Differential scanning calorimetry (DSC)

DSC is commonly used in lipid studies to detect thermotropic phase transitions (most commonly of the gel-fluid sort) in fully hydrated lipid dispersions. Mid-point transition temperature (T_m), measured at the endotherm maximum, provides an indication of the stability of the gel phase, the higher T_m the more stable the gel phase. ∆T_1/2 is the transition width at mid-height, this parameter being related to the transition cooperativity, more cooperative transitions giving rise to narrower endotherms, i.e. smaller ∆T_1/2. ΔH, the change in transition enthalpy, is measured from the endotherm area (more specifically from the integration of c_P vs. T), and is highest for the transitions of a single component⁵⁵. For DSC measurements lipid vesicles were prepared by mixing the desired lipids dissolved in chloroform/methanol (2:1, v/v) and drying the solvent under a steam of nitrogen. The lipid film was kept under high vacuum for 90 minutes to ensure the removal of undesired organic solvent. Multilamellar vesicles (MLV) were formed by hydrating the lipid film with the buffer solution at 90 °C, helping the dispersion with a glass rod. The measurements were performed in a VP-DSC high-sensitivity scanning microcalorimeter (MicroCal, Northampton, MA, USA). Before loading the MLV sample into the appropriate cell both lipid and buffer solutions were degassed. 0.5 mL at 1 mM total lipid concentration was loaded into the calorimeter, performing 8−10 heating scans at a 45 °C/h rate, between 10 and 100 °C for all samples. bCrb concentration and sample volume were known for each sample, and used together with data from the last scan to obtain normalized thermograms. The software Origin 7.0 (MicroCal), provided with the calorimeter, was used to determine the different thermodynamic parameters (T_m, ∆T_1/2 and ΔH). The onset and completion temperatures of the transition were estimated from the scans, as the T at which the C_p values reached respectively 5% and 95% of the maximum C_p. Temperatures at the onset and completion of the endothermic phase transitions were used to build the phase diagrams.