2.4.2. Encapsulation Efficiency and Drug Loading

The encapsulation efficiency was determined by modified ultrafiltration centrifugation method [27]. In brief, 200 μL of initial L-OHP liposomes without dialysis was diluted 10 folds with 9% sucrose, then centrifuged at speed of 12000 rpm for 10 min at 4°C using Amicon® ultra-2 filter devices (Merck Millipore, 10,000 MWCO). The filtrate was collected and used to assess free L-OHP concentration. To evaluate the efficiency of this method, we quantified the recovery of L-OHP from free drug solutions and from the mixture of blank liposomes with free L-OHP (1 mg/mL) using the Amicon® devices. Another 200 μL from L-OHP-liposomal suspensions without dialysis was disturbed by 10% Triton X-100 at 60°C for 10 min to ensure the complete release of encapsulated L-OHP. After cooling at room temperature, the solution was diluted and the concentration of this solution was used to measure total L-OHP concentration. Free and total L-OHP were analyzed using a validated HPLC method (supplementary materials). The separation was performed in inertsil ODS C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was a mixture of water and methanol (95 : 5, v/v) at a flow rate of 1 mL/min. The detection wavelength was 250 nm, and the injection volume was 20 μL. C_free and C_tot are the concentration of nonencapsulated L-OHP and the total L-OHP concentration in the liposomal suspension without dialysis, respectively. The percentage of encapsulation efficiency (EE) was calculated as follows:

The drug loading (DL) was calculated according to the below equation:

where W_free and W_tot are the amount of nonencapsulated and total L-OHP, respectively, and W_lip is the weight of lyophilized liposomes.