2.2. Preparation of Drug-Loaded Liposomes

Thermo-sensitive and thermo-resistant liposomes were prepared by the lipid film hydration and extrusion method. The lipid mixtures containing DPPC, HSPC and DSPE-PEG2000 in the weight ratios summarized in Table 1 were dissolved in chloroform and dried to a thin lipid film under a stream of N₂ gas, followed by incubation overnight under vacuum to remove residual solvent.

Lipid composition of the prepared liposomes in weight ratios. All liposomes are drug loaded.

Next, 100 mM (or 10 mM, 300 mM for sample LIPO1) of CuSO₄ solution was used to hydrate the lipid films to gain a total lipid concentration of 35 mg/3 mL, while mixing with a magnetic stirrer in a 60 °C water bath for 30 min. The resulting multilamellar vesicle (MLV) suspension was subjected to five cycles of freeze-and-thaw (5 min. each, freezing in liquid nitrogen, and thawing at 60 °C) before being extruded 13 times at 60 °C through a 100 nm polycarbonate membrane filter (Whatman, Springfield Mill, UK) via a LIPEX^® Extruder (LIPEX^® Biomembranes, Burnaby, BC, Canada) to generate large unilamellar vesicles (LUVs) encapsulating the Cu salt. Residual un-encapsulated Cu was removed by size exclusion chromatography by passing 2.5 mL liposome suspension through a desalting PD-10 column and eluting with 3.5 mL 100 mM HEPES buffer (pH = 7.8), prepared from Hepes-Na salt, and setting the pH by hydrochloric acid. The pH of the buffer was manually adjusted using S20 SevenEasy™ pH-meter (Mettler Toledo, Budapest, Hungary). The resulting liposomal formulations contained the indicated copper ion solution (typically an unbuffered solution that was at pH 4.5) inside, and the HEPES buffer pH 7.8 outside [35]. After the separation process, 0.1 M neocuproine hydrochloric acid (dissolved in MQ water) was added in 0.2:1 mol drug-to-mol phospholipid ratio and incubated overnight at room temperature for loading of the neocuproine into the liposome. The unencapsulated neocuproine, or copper-neocuproine complex was removed by loading 1 mL sample on a G-25 Midi-Trap column and eluting the purified liposome fraction with 1.5 mL sterile 0.9% NaCl solution. Dynamic Light Scattering (DLS), Microfluidic Resistive Pulse Sensing (MRPS), Differential Scanning Calorimetry (DSC), IR spectroscopy, and UV-VIS spectrophotometry were used to characterize the prepared liposomes. For the Cu determination, the liposomes were dissolved in methanol and diluted 10-fold by Milli-Q water. The copper content of the samples was measured by the TXRF method (see Section 2.12) using 1500 ng Ga (Gallium standard solution) as an internal standard. The neocuproine content and encapsulation efficacy of the neocuproine was measured by spectrophotometry. An aliquot (20 μL) was dissolved in 50 μL methanol and then diluted with deionized water to 100 µL to release encapsulated Cu and neocuproine from liposomes. The complex was reduced with ascorbic acid and the absorption of the samples was measured at 450 nm. Calibrating solutions were prepared with the Cu-neocuproine ratio 1:1. The encapsulation efficacy was calculated as a proportion of the neocuproine administered and recovered after column filtration. Neocuproine free liposomes were also prepared for IR study, apart from these, all of the liposomes in the following are drug loaded and PEGylated.