2.2. Preparation of liposomes and Dox loading

Temperature-sensitive liposomes (TSL) were made from DPPC:DSPE-PEG2k:MPPC (86:4:10, molar ratio). Liposomes were prepared by the hydration and extrusion method as described previously. Lipids, at the indicated ratios, were dissolved in chloroform. The chloroform was removed under a gentle stream of nitrogen gas and, subsequently, the residual solvent was removed under vacuum overnight. The dried lipid was hydrated in 0.3 mL of 100 mM copper (II) gluconate including triethanolamine at 540 mM (pH 8.4) to prepare copper-TSL (Cu-TSL). The multi-lamellar lipid solution at a final concentration of 50 mg/mL was extruded above the phase transition temperature of the lipid mixture through a polycarbonate membrane with a pore diameter of 100 nm. Cu-TSL was separated from non-encapsulated copper/TEA by passing the extruded liposomal suspension through a spin column of Sephadex G-75 (5 × 1 cm, GE Healthcare, Biosciences, Piscataway, NJ) equilibrated with saline (0.9% sodium chloride). The hydrodynamic size and zeta potential values were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The average size and the polydispersity index (PDI) of the purified Cu-TSL were 108 nm and 0.078, respectively, and the zeta value was −25 ± 10 mV. Lipid concentration was measured using the Phospholipids C assay kit (Wako Chemicals USA, Richmond, VA) according to manufacturer’s instructions.

Following separation of Cu-TSL using a Sephadex G-75 column, a transmembrane gradient was created across the membrane that allows highly membrane permeable TEA to diffuse to the external liposomal environment, leaving Cu (P < 10⁻¹¹ cm/s) entrapped within the core of the liposomes. Upon incubation of Cu-TSL with Dox at a drug-to-lipid ratio of 0.2:1 (wt:wt), TEA diffuses through the lipid bilayer into the external liposomal environment and facilitates active loading of Dox into the core of the liposomes where it forms a complex with Cu at neutral pH and results in CuDox-TSL. A loading of 100% was achieved when Dox was added to Cu-TSL at a drug-to-lipid ratio of 0.2:1 (wt:wt) and incubated at 37°C for 1.5 h [25]. The resulting CuDox-TSL were then separated from non-encapsulated Dox using Sephadex G-75 spin columns. Loading of Dox into Cu-TSL did not affect the size and surface charge of the liposomes. The purified CuDox-TSL exhibited a rapid release of ~90% of Dox within 2 min at 42°C as assayed by fluorescence in a reduced pH buffer [25,33]. Additional details are provided in the supporting information.