Increased Bilayer Fluidity Research Articles

Effect of DMSO on Structural Properties of DMPC and DPPC Liposome Suspensions.

The study and characterization of the biophysical properties of membranes and drug-membrane interactions represent a critical step in drug development, as biological membranes act as a barrier that the drug must overcome to reach its active site. Liposomes are widely used in drug delivery to circumvent the poor aqueous solubility of most drugs, improving systemic bioavailability and pharmacokinetics. Further, they can be targeted to deliver to specific disease sites, thus decreasing drug load, and reducing side effects and poor adherence to treatment. To improve drug solubility during liposome preparation, DMSO is the most widely used solvent. This raises concern about the potential effect of DMSO on membranes and leads us to investigate, using DSC and EPR, the influence of DMSO on the behavior of lipid model membranes of DMPC and DPPC. In addition, we tested the influence of DMSO on drug-membrane interaction, using compounds with different hydrophobicity and varying DMSO content, using the same experimental techniques. Overall, it was found that with up to 10% DMSO, changes in the bilayer fluidity or the thermotropic properties of the studied liposomes were not significant, within the experimental uncertainty. For higher concentrations of DMSO, there is a stabilization of both the gel and the rippled gel phases, and increased bilayer fluidity of DMPC and DPPC liposomes leading to an increase in membrane permeability.

Lipid Corona Formation from Nanoparticle Interactions with Bilayers

Summary Although mixing nanoparticles with certain biological molecules can result in coronas that afford some control over how engineered nanomaterials interact with living systems, corona formation mechanisms remain enigmatic. Here, we report results from experiments and computer simulations that provide concrete lines of evidence for spontaneous lipid corona formation without active mixing upon attachment to stationary and suspended lipid bilayer membranes. Experiments show that polycation-wrapped particles disrupt the tails of zwitterionic lipids, increase bilayer fluidity, and leave the membrane with reduced ζ potentials. Computer simulations suggest that the contact ion pairing between the lipid head groups and the polycations' ammonium groups leads to the formation of stable, albeit fragmented, lipid bilayer coronas. The mechanistic insight regarding lipid corona formation can be used to improve control over nano-bio interactions and to help understand why some nanomaterial-ligand combinations are detrimental to organisms but others are not.

Correlating Membrane Morphological Responses with Micellar Aggregation Behavior of Capric Acid and Monocaprin.

The interaction of single-chain lipid amphiphiles with phospholipid membranes is relevant to many scientific fields, including molecular evolution, medicine, and biofuels. Two widely studied compounds within this class are the medium-chain saturated fatty acid, capric acid, and its monoglyceride derivative, monocaprin. To date, most studies about these compounds have involved in vitro evaluation of their biological activities, while mechanistic details of how capric acid and monocaprin interact with phospholipid bilayers remain elusive. Herein, we investigated the effect of these two compounds on the morphological and fluidic properties of prefabricated, supported lipid bilayers (SLBs). The critical micelle concentration (CMC) of each compound was determined by fluorescence spectroscopy measurements. At or above its CMC, capric acid induced the formation of elongated tubules protruding from the SLB, as determined by quartz crystal microbalance-dissipation and fluorescence microscopy experiments. By contrast, monocaprin induced the formation of elongated tubules or membrane buds below and above its CMC, respectively. Fluorescence recovery after photobleaching (FRAP) experiments indicated that capric acid increased bilayer fluidity only above its CMC, whereas monocaprin increased bilayer fluidity both above and below its CMC. We discuss these findings in the context of the two compounds' structural properties, including net charge, molecular length and hydrogen-bonding capacity. Collectively, the findings demonstrate that capric acid and monocaprin differentially affect the morphological and fluidic properties of SLBs, and that the aggregation state of the compounds plays a critical role in modulating their interactions with phospholipid membranes.

Effect of Incorporating Cholesterol into DDA:TDB Liposomal Adjuvants on Bilayer Properties, Biodistribution, and Immune Responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses.

Effect of a 2-hydroxylated fatty acid on Cholesterol-rich membrane domains

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.

Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition

Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [gamma-32P]GTP and subjected to physiological levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0-30 dynes/cm2) activated G proteins in dose-dependent manner (0.48-4.6 pmol/min per microg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.

Changes in lipid composition and fluidity of human placental basal membrane and modulation of bilayer protein functions with progress of gestation.

Human placental syncytiotrophoblast basal membrane plays an important role in transfer of nutrients from the mother to the growing fetus all throughout gestation. The membrane lipid composition together with the bilayer fluidity is found to be the major index in modulation of these transport processes. In the present study, the effects of changing lipid composition on the placental basal membrane fluidity and the modulating influence of the latter on membrane enzyme and transport functions with progress of gestation,were investigated. Steady-state fluorescence analysis using 1,6-diphenyl- 1,3,5 hexatriene as the probe, indicated a decrease in fluorescence anisotropy of both labeled native membrane vesicles and liposomes prepared from lipids extracted from the basal membrane vesicles, signifying increased bilayer fluidity with progress of gestation. This in turn, was successfully correlated to the lowering of cholesterol content and enhanced phospholipid concentration with a steady decrease in cholesterol/phospholipid ratio during placental development. Enhanced Na+-K+-ATPase activity and steady-state glucose uptake across basal membrane with gestational progress suggested modulation of membrane protein functions by the fluidity, which was further corroborated by the increased bilayer fluidity and enzyme activity in benzyl alcohol treated basal membrane in each gestational age group.

Effects of octane on the fatty acid composition and transition temperature of Pseudomonas oleovorans membrane lipids during growth in two-liquid-phase continuous cultures

Growth of Pseudomonas oleovorans GPol in continuous culture containing a bulk n-octane phase resulted in changes of the fatty acid composition of the membrane lipids. Compared to citrate-grown cells, the ratio of C 18 to C 16 fatty acids and the ratio of unsaturated to saturated fatty acids increased as a result of growth on octane. Trans-unsaturated fatty acids, which are rarely found in bacteria, were formed during continuous growth of P. oleovorans on octane. Moreover, the mean acyl chain length and unsaturated fatty acids also increased as the growth rates increased both in octane-grown and citrate-grown cells. Differential scanning calorimetry measurements of extracted lipids showed the transition temperature of membrane lipids from octane-grown cells increased from about 24°C to 32°C as the growth rate increased, whereas cells grown on citrate showed a constant transition temperature of about 6°C at all growth rates tested, indicating a decrease of membrane lipid fluidity in octane-grown cells. Because alkanes are known to increase bilayer fluidity by intercalating between lipid fatty acyl chains, the increased transition temperature of the lipids of cells grown on octane may be a physiological response of P. oleovorans to compensate for the direct effects of octane on its cellular membranes.

Direct force measurements of specific and nonspecific protein interactions.

Streptavidin-biotin (receptor-ligand) interaction forces were measured directly as a function of their intermolecular separation in various salt solutions and at various temperatures with a surface forces apparatus. Electrostatic and van der Waals forces were found to dominate the long-range streptavidin-biotin interaction at > 20 A. At intermediate separations, down to approximately 10 A, the interaction is governed by repulsive steric and attractive van der Waals and hydrophobic forces. A much stronger short-range attraction giving rise to the strong, specific adhesive binding was measured at molecular separations of less than 5 A. A decrease in the pH from 7.2 to 6.0 resulted in complete charge reversal on the binding surface of streptavidin (pK approximately 6) from net negative to net positive, while leaving the negatively charged biotin surface (pK approximately 3.0) unchanged, and the long-range interaction switched from repulsive to attractive. This observed behavior can be attributed to the titration of two histidines on the biotin binding surface of streptavidin. These results reveal a strong sensitivity of the long-range interaction forces to the detailed amino acid composition of the biotin binding surface. They also demonstrate the powerful regulatory potential conferred by small changes in local surface ionic conditions on protein interaction forces over different distance regimes. The effects of temperature on receptor-ligand dynamics and on the strength of intermembrane adhesion forces were studied by measuring the long-range force profiles and short-range adhesion forces above and below the chain melting temperature (Tc approximately 30 degrees C) of the lipids in the supporting bilayers. Increased bilayer fluidity due to a temperature increase to 33 degrees C (T > Tc) increased short-range adhesion by 7-fold relative to bilayers in the gel state at 25 degrees C (T < Tc). This effect was attributed to the enhanced rates of lateral diffusion and molecular rearrangements on the more fluid bilayer surfaces, which resulted in greater and more rapid intermembrane bond formation. A change in the rates of molecular rearrangements was also found to affect the repulsive part of the interaction potential at intermediate separations (10-20 A) via modulation of the steric repulsion between streptavidin and the highly flexible, polymer-like biotin molecules. This is expected to have a large effect on the association rates of receptor-ligand binding, even if it does not change the equilibrium binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.