1100, Agilent, Waldbronn, Germany) with Agilent Eclipse column (5 micron, 4.6 × 150 mm C −18) at 50 °C. The mobile phase of water, acetonitrile, and methanol (15:45:40) was flowed at a rate of 1.0 mL/min. The profile was monitored at 278 nm wavelength.