2.3. Varying the sonication time during liposome preparation

DPPC liposomes were prepared at a total lipid composition of 25.0 mg/mL. Five samples were weighed out to a mass of 25.0 mg. The lipids were transferred to a 2.0 mL glass vial and 1 mL of buffer was added. To generate liposomes, the mixtures were sonicated with the probe-tip sonicator for 6, 12, 20, 28, and 36 min set to 20% duty cycle with a pulse length time of 2 s and a rest period 5 s for a total of 4 min. This was repeated one and a half times for the 6-min sample, three times for the 12-min sample, five times for the 20-min sample, seven times for the 28-min sample, and nine times for the 36-min sample. To avoid excessive heating the samples were rotated through one sonication cycle after 4 min. A 14-min rest was introduced in between the last two cycles for the 36-min sample to avoid excessive heating. The DPPC liposome solutions were then transferred to a 2 mL Eppendorf tube and centrifuged using a microcentrifuge (Eppendorf, Model 5424) at 10,000 rpm for 3 min to remove titanium particles introduced during sonication. The supernatant was transferred to a new 2 mL Eppendorf tube and samples were stored overnight at 4 °C. DSC measurements were carried out the following day. Samples were not stored longer than 16–20 h prior to carrying out the DSC measurements to preserve sample integrity and minimize liposome degradation. The stored liposome samples were removed from the refrigerator, left to equilibrate at room temperature for at least 1 h, centrifuged at 10,000 rpm for 3 min and degassed along with buffer for 30 min.

DSC measurements were carried out as described with a scanning temperature rate of 60 °C/hr beginning at 20 °C and ending at 70 °C. Samples were pre-equilibrated for five to 10 min at 20 °C prior to the initial scan.

After each measurement the raw tabulated data was imported to KaleidaGraph version 4.5 (Synergy, Reading, PA) and plotted. Baseline subtraction was carried out manually prior to generating thermograms and extracting the thermodynamic properties. The main phase transition temperature (T_m) was extracted and the peak morphology in each thermogram was analyzed for changes in the main phase transition temperature. Peaks arising from additional phase transitions were reported and summarized along with their thermodynamic parameters.