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.

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.

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.

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.

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.

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.

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.

The Influence of Calcium Traces in Ultrapure Water on the Lateral Organization in Tetramyristoyl Cardiolipin Monolayers.

Cardiolipin (CL) plays an important role in administering the structural organization of biological membranes and therefore helps maintaining the integer membrane functionality. CL has a dimeric structure consisting of four acyl chains and two phosphate groups. With its unusual structure, the phospholipid is responsible for curvature formation in CL containing biological membranes. The acidic properties of the phosphate head groups are still not sufficiently investigated since there are controversial results in literature. The main aim of this work was to gain deeper insights into the influence of the pH on the ionization degree of CL. During the experiments, it became clear that the used ultrapure water contained traces of calcium. These unexpected calcium ions had a major impact on the behavior of CL monolayers. Therefore, the focus was put on the analysis of CL layers without and with EDTA in the subphase used to complex divalent calcium ions. For the analyses, traditional surface pressure (π) - molecular area (A) isotherm experiments combined with total reflection x-ray fluorescence (TRXF) and grazing incidence x-ray diffraction (GIXD) have been used.

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.

Investigating Ions at Amphiphilic Monolayers with X-ray Fluorescence.

Amphiphilic monolayers formed at the soft air/liquid interface are easy-to-handle and versatile model systems for material and life sciences. Helmuth Möhwald was one of the pioneers in this field. Over the last few decades, total-reflection X-ray fluorescence (TRXF) has become an important analytical tool for the investigation of monolayer interactions with ions. Here, the theoretical background of TRXF is described, and practical aspects are discussed. The experimentally determined fluorescence intensity from the adsorbed ions can be interpreted quantitatively either by a calibration procedure utilizing monolayers with known charge density or by calibration with respect to the bare aqueous surface. Both calibration approaches yield quantitatively consistent results within <10% accuracy. Some examples demonstrating the power of TRXF for the study of ion adsorption to charged and noncharged monolayers as well as for the characterization of the physicochemical properties of novel cationic lipids used for improved gene delivery are given.

DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical-chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air-water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

Synthesis and Biophysical Characterization of an Odd-Numbered 1,3-Diamidophospholipid.

Nanomedicine suffers from low drug delivery efficiencies. Mechanoresponsive vesicles could provide an alternative way to release active compounds triggered by the basic physics of the human body. 1,3-Diamidophospholipids with C16 tails proved to be an effective building block for mechanoresponsive vesicles, but their low main phase transition temperature prevents an effective application in humans. As the main phase transition temperature of a membrane depends on the fatty acyl chain length, we synthesized a C17 homologue of a 1,3-diamidophospholipid: Rad-PC-Rad. The elevated main phase transition temperature of Rad-PC-Rad allows mechanoresponsive drug delivery at body temperature. Herein, we report the biophysical properties of Rad-PC-Rad monolayer and bilayer membranes. Rad-PC-Rad is an ideal candidate for advancing the concept of physically triggered drug release.

Interactions of Cationic Lipids with DNA: A Structural Approach.

Colloidal nucleic acid carrier systems based on cationic lipids are a promising pharmaceutical tool in the implementation of gene therapeutic strategies. This study demonstrates the complex behavior of DNA at the lipid-solvent interface facilitating structural changes of the lyotropic liquid-crystalline phases. For this study, the structural properties of six malonic acid based cationic lipids were determined using small- and wide-angle X-ray scattering (SAXS and WAXS) as well as differential scanning calorimetry (DSC). Selected lipids (lipid 3 and lipid 6) with high nucleic acid transfer activity have been investigated in detail because of the strong influence of the zwitterionic helper lipid 1,2-di(9 Z-octadecenoyl)- sn-glycero-3-phosphoethanolamine (DOPE) on the structural properties as well as of the complex formation of lipid-DNA complexes (lipoplexes). In the case of lipid 3, DNA stabilizes a metastable cubic mesophase with Im3 m symmetry and an Im3 m Qαc lipoplex is formed, which is rarely described for DNA lipoplexes in literature. In the case of lipid 6, a cubic mesophase with Im3 m symmetry turns into a fluid lamellar phase while mixing with DOPE and complexing DNA.

Against the rules: pressure induced transition from high to reduced order.

Envisioning the next generation of drug delivery nanocontainers requires more in-depth information on the fundamental physical forces at play in bilayer membranes. In order to achieve this, we combine chemical synthesis with physical-chemical analytical methods and probe the relationship between a molecular structure and its biophysical properties. With the aim of increasing the number of hydrogen bond donors compared to natural phospholipids, a phospholipid compound bearing urea moieties has been synthesized. The new molecules form interdigitated bilayers in aqueous dispersions and self-assemble at soft interfaces in thin layers with distinctive structural order. At lower temperatures, endothermic and exothermic transitions are observed during compression. The LC1 phase is dominated by an intermolecular hydrogen bond network of the urea moieties leading to a very high chain tilt of 52°. During compression and at higher temperatures, presumably this hydrogen bond network is broken allowing a much lower chain tilt of 35°. The extremely different monolayer thicknesses violate the two-dimensional Clausius-Clapeyron equation.

Lysine-based amino-functionalized lipids for gene transfection: the influence of the chain composition on 2D properties.

The influence of the chain composition on the physical-chemical properties will be discussed for five transfection lipids containing the same lysine-based head group. For this purpose, the chain composition will be gradually varied from saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains to longer but unsaturated oleyl (C18:1) chains with double bonds in the cis configuration. In this work, we investigated the lipids as Langmuir monolayers at the air-water-interface in the absence and presence of calf thymus DNA applying different techniques such as infrared reflection absorption spectroscopy (IRRAS) and grazing incidence X-ray diffraction (GIXD). The replacement of saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains by unsaturated oleyl (C18:1) chains increases the fluidity of the lipid monolayer: TH10 < TT10 < OH10 < OT10 < OO10 resulting in a smaller packing density. TH10 forms the stiffest and OO10 the most fluid monolayer in this structure-property study. OO10 has a higher protonation degree compared to the saturated lipids TT10 and TH10 as well as to the hybrids OT10 and OH10 because of a better accessibility of the amine groups. Depending on the bulk pH, different scenarios of DNA coupling to the lipid monolayers have been proposed.

Lysine-based amino-functionalized lipids for gene transfection: 3D phase behaviour and transfection performance.

Based on previous work, the influence of the chain composition on the physical-chemical properties of five new transfection lipids (TH10, TT10, OH10, OT10 and OO10) containing the same lysine-based head group has been investigated in aqueous dispersions. For this purpose, the chain composition has been gradually varied from saturated tetradecyl (T, C14:0) and hexadecyl (H, C16:0) chains to longer but unsaturated oleyl (O, C18:1) chains with double bonds in the cis configuration. In this work, the lipid dispersions have been investigated in the absence and presence of the helper lipid DOPE and calf thymus DNA by small-angle and wide-angle X-ray scattering (SAXS/WAXS) supplemented by differential scanning calorimetry (DSC), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and Fourier-transform Raman spectroscopy (FTRS). Lamellar and inverted hexagonal mesophases have been observed in single-component systems. In the binary mixtures, the aggregation behaviour changes with an increasing amount of DOPE from lamellar to cubic. The lipid mixtures with DNA show a panoply of mesophases. Interestingly, TT10 and OT10 form cubic lipoplexes, whereas OO10 complexes the DNA sandwich-like between lipid bilayers in a lamellar lipoplex. Surprisingly, the latter is the most effective lipoplex.

Interaction of DNA with Cationic Lipid Mixtures-Investigation at Langmuir Lipid Monolayers.

Four different binary lipid mixtures composed of a cationic lipid and the zwitterionic colipids DOPE or DPPC, which show different DNA transfer activities in cell culture models, were investigated at the soft air/water interface to identify transfection efficiency determining characteristics. Langmuir films are useful models to investigate the interaction between DNA and lipid mixtures in a two-dimensional model system by using different surface sensitive techniques, namely, epifluorescence microscopy and infrared reflection-absorption spectroscopy. Especially, the effect of adsorbed DNA on the properties of the lipid mixtures has been examined. Distinct differences between the lipid composites were found which are caused by the different colipids of the mixtures. DOPE containing lipid mixtures form fluid monolayers with a uniform distribution of the fluorescent probe in the presence and absence of DNA at physiologically relevant surface pressures. Only at high nonphysiological pressures, the lipid monolayer collapses and phase separation was observed if DNA was present in the subphase. In contrast, DPPC containing lipid mixtures show domains in the liquid condensed phase state in the presence and absence of DNA in the subphase. The adsorption of DNA at the positively charged mixed lipid monolayer induces phase separation which is expressed in the morphology and the point of appearance of these domains.

Lysine-based amino-functionalized lipids for gene transfection: the protonation state in monolayers at the air-liquid interface.

Cationic lipids are considered as non-viral carriers for genetic material used in gene therapy. They have no carcinogenic potential and cause low immune response compared to existing viral systems. The protonation degree of these cationic lipids is a crucial parameter for the binding behavior of polynucleotides (e.g., DNA). Newly synthesized peptide-mimic lysine-based amino-functionalized lipids have been investigated in 2D models as monolayers at the air-liquid interface. Standard surface pressure - area isotherms have been measured to prove the layer stability. Total reflection X-ray fluorescence (TRXF) has been used as a surface sensitive analytical method to estimate the amount of counterions at the head groups. Using a standard sample as a reference, the protonation degree of these cationic lipids can be quantified on buffers with different pH values. It is found that the protonation degree depends linearly on the packing density of the lipid monolayer.