Understanding interactions of plasticisers with a phospholipid monolayer.

The use of DEHP (diethylhexyl phthalate) is now banned for most applications in Europe; the exception is for blood bags, where its toxicity is overshadowed by its ability to extend the storage life of red blood cells. Another plasticiser, BTHC (butanoyl trihexyl citrate), is used in paediatric blood bags but does not stabilise blood cells as effectively. Interactions between plasticisers and lipids are investigated with a phospholipid, DMPC, to understand the increased stability of blood cells in the presence of DEHP as well as bioaccumulation and identify differences with BTHC. Mixed monolayers of DMPC and DEHP or BTHC were studied on Langmuir troughs where surface pressure/area isotherms can be measured. Neutron reflection measurements were made to determine the composition and structure of these mixed layers. A large amount of plasticiser can be incorporated into a DMPC monolayer but once an upper limit is reached, plasticiser is selectively removed from the interface at high surface pressures. The upper limit is found to occur between 40-60 mol% for DEHP and 20-40 mol% for BTHC. The areas per molecule are also different with DEHP being in the range of 50-100 Å2 and BTHC being 65-120 Å2. Results indicate that BTHC does not fit as well as DEHP in DMPC monolayers which could help explain the differences observed with regards to the stability of blood cells.

Investigating the Orientation of an Interfacially Adsorbed Monoclonal Antibody and Its Fragments Using Neutron Reflection.

Interfacial adsorption is a molecular process occurring during the production, purification, transport, and storage of antibodies, with a direct impact on their structural stability and subsequent implications on their bioactivities. While the average conformational orientation of an adsorbed protein can be readily determined, its associated structures are more complex to characterize. Neutron reflection has been used in this work to investigate the conformational orientations of the monoclonal antibody COE-3 and its Fab and Fc fragments at the oil/water and air/water interfaces. Rigid body rotation modeling was found to be suitable for globular and relatively rigid proteins such as the Fab and Fc fragments but less so for relatively flexible proteins such as full COE-3. Fab and Fc fragments adopted a 'flat-on' orientation at the air/water interface, minimizing the thickness of the protein layer, but they adopted a substantially tilted orientation at the oil/water interface with increased layer thickness. In contrast, COE-3 was found to adsorb in tilted orientations at both interfaces, with one fragment protruding into the solution. This work demonstrates that rigid-body modeling can provide additional insights into protein layers at various interfaces relevant to bioprocess engineering.

Aqueous Radical Initiated Oxidation of an Organic Monolayer at the Air-Water Interface as a Proxy for Thin Films on Atmospheric Aerosol Studied with Neutron Reflectometry.

Neutron reflectometry has been used to study the radical initiated oxidation of a monolayer of the lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at the air-solution interface by aqueous-phase hydroxyl, sulfate, and nitrate radicals. The oxidation of organic films at the surface of atmospheric aqueous aerosols can influence the optical properties of the aerosol and consequently can impact Earth's radiative balance and contribute to modern climate change. The amount of material at the air-solution interface was found to decrease on exposure to aqueous-phase radicals which was consistent with a multistep degradation mechanism, i.e., the products of reaction of the DSPC film with aqueous radicals were also surface active. The multistep degradation mechanism suggests that lipid molecules in the thin film degrade to form progressively shorter chain surface active products and several reactive steps are required to remove the film from the air-solution interface. Bimolecular rate constants for oxidation via the aqueous phase OH radical cluster around 1010 dm3 mol-1 s-1. Calculations to determine the film lifetime indicate that it will take ∼4-5 days for the film to degrade to 50% of its initial amount in the atmosphere, and therefore attack by aqueous radicals on organic films could be atmospherically important relative to typical atmospheric aerosol lifetimes.

The reaction of oleic acid monolayers with gas-phase ozone at the air water interface: the effect of sub-phase viscosity, and inert secondary components.

Organic films that form on atmospheric particulate matter change the optical and cloud condensation nucleation properties of the particulate matter and consequently have implications for modern climate and climate models. The organic films are subject to attack from gas-phase oxidants present in ambient air. Here we revisit in greater detail the oxidation of a monolayer of oleic acid by gas-phase ozone at the air-water interface as this provides a model system for the oxidation reactions that occur at the air-water interface of aqueous atmospheric aerosol. Experiments were performed on monolayers of oleic acid at the air-liquid interface at atmospherically relevant ozone concentrations to investigate if the viscosity of the sub-phase influences the rate of the reaction and to determine the effect of the presence of a second component within the monolayer, stearic acid, which is generally considered to be non-reactive towards ozone, on the reaction kinetics as determined by neutron reflectometry measurements. Atmospheric aerosol can be extremely viscous. The kinetics of the reaction were found to be independent of the viscosity of the sub-phase below the monolayer over a range of moderate viscosities, , demonstrating no involvement of aqueous sub-phase oxidants in the rate determining step. The kinetics of oxidation of monolayers of pure oleic acid were found to depend on the surface coverage with different behaviour observed above and below a surface coverage of oleic acid of ∼1 × 1018 molecule m-2. Atmospheric aerosol are typically complex mixtures, and the presence of an additional compound in the monolayer that is inert to direct ozone oxidation, stearic acid, did not significantly change the reaction kinetics. It is demonstrated that oleic acid monolayers at the air-water interface do not leave any detectable material at the air-water interface, contradicting the previous work published in this journal which the authors now believe to be erroneous. The combined results presented here indicate that the kinetics, and thus the atmospheric chemical lifetime for unsaturated surface active materials at the air-water interface to loss by reaction with gas-phase ozone, can be considered to be independent of other materials present at either the air-water interface or in the aqueous sub-phase.

Physical Properties of Bacterial Outer Membrane Models: Neutron Reflectometry & Molecular Simulation.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.

Self-Assembled Fluid Phase Floating Membranes with Tunable Water Interlayers.

We present a reliable method for the fabrication of fluid phase, unsaturated lipid bilayers by self-assembly onto charged Self-Assembled Monolayer (SAM) surfaces with tunable membrane to surface aqueous interlayers. Initially, the formation of water interlayers between membranes and charged surfaces was characterized using a comparative series of bilayers deposited onto charged, self-assembled monolayers by sequential layer deposition. Using neutron reflectometry, a bilayer to surface water interlayer of ∼8 Å was found between the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane and an anionic carboxyl terminated grafted SAM with the formation of this layer attributed to bilayer repulsion by hydration water on the SAM surface. Furthermore, we found we could significantly reduce the technical complexity of sample fabrication through self-assembly of planar membranes onto the SAM coated surfaces. Vesicle fusion onto carboxyl-terminated monolayers yielded high coverage (>95%) bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) which floated on a 7-11 Å solution interlayer between the membrane and the surface. The surface to membrane distance was then tuned via the addition of 200 mM NaCl to the bulk solution immersing a POPC floating membrane, which caused the water interlayer to swell reversibly to ∼33 Å. This study reveals that biomimetic membrane models can be readily self-assembled from solution onto functionalized surfaces without the use of polymer supports or tethers. Once assembled, surface to membrane distance can be tailored to the experimental requirements using physiological concentrations of electrolytes. These planar bilayers only very weakly interact with the substrate and are ideally suited for use as biomimetic models for accurate in vitro biochemical and biophysical studies, as well as for technological applications, such as biosensors.

Structural Features of Reconstituted Cuticular Wax Films upon Interaction with Nonionic Surfactant C12E6.

The interaction of nonionic surfactant hexaethylene glycol monododecyl ether (C12E6) with a reconstituted cuticular wheat wax film has been investigated by spectroscopic ellipsometry and neutron reflection (NR) to help understand the role of the leaf wax barrier during pesticide uptake, focusing on the mimicry of the actions adjuvants impose on the physical integrity and transport of the cuticular wax films against surfactant concentration. As the C12E6 concentration was increased up to the critical micelle concentration (CMC = 0.067 mM), an increasing amount of surfactant mass was deposited onto the wax film. Alongside surface adsorption, C12E6 was also observed to penetrate the wax film, which is evident from the NR measurements using fully protonated and chain-deuterated surfactants. Furthermore, surfactant action upon the model wax film was found to be physically reversible below the CMC, as water rinsing could readily remove the adsorbed surfactant, leaving the wax film in its original state. Above the CMC, the detergency action of the surfactant became dominant, and a significant proportion of the wax film was removed, causing structural damage. The results thus reveal that both water and C12E6 could easily penetrate the wax film throughout the concentration range measured, indicating a clear pathway for the transport of active ingredients while the removal of the wax components above the CMC must have enhanced the transport process. As the partial removal of the wax film could also expose the underlying cutaneous substrate to the environment and undermine the plant's health, this study has a broad implication to the roles of surfactants in crop care.

Correction: Predicting oligomer/polymer compatibility and the impact on nanoscale segregation in thin films.

Correction for 'Predicting oligomer/polymer compatibility and the impact on nanoscale segregation in thin films' by Elise F. D. Sabattié et al., Soft Matter, 2017, 13, 3580-3591.

Predicting oligomer/polymer compatibility and the impact on nanoscale segregation in thin films.

Compatibility between oligomers and polymers was systematically assessed using differential scanning calorimetry (DSC) and was correlated with similarity in saturation and solubility parameter. These measurements enabled validation of detailed volume of mixing calculations using Statistical Association Fluid Theory (SAFT-γ Mie) and molecular dynamics (MD) simulations, which can be used to predict behaviour beyond the experimentally accessible conditions. These simulations confirmed that squalane is somewhat more compatible with poly(isoprene), "PI" than poly(butadiene), "PB", and further enabled prediction of the temperature dependence of compatibility. Surface and interfacial segregation of a series of deuterated oligomers was quantified in rubbery polymer films: PI, PB and hydrogenated poly(isoprene) "hPI". A striking correlation was established between surface wetting transition and mixtures of low compatibility, such as oligo-dIB in PB or PI. Segregation was quantified normal to the surface by ion beam analysis and neutron reflectometry and in some cases lateral segregation was observable by AFM. While surface segregation is driven by disparity in molecular weight in highly compatible systems this trend reverses as critical point is approached, and surface segregation increases with increasing oligomer molecular weight.

Surfactant and Plasticizer Segregation in Thin Poly(vinyl alcohol) Films.

The vertical depth distributions of individual additive components [cetyltrimethylammonium bromide (CTAB), deuterated pentaethylene glycol monododecyl ether (d25-C12E5), and deuterated glycerol (d-glycerol)] in PVA films have been isolated and explored by ion beam analysis techniques and neutron reflectometry. The additives display an unexpectedly rich variety of surface and interfacial behaviors in spin-cast films. In separate binary films with PVA, both d-glycerol and CTAB were evenly distributed, whereas d25-C12E5 showed clear evidence for surface and interfacial segregation. The behavior of each surfactant in PVA was reversed when the plasticizer (glycerol) was also incorporated into the films. With increasing plasticizer content, the surface activity of d25-C12E5 systematically decreased, but remarkably, when glycerol and CTAB were present in PVA, the surface and interfacial activities of CTAB increased dramatically in the presence of glycerol. Quantification of the surface excess by ion beam analysis revealed that, in many cases, the adsorbed quantity far exceeded what could reasonably be explained by a single layer, thus indicating a wetting transition of the small molecules at the surface or interface of the film. It appears that the surface and interfacial behaviors are partly driven by the relative surface energies of the components, but are also significantly augmented by the incompatibility of the components.

Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B).

Lung surfactant protein B (SP-B) is an essential protein found in the surfactant fluid at the air-water interface of the lung. Exposure to the air pollutant ozone could potentially damage SP-B and lead to respiratory distress. We have studied two peptides, one consisting of the N-terminus of SP-B [SP-B(1-25)] and the other a construct of the N- and C-termini of SP-B [SP-B(1-25,63-78)], called SMB. Exposure to dilute levels of ozone (~2 ppm) of monolayers of each peptide at the air-water interface leads to a rapid reaction, which is evident from an increase in the surface tension. Fluorescence experiments revealed that this increase in surface tension is accompanied by a loss of fluorescence from the tryptophan residue at the interface. Neutron and X-ray reflectivity experiments show that, in contrast to suggestions in the literature, the peptides are not solubilized upon oxidation but rather remain at the interface with little change in their hydration. Analysis of the product material reveals that no cleavage of the peptides occurs, but a more hydrophobic product is slowly formed together with an increased level of oligomerization. We attributed this to partial unfolding of the peptides. Experiments conducted in the presence of phospholipids reveal that the presence of the lipids does not prevent oxidation of the peptides. Our results strongly suggest that exposure to low levels of ozone gas will damage SP-B, leading to a change in its structure. The implication is that the oxidized protein will be impaired in its ability to interact at the air-water interface with negatively charged phosphoglycerol lipids, thus compromising what is thought to be its main biological function.

Neutron reflection study of the adsorption of the phosphate surfactant NaDEHP onto alumina from water.

The adsorption of a phosphorus analogue of the surfactant AOT, sodium bis(2-ethylhexyl) phosphate (NaDEHP), at the water/alumina interface is described. The material is found to adsorb as an essentially water-free bilayer from neutron reflection measurements. This is similar to the behavior of AOT under comparable conditions, although AOT forms a thicker, more hydrated layer. The NaDEHP shows rather little variation with added salt, but a small thickening of the layer on increasing the pH, in contrast to the behavior of AOT.

An accurate in vitro model of the E.†coli envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers.

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20-27 Šthick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Degradation and rearrangement of a lung surfactant lipid at the air-water interface during exposure to the pollutant gas ozone.

The presence of unsaturated lipids in lung surfactant is important for proper respiratory function. In this work, we have used neutron reflection and surface pressure measurements to study the reaction of the ubiquitous pollutant gas-phase ozone, O3, with pure and mixed phospholipid monolayers at the air-water interface. The results reveal that the reaction of the unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, with ozone leads to the rapid loss of the terminal C9 portion of the oleoyl strand of POPC from the air-water interface. The loss of the C9 portion from the interface is accompanied by an increase in the surface pressure (decrease in surface tension) of the film at the air-water interface. The results suggest that the portion of the oxidized oleoyl strand that is still attached to the lipid headgroup rapidly reverses its orientation and penetrates the air-water interface alongside the original headgroup, thus increasing the surface pressure. The reaction of POPC with ozone also leads to a loss of material from the palmitoyl strand, but the loss of palmitoyl material occurs after the loss of the terminal C9 portion from the oleoyl strand of the molecule, suggesting that the palmitoyl material is lost in a secondary reaction step. Further experiments studying the reaction of mixed monolayers composed of unsaturated lipid POPC and saturated lipid dipalmitoyl-sn-glycero-3-phosphocholine, DPPC, revealed that no loss of DPPC from the air-water interface occurs, eliminating the possibility that a reactive species such as an OH radical is formed and is able to attack nearby lipid chains. The reaction of ozone with the mixed films does cause a significant change in the surface pressure of the air-water interface. Thus, the reaction of unsaturated lipids in lung surfactant changes and impairs the physical properties of the film at the air-water interface.

Advice on describing Bayesian analysis of neutron and X-ray reflectometry.

As a result of the availability of modern software and hardware, Bayesian analysis is becoming more popular in neutron and X-ray reflectometry analysis. The understandability and replicability of these analyses may be harmed by inconsistencies in how the probability distributions central to Bayesian methods are represented in the literature. Herein advice is provided on how to report the results of Bayesian analysis as applied to neutron and X-ray reflectometry. This includes the clear reporting of initial starting conditions, the prior probabilities, the results of any analysis and the posterior probabilities that are the Bayesian equivalent of the error bar, to enable replicability and improve understanding. It is believed that this advice, grounded in the authors' experience working in the field, will enable greater analytical reproducibility in the work of the reflectometry community, and improve the quality and usability of results.

Unraveling dendrimer translocation across cell membrane mimics.

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.

RNA and DNA association to zwitterionic and charged monolayers at the air-liquid interface.

The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.

Adsorption of aerosol-OT to sapphire: lamellar structures studied with neutrons.

The adsorption of sodium bis 2-ethylhexyl sulfosuccinate, NaAOT, to a sapphire surface from aqueous solution has been studied by neutron reflection at concentrations above the critical micelle concentration (cmc). Complementary measurements of the bulk structure were made with small-angle neutron scattering and grazing incidence small-angle neutron scattering. At a concentration of about 1% wt (10 × cmc), lamellar phase NaAOT was observed both at the surface and in the bulk. The structure seen at the interface for a solution of 2% wt NaAOT is a 35 ± 2 Å thick bilayer adsorbed to the sapphire surface at maximum packing density, followed by an aligned stack of fluctuating bilayers of thickness 51 ± 2 Å and with an area per molecule of 40 ± 2 Å(2). Each bilayer is separated by a water: at 25 °C, this layer is 148 ± 2 Å. A simple model for the reflectivity from fluctuating layers is presented, and for 2.0% wt NaAOT the fluctuations were found to have an amplitude of 25 ± 5 Å. The temperature sensitivity of the structure at the surface was investigated in the range 15-30 °C. The effect of temperature was pronounced, with the solvent layer becoming thinner and the volume occupied by the NaAOT molecules in a bilayer increasing with temperature. The amplitude of the fluctuations, however, is approximately temperature independent in this range. The adsorption of NaAOT at the sapphire surface resembles that previously found at hydrophilic and hydrophobic silica surfaces. The coexisting bulk lamellar phase has a spacing of layers similar to that observed at the surface. These observations are an indication that the major driving force for adsorption is self-assembly, independent of the chemical nature of the interface.