2.2.3. Liposomes’ Drug Loading and Release Characterization

Two different assays were performed to assess the pharmacokinetics of the nanoparticles. Drug loading was performed to determine the quantity of IQ integrated into the lipidic membrane and was obtained with centrifugal concentrators (Sartorius, Göttingen, Germany; MWCO of 2 kDa). A standard calibration curve was established with different concentrations of IQ to further determine the amount of drug incorporated in the liposomes. The absorbance of the standard samples was measured by spectrophotometry at a wavelength of 318 nm with a UV–Vis spectrophotometer (Thermo Scientific™Evolution 220, Waltham, MA, USA) [28]. For the drug release assay, a standard calibration curve was established with different concentrations of IQ to further determine the exact amount of the drug released by the liposomes. The fluorescence of the standard samples was measured with a Horiba FluoroMax^® 4 Fluorometer, Osaka, Japan. A total of 100 μL of Lipo IQ were inserted in a Slide-a-LyzerTM (Thermo Fisher Scientific™, Waltham, MA, USA; MWCO of 3.5 kDa), and the dialysis device was further inserted in a 1.5 mL Eppendorf previously filled with 1 mL of a phosphate-buffered saline solution (PBS). The samples were under agitation at room temperature using a PTR-35 Multi-Rotator (Thermo Fisher Scientific™, Waltham, MA, USA), and 100 μL of the buffer was collected and replaced at different time points (0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, 24 h, and 72 h). The fluorescence of the collected samples (λ_ex = 318 nm and λ_em = 333 nm) was measured to further calculate the percentage of the drug released.