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

Abstract

Ceramide 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 X cer = 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 X cer the pretransition temperature T p first increases, whereafter at X cer > 0.06 it can no longer be resolved. The main transition enthalpy Δ H remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to ∼63°C at X cer = 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 T m for DMPC. At X cer ≥ 0.03 a second endotherm is evident at higher temperatures, starting at 32.1°C and reaching 54.6°C at X cer = 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 X cer < 0.18, together with an increase in T p. In the gel phase the lamellar repeat distance d increases from ∼61 Å at X cer = 0.03, to 67 Å at X cer = 0.35. In the fluid phase increasing X cer from 0.06 to 0.35 augments d from 61 Å to 64 Å. An L β′ /L α (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5°C) when X cer > 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 X cer = 0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity.