Lipid Rearrangement in DSPC/DMPC Bilayers: A Neutron Reflectometry Study

Lipid translocation in membranes is still far from being understood and well characterized for natural cell membranes as well as for simpler bilayer model systems. Several discrepancies with respect to its occurrence and its characteristic time scale are present in the literature. In the current work, the structural changes induced by lipid rearrangement in a distearoyl-/dimyristoyl-phosphocholine binary lipid system have been addressed by means of neutron reflectivity. It has been shown that a fast, spontaneous compositional reorganization with lipid transfer between the two leaflets of the bilayer takes place only when the lipid species are both in the fluid phase. This process has been identified as the so-called lipid flip-flop. Moreover, the influence of the preparation protocol on the structural properties of the system has been investigated

Effect of Functionalized Gold Nanoparticles on Floating Lipid Bilayers

The development of novel nano-engineered materials poses important questions regarding the impact of these new materials on living systems. Possible adverse effects must be assessed in order to prevent risks for health and the environment. On the other hand, a thorough understanding of their interaction with biological systems might also result in the creation of novel biomedical applications. We present a study on the interaction of model lipid membranes with gold nanoparticles (AuNP) of different surface modifications. Neutron reflectometry experiments on zwitterionic lipid double bilayers were performed in the presence of AuNP functionalized with cationic and anionic head groups. Structural information was obtained that provided insight into the fate of the AuNPs with regard to the integrity of the model cell membranes. The AuNPs functionalized with cationic head groups penetrate into the hydrophobic moiety of the lipid bilayers and cause membrane disruption at an increased concentration. In contrast, the AuNPs functionalized with anionic head groups do not enter but seem to impede the destruction of the lipid bilayer at an alkaline pH. The information obtained might influence the strategy for a better nanoparticle risk assessment based on a surface charge evaluation and contribute to nano-safety considerations during their design

Generic Role of Polymer Supports in the Fine Adjustment of Interfacial Interactions between Solid Substrates and Model Cell Membranes

To understand the generic role of soft, hydrated biopolymers in adjusting interfacial interactions at biological interfaces, we designed a defined model of the cell–extracellular matrix contacts based on planar lipid membranes deposited on polymer supports (polymer-supported membranes). Highly uniform polymer supports made out of regenerated cellulose allow for the control of film thickness without changing the surface roughness and without osmotic dehydration. The complementary combination of specular neutron reflectivity and high-energy specular X-ray reflectivity yields the equilibrium membrane–substrate distances, which can quantitatively be modeled by computing the interplay of van der Waals interaction, hydration repulsion, and repulsion caused by the thermal undulation of membranes. The obtained results help to understand the role of a biopolymer in the interfacial interactions of cell membranes from a physical point of view and also open a large potential to generally bridge soft, biological matter and hard inorganic materials

Neutron Reflectometry Elucidates Protein Adsorption from Human Blood Serum onto PEG Brushes

Poly­(ethylene glycol) (PEG) brushes are reputed for their ability to prevent undesired protein adsorption to material surfaces exposed to biological fluids. Here, protein adsorption out of human blood serum onto PEG brushes anchored to solid-supported lipid monolayers was characterized by neutron reflectometry, yielding volume fraction profiles of lipid headgroups, PEG, and adsorbed proteins at subnanometer resolution. For both PEGylated and non-PEGylated lipid surfaces, serum proteins adsorb as a thin layer of approximately 10 Å, overlapping with the headgroup region. This layer corresponds to primary adsorption at the grafting surface and resists rinsing. A second diffuse protein layer overlaps with the periphery of the PEG brush and is attributed to ternary adsorption due to protein–PEG attraction. This second layer disappears upon rinsing, thus providing a first observation of the structural effect of rinsing on protein adsorption to PEG brushes

Structure and Stability of Phospholipid Bilayers Hydrated by a Room-Temperature Ionic Liquid/Water Solution: A Neutron Reflectometry Study

Neutron reflectometry (NR) measurements were carried out to probe the structure and stability of two model biomembranes consisting of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DMPC) phospholipid bilayers hydrated by water solutions of two prototypical room-temperature ionic liquids (RTILs), namely, 1-butyl-3-methyl-imidazolium chloride ([bmim]­[Cl]) and choline chloride ([Chol]­[Cl]) at concentrations of 0.1 M and 0.5 M, respectively. The raw data were analyzed by fitting a distribution of scattering length densities arising from the different chemical species in the system. The results of this analysis show that (a) for all systems and concentrations that we considered, the thickness of the bilayers shrinks by ∼1 Å upon dissolving the ionic liquid into water and that (b) the RTIL ions enter the bilayer, finding their way to a preferred location in the lipid range that is nearly independent of the lipid and of the [bimim]⁺ or [Chol]⁺ choice. The volume fraction of RTIL sorbed in/on the bilayer, however, does depend on the lipid, but, again, is the same for [bmim]­[Cl] and for [Chol]­[Cl]. Thus, the RTIL occupies ∼5% of the bilayer volume in POPC, rising to ∼10% in DMPC. Repeating the measurements and data analysis after rinsing in pure water shows that the changes in the bilayer due to the RTIL sorption are irreversible and that a measurable amount of IL remains in the lipid fraction, that is, ∼2.5% of the bilayer volume in POPC and ∼8% in DMPC

Neutron Reflectometry Elucidates Density Profiles of Deuterated Proteins Adsorbed onto Surfaces Displaying Poly(ethylene glycol) Brushes: Evidence for Primary Adsorption

The concentration profile of deuterated myoglobin (Mb) adsorbed onto polystyrene substrates displaying poly­(ethylene glycol) (PEG) brushes is characterized by neutron reflectometry (NR). The method allows to directly distinguish among primary adsorption at the grafting surface, ternary adsorption within the brush, and secondary adsorption at the brush outer edge. It complements depth-insensitive standard techniques, such as ellipsometry, radioactive labeling, and quartz crystal microbalance. The study explores the effect of the PEG polymerization degree, <i>N</i>, and the grafting density, σ, on Mb adsorption. In the studied systems there is no indication of secondary or ternary adsorption, but there is evidence of primary adsorption involving a dense inner layer at the polystyrene surface. For sparsely grafted brushes the primary adsorption involves an additional dilute outer protein layer on top of the inner layer. The amount of protein adsorbed in the inner layer is independent of <i>N</i> but varies with σ, while for the outer layer it is correlated to the amount of grafted PEG and is thus sensitive to both <i>N</i> and σ. The use of deuterated proteins enhances the sensitivity of NR and enables monitoring exchange between deuterated and hydrogenated species

Ratio (upper part) of C16/C18 fatty acids and (lower part) of unsaturated/saturated fatty acid (UFA/SFA) when <i>Pichia pastoris</i> cells were grown in hydrogenated (H) and deuterated (D) media at 18 and 30°C.

a<p>: Total, total fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; CL, cardiolipine.</p

Fatty acid distribution of the main phospholipids produced by <i>Pichia pastoris</i> cells grown in a hydrogenated medium at 30°C (red) and at 18°C (green) and in a deuterated environment at 30°C (blue) and 18°C (cyan).

<p>In all individual phospholipids, the deuterated environment triggers the enrichment in C18:1 fatty acids PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. Errors bars represent the standard deviation from three different phospholipids extractions and separations. Data represent mean values ± s.d (n = 3). In histograms, *<i>P</i><0.05 from Student's <i>t</i>-test, assuming equal variance. PC: for C16:0, the difference is significant between H30°C and H18°C, between H30°C and D30°C and between H30°C and D18°C. For C16:1, there is a significant difference between H30°C and D30°C and between H18°C and D18°C. For C18:0, there is a significant difference between H18°C and D18°C, between H18°C and H30°C and between H18°C and D30°C. For C18:1, the difference is significant between all the different temperatures and isotopic contents. For C18:2, there is a significant difference between D30°C and D18°C, between D30°C and H30°C and between D30°C and H18°C. For C18:3, the difference is significant between all the different temperatures and isotopic contents. PE: for C16:0, the difference is significant between H30°C and D30°C, between H30°C and H18°C and between H30°C and D18°C. For C18:1 and C18:3, the difference is significant between all the different temperatures and isotopic contents. PS and PI: for C16:0, there is a significant difference between H30° and D30°C and between H30° and D18°C. For C16:2 and C18:1, there is a significant difference between H30°C and D30°C, between H18°C and D18°C and between D18°C and D30°C. For C18:2 and C18:3, there is a significant difference between H30°C and D30°C and between H18°C and D18°C.</p

GC-MS analysis of ergosterol isotopomers from <i>Pichia pastoris</i> unsaponifiable extracts.

<p>A, TIC of an acetylated extract from cells grown in hydrogenated medium. The peak at RT = 36.10 min is ergosterol. B, TIC of an acetylated extract from cells grown in deuterated medium. The peak at RT = 35.26 min is deuterioergosterol. C, mass spectrum of ergosteryl acetate. Prominent ions and interpretation of the fragmentation pattern: M⁺(438), M⁺-acetate-H (378), M⁺-acetate-H-CH₃ (363), M+-acetate-H-side chain (253). D, mass spectrum of deuterioergosteryl acetate. Prominent ions and interpretation of the fragmentation pattern: M⁺(481), M⁺-acetate-D (420), M⁺-acetate-D-CD₃ (402), M⁺-acetate-D-side chain (278).</p

Total fatty acid distribution in <i>Pichia pastoris</i> cells grown in a hydrogenated environment at 30°C (red) and 18°C (green) and in a deuterated environment at 30°C (blue) and 18°C (cyan).

<p>Data represent mean values ± s.d (n = 3). In histograms, *<i>P</i><0.05 from Student's <i>t</i>-test, assuming equal variance. For C16:0, there is a significant difference between H 30°C and H 18°C, between H 30°C and D30°C, H 30°C and D18°C, between H 18°C and D 30°C but not between H18°C and D18°C. For C16:1, there is a significant difference for all 4 different temperatures and isotopic contents. For C16:2, there is a significant difference between D30°C and D18°C. For C18:1, there is a significant difference for all 4 different temperatures and isotopic contents. For C18:2, there is a significant difference between D30°C and H30°C, between D30°C and D18°C, between D30°C and H18°C. For C18:3, there is a significant difference for all 4 different temperatures and isotopic contents.</p