Arwel V. Hughes

Floating lipid bilayers deposited on chemically grafted phosphatidylcholine surfaces

Floating supported bilayers (FSBs) are new systems which have emerged over the past few years to produce supported membrane mimics, where the bilayers remain associated with the substrate, but are cushioned from the substrates constraining influence by a large hydration layer. In this paper we describe a new approach to fabricating FSBs using a chemically grafted phospholipid layer as the support for the floating membrane. The grafted lipid layer was produced using a Langmuir-Schaeffer transfer of acryloyl-functionalized lipid onto a pre-prepared substrate, with AIBN-induced cross-polymerization to permanently bind the lipids in place. A bilayer of DSPC was then deposited onto this grafted monolayer using a combination of Langmuir-Blodgett and Langmuir-Schaeffer transfer. The resulting system was characterized by neutron reflection under two water contrasts, and we show that the new system shows a hydrating layer of approximately 17.5 A in the gel phase, which is comparable to previously described FSB systems. We provide evidence that the grafted substrate is reusable after cleaning and suggest that this greatly simplifies the fabrication and characterization of FSBs compared to previous methods.

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.

Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions.

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.

Reaction of a Phospholipid Monolayer with Gas-Phase Ozone at the Air—Water Interface: Measurement of Surface Excess and Surface Pressure in Real Time

The reaction between gas-phase ozone and monolayers of the unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, on aqueous solutions has been studied in real time using neutron reflection and surface pressure measurements. The reaction between ozone and lung surfactant, which contains POPC, leads to decreased pulmonary function, but little is known about the changes that occur to the interfacial material as a result of oxidation. The results reveal that the initial reaction of ozone with POPC leads to a rapid increase in surface pressure followed by a slow decrease to very low values. The neutron reflection measurements, performed on an isotopologue of POPC with a selectively deuterated palmitoyl strand, reveal that the reaction leads to loss of this strand from the air-water interface, suggesting either solubilization of the product lipid or degradation of the palmitoyl strand by a reactive species. Reactions of (1)H-POPC on D(2)O reveal that the headgroup region of the lipids in aqueous solution is not dramatically perturbed by the reaction of POPC monolayers with ozone supporting degradation of the palmitoyl strand rather than solubilization. The results are consistent with the reaction of ozone with the oleoyl strand of POPC at the air-water interface leading to the formation of OH radicals. The highly reactive OH radicals produced can then go on to react with the saturated palmitoyl strands leading to the formation of oxidized lipids with shorter alkyl tails.

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.

Structural studies on surfactant-templated silica films grown at the air/water interface

In situ characterisation of a growing surfactant-templated silica film has been carried out by X-ray reflectivity, diffuse X-ray scattering from the surface, Brewster angle microscopy and surface pressure measurements. The results indicate an unexpected film growth mechanism where layered structures form in solution and diffuse to the interface after some critical induction period.

The effect of neutral helper lipids on the structure of cationic lipid monolayers

Successful drug delivery via lipid-based systems has often been aided by the incorporation of ‘helper lipids’. While these neutral lipids enhance the effectiveness of cationic lipid-based delivery formulations, many questions remain about the nature of their beneficial effects. The structure of monolayers of the cationic lipid dimethyldioctadecylammonium bromide (DODAB) alone, and mixed with a neutral helper lipid, either diolelyphosphatidylethanolamine or cholesterol at a 1 : 1 molar ratio was investigated at the air–water interface using a combination of surface pressure–area isotherms, Brewster angle microscopy (BAM) and specular neutron reflectivity in combination with contrast variation. BAM studies showed that while pure DODAB and DODAB with cholesterol monolayers showed fairly homogeneous surfaces, except in the regions of phase transition, monolayers of DODAB with diolelyphosphatidylethanolamine were, in contrast, inhomogeneous exhibiting irregular bean-shaped domains throughout. Neutron reflectivity data showed that while the thickness of the DODAB monolayer increased from 17 to 24 Å as it was compressed from a surface pressure of 5–40 mN m−1, the thickness of the helper lipid-containing monolayers, over the same range of surface pressures, was relatively invariant at between 25 and 27 Å. In addition, the monolayers containing diolelyphosphatidylethanolamine were found to be more heavily hydrated than the monolayers of cationic lipid, alone or in combination with cholesterol, with hydration levels of 18 molecules of water per molecule of lipid being recorded for the diolelyphosphatidylethanolamine-containing monolayers at a surface pressure of 30 mN m−1 compared with only six and eight molecules of water per molecule of lipid for the pure DODAB monolayer and the cholesterol-containing DODAB monolayer, respectively.

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.

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.