3.2. Liposome Preparation

Liposomes were prepared using the thin film hydration method. Briefly, lipids dissolved in chloroform with a total lipid mass of 5 mg were mixed in a round flask. The solvent was dried under vacuum at 50 °C on a rotatory evaporator. The lipid film obtained was hydrated with 1 mL of sterile PBS, while stirring and heated above the lipid melting point. This resulted in the formation of MultiLamellar Vesicles (MLVs) with various sizes and number of layers. Six freeze–thaw cycles in liquid nitrogen were then applied to the prepared liposomes in order to burst the MLVs into Large Unilamellar Vesicles (LUVs). The LUVs size was defined by extrusion through a porous membrane with a Mini-Extruder (Avanti Polar Lipids, Alabaster, AL, USA). Liposomes were heated above their phase-transition temperature (Tm), extruded through a 400 nm and then, a 100 nm pore diameter polycarbonate membrane using a MiniExtruder (Avanti Polar Lipids, Alabaster, AL, USA). The final liposome solution was stored at 4 °C for 4 weeks, without further extrusion.

MMAE-derivative was added to the lipid mixture in chloroform prior to drying. Liposomes containing a final concentration of 5 µM MMAE-derivative were prepared. This corresponded to 0.12% of the total lipid mass and a lipid/MMAE derivative molar ratio of 2500:1, at a molar percentage of 0.04%. Liposomes contained 20 molar % of fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 79.06% of phosphatidylcholine as follows 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), for DS, DP, DM and PO-MMAE preparations, respectively. Detailed liposome composition is given in Table 1.