Thermosensitive Liposomal Doxorubicin Preparation

Thermosensitive Liposomal Doxorubicin (TSL-Dox) was prepared as described earlier [24 (link)]. Briefly 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), monostearoylphosphatidylcholine (MSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) (Avanti Lipids, Alabaster, AL, USA) (DPPC:MSPC:DSPE-PEG2000 = 85.3:9.7:5.0 mol%) were dissolved in chloroform and dried under a stream of atmospheric air at room temperature for forming a thin film. The lipids were then hydrated with 300 mM citric acid buffer (pH 4.0) and extruded 5 times at 55 °C (Thermobarrel extruder; Northern Lipids, BC, Canada) through a 100 nm filter. Active loading of doxorubicin into the liposomes was carried out by pH gradient with phosphate buffered saline (PBS, pH 7.4) outside the liposomes. The release kinetics of doxorubicin from the TSL was measured between 37 and 45 °C by a millifluidic device, as described earlier [42 (link),43 ].
A second batch of TSL was prepared based on the same lipid composition as above, with the addition of 0.1 mol% of Rhodamine headgroup-labeled 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (Rho-DPPE) to produce empty, fluorescence-labeled TSL. This second batch was used to quantify the removal of TSL by the filter.