2.3. Preparation of PTX-Encapsulated Thermosensitive Liposomes

PTX-loaded liposomes were prepared by combining DPPG, DSPG, PC, cholesterol, and PEG-1500 in a molar ratio of 80:5:5:2.5:7.5 by the thin-film hydration (TFH) method, according to Romana et al. [39] and Bangham et al. [44]. The lipids (100 mg) and PTX (10, 15, 20, and 25 mg) were dissolved in a mixture of methanol, chloroform, and water (10 mL) with a ratio of 1:5:0.2 in a round bottom flask by gentle handshaking and sonication (1–2 min continuous sonication at 20,000 Hz in direct mode) to form a clear solution. The excipient-solvent mixture was subjected to vacuum evaporation at 60 °C for 3 h (BUCHI rotavapor R-124 and BUCHI water bath B-480) until complete evaporation of the solvents produced a thin drug-lipid film. This process was above the phase transition temperature (Tc) of the lipids (55 °C). The rotation speed was kept at 60 rpm.

The homogenous thin lipid film was further dried for an hour with flowing N₂ gas and kept under a vacuum in a hood overnight to remove the solvents completely. The resultant film was hydrated with 0.9% (w/v) sucrose in phosphate-buffered saline (PBS) for 2 h in a water bath (60 °C) with constant rotation at a slow speed. The hydrated lipid mixture was subsequently sonicated in an ice bath for 10 min. The drug-loaded liposomes were separated from the unencapsulated free drug by ultracentrifugation (32,000 rpm at 4 °C, Avanti JXN-30, Beckman Coulter Life Science, NSW, Australia). The collected pellets were suspended in the hydration media and extruded through polycarbonate filters (400–800 nm pore diameter) 8–10 times to obtain highly monodispersed (PDI < 0.25) and unilamellar liposomes. The final liposome product appeared as a clear, transparent, light blue-white suspension. The liposome suspension was stored in refrigerator conditions (4 °C) until used.