N4 coulter particle size analyzer

Particle size and zeta potential were measured using an N4 Coulter Particle Size Analyzer and Zetaplus (Brookhaven Instruments Corporation, Holtsville, NY), respectively. The particle size and surface morphology was further confirmed with a uranyl acetate stain using transmission electron microscopy (TEM). The liposomal drug concentrations were determined by reverse phase high performance liquid chromatography using an X-bridge C18 column on a Hitachi Elite LaChrom HPLC system. A mixture of 10 mM ammonium acetate buffer (pH = 4) (40%) and acetonitrile (60%) was used as the mobile phase with a 1 mL/min flow rate. The tariquidar had a retention time of about 3.2 minutes while that of the paclitaxel was about 5.3 minutes. Detection of both drugs was carried out using a UV detector (228 nm). Liposomal drug concentrations were calculated by comparison against a standard curve of each drug (0–20 µg/mL). The characterization of drug-loaded liposomes was described in Supplementary Table S1.

Particle size and zeta potential analysis was carried out using an N4 Coulter particle size analyzer (West Lafayette, IN) and Zetaplus (Brookhaven Instruments Corporation, Holtsville, NY) respectively. For particle size analysis, 5μL of the liposomal solution was mixed with 990μL of 1mM KCl while for zeta potential, 50μL was mixed with 1.5mL of 1mM KCl. For the determination of liposomal Dox content, the liposomes were dissolved in methanol at a dilution factor of 50 and the absorbance was measured at 480 nm. The drug concentration was determined by comparison with a standard curve of free Dox in methanol (0–60 μg/mL). To characterize the pH-dependent release of Dox from the liposomes, 0.8 mL of the liposomal Dox solution was dialyzed against 40 mL of either 300mM citrate buffer pH 5.0 or PBS pH 7.4 using a MWCO membrane of 12–14kDa for 96 hours. The release samples were taken at the mentioned time points while being replaced with equal volumes of fresh buffer and the Dox content in the release buffer was determined using HPLC (Hitachi Elite LaChrom, Pleasanton, CA). For the HPLC method, a Waters (Milford, MA) Cortecs^™ C18 column (2.7 μm, 4.6 mm × 150 mm) was used with a mobile phase of 60% (v/v) acetonitrile and 40% (v/v) of 0.1% trifluoroacetic acid in water with a flow rate of 1 mL/min. The Dox peak was determined by fluorescence (Ex/Em 445/550 nm).