Dimyristoylphosphatidylcholine/C16:0-Ceramide Binary Liposomes Studied by Differential Scanning Calorimetry and Wide- and Small-Angle X-Ray Scattering

AbstractCeramide has recently been established as a central messenger in the signaling cascades controlling cell behavior. Physicochemical studies have revealed a strong tendency of this lipid toward phase separation in mixtures with phosphatidylcholines. The thermal phase behavior and structure of fully hydrated binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and N-palmitoyl-ceramide (C16:0-ceramide, up to a mole fraction Xcer=0.35) were resolved in further detail by high-sensitivity differential scanning calorimetry (DSC) and x-ray diffraction. Both methods reveal very strong hysteresis in the thermal phase behavior of ceramide-containing membranes. A partial phase diagram was constructed based on results from a combination of these two methods. DSC heating scans show that with increased Xcer the pretransition temperature Tp first increases, whereafter at Xcer>0.06 it can no longer be resolved. The main transition enthalpy ΔH remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to ∼63°C at Xcer=0.30. Upon cooling, profound phase separation is evident, and for all of the studied compositions there is an endotherm in the region close to the Tm for DMPC. At Xcer≥0.03 a second endotherm is evident at higher temperatures, starting at 32.1°C and reaching 54.6°C at Xcer=0.30. X-ray small-angle reflection heating scans reveal a lamellar phase within the temperature range of 15–60°C, regardless of composition. The pretransition is observed up to Xcer<0.18, together with an increase in Tp. In the gel phase the lamellar repeat distance d increases from ∼61Å at Xcer=0.03, to 67Å at Xcer=0.35. In the fluid phase increasing Xcer from 0.06 to 0.35 augments d from 61Å to 64Å. An Lβ′/Lα (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5°C) when Xcer>0.03. With cooling from temperatures above 50°C we observe a slow increase in d as the coexistence region is entered. A sudden solidification into a metastable, modulated gel phase with high d values is observed for all compositions at ∼24°C. The anomalous swelling for up to Xcer=0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity