Cholesterol Mole Fraction Research Articles

Lipid redistribution due to a cell-cell fusion pore

We consider the redistribution of lipids comprising the plasma membranes during cell-cell fusion, particularly due to the presence of a fusion pore. Assuming the membranes are of constant thickness, we find that the mole fraction of cholesterol increases in the directly apposed exoplasmic leaflets, and is decreased in the cytoplasmic leaflets. The redistribution of the phospholipids is obtained as well.

Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of nonideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (μc) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, referenced to crystalline cholesterol. μc is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase separated mixture. It is simply related to concentration under conditions of ideal mixing, but is far from ideal for the majority of lipid mixtures investigated here. Measurements of μc can vary with phospholipid composition by 1.5 kBT at constant cholesterol mole fraction implying a more than fivefold change in its availability for binding receptors and other reactions. Experimental measurements are fit to thermodynamic models including cholesterol-DPPC complexes or pairwise interactions between lipid species to provide intuition about the magnitude of interactions. These findings reinforce that μc depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration.

Extent of raft composition in a model plasma membrane

“Rafts” are nanometer-size inhomogeneities in the plasma membrane that, in the outer leaflet, are enriched in sphingomyelin and cholesterol. They are thought to provide a platform for proteins to carry out biological processes. Here, we employ a model asymmetric plasma membrane to address the question of the range of sphingomyelin and cholesterol compositions in which one would expect the formation of rafts. We define a weight for the likelihood of raft formation and evaluate it as a function of the sphingomyelin mole fraction in the outer leaflet for three bilayers with total cholesterol mole fractions of 0.30, 0.40, and 0.50. Not surprisingly, the weight decreases when there is little sphingomyelin. Less expected, we find that the weight also decreases when there is a large mole fraction of sphingomyelin. The weight is largest in the bilayer with a total cholesterol mole fraction of 0.30 and decreases rapidly with increasing total cholesterol. We explicate the reasons for these behaviors. In the 0.30 cholesterol bilayer, the largest weight occurs at a sphingomyelin mole fraction in the outer leaflet of approximately 0.23. The weight falls to one half its maximum value at sphingomyelin mole fractions of 0.15 and 0.33. In terms of the sphingomyelin mole fraction of the asymmetric bilayer, the maximum weight occurs at 0.12 and falls to half maximum at 0.08 and 0.17.

The effect of cholesterol on highly curved membranes measured by nanosecond Fluorescence Correlation Spectroscopy

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τ r) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τ r) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%, τ r increases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Quantification of pulsed electric field for the rupture of giant vesicles with various surface charges, cholesterols and osmotic pressures.

Electropermeabilization is a promising phenomenon that occurs when pulsed electric field with high frequency is applied to cells/vesicles. We quantify the required values of pulsed electric fields for the rupture of cell-sized giant unilamellar vesicles (GUVs) which are prepared under various surface charges, cholesterol contents and osmotic pressures. The probability of rupture and the average time of rupture are evaluated under these conditions. The electric field changes from 500 to 410 Vcm-1 by varying the anionic lipid mole fraction from 0 to 0.60 for getting the maximum probability of rupture (i.e., 1.0). In contrast, the same probability of rupture is obtained for changing the electric field from 410 to 630 Vcm-1 by varying the cholesterol mole fraction in the membranes from 0 to 0.40. These results suggest that the required electric field for the rupture decreases with the increase of surface charge density but increases with the increase of cholesterol. We also quantify the electric field for the rupture of GUVs containing anionic mole fraction of 0.40 under various osmotic pressures. In the absence of osmotic pressure, the electric field for the rupture is obtained 430 Vcm-1, whereas the field is 300 Vcm-1 in the presence of 17 mOsmL-1, indicating the instability of GUVs at higher osmotic pressures. These investigations open an avenue of possibilities for finding the electric field dependent rupture of cell-like vesicles along with the insight of biophysical and biochemical processes.

Interaction between Hydrophilic Ionic Liquid and Phospholipid/Cholesterol Mixed Film.

Surface pressure (π)-area (A) isotherms were studied to analyze the interactions between a hydrophilic ionic liquid (IL) (ethyl(2-hydroxyethyl)dimethylammonium methanesulfonate) and a pure dipalmitoylphosphatidylcholine (DPPC) film or a DPPC-cholesterol mixed film. When the hydrophilic IL was added to an underlayer solution, the isotherm shifted toward higher areas. Intriguingly, when the hydrophilic IL was added, the packing of the film materials became loose and the elastic modulus decreased, resulting in increased flexibility. This phenomenon was most evident under a cholesterol mole fraction of 0.2. This composition resembles that of cell membranes, which typically comprise phospholipids and cholesterol, suggesting that this hydrophilic IL may be able to interact significantly with biological membranes.

Axonal plasma membrane-mediated toxicity of cholesterol in Alzheimer's disease: A microsecond molecular dynamics study

Alzheimer's disease is increasingly being recognized as an immune-mediated disease of brain. Since physiological brain health and brain immune function is dependent upon homeostatic neuronal membrane structure and function, alterations in membrane lipid biochemistry may predispose to disease. Brain is rich in cholesterol, and cholesterol metabolism dysfunction is a known risk factor for AD. Employing extensive microsecond all-atom molecular dynamics simulations, we investigated the properties of model neuronal membranes as a function of cholesterol concentration; phospholipid and phospholipid/cholesterol bilayers were also simulated to compare against available experimental data. Increased cholesterol concentrations compact and stiffen the lipid membrane, reducing permeability while modulating local water densities in the peri-membranous environment. Conversely, lower cholesterol mole fraction yields membranes with increased molecular disorder, enhanced fluidity, higher molecular tilting, and augmented interdigitation between bilayer leaflet lipids. Our findings provide a molecular insight on effect of cholesterol composition on various biochemical processes occurring at neuronal axon plasma membrane. These calculations also endeavor to establish a membrane-based link between cholesterol as an AD risk factor and possible AD pathology.

Unravelling the Effects of Cholesterol on the Second-Order Nonlinear Optical Responses of Di-8-ANEPPS Dye Embedded in Phosphatidylcholine Lipid Bilayers.

Cholesterol is known for its role in maintaining the correct fluidity and rigidity of the animals cell membranes and thus their functions. Assessing the content and the role of cholesterol in lipid bilayers is therefore of crucial importance for a deeper understanding and control of membrane functioning. In this computational work, we investigate bilayers built from three types of glycerophospholipid phosphatidylcholine (PC) lipids, namely dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and dioleoylphosphatidylcholine (DOPC), and containing different amounts of cholesterol by analyzing the second-harmonic generation (SHG) nonlinear optical (NLO) response of a probe molecule, di-8-ANEPPS, inserted into the membranes. This molecular property presents the advantage to be specific to interfacial regions such as lipid bilayers. To unravel these effects, Molecular Dynamics (MD) simulations have been performed on both DPPC and DOPC lipids by varying the cholesterol mole fraction (from 0 to 0.66), while POPC was only considered as a pure bilayer. In the case of the structural properties of the bilayers, all the analyses converge toward the same conclusion: as the mole fraction of cholesterol increases, the systems become more rigid, confirming the condensing effect of cholesterol. In addition, the chromophore is progressively more aligned with respect to the normal to the bilayer. On the contrary, addition of unsaturation disorders the lipid bilayers, with barely no impact on the alignment of the chromophore. Then, using the frames obtained from the MD simulations, the first hyperpolarizability β of the dye in its environment has been computed at the TDDFT level. On the one hand, the addition of cholesterol induces a progressive increase of the diagonal component the β tensor parallel to the bilayer normal. On the other hand, larger β values have been calculated for the unsaturated than for the saturated lipid systems. In summary, this study illustrates the relationship between the composition and structure of the bilayers and the NLO responses of the embedded dye.

Cholesterol Levels Affect the Performance of AuNPs-Decorated Thermo-Sensitive Liposomes as Nanocarriers for Controlled Doxorubicin Delivery

Stimulus-responsive liposomes (L) for triggering drug release to the target site are particularly useful in cancer therapy. This research was focused on the evaluation of the effects of cholesterol levels in the performance of gold nanoparticles (AuNPs)-functionalized L for controlled doxorubicin (D) delivery. Their interfacial and morphological properties, drug release behavior against temperature changes and cytotoxic activity against breast and ovarian cancer cells were studied. Langmuir isotherms were performed to identify the most stable combination of lipid components. Two mole fractions of cholesterol (3.35 mol% and 40 mol%, L1 and L2 series, respectively) were evaluated. Thin-film hydration and transmembrane pH-gradient methods were used for preparing the L and for D loading, respectively. The cationic surface of L allowed the anchoring of negatively charged AuNPs by electrostatic interactions, even inducing a shift in the zeta potential of the L2 series. L exhibited nanometric sizes and spherical shape. The higher the proportion of cholesterol, the higher the drug loading. D was released in a controlled manner by diffusion-controlled mechanisms, and the proportions of cholesterol and temperature of release media influenced its release profiles. D-encapsulated L preserved its antiproliferative activity against cancer cells. The developed liposomal formulations exhibit promising properties for cancer treatment and potential for hyperthermia therapy.

Effects of Cholesterol on the Partitioning of a Drug Molecule in Lipid Bilayers.

Drug molecules either bind to membrane-bound targets or permeate through cell membranes to reach intracellular targets, and hence, their membrane partition and permeation are of great importance. Here, we studied the effects of cholesterol on the partition of amantadine, an antiflu drug molecule, into 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers using molecular dynamics simulations. The membrane partition of amantadine is sensitive to the cholesterol mole fraction (xchol). In the absence of cholesterol, amantadine is stably bound in membranes, but at xchol = 32%, it can escape to the aqueous phase, in agreement with recent experiments. The reduced membrane partition of amantadine at a high cholesterol content is mainly due to the perturbation of the bilayer structure and dynamics. Surrounding lipids stabilize amantadine by having their tails wrapped around the drug molecule, and this ability is compromised when cholesterol is present to increase the order in lipid tails. The atomic details on interactions with lipids and perturbations by cholesterol revealed here provide insight into membrane partition and delivery of drug molecules to their targets.

Raman spectroscopy and DSC assay of the phase coexistence in binary DMPC/cholesterol multilamellar vesicles

The phospholipid/cholesterol binary model systems are an example of simple models whose structure has caused controversy and genuine interest over many decades. The cornerstone underlying the description of such models is the answer to the question of whether these membranes are separated into coexisting phases or domains. Here, we apply label-free Raman spectroscopy and differential scanning calorimetry (DSC) to verify the phase coexistence in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol binary model. Raman spectra demonstrate the peculiarity at 30% molar fraction of cholesterol. Above this concentration, Raman data demonstrate similar characteristics at T = 291, 298, 303 K. At lower molar fractions, at 303 K, we found the agreement of Raman spectra with the predictions of the lever rule of cholesterol. Taken together, low cooperativity of the transition at 30 mol% and the fulfillment of the lever rule suggest the existence of nanoclusters composed of approximately 4 DMPC and 2 cholesterol molecules. At 298 K, the compliance of the lever rule was found in the range from 0 to 20 mol% of cholesterol. At 291 K, the addition of 5% cholesterol leads to the abrupt change of Raman spectra parameters and their continuous evolution with the further increase of cholesterol molar fraction. It seems that cholesterol plays a twofold role in binary mixtures; it reduces the intermolecular cooperativity and forms clusters whose size and DMPC-to-cholesterol ratio depend on cholesterol concentration and temperature.

Structure of supported DPPC/cholesterol bilayers studied via X-ray reflectivity.

The electron density profile of bilayers of DPPC/cholesterol mixtures supported on semiconductor grade silicon substrates were studied with the objective of determining how the proximity of a solid interface modifies the phase diagram of mixed bilayers. The bilayers were studied in situ immersed in water via synchrotron X-ray reflectivity (XRR). Measurements were performed as a function of temperature through the main phase transition and cholesterol mole fractions up to 40%. Analysis of XRR yields the bilayer thickness, roughness and leaflet asymmetry. We find that the structure of the pure DPPC bilayers in the gel phase is in agreement with previous X-ray measurements of supported bilayers deposited via vesicle fusion and multilamellar vesicles but show more clearly defined features than measurements made on films formed using Langmuir-Blodget Langmuir-Shaffer (LB) deposition. Examination of bilayer thickness vs. temperature shows that the melting temperature for supported bilayers is shifted upwards by approximately 4 °C relative to multilamellar vesicles and that the melting temperature decreases with increasing cholesterol content up to 20%. For pure DPPC bilayers the leaflets melt in two stages with the distal leaflet melting first. For cholesterol concentrations of 10% and 20% there is no clear indication of separate melting. For 33% and 40% cholesterol content no clear transition is seen in the bilayer thickness, but an abrupt change in roughness indicates possible microdomain formation in the 40% cholesterol sample.

A new approach for investigating the response of lipid membranes to electrocompression by coupling droplet mechanics and membrane biophysics.

A new method for quantifying lipid-lipid interactions within biomimetic membranes undergoing electrocompression is demonstrated by coupling droplet mechanics and membrane biophysics. The membrane properties are varied by altering the lipid packing through the introduction of cholesterol. Pendant drop tensiometry is used to measure the lipid monolayer tension at an oil-water interface. Next, two lipid-coated aqueous droplets are manipulated into contact to form a bilayer membrane at their adhered interface. The droplet geometries are captured from two angles to provide accurate measurements of both the membrane area and the contact angle between the adhered droplets. Combining the monolayer tension and contact angle measurements enables estimations of the membrane tension with respect to lipid composition. Then, the membrane is electromechanically compressed using a transmembrane voltage. Electrostatic pressure, membrane tension and the work necessary for bilayer thinning are tracked, and a model is proposed to capture the mechanics of membrane compression. The results highlight that a previously unaccounted for energetic term is produced during compression, potentially reflecting changes in the lateral membrane structure. This residual energy is eliminated in cases with cholesterol mole fractions of 0.2 and higher, suggesting that cholesterol diminishes these adjustments.

Complexes of Anionic Cholesterol-Containing Liposomes and Cationic Chitosan Microparticles

The preparation of multiliposomal complexes by adsorption of anionic cholesterol-containing liposomes on the surface of cationic chitosan microparticles has been described. The use of liposomes with a mole fraction of cholesterol in the liposomal membrane of 0.3 forms a complex in which each microparticle binds 150 intact liposomes. The size of complex particles (250–400 nm) allows recommending them as a potential carrier of medicinal substances that can penetrate into the cells through the mechanism of passive transport.

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol-Dependent Bending Energy Is Important in Cholesterol Distribution of the Plasma Membrane

We consider the plasma membrane that contains a cholesterol molar fraction of 0.4 and ask how that cholesterol is distributed between the two leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical potentials in the two leaves. When we consider only the contributions of entropy and interactions to the cholesterol chemical potential in our model system, we find, not surprisingly, that the cholesterol is mostly in the outer leaf because of the strong attraction between cholesterol and sphingomyelin (SM), which is predominantly in that leaf. We find 72% there. We then include the contribution from the bending energy in each leaf that must be overcome to join the leaves in a flat bilayer. The product of bending modulus and spontaneous curvature is obtained from simulation. We find that the addition of cholesterol to the outer leaf reduces the spontaneous curvature, which is initially positive, until it passes through zero when the molar fraction of cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward the inner leaf by the sphingomyelin phosphatidylcholine mixture. This is resisted by the bending energy contribution to the inner leaf. We find, again by simulation, that the addition of cholesterol monotonically increases the magnitude of the spontaneous curvature of the inner leaf, which is negative. This increases its bending energy. We conclude that, as a result of these competing effects, the percentage of cholesterol in the outer leaf is reduced to ∼63 ± 6%.

Redox-Triggered Disassembly of Nanosized Liposomes Containing Ferrocene-Appended Amphiphiles.

We report a redox-responsive liposomal system capable of oxidatively triggered disassembly. We describe the synthesis, electrochemical characterization, and incorporation into vesicles of an alternative redox lipid with significantly improved synthetic efficiency and scalability compared to a ferrocene-appended phospholipid previously employed by our group in giant vesicles. The redox-triggered disassembly of both redox lipids is examined in nanosized liposomes as well as the influence of cholesterol mole fraction on liposome disassembly and suitability of various chemical oxidants for in vitro disassembly experiments. Electronic structure density functional theory calculations of membrane-embedded ferrocenes are provided to characterize the role of charge redistribution in the initial stages of the disassembly process.

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.

Structural Change in Dimyristoyl Phosphatidyl Choline (DMPC) Molecule Alkyl Chains on the Addition of Cholesterol to DMPC Monolayer: Specific Addition Effect of Cholesterol

We have investigated the morphology of dimyristoyl phosphatidyl choline (DMPC)—cholesterol mixed monolayer formed on the water surface by dropping method using surface tension measurement (STm), Brewster angle microscopy (BAM), and infrared external reflection spectroscopy (IERS). STm results showed negative deviation of the limiting molecular area (A0) of cholesterol occurred when cholesterol was added to the DMPC monolayer. BAM images showed the expandable DMPC monolayer changed to the condensed rigid monolayer at more than cholesterol mole fraction (xChol) 0.4. IERS recordings showed that the addition of cholesterol at xChol = 0.4 occurred structural change from gauche- to trans- conformation of two DMPC molecule alkyl chains. From these results, it is found that cholesterol molecule has specific properties that cause structural transition of DMPC molecule alkyl chains.

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld) + liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld + Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.