Inverted Hexagonal Phase Research Articles

Interactions of Mono- and Di-Saccharides with Three Lipid Phases

Sugars play important biological roles in lipid membranes; however, a quantitative picture of their interactions is still being developed. In particular, we have studied the effects of fructose, glucose, sucrose and trehalose. We present phase diagrams showing the effects of these mono- and di-saccharides on the gel-lamellar, fluid-lamellar and inverted hexagonal phases of the lipid SOPE. Our results suggest that all the sugars we have tested are all excluded from a thin layer immediately in front of the lipid head groups. Consequently, they are entirely excluded in the gel-lamellar phase and only partially included in the gaps between head groups in the fluid lamellar phase. The amount of inclusion in the fluid lamellar phase is similar among the monosaccharides and among the disaccharides, with the monosaccharides showing a higher degree of inclusion. In the inverted hexagonal phase, for all of the sugars, there appears to be the aforementioned exclusion zone which surrounds a core of sugar and water which matches the bulk concentration.

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Line tension of branching junctions of bilayer membranes

Branching of bilayer membranes appear in the inverted hexagonal phase as well as in metastable states of the lamellar phase such as membrane fusion intermediates. A method for estimating the line tension of the branching junction is proposed for molecular simulations. The line tension is calculated from the pressure tensor of equiangularly branched membranes. The simulation results agree very well with the theoretical prediction of Hamm and Kozlov's tilt model. The transition between the lamellar and inverted hexagonal phases is also investigated using the tilt model.

An Amino Acid-based Amphoteric Liposomal Delivery System for Systemic Administration of siRNA

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA₂ compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA₂-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA₂, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA₂-based liposomes demonstrate a linear dose-response with an ED₅₀ of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Aggregatibacter Actinomycetemcomitans Leukotoxin Disrupts Membranes By Inducing the Formation of An Inverted Hexagonal Lipid Phase

The leukotoxin (LtxA) secreted by Aggregatibacter actinomycetemcomitans is a member of the repeats-in-toxin (RTX) family, and like the other members of the family, is a virulence protein which destroys host cells. LtxA exhibits specificity to human white blood cells, thereby allowing A. actinomycetemcomitans to flourish in the human upper aerodigestive track. Scanning electron micrographs of LtxA-treated human immune cell lines showed that LtxA induces the formation of large pores, which appear to be caused by membrane bending. To understand the molecular mechanism of this interaction, we analyzed the behavior of LtxA-membrane interactions in model membranes. Freeze-fracture transmission electron microscopy revealed the presence of unique structures, such as nanotubules, in multilamellar liposomes treated with LtxA. Formation of these structures indicated that the toxin acts by bending the membrane, possibly by inducing a bilayer-to-nonbilayer transition. This phase transition was quantified using differential scanning calorimetry, and it was found that LtxA is a potent inverted hexagonal (HII) phase promoter. The relationship between HII phase induction and membrane disruption was determined with a calcein leakage assay. LtxA-induced leakage from calcein-encapsulating liposomes composed of lipids with negative curvature, favoring HII phase formation, was significantly enhanced compared to leakage from liposomes composed of lipids with neutral curvature, favoring bilayer formation. Addition of lipids with positive curvature, inhibiters of HII phase formation, or cholesterol sulfate, a bilayer stabilizer, completely eliminated calcein leakage. It appears that LtxA causes membrane disruption by inducing the formation of the HII phase, and its toxic effect is therefore highly dependent on lipid curvature. Supported by NIH DE09517.

Mediterranean-Style Diet Effect on the Structural Properties of the Erythrocyte Cell Membrane of Hypertensive Patients

A currently ongoing randomized trial has revealed that the Mediterranean diet, rich in virgin olive oil or nuts, reduces systolic blood pressure in high-risk cardiovascular patients. Here, we present a structural substudy to assess the effect of a Mediterranean-style diet supplemented with nuts or virgin olive oil on erythrocyte membrane properties in 36 hypertensive participants after 1 year of intervention. Erythrocyte membrane lipid composition, structural properties of reconstituted erythrocyte membranes, and serum concentrations of inflammatory markers are reported. After the intervention, the membrane cholesterol content decreased, whereas that of phospholipids increased in all of the dietary groups; the diminishing cholesterol:phospholipid ratio could be associated with an increase in the membrane fluidity. Moreover, reconstituted membranes from the nuts and virgin olive oil groups showed a higher propensity to form a nonlamellar inverted hexagonal phase structure that was related to an increase in phosphatidylethanolamine lipid class. These data suggest that the Mediterranean-style diet affects the lipid metabolism that is altered in hypertensive patients, influencing the structural membrane properties. The erythrocyte membrane modulation described provides insight in the structural bases underlying the beneficial effect of a Mediterranean-style diet in hypertensive subjects.

Anionic Nucleotide−Lipids for In Vitro DNA Transfection

A family of new anionic nucleotide based lipids featuring thymidine-3'-monophosphate as nucleotide and 1,2-diacyl-sn-glycerol as lipid moiety for in vitro delivery of nucleic acids is described. The nucleotide lipids were prepared in three steps starting from 1,2-diacyl-sn-glycerols and 2'-deoxythymidine-3'-phosphoramidite. Gel electrophoresis experiments show that nucleotide-based lipid-DNA complexes are observed at Ca(2+) concentration higher than 1 mM. The transfection experiments carried out on mammalian Hek cell lines clearly demonstrate that the nucleotide moiety enhances the transfection efficacy of the natural anionic DPPA and DPPG lipids. SAXS studies indicate that the enhancement in transfection for nucleotide-based lipid formulations compared to those of the abasic natural derivative (DPPA) is likely due to the presence of the 2D columnar inverted hexagonal phase (H(II)) with a unit cell parameter a = 69.1 A in the nucleotide lipid formulations. The cytotoxicity studies of lipoplexes, evaluated against Hek cells using an MTS assay, revealed that palmitoyl nucleotide derivative complexes were not toxic even after 4 h of incubation, thus indicating that the anionic nucleotide lipids presented in this work offer an alternative to cationic transfection reagents.

Aqueous Phase Behavior of Lipids with Isoprenoid Type Hydrophobic Chains

Aqueous phase behavior of lipids with isoprenoid type hydrophobic chains (hereafter referred to as "isoprenoid-chained lipids") as functions of the chain length and the hydrophilic headgroup type has been examined over a chain length span from 12 to 16 carbon atoms long and a temperature range from -40 to 65 degrees C by using optical microscopy, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering) techniques. Characteristic phase behavior that arises from the unique "isoprenoid-chain" structure was discussed. This work together with previous studies (Prog. Colloid Polym. Sci. 2004, 123, 56-60 and J. Phys. Chem. B 2008, 112, 12286-12296) has clarified the molecular correlation of the aqueous phase behavior of "isoprenoid-chained lipids" over the chain lengths from 12 to 18 carbon atoms and introduced a new lipid library which can form a range of liquid crystals, such as an HII (an inverted hexagonal phase), a QII (an inverted cubic phase), and an Lalpha (a lamellar phase), that are stable over a wide temperature span from 0 degrees C.

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above − 30 °C and that a phase transition occurs only above 45 °C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases – upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 °C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 °C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.

New Lipid Family That Forms Inverted Cubic Phases in Equilibrium with Excess Water: Molecular Structure−Aqueous Phase Structure Relationship for Lipids with 5,9,13,17-Tetramethyloctadecyl and 5,9,13,17-Tetramethyloctadecanoyl Chains

With a view to discovering a new family of lipids that form inverted cubic phases, the aqueous phase behavior of a series of lipids with isoprenoid-type hydrophobic chains has been examined over a temperature range from -40 to 65 degrees C by using optical microscopy, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering) techniques. The lipids examined are those with 5,9,13,17-tetramethyloctadecyl and 5,9,13,17-tetramethyloctadecanoyl chains linked to a series of headgroups, that is, erythritol, pentaerythritol, xylose, and glucose. All of the lipid/water systems displayed a "water + liquid crystalline phase" two-phase coexistence state when sufficiently diluted. The aqueous phase structures of the most diluted liquid crystalline phases in equilibrium with excess water depend both on the lipid molecular structure and on the temperature. Given an isoprenoid chain, the preferred phase consistently follows a phase sequence of an H II (an inverted hexagonal phase) to a Q II (an inverted bicontinuous cubic phase) to an L alpha (a lamellar phase) as A* (cross-section area of the headgroup) increases. For a given lipid/water system, the phase sequence observed as the temperature increases is L alpha to Q II to H II. The present study allowed us to find four cubic phase-forming lipid species, PEOC 18+4 [mono- O-(5,9,13,17-tetramethyloctadecyl)pentaerythritol], beta-XylOC 18+4 [1- O-(5,9,13,17-tetramethyloctadecyl)-beta- d-xylopyranoside], EROCOC 17+4 [1- O-(5,9,13,17-tetramethyloctadecanoyl)erythritol], and PEOCOC 17+4 [mono- O-(5,9,13,17-tetramethyloctadecanoyl)pentaerythritol]. The values of T K (hydrated solid-liquid crystalline phase transition temperature) of the cubic phase-forming lipids are all below 0 degrees C. Quantitative analyses of the lipid molecular structure-aqueous phase structure relationship in terms of the experimentally evaluated "surfactant parameter" allow us to rationally select an optimum combination of hydrophilic/hydrophobic part of a lipid molecule that will form a desired phase in a desired temperature range.

Conformation Transformation Determined by Different Self-Assembled Phases in a DNA Complex with Cationic Polyhedral Oligomeric Silsesquioxane Lipid

In this work, a novel cube-shaped cationic lipid based on the imidazolium salt of polyhedral oligomeric silsesquioxane (POSS) was complexed with double-stranded DNA. Because of the negative spontaneous curvature of the cationic POSS imidazolium lipid, an inverted hexagonal phase resulted above the melting point of POSS crystals. Depending on the competition between the crystallization of POSS molecules and the negative spontaneous curvature of cationic POSS imidazolium lipids, different self-assembled phase morphologies were obtained. A lamellar phase was obtained when the POSS crystallization was relatively slow. When the POSS crystallization was fast, an inverted hexagonal phase was obtained with POSS lamellar crystals grown in the interstitials of DNA cylinders. On the basis of a circular dichroism study, double-stranded DNA adopted the B-form helical conformation in the inverted hexagonal phase, whereas the helical conformation was largely destroyed in the lamellar phase.

Electrostatics of DNA Complexes with Cationic Lipid Membranes

We present the exact solutions of the linear Poisson-Boltzmann equation for several problems relevant to electrostatics of DNA complexes with cationic lipids. We calculate the electrostatic potential and electrostatic energy for lamellar and inverted hexagonal phases, concentrating on the effects of dielectric boundaries. We compare our results for the complex energy with the known results of numerical solution of the nonlinear Poisson-Boltzmann equation. Using the solution for the lamellar phase, we calculate the compressibility modulus and compare our findings with the experimental data available. Also, we treat charge-charge interactions across, along, and between two low-dielectric membranes. We obtain an estimate for the strength of electrostatic interactions of one-dimensional DNA smectic layers across the lipid membrane. We discuss in the end some aspects of two-dimensional DNA condensation and DNA-DNA attraction in the DNA-lipid lamellar phase in the presence of di- and trivalent cations. We analyze the equilibrium DNA-DNA separations in lamellar complexes using the recently developed theory of electrostatic interactions of DNA helical charge motifs.

Structure and Gene Silencing Activities of Monovalent and Pentavalent Cationic Lipid Vectors Complexed with siRNA

Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (rhochg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given rhochg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and rhochg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.

Cryo-TEM investigation of phase behaviour and aggregate structure in dilute dispersions of monoolein and oleic acid

Cryo-transmission electron microscopy (cryo-TEM) was used to image the microstructure in dilute sonicated dispersions of monoolein and oleic acid. The aim of the study was to explore how different experimental parameters, such as sample composition, total lipid concentration, pH, and ageing affect the phase behaviour and aggregate structure. Our investigations show that a rich variety of lamellar and non-lamellar structures, including liposomes and particles of cubic and inverted hexagonal phase, may form depending on the experimental conditions. The results are analyzed and discussed in relation to existing phase diagrams and earlier investigations concerning phase- and structural behaviour in monoolein/oleic acid/water systems.

Phase Behavior of a Phospholipid/Fatty Acid/Water Mixture Studied in Atomic Detail

Molecular dynamics simulations have been used to study the phase behavior of a dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA)/water 1:2:20 mixture in atomic detail. Starting from a random solution of DPPC and PA in water, the system adopts either a gel phase at temperatures below approximately 330 K or an inverted hexagonal phase above approximately 330 K in good agreement with experiment. It has also been possible to observe the direct transformation from a gel to an inverted hexagonal phase at elevated temperature (approximately 390 K). During this transformation, a metastable fluid lamellar intermediate is observed. Interlamellar connections or stalks form spontaneously on a nanosecond time scale and subsequently elongate, leading to the formation of an inverted hexagonal phase. This work opens the possibility of studying in detail how the formation of nonlamellar phases is affected by lipid composition and (fusion) peptides and, thus, is an important step toward understanding related biological processes, such as membrane fusion.

Polymorphism of DNA–anionic liposome complexes reveals hierarchy of ion-mediated interactions

Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid-DNA complexes because of their low cytotoxicity. We investigate AL-DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the self-assembled structures and phase behavior of AL-DNA complexes. The governing interactions in AL-DNA systems are complex: divalent ions can mediate strong attractions between different combinations of the components (such as DNA-DNA and membrane-membrane). Moreover, divalent cations can coordinate non-electrostatically with lipids and modify the resultant membrane structure. We find that at low membrane charge densities AL-DNA complexes organize into a lamellar structure of alternating DNA and membrane layers crosslinked by ions. At high membrane charge densities, a new phase with no analog in cationic lipid-DNA systems is observed: DNA is expelled from the complex, and a lamellar stack of membranes and intercalated ions is formed. For a subset of the ionic species, high ion concentrations generate an inverted hexagonal phase comprised of DNA strands wrapped by ion-coated lipid tubes. A simple theoretical model that takes into account the electrostatic and membrane elastic contributions to the free energy shows that this transition is consistent with an ion-induced change in the membrane spontaneous curvature, c0. Moreover, the crossover between the lamellar and inverted hexagonal phases occurs at a critical c0 that agrees well with experimental values.

Molecular View of Hexagonal Phase Formation in Phospholipid Membranes

Important biological processes, such as vesicle fusion or budding, require the cell matrix to undergo a transition from a lamellar to a nonlamellar state. Although equilibrium properties of membranes are amenable to detailed theoretical studies, collective rearrangements involved in phase transitions have thus far only been modeled on a qualitative level. Here, for the first time, the complete transition pathway from a multilamellar to an inverted hexagonal phase is elucidated at near-atomic detail using a recently developed coarse-grained molecular dynamics simulation model. Insight is provided into experimentally inaccessible data such as the molecular structure of the intermediates and the kinetics involved. Starting from multilamellar configurations, the spontaneous formation of stalks between the bilayers is observed on a nanosecond timescale at elevated temperatures or reduced hydration levels. The stalks subsequently elongate in a cooperative manner leading to the formation of an inverted hexagonal phase. The rate of stalk elongation is ∼0.1 nm ns −1. Within a narrow hydration/temperature/composition range the stalks appear stable and rearrange into the rhombohedral phase.

Distorted Hexagonal Phase Studied by Neutron Diffraction: Lipid Components Demixed in a Bent Monolayer

The recent discovery of a distorted hexagonal phase in 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine/1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPE/DOPC) mixtures raised the intriguing question as to whether lipid mixtures demix in a bent monolayer. We performed neutron diffraction on a mixture of headgroup deuterated DOPC-d(13) and nondeuterated DOPE to study the lipid distribution in the distorted hexagonal phase. The 1:1 lipid mixture in full hydration and 25 degrees C was in a homogeneous lamellar phase. Upon dehydration the mixture transformed to a rhombohedral phase, then to a distorted inverted hexagonal phase, and finally to a regular inverted hexagonal phase. In the distorted hexagonal phase, the diffraction pattern showed a two-dimensional monoclinic lattice with two reciprocal vectors of equal length (1.5 nm(-)(1)) forming an angle 53 degrees between them. Diffraction intensities measured while varying the D(2)O/H(2)O ratio in the humidity was used to solve the phase problem. The neutron scattering length density distribution of the distorted hexagonal phase was constructed. The constant density contours are approximately elliptical. The difference in the eccentricities of the contours between the water and lipid distributions indicates that the DOPE/DOPC ratio is not uniform around the elliptical lipid tube in the unit cell. DOPE is preferentially distributed at the vertex regions where the curvature is the highest. Thus for the first time it is shown that when a monolayer of a homogeneous lipid mixture is bent, the lipid components are partially demixed in reaching the free energy minimum.

Field Theoretic Study of Bilayer Membrane Fusion. I. Hemifusion Mechanism

Self-consistent field theory is used to determine structural and energetic properties of metastable intermediates and unstable transition states involved in the standard stalk mechanism of bilayer membrane fusion. A microscopic model of flexible amphiphilic chains dissolved in hydrophilic solvent is employed to describe these self-assembled structures. We find that the barrier to formation of the initial stalk is much smaller than previously estimated by phenomenological theories. Therefore its creation it is not the rate-limiting process. The relevant barrier is associated with the rather limited radial expansion of the stalk into a hemifusion diaphragm. It is strongly affected by the architecture of the amphiphile, decreasing as the effective spontaneous curvature of the amphiphile is made more negative. It is also reduced when the tension is increased. At high tension the fusion pore, created when a hole forms in the hemifusion diaphragm, expands without bound. At very low membrane tension, small fusion pores can be trapped in a flickering metastable state. Successful fusion is severely limited by the architecture of the lipids. If the effective spontaneous curvature is not sufficiently negative, fusion does not occur because metastable stalks, whose existence is a seemingly necessary prerequisite, do not form at all. However if the spontaneous curvature is too negative, stalks are so stable that fusion does not occur because the system is unstable either to a phase of stable radial stalks, or to an inverted-hexagonal phase induced by stable linear stalks. Our results on the architecture and tension needed for successful fusion are summarized in a phase diagram.