Gel-gel phase separation within milk sphingomyelin domains revealed at the nanoscale using atomic force microscopy.

The milk sphingomyelin (MSM) is involved in the formation of ordered lipid domains in the biological milk fat globule membrane (MFGM), where it accounts for about 30%wt of the polar lipids. Moreover, MSM exhibits a large variety in saturated acyl chain lengths (from C16:0 to C24:0-SM) compared to other natural sphingomyelins, which may impact the packing of MSM molecular species in the gel phase domains and the topography of the MFGM. To investigate this, supported lipid bilayers of synthetic sphingomyelins or of MSM-containing mixtures, including a MFGM polar lipid extract, were imaged at temperatures below the Tm of MSM (i.e. <34°C for which MSM is in the gel phase) in hydrated conditions using atomic force microscopy. In all compositions containing MSM, the MSM-rich gel phase domains exhibited lower and upper height levels H, interpreted as two distinct gel phases with ∆H~0.5-1.1nm. Two (lower and upper) gel phases were also found for pure C24:0-SM bilayers or for bilayers of a C16:0-SM/C24:0-SM equimolar mixture, while C16:0-SM bilayers were uniformly flat and less thick than C24:0-SM bilayers. The upper gel phase of MSM-containing bilayers was interpreted as mixed interdigitated C24:0-SM molecules, while the lower gel phase was attributed both to fully interdigitated C24:0-SM molecules and non-interdigitated C16:0-SM molecules. These results show that the composition of natural sphingomyelins, inducing a mismatch between the d18:1 sphingosine and the acyl chains, is important in both the internal organization and the topography of biological membranes, especially that of the MFGM. This organization could be involved in specific biological functions, e.g. the insertion of proteins.

Mechanical Properties of Membranes Composed of Gel-Phase or Fluid-Phase Phospholipids Probed on Liposomes by Atomic Force Spectroscopy.

In many liposome applications, the nanomechanical properties of the membrane envelope are essential to ensure, e.g., physical stability, protection, or penetration into tissues. Of all factors, the lipid composition and its phase behavior are susceptible to tune the mechanical properties of membranes. To investigate this, small unilamellar vesicles (SUV; diameter < 200 nm), referred to as liposomes, were produced using either unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in aqueous buffer at pH 6.7. The respective melting temperatures of these phospholipids were -20 and 41 °C. X-ray diffraction analysis confirmed that at 20 °C DOPC was in the fluid phase and DPPC was in the gel phase. After adsorption of the liposomes onto flat silicon substrates, atomic force microscopy (AFM) was used to image and probe the mechanical properties of the liposome membrane. The resulting force-distance curves were treated using an analytical model based on the shell theory to yield the Young's modulus (E) and the bending rigidity (kC) of the curved membranes. The mechanical investigation showed that DPPC membranes were much stiffer (E = 116 ± 45 MPa) than those of DOPC (E = 13 ± 9 MPa) at 20 °C. The study demonstrates that the employed methodology allows discrimination of the respective properties of gel- or fluid-phase membranes when in the shape of liposomes. It opens perspectives to map the mechanical properties of liposomes containing both fluid and gel phases or of biological systems.

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid-ordered phase, determined using AFM force spectroscopy.

The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10-20 MPa and 4-6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0-5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.

Mechanical properties of milk sphingomyelin bilayer membranes in the gel phase: Effects of naturally complex heterogeneity, saturation and acyl chain length investigated on liposomes using AFM.

Sphingomyelin (SM) molecules are major lipid components of plasma membranes that are involved in functional domains. Among natural SMs, that found in milk (milk-SM) exhibits important acyl chain heterogeneities in terms of length and saturation, which could affect the biophysical properties and biological functions of the milk fat globule membrane or of liposome carriers. In this study, the thermotropic and mechanical properties of milk-SM, synthetic C16:0-SM, C24:0-SM and the binary mixtures C16:0-SM/C24:0-SM (50:50% mol) and C24:0-SM/C24:1-SM (95:5% mol) bilayer membranes were investigated using differential scanning calorimetry and atomic force microscopy, respectively. Results showed that acyl chain length, heterogeneity and unsaturation affected i) the temperature of phase transition of SM bilayers, and ii) the mechanical properties of liposome (diameter<200nm) membranes in the gel phase, e.g. the Young modulus E and the bending rigidity kC. This study increases our knowledge about the key role of naturally complex lipid compositions in tailoring the physical properties of biological membranes. It could be also used in liposomes development e.g. to select the suitable lipid composition according to usage.

Lipid domains in the milk fat globule membrane: Dynamics investigated in situ in milk in relation to temperature and time.

The microstructure of the milk fat globule membrane (MFGM) is still poorly understood. The aim of this study was to investigate the dynamics of the MFGM at the surface of milk fat globules in relation to temperature and time, and in relation to the respective lipid compositions of the MFGM from bovine, goat and sheep milks. In-situ structural investigations were performed using confocal microscopy. Lipid domains were observed over a wide range of temperatures (4-60°C). We demonstrated that rapid cooling of milk enhances the mechanisms of nucleation and that extended storage induces lipid reorganization within the MFGM with growth, leading to circular lipid domains. Diffusion of the lipid domains, coalescence and reduction in domain size were observed upon heating. Different MFGM features could be related to the respective cholesterol/sphingomyelin molar ratio in the three milk species. These structural changes may affect the interfacial properties of the MFGM, with consequences for the functional properties of fat globules and the mechanisms of their digestion.