2.2. Liposomal Preparation

The preparation of Cu(DDC)₂ liposomes followed the idea of Wehbe et al. (2016) and was modified as indicated (Figure 1) [8]. Liposomes composed of DSPC:Chol [55:45 molar ratio] and DSPC:Chol:DSPE-mPEG₂₀₀₀ [50:45:5 molar ratio] were prepared by the thin film hydration method [22] with subsequent hand extrusion [23]. Briefly, organic stock solutions were transferred at the indicated ratios into a round bottom flask and the solvent was removed via rotary evaporation. The flask was placed under vacuum for at least 2 h to remove residual solvent. The lipid film was hydrated with 1 mL of an aqueous CuSO₄ solution (Cu²⁺, 150 mM in aqua destillata, pH 3.5) resulting in a lipid concentration of 40 mM. After 30 min of swelling and agitation, the formed liposomes were extruded for 41 passages through a polycarbonate membrane with a pore diameter of 80 nm (Nuclepore^®, GE Healthcare Life Science, Chicago, IL, USA). Solvent removal, film hydration, and extrusion were performed at 65 °C which is 10 °C above the phase transition temperature of DSPC. Prior to the addition of DDC⁻, Cu²⁺ liposomes were separated from non-encapsulated Cu²⁺ via size exclusion chromatography (SEC). The SEC column, prepared with Sephadex G-50, was equilibrated with an EDTA containing sucrose buffer (SHE, 300 mM sucrose, 20 mM HEPES, 30 mM EDTA, pH 7.4). Hereafter, buffer exchange with an EDTA-free sucrose buffer (SH, 300 mM sucrose, 20 mM HEPES, pH 7.4) was performed via three centrifugation steps at room temperature (RT) and 3000× g for 1 h using Vivaspin^® Turbo 4 filtration units (100 kDa MWCO, Sartorius, Germany). To allow the diffusion of DDC⁻ into the liposomal interior, followed by complexation of DDC⁻ with the liposomal encapsulated Cu²⁺, the preparation was left at 25 °C for 10 min at 300 rpm in a shaker. Free DDC⁻ was removed via filtration units as described above with the final SH formulation buffer. The resulting Cu(DDC)₂ liposomes were prefiltered via a cellulose acetate (CA) 0.45 µm filter (VWR International, USA) to remove non-incorporated and precipitated Cu(DDC)₂. For cell experiments, the liposomes were sterile filtered under aseptic conditions through a 0.2-µm CA filter. Fluorescent-labeled liposomes (with 0.5% DiO, related to the total lipid amount) were used to perform flow cytometry experiments. For the DiO liposome preparation, DiO was added from an organic stock solution to the residual liposomal components in a round bottom flask. The lipid film was treated as described above. In this case, the lipid film was hydrated with 1 mL HEPES buffered saline (HBS, 10 mM HEPES, 140 mM NaCl, pH 7.4), resulting in a final lipid concentration of 20 mM. The liposomal dispersion was extruded through an 80-nm polycarbonate membrane (Nuclepore^®, GE Healthcare Life Science, USA) after 30 min of swelling and agitation and stored at 4–6 °C protected from light until use.

Schematic representation of Cu(DDC)₂ liposome preparation by generating Cu²⁺ liposomes followed by remote loading of DDC⁻. This method was modified from Wehbe et al. (2016) [8].