Neutron reflectivity measurements at the oil/water interface for the study of stimuli-responsive emulsions⋆.

Stable stimuli-responsive emulsions between oil and water are formed with an amphiphilic block copolymer bearing polystyrene (PS) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) moieties. Different kinds of emulsions like direct, multiple or inverse ones are reproducibly formed as a function of chemical parameters such as p H and salt concentration. To test the correlation between the different nature of the emulsion and the conformation of the polymer chain at the interface, neutron reflectometry at the oil/water interface was carried out. An original sample cell was built and the procedure to get reliable results with it on the FIGARO reflectometer at the Institut Laue-Langevin is described. Results show that for direct emulsions, the copolymer is much more extended on the water side than on the oil side. In the case where multiple emulsions are stabilized, the conformation is strongly modified and is compatible with a more equilibrated extension of the chain on both sides. The inverse case shows that the extension in oil is stronger than in water. These results are discussed in term of polymer brushes (charged or neutral) extension with respect to salt addition and hydrophobic interactions.

Directional K+ channel insertion in a single phospholipid bilayer: Neutron reflectometry and electrophysiology in the joint exploration of a model membrane functional platform.

We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Structure of a liquid/liquid interface during solvent extraction combining X-ray and neutron reflectivity measurements.

We have resolved the molecular structure of a bulk oil/water interface that contains amphiphilic ligand molecules using a combination of X-ray and neutron reflectivity measurements for the first time. This new capability can greatly impact future work in the field of ion separation by phase transfer, i.e. liquid/liquid extraction.

Interaction of amphotericin B with lipid monolayers.

Langmuir isotherm, neutron reflectivity, and Brewster angle microscopy experiments have been performed to study the interaction of amphotericin B (AmB) with monolayers prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and mixtures of this lipid with cholesterol or ergosterol to mimic mammalian and fungal cell membranes, respectively. Isotherm data show that AmB causes a more pronounced change in surface pressure in the POPC/ergosterol system than in the POPC and POPC/cholesterol systems, and its interaction with the POPC/ergosterol monolayer is also more rapid than with the POPC and POPC/cholesterol monolayers. Brewster angle microscopy shows that, in interaction with POPC monolayers, AmB causes the formation of small domains which shrink and disappear within a few minutes. The drug also causes domain formation in the POPC/cholesterol and POPC/ergosterol monolayers; in the former case, these are formed more slowly than is seen with the POPC monolayers and are ultimately much smaller; in the latter case, they are formed rather more quickly and are more heterogeneous in size. Neutron reflectivity data show that the changes in monolayer structure following interaction with AmB are the same for all three systems studied: the data are consistent with the drug inserting into the monolayers with its macrocyclic ring intercalated among the lipid acyl chains and sterol ring systems, with its mycosamine moiety colocalizing with the sterol hydroxyl and POPC head groups. On the basis of these studies, it is concluded that AmB inserts in a similar manner into POPC, POPC/cholesterol, and POPC/ergosterol monolayers but does so with differing kinetics and with the formation of quite different in-plane structures. The more rapid time scale for interaction of the drug with the POPC/ergosterol monolayer, its more pronounced effect on monolayer surface pressure, and its more marked changes as regards domain formation are all consistent with the drug's selectivity for fungal vs mammalian cell membranes.

Neutrons for rafts, rafts for neutrons.

The determination of the structure of membrane rafts is a challenging issue in biology. The selection of membrane components both in the longitudinal and transverse directions plays a major role as it determines the creation of stable or tunable platforms that host interactions with components of the outer environment. We focus here on the possibility to apply neutron scattering to the study of raft mimics. With this aim, we realized two extreme experimental models for the same complex membrane system (phospholipid : cholesterol : ganglioside GM1), involving two of the characteristic components of glycolipid-enriched rafts. One consists of a thick stack of tightly packed membranes, mixed and symmetric in composition, deposited on a silicon wafer and analyzed by neutron diffraction. The other consists of a free floating individual membrane, mixed and asymmetric in composition in the two layers, studied by neutron reflection. We present here results on the ganglioside-cholesterol coupling. Ganglioside GM1 is found to force the redistribution of cholesterol between the two layers of the model membranes. This causes cholesterol exclusion from compositionally symmetric ganglioside-containing membranes, or, alternatively, asymmetric cholesterol enrichment in raft-mimics, where gangliosides reside into the opposite layer.

Neutron reflectivity of supported membranes incorporating terminally anchored polymers: Protrusions vs. blisters.

The effect of terminally anchored chains on the structure of lipid bilayers adsorbed at the solid/water interface was characterized by neutron reflectivity. In the studied system, the inner leaflet, closer to the substrate, consisted of head-deuterated 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and the outer leaflet comprised a mixture of DSPC and polyethylene glycol (PEG) functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine. The DSPC headgroups were deuterated to enhance sensitivity and demarcate the bilayer/water interface. The effect on the inner and outer headgroup layers was characterized by w(1/2), the width at half-height of the scattering length density profile. The inner headgroup layer was essentially unperturbed while w(1/2) of the outer layer increased significantly. This suggests that the anchored PEG chains give rise to headgroup protrusions rather than to blister-like membrane deformations.

Membrane thickness and the mechanism of action of the short peptaibol trichogin GA IV.

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane.

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.

Biological physics near surfaces/interfaces: a perspective.

Biological physics at interfaces is an expanding field of research where a wide variety of disciplines fruitfully cross-link. The high degree of complexity of the involved systems requires the concerted application of many different and complementary techniques. Future success relies both upon development of new instrumentation and a better interaction of material scientists, physicists, molecular biologists and chemists.

Interfacial behavior of a hairpin DNA probe immobilized on gold surfaces.

Neutron reflection has been used in combination with electrochemical and quartz microbalance techniques to characterize a mixed monolayer of thiolated hairpin ss-DNA and 4-mercaptobutan-1-ol monolayer self-assembled on gold before and after hybridization with a cDNA target. Neutron reflection has revealed the opening of the stem-loop configuration of the probe associated with helix formation. This change in conformation correlates to the modification of the electron-transfer resistance associated with the [Fe(CN)6](3-/4-) redox marker present in solution.

Interaction of cationic lipid/DNA complexes with model membranes as determined by neutron reflectivity.

Transfection of cells by DNA for the purposes of gene therapy can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these complexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of these lipoplexes with model membranes composed primarily of zwitterionic lipid 1,2-dimyristoylphosphatidylcholine (DMPC) together with 10 mol % 1,2-dipalmitoylphosphatidylserine (DPPS). Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and complexed with a 1:1 charge ratio of DNA. The interaction of these complexes with the supported phospholipid bilayer was determined. DOPE-containing lipoplexes were found to interact faster with the model cell membrane than those containing cholesterol, and complexes containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the lipoplexes and the model membrane was characterized by an exchange of lipid between the membrane and the lipid/DNA aggregates in solution; the deposition of(additional) lipid on the surface of the model cell membrane was not apparent.

Lamellar stacking split by in-membrane clustering of bulky glycolipids.

We developed a simple model to investigate the effect of lipid clustering on the local interlayer distance in a cluster of interacting lamellae. The model, based on nonequilibrium thermodynamics and linear stability theories, explores the early stages of the lamella-lamella phase separation process where the lateral diffusion is much faster than the interlamellar lipid exchange. Results indicate, in the early stages, the presence of locally distorted regions with a higher concentration of one lipid component and an anomalous repeat distance. Experimental cases are presented, consisting of multilamellar-oriented depositions of phospholipids containing minority amounts of ganglioside or sphingomyelin under a low-hydration condition. The minority components are known to form domains within the phospholipid bilayer matrix. The low water content inhibits the lipid exchange among nearby lamellae and strengthens lamella-lamella interaction, allowing for a straightforward comparison with the model. Small-angle and wide-angle neutron diffraction experiments were performed in order to detect interlayer distances and local chain order, respectively. Lamellar stacking splitting has been observed for the ganglioside-containing lamellae, induced by in-phase lipid clustering. In excess water and after long equilibration times, these local structures may further evolve, leading to coexisting lamellar phases with different lipid compositions and interlayer distances.

Specular neutron reflectivity studies of the interaction of cytochrome c with supported phosphatidylcholine bilayers doped with phosphatidylserine.

Specular neutron reflectivity was used to study the time course and nature of the interaction of the positively charged, peripheral membrane protein cytochrome c with supported bilayers of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) containing the anionic lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine (POPS). The supported bilayers were prepared by deposition on silicon blocks of two monolayers of DOPC, the second of which contained either 10 or 20 mol % POPS at surface pressures of either 15 or 20 mN/m using a combination of Langmuir-Blodgett and Schaefer deposition techniques. Each supported bilayer was initially characterized by specular neutron reflectivity using subphases of 10 mM NaCl aqueous solutions. Regardless of POPS content and bilayer deposition pressure, the molecular architecture of the bilayers was similar. The addition of cytochrome c resulted in an almost immediate change in reflectivity, which was well modeled by assuming that an additional layer was present next to the outer leaflet of the bilayer. The thickness of this layer, which contained the volume fraction of approximately 15% protein, was approximately 30 A (consistent with the cross-section of a single cytochrome c molecule). The addition of cytochrome c to the subphase also resulted in a change in the structure of the phospholipid bilayer, suggesting some penetration of cytochrome c into the bilayer. Specular neutron reflectivity studies after careful washing with solvent showed that although most of the protein was washed off by flushing 10 mM NaCl D2O through the cell a small amount remained both within the bilayer and bound to the membrane surface.

Hydrated cholesterol: phospholipid domains probed by synchrotron radiation.

X-ray scattering experiments on mixed films of cholesterol and phospholipids at air-water and Si solid-water interfaces were undertaken to glean information on pathological crystallization of cholesterol bilayers. Grazing-incidence X-ray diffraction patterns at the air-water interface of various cholesterol:dipalmitoyl-phosphatidylcholine (Ch:DPPC) monolayer mixtures compressed beyond monolayer collapse yielded the established 10 x 7.5 Ų Ch bilayer motif, for Ch:DPPC molar ratios higher than 2.5:1. Attempts to obtain a diffraction signal from various Ch:phospholipid film mixtures at the Si solid-water interface, indicative of the presence of the Ch bilayer motif, were unsuccessful. Only after removal of sufficient water from the cell was a weak diffraction signal obtained suggestive of a cholesterol film two bilayers thick. Off-specular X-ray reflectivity measurements made on a 1.75:1 mixture of Ch and bovine cardiac phosphatidylcholine (BCPC) deposited as a bilayer on a Si wafer and placed in a cell filled with water yielded positive results. The derived electron density profile showed the presence of a bilayer mixture consistent with a phase separation of cholesterol and BCPC, and possible formation of a crystalline cholesterol bilayer within the hydrated mixed bilayer, but not a proof thereof.

Fibronectin adsorption studied using neutron reflectometry and complementary techniques.

In implantology it is known that fibronectin affects cell-substrate adhesion, consequently, the structure and composition of the initially adsorbed fibronectin layer to a large extent determines the biological response to a biomaterial implanted into the body. In this study we have used neutron reflectometry and quartz-crystal microbalance with dissipation to investigate the amount of fibronectin adsorbed, the layer density, thickness and structure of films adsorbed to polished silicon oxide surfaces. We have cultured MG63 osteoblast-like cells on surfaces coated and uncoated with fibronectin and monitored the cellular response to these surfaces. The results show that at fibronectin concentrations in the range 0.01 to 0.1 mg/ml a single highly hydrated layer of fibronectin approximately 40-50 Å in thickness adsorbs to a polished silicon oxide surface and is likely to correspond to one diffuse monolayer of fibronectin arranged side-on. Cells cultured on this fibronectin layer have dramatically different morphology and growth to those grown on bare surfaces. Using a model silicon oxide surface has enabled us to study the substrate/protein interface, together with the impact of a fibronectin layer on the cellular response using consistent experimental conditions across a unique set of experimental techniques.

Neutron reflectivity study of the kinetics of polymer-polymer interface formation.

We have investigated how the interface width between two thin polymer films approaches its equilibrium state. Neutron reflection for different polyolefin bilayers of various degrees of incompatibility as a function of the annealing time was measured. By tuning the interaction parameter, we have probed both an immiscible polymer couple and systems approaching criticality where the interface is wider. Since polymer chains have slow dynamics, we have observed the slow broadening of the interface connected to the growth at long times of the wavelength capillary-wave modes, which involve large-scale hydrodynamic flows.

Fluctuations and destabilization of single phospholipid bilayers.

Supported phospholipid bilayers are interesting model systems for biologists and present fascinating physical properties. The authors present an extensive experimental study of the dynamic properties of supported bilayers. The structure and the equilibrium properties of single and double supported bilayers were investigated with neutron reflectivity. The submicronic fluctuation spectrum of a nearly free "floating" bilayer was determined using off-specular x-ray scattering: the surface tension of the bilayer, its bending modulus, and the intermembrane potential could be determined. Using fluorescence microscopy, the authors showed that this well-controlled single bilayer can form vesicles. Destabilization occurred either at the main gel-fluid transition of the lipids and could be interpreted in terms of a decrease in the bending rigidity or under an ac low-frequency electric field applied in the fluid phase. In the latter case, the authors also studied the effect of the electric field at the molecular length scale by neutron reflectivity. In both cases, destabilization leads to the formation of relatively monodisperse vesicles. This could give further understanding on the vesicle formation mechanism and on the parameters that determine the vesicle size.

New sources and instrumentation for neutrons in biology.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

Primary versus ternary adsorption of proteins onto PEG brushes.

Polyethylene glycol (PEG) brushes are used to reduce protein adsorption at surfaces. Their design needs to allow for two leading adsorption modes at the brush-coated surface. One is primary adsorption at the surface itself. The second is ternary adsorption within the brush as a result of weak PEG-protein attraction. We present a scaling theory of the equilibrium adsorption isotherms allowing for concurrent primary and ternary adsorption. The analysis concerns the weak adsorption limit when individual PEG chains do not bind proteins. It also addresses two issues of special relevance to brushes of short PEGs: the consequences of large proteins at the surface protruding out of a shallow brush and the possibility of marginal solvent conditions leading to mean-field behavior. The simple expressions for the adsorption isotherms are in semiquantitative agreement with experiments.

Effect of an electric field on a floating lipid bilayer: A neutron reflectivity study.

We present here a neutron reflectivity study of the influence of an alternative electric field on a supported phospholipid double bilayer. We report for the first time a reproducible increase of the fluctuation amplitude leading to the complete unbinding of the floating bilayer. Results are in good agreement with a semi-quantitative interpretation in terms of negative electrostatic surface tension.