Stabilization of DPPC lipid bilayers in the presence of co-solutes: molecular mechanisms and interaction patterns.

We study the interactions between dipalmitoylphosphatidylcholine (DPPC) lipid bilayers in the gel and the fluid phase with ectoine, amino ectoine and water molecules by means of atomistic molecular dynamics (MD) simulations and conceptual density functional theory (DFT) calculations. Our results reveal a pronounced preferential exclusion of both co-solutes from the DPPC lipid bilayer which is stronger for the fluid phase. The corresponding outcomes can be brought into relation with the Kirkwood-Buff theory of solutions in order to provide a thermodynamic rationale for the experimentally observed stabilization of the gel phase. Closely related to preferential exclusion of both co-solutes, our simulations also highlight a preferential hydration behavior as manifested by an increased number of hydrogen bonds between water and DPPC molecules. All results are rationalized by conceptual DFT calculations with regard to differences in the electronic properties between ectoine and amino ectoine.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

Meet the IUPAB Councilor-Hans-Joachim Galla.

As one of the twelve Councilors, it is my pleasure to provide a short biographical sketch for the readers of Biophys. Rev. and for the members of the Biophysical Societies. I have been a member of the council in the former election period. Moreover, I served since decades in the German Biophysical Society (DGfB) as board member, secretary, vice president, and president. I hold a diploma degree in chemistry as well as PhD from the University of Göttingen. The experimental work for both qualifications has been performed at the Max Planck Institute for Biophysical Chemistry in Göttingen under the guidance of Erich Sackmann and the late Herman Träuble. When E. Sackmann moved to the University of Ulm, I joined his group as a research assistant performing my independent research on structure and dynamics of biological and artificial membranes and qualified for the "habilitation" thesis in Biophysical Chemistry. I have spent a research year at Stanford University supported by the Deutsche Forschungsgemeinschaft (DFG) and after coming back to Germany, I was appointed as a Heisenberg Fellow by the DFG and became Professor in Biophysical Chemistry in the Chemistry Department of the University of Darmstadt. Since 1990, I spent my career at the Institute for Biochemistry of the University of Muenster as full Professor and Director of the institute. I have trained numerous undergraduate, 150 graduate, and postdoctoral students from chemistry, physics, and also pharmacy as well as biology resulting in more than 350 published papers including reviews and book articles in excellent collaboration with colleagues from different academic disciplines in our university and also internationally, e.g., as a guest professor at the Chemistry Department of the Chinese Academy of Science in Beijing.

An Imidazolium-Based Lipid Analogue as a Gene Transfer Agent.

A dicationic imidazolium salt is described and investigated towards its application for gene transfer. The polar head group and the long alkyl chains in the backbone contribute to a lipid-like behavior, while an alkyl ammonium group provides the ability for crucial electrostatic interaction for the transfection process. Detailed biophysical studies regarding its impact on biological membrane models and the propensity of vesicle fusion are presented. Fluorescence spectroscopy, atomic force microscopy and confocal fluorescence microscopy show that the imidazolium salt leads to negligible changes in lipid packing, while displaying distinct vesicle fusion properties. Cell culture experiments reveal that mixed liposomes containing the novel imidazolium salt can serve as plasmid DNA delivery vehicles. In contrast, a structurally similar imidazolium salt without a second positive charge showed no ability to support DNA transfection into cultured cells. Thus, we introduce a novel and variable structural motif for cationic lipids, expanding the field of lipofection agents.

Interaction of imidazolium-based lipids with phospholipid bilayer membranes of different complexity.

In recent years, alkylated imidazolium salts have been shown to affect lipid membranes and exhibit general cytotoxicity as well as significant anti-tumor activity. Here, we examined the interactions of a sterically demanding, biophysically unexplored imidazolium salt, 1,3-bis(2,6-diisopropylphenyl)-4,5-diundecylimidazolium bromide (C11IPr), on the physico-chemical properties of various model biomembrane systems. The results are compared with those for the smaller headgroup variant 1,3-dimethyl-4,5-diundecylimidazolium iodide (C11IMe). We studied the influence of these two lipid-based imidazolium salts at concentrations from 1 to about 10 mol% on model biomembrane systems of different complexity, including anionic heterogeneous raft membranes which are closer to natural membranes. Fluorescence spectroscopic, DSC, surface potential and FTIR measurements were carried out to reveal changes in membrane thermotropic phase behavior, lipid conformational order, fluidity and headgroup charge. Complementary AFM and confocal fluorescence microscopy measurements allowed us to detect changes in the lateral organization and membrane morphology. Both lipidated imidazolium salts increase the membrane fluidity and lead to a deterioration of the lateral domain structure of the membrane, in particular for C11IPr owing to its bulkier headgroup. Moreover, partitioning of the lipidated imidazolium salts into the lipid vesicles leads to marked changes in lateral organization, curvature and morphology of the lipid vesicles at high concentrations, with C11IPr having a more pronounced effect than C11IMe. Hence, these compounds seem to be vastly suitable for biochemical and biotechnological engineering, with high potentials for antimicrobial activity, drug delivery and gene transfer.

Effect of ectoine, hydroxyectoine and ß-hydroxybutyrate on the temperature and pressure stability of phospholipid bilayer membranes of different complexity.

Previous research has shown that ectoines fluidize lipid monolayers by increasing the liquid expanded region in DPPC monolayers and also decreasing the line tension responsible for the phase morphology. Here, we explored possible effects of the compatible osmolytes ectoine, hydroxyectoine and ß-hydroxybutyrate on lipid bilayer membranes, including effects of temperature and pressure. The effect of the protective osmolytes on the phase transition of DPPC bilayers was investigated by fluorescence spectroscopy, differential scanning calorimetry and pressure perturbation calorimetry. A slight change of the phase behavior was observed, which resulted in a stabilization of the gel phase, which may be caused by an alteration of the hydration properties at the lipid interface and H-bond and electrostatic interactions in the headgroup region. We then explored the cosolvents' effects on giant unilamellar vesicles (GUVs) formed by lipid mixtures exhibiting phase separation into liquid-ordered (lo) and liquid-disordered (ld) domains using BODIPY-PC and the DiI18 dye as labels. The presence of both, ectoine and hydroxyectoine showed significant effects on the lateral organization increasing the fluid domains. Moreover, we observed a considerable increase in the adhesion behavior of small vesicles onto GUV surfaces. Diffusion studies by fluorescence recovery after photobleaching experiments on POPC giant vesicles quantitatively showed a hydroxyectoine-induced increase of the diffusion coefficient values, clearly demonstrating an increase in the lateral mobility of lipid within the bilayer membrane. This study provides clear evidence for the fluidizing effect of the compatible solutes on bilayer lipid membranes. A marked effect, however, was only detected if phase separated domains exist.

Effect of hyaluronic acid on phospholipid model membranes.

The role of hyaluronic acid (HA) in supporting low friction and low abrasion during movement in synovial joints is still not fully understood. In this study, we set out to investigate the interaction between HA and representative lipid model membranes, bilayers as well as monolayers, in detail using a variety of calorimetric, spectroscopic, scattering and microscopic techniques, to explore their role in lubrication of articular cartridge. We also cover a wide range of pressures to mimic pressures occurring upon joint movement, aiming at elucidating a possible mechanism for the low friction forces in synovial joints. Effects of HA on lipid bilayer membranes, encompassing significant adsorption at the membrane, penetration of the hydrophobic regions of the HA between lipid head groups, or changes of the temperature- and pressure dependent phase behavior of the membrane or mechanical properties could not be observed. High molecular weight HA at physiological NaCl concentrations might rather operate independently, via an entropy-driven excluded volume effect, to control the hydrodynamics of the synovial fluid. Minor effects are observed only at domain boundaries using lipid monolayers. As lubrication of natural joints is a synergistic effect, other components of the synovial fluid, such as proteoglycans, might play a more active role.

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Bridging of membrane surfaces by annexin A2.

The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca2+ regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca2+ dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.

3D Molecular ToF-SIMS Imaging of Artificial Lipid Membranes Using a Discriminant Analysis-Based Algorithm.

Artificial lipid membranes play a growing role in technical applications such as biosensors in pharmacological research and as model systems in the investigation of biological lipid films. In the standard procedure for displaying the distribution of membrane components, fluorescence microscopy, the fluorophores used can influence the distribution of the components and usually not all substances can be displayed at the same time. The discriminant analysis-based algorithm used in combination with scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables marker-free, quantitative, simultaneous recording of all membrane components. These data are used for reconstruction of distribution patterns. In the model system used for this survey, a tear fluid lipid layer, the distribution patterns of all lipids correlate well in calculated ToF-SIMS images and epi-fluorescence microscopic images. All epi-fluorescence microscopically viewable structures are visible when using both positive and negative secondary ions and can be reproduced with high lateral resolution in the submicrometer range despite the very low signal intensity and a very low signal-to-noise ratio. In addition, three-dimensional images can be obtained with a subnanometer depth resolution. Furthermore, structures and the distribution of substances that cannot be made visible by epi-fluorescence microscopy can be displayed. This enables new insights that cannot be gained by epi-fluorescence microscopy alone.

Monocultures of primary porcine brain capillary endothelial cells: Still a functional in vitro model for the blood-brain-barrier.

The main obstacle for the treatment of brain diseases is the restriction of the passage of pharmaceuticals across the blood-brain barrier. Endothelial cells line up the cerebral micro vessels and prevent the uncontrolled transfer of polar substances by intercellular tight junctions. In addition to this physical barrier, active transporters of the multi-drug-resistance prevent the passage of hydrophobic substances by refluxing them back to the blood stream. This paper reviews the development and selected applications of an in vitro porcine brain derived primary cell culture system established in the authors lab that closely resembles the BBB in vivo and could thus be used to study beyond other applications drug delivery to the brain. An essential technique to control the intactness or destruction of the barrier, the impedance spectroscopy, will be introduced. It will be shown that nanoparticles can cross the blood brain barrier by two mechanisms: opening the tight junctions and thus allowing parallel import of substances into the brain as well as receptor mediated endocytosis using brain specific target molecules. However cytotoxic effects have to be considered as well which beside standard cytotoxicity assays could be also determined by impedance technology. Moreover it will be shown that enzymes e.g. for enzyme replacement therapy could be transferred across the barrier by proper tuning or chemical modification of the enzyme. Since this review is based on a conference presentation it will mainly focus on applications of the monoculture system developed in the authors lab which under given culture conditions is useful due to its easy availability, robustness, good reproducibility and also due to its simplicity. Improvements of this model made by other groups will be acknowledged but not discussed here in detail.

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells.

Membrane interactions of ionic liquids and imidazolium salts.

Room-temperature ionic liquids (RTILs) have attracted considerable attention in recent years due to their versatile properties such as negligible volatility, inflammability, high extractive selectivity and thermal stability. In general, RTILs are organic salts with a melting point below ~100 °C determined by the asymmetry of at least one of their ions. Due to their amphiphilic character, strong interactions with biological materials can be expected. However, rising attention has appeared towards their similarity and interaction with biomolecules. By employing structural modifications, the biochemical properties of RTILs can be designed to mimic lipid structures and to tune their hydrophobicity towards a lipophilic behavior. This is evident for the interaction with lipid-membranes where some of these compounds present membrane-disturbing effects or cellular toxicity. Moreover, they can form micelles or lipid-like bilayer structures by themselves. Both aspects, cellular effects and membrane-forming capacities, of a novel class of lipophilic imidazolium salts will be discussed.

Towards quantification of toxicity of lithium ion battery electrolytes - development and validation of a liquid-liquid extraction GC-MS method for the determination of organic carbonates in cell culture materials.

A novel method based on liquid-liquid extraction with subsequent gas chromatography separation and mass spectrometric detection (GC-MS) for the quantification of organic carbonates in cell culture materials is presented. Method parameters including the choice of extraction solvent, of extraction method and of extraction time were optimised and the method was validated. The setup allowed for determination within a linear range of more than two orders of magnitude. The limits of detection (LODs) were between 0.0002 and 0.002 mmol/L and the repeatability precisions were in the range of 1.5-12.9%. It could be shown that no matrix effects were present and recovery rates between 98 and 104% were achieved. The methodology was applied to cell culture models incubated with commercial lithium ion battery (LIB) electrolytes to gain more insight into the potential toxic effects of these compounds. The stability of the organic carbonates in cell culture medium after incubation was studied. In a porcine model of the blood-cerebrospinal fluid (CSF) barrier, it could be shown that a transfer of organic carbonates into the brain facing compartment took place. Graphical abstract Schematic setup for the investigation of toxicity of lithium ion battery electrolytes.

The effects of gold nanoparticles functionalized with ß-amyloid specific peptides on an in vitro model of blood-brain barrier.

We studied the effect of gold nanoparticle (AuNP) size, surface charge, concentration and morphology on the integrity of the blood-brain barrier (BBB) in a well-established in vitro model set-up. We focused on the effect of peptide functionalized hollow gold nanospheres and gold nanorods, which selectively bind to amyloidogenic ß-amyloid structures. These AuNP conjugates have already been successfully tested as photothermal absorbers for potential application in Alzheimer's disease (AD) therapy in an in vitro set-up, but may exhibit a low passage through the BBB due to their overall negative charge. Our results show that: (i) small (1.4 nm) AuNPs strongly affects the BBB integrity, (ii) negative surface charge impedes BBB passage, and (iii) this charge effect caused by the peptide is compensated by covalent coupling to a polyethylene glycol ligand stabilizing the particles in diluted manner.

Influence of the Headgroup of Azolium-Based Lipids on Their Biophysical Properties and Cytotoxicity.

A series of (un-)charged NHC derivatives bearing two pentadecyl chains in the backbone was studied in detail to find cooperative effects between the membrane and the NHC derivative. The tendency to show lipid-like behavior is dependent on the properties of the NHC derivative headgroup, which can be modified on demand. The surface activity was investigated by film balance measurements, epifluorescence microscopy, and differential scanning calorimetry. Additionally the cytotoxicity was evaluated against different cell lines such as eukaryotic tumor cell lines. These novel lipid-like NHC derivatives offer a broad spectrum for biological applications.

Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit Remarkable Properties Forming Lamellar Phases and Giant Vesicles.

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (Cn-IMe·HI, n = 7, 11, 15) and 1,3-dibenzyl-4,5-dialkylimidazolium (Cn-IBn·HBr, n = 7, 11, 15) salts. Diverse alkyl chain lengths and headgroups differing in their steric demand were employed for the membrane interface interaction with bilayer membranes imitating the cellular plasma membrane. Membrane hydration properties and domain fluidization were analyzed by fluorescent bilayer probes in direct comparison to established model membranes in a buffered aqueous environment, which resembles the salt content and pH of the cytosol of living cells. Membrane binding and insertion was analyzed via a quartz crystal microbalance and confocal laser scanning microscopy. We show that short-chain 4,5-dialkylimidazolium salts with a bulky headgroup were able to disintegrate membranes. Long-chain imidazolium salts form bilayer membrane vesicles spontaneously and autonomously without the addition of other lipids. These 4,5-dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.