Monoolein Bilayers Research Articles

Tuning curvature and phase behavior of monoolein bilayers by epigallocatechin-3-gallate: Structural insight and cytotoxicity

The use of glyceryl monooleate (GMO)-based nanoparticles has not yet been explored in overcoming the low bioavailability of Epigallocatechin-3-gallate (EGCG), a green tea polyphenol with a known anticancer activity. Since the inclusion of a guest molecule can affect the curvature and the supramolecular structure of fully hydrated GMO-based phase, the phase behavior of bulk and dispersed liquid crystalline systems containing EGCG were explored by Small Angle Neutron Scattering and X-Ray Diffraction experiments. Molecular Dynamic Simulations showed how the interaction of EGCG with polar heads of GMO strongly affects the curvature and packing of GMO phase. The EGCG encapsulation efficiency was determined in the nanodispersions and their size studied by Dynamic Light Scattering and Atomic Force Microscopy. A nanodispersed formulation has been optimized with a cytotoxic effect more than additive of GMO and EGCG.

Salt-responsive monoolein cubic phase containing polyethyleneimine gel

Monoolein (MO) cubic phases responsive to salt ions were prepared by immobilizing polyethyleneimine (PEI) in its water channel through its electrostatic attraction with octanoic acid (OA) intercalated in MO bilayer and ionically gelling PEI using tripolyphosphoric acid (TPPA). The interfacial tension of PEI/OA solution markedly decreased with increasing concentration and it was typical of the interfacial tension profile of a surfactant solution. TPPA remarkably lowered the transition temperature of MO cubic phase from about 63 °C to below 46 °C, possibly because the hydroxyl groups of TPPA participated in hydrogen bonding with the glycerol residue of MO. The cubic phases showed finger print–like patterns on TEM micrographs and neither OA, nor PEI, nor TPPA affected the structure of the cubic phases. When PBS was used as a release medium, the release degree of a dye (i.e. Rhodamine B) loaded in the cubic phase was more than two times higher than that obtained using distilled water. The salt ions contained in the buffer solution would be able to dissolve PEI gel via ion exchange and promote the release of dye out of the cubic phase.

In vitro Anti-cancer Efficacy and Cellular Interaction of Cubic Phases Containing Cinnamic Acid, Poly(ethyleneimine), and Doxorubicin

Folate-decorated monoolein (MO) cubosomes containing poly(ethyleneimine) (PEI), cinnamic acid (CA), and doxorubicin (DOX) were prepared to deliver the anticancer drug specifically to cancer cells (KB cells) and boost the anti-cancer efficacy without causing an acute toxicity. Hydrophobically modified (Hm) gelatin (Gel)-folate (FA) conjugate (HmGel-FA) was prepared through amidation reaction among Gel, decanoyl chloride, and FA. HmGel-FA and Pluronic F127 were used as stabilizers for the cubosomal particles. According to the measurement of air/water interfacial tension, the surface activity was greater in the order of HmGel > HmGel-FA > Gel. Since it was reported that PEI and CA formed a self-assembly in the water channel of cubosomes and the dissolving property of the self-assembly was dependent on the pH value of medium, they were included in the water channel as a controller for DOX release. On the TEM micrographs of the cubosomes, water channels were surrounded by MO bilayers, and the payload (i.e. PEI, CA, DOX) had little effect on the structure. The in vitro anti-cancer efficacy of folate-decorated cubosomes containing PEI, CA, and DOX was higher than that of folate-free ones. According to flow cytometric analysis and confocal laser scanning microscopy, DOX fluorescence intensity was greater in the order of KB cells treated with folate-decorated cubosomes containing PEI, CA, and DOX > folate-free ones > free DOX. The folate-decorated cubosomes would be able to target the cancer cells and subsequently the receptor-mediated endocytosis would take place.

Complexation-responsive monoolein cubic phase containing extract of Bambusae Caulis in Taeniam

Monoolein (MO) cubic phase (CP) containing Bambusae Caulis in Taeniam extract (BCT), hydrophobically modified alginate (HmAlg), and hydrophobically modified gelatin (HmGel) was prepared by a melt and hydration method. On the TEM micrographs of CP, water channels and MO bilayers were found. The phase transition temperature of MO CP was about 65.3 °C and it deceased by the inclusion of the additives. The release of 4-hydroxybenzaldehyde from CP was markedly suppressed at pH 4.5 but not at pH 5.5 and pH 7.4. The complex coacervate of HmGel and HmAlg was thought to suppress the release in the acidic condition.

Monoolein cubic phase containing alginate/cystamine gel for controlled release of epidermal growth factor

Monoolein (MO) cubic phase including alginate and cystamine in its water channel controlled the release of epidermal growth factor (EGF) by responding to changes in pH value and the reductive conditions of the release medium. The crosslinking degree of alginate gel with cystamine and the complex coacervation of alginate and EGF were investigated by using light scattering. TEM micrographs of cubic phases revealed MO bilayers along with water channels. Differential scanning calorimetry indicated that the cubic-to-hexagonal phase transition took place at 60.2 °C. Additives such as stearyl trimethyl ammonium chloride and cystamine decreased the transition temperature by a few to more than 10 °C. The release of EGF loaded in cubic phases was completed in 5 h and, thereafter, no significant additional release was observed. The release % of EGF loaded in MO cubic phase containing alginate and cystamine increased not only with the increase of pH but also glutathione concentration. The MO cubic phase containing alginate/cystamine gel can be used as a carrier for the delivery of peptide and protein drugs.

Large PAMAM Dendron Induces Formation of Unusual P4332 Mesophase in Monoolein/Water Systems.

Compact macromolecular dendrons have previously been shown to induce the formation of discontinuous inverse micellar assemblies with Fd3 m symmetry in monoolein/water systems. Here, we demonstrate that a large PAMAM dendron (G5: fifth generation) induces the formation of a very unusual mesophase with P4332 symmetry. This mesophase had previously been observed in monoolein/water systems only on addition of cytochrome c. The P4332 mesophase can be considered an intermediate phase between the bicontinuous Ia3 d and discontinuous micellar mesophases. We present a detailed investigation of the phase behavior of monoolein/water as a function of G5 concentration and temperature. Addition of 1% G5 in 85/15 monoolein/water system induces a transition from the Lα to Ia3 d phase. Further increase in G5 concentration to above 2% induces the formation of the P4332 phase. In contrast to this, incorporation of lower generation PAMAM dendrons (G2-G4) in monoolein/water yields a qualitatively different phase diagram with the formation of the reverse micellar Fd3 m phase. PAMAM dendrons of all generations, G2-G5, bear terminal amine groups that interact with the monoolein headgroup. The compact molecular architecture of the dendrons and these attractive interactions induce bending of the monoolein bilayer structure. For smaller dendrons, G2-G4, this results in the formation of the Fd3 m phase. However, the large size of the G5 dendron precludes this and a rare intermediate phase between the Ia3 d and discontinuous micellar phase, and the P4332 mesophase forms instead.

Extension of the LOPLS-AA Force Field for Alcohols, Esters, and Monoolein Bilayers and its Validation by Neutron Scattering Experiments.

The recently presented LOPLS-AA all-atom force field for long hydrocarbon chains, based on the OPLS-AA force field, was extended to alcohols, esters, and glyceryl monooleate (GMO) lipids as a model lipid. Dihedral angles were fitted against high level ab initio calculations, and ester charges were increased to improve their hydration properties. Additionally, the ester Lennard-Jones parameters were readjusted to reproduce experimental liquid bulk properties, densities, and heats of vaporization. This extension enabled the setup of LOPLS-AA parameters for GMO molecules. The properties of the lipid force field were tested for the liquid-crystalline phase of a GMO bilayer. The obtained area per lipid for GMO is in good agreement with experiment. Additionally, the lipid dynamics on the subpicosecond to the nanosecond time scale is in excellent agreement with results from time-of-flight (TOF) quasielastic neutron scattering (QENS) experiments on a multilamellar monoolein system, enabling here for the first time the critical evaluation of the short-time dynamics obtained from a molecular dynamics simulation of a membrane system.

Glucose-Responsive Monoolein Cubic Phase Containing Glucose Oxidase

Glucose-responsive monoolein (MO) cubic phase was prepared by immobilizing proteinoid composed of Asp and Leu (PAL) and hydrophobically modified glucose oxidase (HmGOD) onto the MO bilayers. The hydrodynamic mean diameter of PAL aggregate in aqueous solution decreased with increasing the pH value. The number of pamitic acid residue per one molecule of HmGOD was determined to be 6.3 by a calorimetric method. HmGOD could acidify glucose solution in a few hours, possibly because it converted glucose to gluconic acid. PAL- and HmGOD-immobilized MO cubic phase was prepared by hydrating MO melt with the mixture aqueous solution of PAL and HmGOD. The cubic phase exhibited its phase transition around 62.5°C, determined by polarizing microscopy. The release of carboxylic fluorescein (CF) from the cubic phase was suppressed when the pH value of release medium decreased, possibly because PAL can aggregate more at a lower pH value. The release was suppressed when glucose concentration increased, possibly because the release medium can be more acidified and PAL will be more aggregated at a higher glucose concentration. The cubic phase could be used as a drug carrier which releases its content in a sustained manner when the glucose concentration is abnormally high.

Hydrogen Bonding of Cholesterol in the Lipidic Cubic Phase

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.

Structure and dynamics of a sponge phase in the methyl δ-aminolevulinate/monoolein/water/propylene glycol system

The structural effect caused by the addition of up to 16% (w/w) of the hydrochloride salt of δ-aminolevulinic acid (ALA, HOOC–CH 2–CH 2–CO–CH 2–NH 2⋅HCl) or its methyl ester (m-ALA) to the sponge phase formed of monoolein/water/propylene glycol was investigated by means of crossed polarizers, small angle X-ray diffraction (SAXD) and nuclear magnetic resonance diffusometry (NMRD). Inspection with crossed polarizers revealed that additions of 4–16% (w/w) m-ALA transformed the isotropic bicontinuous sponge phase partly (4–10%) or completely (13 and 16%) into an anisotropic lamellar phase, indicating that m-ALA has a flattening effect on the bilayer curvature. The addition of 16% (w/w) ALA did not show any effect on the sponge phase. By addition of water to the anisotropic m-ALA samples, isotropic liquids were re-formed. The SAXD data for the isotropic liquids showed a diffuse Bragg peak and the NMRD self-diffusion coefficients for the drug (m-ALA) and the components of the original sponge phase (monoolein, water and propylene glycol) were shown to be essentially constant for 0–16% (w/w) added m-ALA. These results confirmed the hypothesis that the re-formed isotropic phases were indeed sponge phases. Water, for example, showed a diffusion coefficient of 3.1 – 3.9 × 10 −10 m 2 s −1 in the sponge phase, compared to 5.3 – 5.7 × 10 −10 m 2 s −1 in relevant water/propylene glycol solutions or 2.3 × 10 −9 m 2 s −1 in pure water. The reduction can be explained as a consequence of the microstructure (congruent monoolein bilayer) of the sponge phase and of the viscosity effect caused by propylene glycol and m-ALA.

Tuning curvature and stability of monoolein bilayers by amphiphilic designer peptides

Tuning curvature and stability of monoolein bilayers by amphiphilic designer peptides

Tuning curvature and stability of monoolein bilayers by designer lipid-like peptide surfactants.

This study reports the effect of loading four different charged designer lipid-like short anionic and cationic peptide surfactants on the fully hydrated monoolein (MO)-based Pn3m phase (Q224). The studied peptide surfactants comprise seven amino acid residues, namely A6D, DA6, A6K, and KA6. D (aspartic acid) bears two negative charges, K (lysine) bears one positive charge, and A (alanine) constitutes the hydrophobic tail. To elucidate the impact of these peptide surfactants, the ternary MO/peptide/water system has been investigated using small-angle X-ray scattering (SAXS), within a certain range of peptide concentrations (R≤0.2) and temperatures (25 to 70°C). We demonstrate that the bilayer curvature and the stability are modulated by: i) the peptide/lipid molar ratio, ii) the peptide molecular structure (the degree of hydrophobicity, the type of the hydrophilic amino acid, and the headgroup location), and iii) the temperature. The anionic peptide surfactants, A6D and DA6, exhibit the strongest surface activity. At low peptide concentrations (R = 0.01), the Pn3m structure is still preserved, but its lattice increases due to the strong electrostatic repulsion between the negatively charged peptide molecules, which are incorporated into the interface. This means that the anionic peptides have the effect of enlarging the water channels and thus they serve to enhance the accommodation of positively charged water-soluble active molecules in the Pn3m phase. At higher peptide concentration (R = 0.10), the lipid bilayers are destabilized and the structural transition from the Pn3m to the inverted hexagonal phase (H2) is induced. For the cationic peptides, our study illustrates how even minor modifications, such as changing the location of the headgroup (A6K vs. KA6), affects significantly the peptide's effectiveness. Only KA6 displays a propensity to promote the formation of H2, which suggests that KA6 molecules have a higher degree of incorporation in the interface than those of A6K.

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid-detergent matrices.

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic-detergent matrices. Detergent-soluble AChR complexed with alpha-bungarotoxin (alphaBTx), a polypeptidic competitive antagonist, was purified. The AChR-alphaBTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The alphaBTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.

Interactions of cyclic AMP and its dibutyryl analogue with model membrane: X-ray diffraction and Raman spectroscopic study using cubic liquid–crystalline phases of monoolein

Interactions of adenosine 3′:5′-cyclic monophosphate (cAMP) and its dibutyryl analogue, N 6,2′- O-dibutyryladenosine 3′:5′-cyclic monophosphate (dbcAMP), with a lipid bilayer were studied by small-angle X-ray diffraction (SAXD) and Raman spectroscopy. The cubic Pn3m phase of monoolein (MO) served as a bilayer-based model system. SAXD measurements have indicated that incorporation of approximately 3 wt.% cAMP leaves the phase parameters practically unaltered, whereas the same content of dbcAMP induces the intercubic Pn3m→ Ia3d transition. By applying the concepts of lipid shape parameter and infinite periodic minimal surface to these MO phases, we have suggested that, as opposed to cAMP, dbcAMP associates with the MO bilayer. This conclusion has been supported by the different effects of phase matrix on the Raman shifts of the adenine and phosphate vibrational modes of these two nucleotides. Moreover, Raman spectra have indicated that dbcAMP inserts into the bilayer through the butyryladenine group, positioning dbcAMP preferentially at the polar/apolar interface.

Gramicidin tryptophans mediate formamidinium-induced channel stabilization

Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl.

The transport of ions across monoolein bilayers mediated by synthetic macrocyclic carriers

The efficiency as carriers for the alkali and the alkaline earth ions across monoolein bilayers of three macrocyclic compounds has been investigated by conductometric measurements. The results show that although the three compounds have the optimum cavity size for fitting potassium and barium cations, their efficiency in ionic transport is quite different. These discrepancies are rationalized on the basis of the overlap of the water-diazacrown and cation-ligand interactions on the carrier mechanism.

Optical and electrical properties of thin monoolein lipid bilayers.

Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K+ and valinomycin-Rb+ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K+ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb+ complex.

The influence of n-alkanols and cholesterol on the duration and conductance of gramicidin single channels in monoolein bilayers

The mean lifetime of gramicidin A channels in bilayers formed from monoolein and squalane was sharply reduced by the adsorption of a range of n-alkanols and cholesterol. Results are shown for n-hexanol, n-octanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, n-octadecanol and cholesterol. The longer chain n-alkanols were apparently more effective than the shorter members and cholesterol was the most effective of the substances examined. The single channel conductance was also affected, though to a much lesser extent than the mean channel lifetime, the n-alkanols producing increases and cholesterol a decrease. It is suggested that membrane fluidity changes are not likely to be primarily responsible for the reductions in channel lifetimes but that the bilayer tension, which is known to be increased by n-octanol, could be significant.

The thickness of monoolein lipid bilayers as determined from reflectance measurements

Measurements of the reflectance of monoolein n- alkane and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was 0.5 ± 0.1 nm , independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When n- tetradecane and n- hexadecane were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.

Electrical capacity of black lipid films and of lipid bilayers made from monolayers

Planar bilayer membranes were formed from monolayers of a series of monounsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 μF/cm 2 which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride. The specific capacity ( C m ) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the C m increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with n- decane as a solvent.