Anion Competition at Positively Charged Surfactant Monolayers.

Interactions of anions with hydrophobic surfaces of proteins and water-soluble polymers depend on the ability of the ions to shed their hydration shells. At positively charged surfactant monolayers, the interactions of anions are less well understood. Due to the interplay of electrostatic surface forces, hydration effects, and ion-ion interactions in the electrostatic double layer, a comprehensive microscopic picture remains elusive. Herein, we study the interactions of chloride, bromide, and a mixture of these two anions at the aqueous interface of dihexadecyldimethylammonium (DHDA+) and dioctadecyldimethylammonium (DODA+) cationic monolayers. Using molecular dynamics simulations and three surface-sensitive X-ray scattering techniques, we demonstrate that bromide interacts preferentially over chloride with both monolayers. The structure of the two monolayers and their interfacial electron density profiles obtained from the simulations quantitatively reproduce the experimental data. We observe that chloride and bromide form contact ion pairs with the quaternary ammonium groups on both monolayers. However, ion pairing with bromide leads to a greater reduction in the number of water molecules hydrating the anion, resulting in more energetically stable ion pairs. This leads to long-range (>3 nm) lateral correlations between bromide ions on the structured DODA+ monolayer. These observations indicate that ion hydration is the dominant factor determining the interfacial electrolyte structure.

Structural analysis of the NK-lysin-derived peptide NK-2 upon interaction with bacterial membrane mimetics consisting of phosphatidylethanolamine and phosphatidylglycerol.

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil - helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.

Cross-linking reactions in Langmuir monolayers of specially designed aminolipids - a toolbox for the customized production of amphiphilic nanosheets.

Synthetic amino lipids, already known as highly efficient gene therapy tool, are used in a novel way to create cross-linked stable one-molecule-thin films envisioned for future (bio)-materials applications. The films are prepared as Langmuir monolayers at the air/water interface and cross-linked 'in situ' via dynamic imine chemistry. The cross-linking process and the film characteristics are monitored by various surface-sensitive techniques such as grazing incidence X-ray diffraction, X-ray reflectivity, and infrared reflection-absorption spectroscopy. After transfer onto carbon grids, the cross-linked films are investigated by transmission and scanning electron microscopy. The obtained micrographs display mechanically self-supported nanosheets with area dimensions over several micrometers and, thus, an undeniable visual proof of successful cross-linking. The cross-linking process at the air/water interface allows to obtain Janus-faced sheets with a hydrophobic side characterized by aliphatic alkyl chains and a hydrophilic side characterized by nucleophilic groups like amines, hydroxyl groups and imine.

Interaction of Lysozyme with Poly(L-lysine)/Hyaluronic Acid Multilayers: An ATR-FTIR Study.

Polyelectrolyte multilayers (PEM) loaded with bioactive molecules such as proteins serve as excellent mimics of an extracellular matrix and may find applications in fields such as biomedicine and cell biology. A question which is crucial to the successful employment of PEMs is whether conformation and bioactivity of the loaded proteins is preserved. In this work, the polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) technique is applied to investigate the conformation of the protein lysozyme (Lys) loaded into the poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers. Spectra are taken from the protein in the PEMs coated onto an ATR crystal during protein adsorption and desorption. For comparison, a similar investigation is performed for the case of Lys in contact with the uncoated crystal. The study highlights the presence of both "tightly" and "poorly bound" Lys fractions in the PEM. These fractions differ in their conformation and release behavior from the PEM upon washing. Comparison of spectra recorded with different polarizations suggests preferential orientation of alpha helical structures, beta sheets and turns in the "tightly bound" Lys. In contrast, the "poorly bound" fraction shows isotropic orientation and its conformation is well preserved.

Characterization of lipid bilayers adsorbed to functionalized air/water interfaces.

Lipid bilayers immobilized in planar geometries, such as solid-supported or "floating" bilayers, have enabled detailed studies of biological membranes with numerous experimental techniques, notably X-ray and neutron reflectometry. However, the presence of a solid support also has disadvantages as it complicates the use of spectroscopic techniques as well as surface rheological measurements that would require surface deformations. Here, in order to overcome these limitations, we investigate lipid bilayers adsorbed to inherently soft and experimentally well accessible air/water interfaces that are functionalized with Langmuir monolayers of amphiphiles. The bilayers are characterized with ellipsometry, X-ray scattering, and X-ray fluorescence. Grazing-incidence X-ray diffraction reveals that lipid bilayers in a chain-ordered state can have significantly different structural features than regular Langmuir monolayers of the same composition. Our results suggest that bilayers at air/water interfaces may be well suited for fundamental studies in the field of membrane biophysics.

The Complete Phase Diagram of Monolayers of Enantiomeric N-Stearoyl-threonine Mixtures with Preferred Heterochiral Interactions.

Langmuir monolayers of chiral amphiphiles are well-controlled model systems for the investigation of phenomena related to stereochemistry. Here, we have investigated mixed monolayers of one pair of enantiomers (l and d) of the amino-acid-based amphiphile N-stearoyl-threonine. The monolayer characteristics were studied by pressure-area isotherm measurements and grazing incidence X-ray diffraction (GIXD) over a wide range of mixing ratios defined by the d-enantiomer mole fraction xD. While the isotherms provide insights into thermodynamical aspects, such as transition pressure, compression/decompression hysteresis, and preferential homo- and heterochiral interactions, GIXD reveals the molecular structural arrangements on the Ångström scale. Dominant heterochiral interactions in the racemic mixture lead to compound formation and the appearance of a nonchiral rectangular lattice, although the pure enantiomers form a chiral oblique lattice. Miscibility was found to be limited to mixtures with 0.27 ≲ xD ≲ 0.73, as well as to both outer edges (xD ≲ 0.08 and xD ≳ 0.92). Beyond this range, coexistence of oblique and rectangular lattices occurs, as is clearly seen in the GIXD patterns. Based on the results, a complete phase diagram with two eutectic points at xD ≈ 0.25 and xD ≈ 0.75 is proposed. Moreover, N-stearoyl-threonine was found to have a strong tendency to form a hydrogen-bonding network between the headgroups, which promotes superlattice formation.

pH-Dependent physicochemical properties of ornithine lipid in mono- and bilayers.

In certain bacteria, phosphatidylethanolamine lipids (PEL) get largely replaced by phosphate-free ornithine lipids (OL) under conditions of phosphate starvation. It has so far been unknown how much these two lipid types deviate in their physicochemical properties, and how strongly bacteria thus have to adapt in order to compensate for the difference. Here, we use differential scanning calorimetry, X-ray scattering, and X-ray fluorescence to investigate the properties of OL with saturated C14 alkyl chains in mono- and bilayers. OL is found to have a greater tendency than chain-analogous PEL to form ordered structures and, in contrast to PEL, even a molecular superlattice based on a hydrogen bonding network between the headgroups. This superlattice is virtually electrically uncharged and persists over a wide pH range. Our results indicate that OL and PEL behave very differently in ordered single-component membranes but may behave more similarly in fluid multicomponent membranes.

Phase Behavior and Miscibility in Two-Component Glycolipid Monolayers.

Glycolipids are known to be involved in the formation of ordered functional domains in biological membranes. Since the structural characterization of such domains is difficult, most studies have so far dealt with lipid mixtures containing only one glycolipid component at a time, although biological membranes usually contain several glycolipid species, which can result in more complex structures and phase behavior. Here, we combine classical isotherm measurements with surface-sensitive grazing-incidence X-ray diffraction to investigate the phase behavior and miscibility in Langmuir monolayers of binary glycolipid mixtures. We find that the phase behavior has a subtle dependence on the saccharide headgroup chemistry. For compatible chemistries, molecular superlattice structures formed by one of the glycolipid species are conserved and can host foreign glycolipids up to a defined stoichiometry. In contrast, for sterically incompatible saccharide chemistries, the superlattice is lost even if both species are able to form such structures in their pure forms. Our results suggest that related phenomena may play important roles also in biological contexts.

The Effect of pH on the Structure and Lateral Organization of Cardiolipin in Langmuir Monolayers.

Cardiolipin (CL) is a unique phospholipid featuring a dimeric structure. With its four alkyl chains, it has a large hydrophobic region and the charged hydrophilic head group is relatively small. Biological membranes exhibit CL exclusively in the inner bacterial and mitochondrial membranes. Alteration of CL packing can lead to structural changes and membrane instabilities. One environmental influence is the change in pH. Since the acidic properties of the phosphate head groups remain still controversial in literature, this work focusses on the influence of pH on the ionization degree of CL. For the analyses, surface pressure (π) - molecular area (A) isotherm experiments were combined with total reflection X-ray fluorescence (TRXF) and grazing incidence X-ray diffraction (GIXD). Continuous ionization with a high CL packing density was observed in the monolayer over a wide pH range. No individual pKa values can be assigned to the two phosphate groups, but mutual influence is observed.

Phase behavior and miscibility in lipid monolayers containing glycolipids.

HYPOTHESIS: Glycolipids in biological membranes are ubiquitous and believed to be involved in the formation of ordered functional domains. However, our current knowledge about such glycolipid-enriched domains is limited because they are inherently difficult to characterize. EXPERIMENTS: We use grazing-incidence X-ray diffraction, isotherm measurements, and Brewster angle microscopy to investigate the phase behavior and miscibility in Langmuir lipid monolayers containing glycolipids. FINDINGS: Glycolipid-enriched domains give rise to distinct diffraction patterns that allow for a systematic structural investigation and reveal a rich phenomenology, ranging from near-complete demixing to the formation of mixed domains with unique features. The phase behavior is governed by the headgroup chemistry and by the length and saturation of the tails.

Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces.

The stratum corneum (SC) is the largest physical barrier of the human body. It protects against physical, chemical and biological damages, and avoids evaporation of water from the deepest skin layers. For its correct functioning, the homeostasis of the SC lipid matrix is fundamental. An alteration of the lipid matrix composition and in particular of its ceramide (CER) fraction can lead to the development of pathologies such as atopic dermatitis and psoriasis. Different studies showed that the direct replenishment of SC lipids on damaged skin had positive effects on the recovery of its barrier properties. In this work, cerosomes, i.e. liposomes composed of SC lipids, have been successfully prepared in order to investigate the mechanism of interaction with a model SC lipid matrix. The cerosomes contain CER[NP], D-CER[AP], stearic acid and cholesterol. In addition, hydrogenated soybean phospholipids have been added to one of the formulations leading to an increased stability at neutral pH. For the mode of action studies, monolayer models at the air-water interface and on solid support have been deployed. The results indicated that a strong interaction occurred between SC monolayers and the cerosomes. Since both systems were negatively charged, the driving force for the interaction must be based on the ability of CERs head groups to establish intermolecular hydrogen bonding networks that energetically prevailed against the electrostatic repulsion. This work proved for the first time the mode of action by which cerosomes exploit their function as skin barrier repairing agents on the SC.

Two- and Three-Dimensional Physical-Chemical Characterization of CER[AP]: A Study of Stereochemistry and Chain Symmetry.

The stratum corneum represents the first skin barrier against chemical and physical damage. These unique properties are based on its peculiar lipid composition with ceramides (CERs) as the main protagonists. In this study, the structural and chemical properties of the α-OH phytosphingosine [AP] CER class have been investigated. α-OH CERs are present in the stratum corneum in their d-forms; however, in most model systems the diastereomer mixture with the synthetically produced l-form is used. The d-form is well-known to form a hydrogen bonding network that helps to reduce the permeability of the lipid matrix, while the l-form does not show any hydrogen bonding network formation. In this paper, 2D (monolayers) and 3D (aqueous dispersions) models have been used to thoroughly study the physical-chemical behaviors of CER[AP] diastereomers taking into account how the symmetry of the chain pattern influences the behavior of the molecules. The chains of both diastereomers arrange in an oblique unit cell, but only the d-CER[AP] forms a supramolecular lattice (subgel phase) in both model systems. Interestingly, the chain pattern does not play any role in structure formation since the hydrogen bonding network dictates the packing properties. The 1:1 mixture of the diastereomers phase separates into two domains: one is composed of practically pure d-form and the other one is composed of a mixture of the l-form with a certain amount of d-form molecules.

Zwitterionic Character and Lipid Composition Determine the Behaviour of Glycosylphosphatidylinositol Fragments in Monolayers.

Glycosylphosphatidylinositols (GPIs) are complex glycolipids found in free form or anchoring proteins to the outer leaflet of the cell membrane in eukaryotes. GPIs have been associated with the formation of lipid rafts and protein sorting on membranes. The presence of a conserved glycan core with cell-specific modifications together with lipid remodelling during biosynthesis suggest that the properties of the glycolipids are being fine-tuned. We synthesized a series of GPI fragments and evaluated the interactions and arrangement of these glycolipids in monolayers as a 2-D membrane model. GIXD and IRRAS analyses showed the need of N-acetylglucosamine deacetylation for the formation of hydrogen bonds to obtain highly structured domains in the monolayers and an effect of the unsaturated lipids in formation and localization of the glycolipids within or between membrane microdomains. These results contribute to understand the role of these glycolipids and their modifications in the organization of membranes.

Non-ionic surfactants as innovative skin penetration enhancers: insight in the mechanism of interaction with simple 2D stratum corneum model system.

Transdermal drug delivery is a passive diffusion process of an active compound through the skin which is affected by drug solubility in the multilamellar lipidic matrix of the stratum corneum (SC). Widely used non-ionic surfactants (NIS) can be added into transdermal formulations to enhance the penetration of drugs by influencing the packing of the stratum corneum lipidic matrix. Objective of our study was to analyse the interaction between selected NIS and a simple SC lipidic matrix model system using a variety of surface-sensitive techniques based on the application of Langmuir monolayers. In this work, the well-known surfactant Polysorbate 80 was compared with a modern surfactant Sucrose monolaurate. Infrared reflection-absorption spectroscopy (IRRAS) and epifluorescence microscopy provide information about the effects of those surfactants on the SC model system. Monolayer isotherms of the SC model mixture indicate a very stiff and well-packed layer, however, packing defects are evidenced in epifluorescence studies. The injection of the two NIS underneath the SC monolayers proved their potential to penetrate into the SC model at the air-water interface having a maximum insertion pressure (MIP) above the assumed lateral pressure of biological membranes. The NIS adsorbed preferentially into packing defects seen in epifluorescence microscopy studies with Sucrose monolaurate being more active than Polysorbate 80 in disordering the SC monolayer.

Tuning the Thickness of a Biomembrane by Stapling Diamidophospholipids with Bolalipids.

In a biological membrane, proteins require specific lipids of distinctive length and chain saturation surrounding them. The active tuning of the membrane thickness therefore opens new possibilities in the study and manipulation of membrane proteins. Here, we introduce the concept of stapling phospholipids to different degrees of interdigitation depth by mixing 1,3-diamidophospholipids with single-chain bolalipids. The mixed membranes were studied by calorimetric assays, electron microscopy, X-ray, and infrared measurements to provide a complete biophysical characterization of membrane stapling. The matching between the diamidophospholipids and the bolalipids can be so strong as to completely induce a new phase that is more stable than the gel phase of the individual components.

Phase Diagram for a Lysyl-Phosphatidylglycerol Analogue in Biomimetic Mixed Monolayers with Phosphatidylglycerol: Insights into the Tunable Properties of Bacterial Membranes.

Ion pairing between the major phospholipids of the Staphylococcus aureus plasma membrane (phosphatidylglycerol - PG and lysyl-phosphatidylglycerol - LPG) confers resistance to antimicrobial peptides and other antibiotics. We developed 3adLPG, a stable synthetic analogue which can substitute for the highy-labile native LPG, in biophysical experiments examining the membrane-protecting role of lipid ion pairing, in S. aureus and other important bacteria. Here we examine the surface charge and lipid packing characteristics of synthetic biomimetic mixtures of DPPG and DP3adLPG in Langmuir monolayers, using a combination of complementary surface-probing techniques such as infrared reflection-absorption spectroscopy and grazing-incidence x-ray diffraction. The resultant phase diagram for the ion paired lipids sheds light on the mixing behavior of lipids in monolayer models of resistant phenotype bacterial membranes, and provides a platform for future biophysical studies.

Thermodynamic and Structural Behavior of α-Galactosylceramide and C6-Functionalized α-GalCer in 2D Layers at the Air-Liquid Interface.

α-Galactosylceramide (α-GalCer; KRN7000) is a ligand for the glycoprotein CD1d that presents lipid antigens to natural killer T cells. Therefore, KRN7000 as well as some modified versions thereof have been widely investigated as part of novel immunotherapies. To examine the impact of structural modification, we investigated KRN7000 and C6-modified KRN7000 at the air-liquid interface using monolayer isotherms, BAM, IRRAS, GIXD, and TRXF. The amino group has no influence on the highly ordered sub-gel structures found at lateral pressures relevant for biological membranes. Neither lateral compression nor the protonation state of the amino group has a measurable effect on the lattice structure, which is defined by strong and rigid intermolecular hydrogen bonds. However, the first-order phase transition found for the C6-functionalized α-GalCer is connected with an extraordinary surface-inhibited nucleation. Our study demonstrates that KRN7000 can be functionalized at C6 without significantly changing the structural properties.

A triple chain polycationic peptide-mimicking amphiphile - efficient DNA-transfer without co-lipids.

Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.

Enhanced chain packing achieved via putative headgroup ion-triplet formation in binary anionic lipid/cationic surfactant mixed monolayers.

Lipid/surfactant miscibility was investigated in monolayers composed of binary mixtures of dipalmitoylphosphatidylglycerol (DPPG) and dihexadecyldimethylammonium bromide (DHDAB). Langmuir monolayers formed from biomimetic DPPG/DHDAB mixtures based on the anionic:cationic lipid ratios observed in the bacterium Staphylococcus aureus (7:3 and 1:1) were examined alongside those of the pure amphiphiles and a surfactant rich 3:7 mixture. Using a combination of GIXD, TRXF and IRRAS, DPPG/DHDAB 1:1 monolayers were found to form a more stabilised condensed phase compared to pure DPPG, which was composed entirely of electrostatically neutral ion pairs, analogous to the so-called catanionic amphiphiles spontaneously formed by single-chain surfactants with opposing headgroup charges. Despite the lack of lateral charge repulsion the ion paired phase of DPPG/DHDAB exhibited slightly looser chain packing that was observed for DPPG indicating a significant steric effect on packing geometry caused by ion pair formation. Surprisingly, the 7:3 mixture of DPPG/DHDAB formed a completely condensed phase, with no isotherm transitions, in which the chain packing was significantly closer than was found for either DPPG or the totally ion paired monolayer. It is postulated that this mixture forms a distinct DPPG/DHDAB/DPPG ion triplet phase in which the overall negative charge is delocalised across the headgroups. Vesicles composed from the 7:3 mixture formed highly stable dispersions with an increased gel to liquid crystalline phase transition temperature with respect to its pure components. Increasing the proportion of DHDAB above 50 mol% led to demixing between the condensed ion paired phase and the more fluid surfactant, as was clearly observed in epifluorescence images taken of the surface films.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.