Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Membrane thickness and acyl chain length

It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.     

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

Localized density pulses in lipid membranes on the piсosecond time scale

Physical mechanisms for the modulation of lipid nanodomain dynamics and transport of small molecules across the lipid bilayer of biological membranes can have considerable functional significance. The longitudinal propagating compression–expansion mode in a single-component lipid bilayer is considered on a spatial scale comparable to the bilayer thickness. The expression used for free energy per lipid molecule includes a term with the gradient of the area per lipid molecule. The finite character of the stress relaxation time in the lipid bilayer, which causes significant attenuation of viscous damping at sufficiently high frequencies, is taken into account. A hydrodynamic ad hoc model that describes soliton-like excitations in the bilayer is proposed. The possibility of formation and propagation of nanoscale pulses of lipid density with velocities determined by surface compressibility of the bilayer (similarly to the propagation of sound) is demonstrated for the picosecond time scale.     

Molecular modeling of the reactive configuration of peroxidized lipid and α-tocopherol in the membrane

The mutual arrangement of a phospholipid molecule containing a peroxyl radical and a molecule of membrane-acting antioxidant α-tocopherol (vitamin E) in the lipid bilayer has been studied by molecular dynamics simulation. The geometry of molecules in the membrane is revealed at which the hydrogen atom can be transferred from the exocyclic hydroxyl of α-tocopherol to the peroxyl lipid radical. It is shown that, under equilibrium conditions, the peroxidized fatty acid segment rises nearer to the polar surface of the membrane, while α-tocopherol submerges into the hydrophobic part of the lipid bilayer.     

Dynamical and structural properties of charged and uncharged lidocaine in a lipid bilayer

Molecular dynamics computer simulations have been performed to investigate dynamical and structural properties of a lidocaine local anesthetic. Both charged and uncharged forms of the lidocaine molecule were investigated. Properties such as membrane area per lipid, diffusion, mass density, bilayer penetration and order parameters have been examined. An analysis of the lidocaine interaction with the lipid surrounding according to a simple mean field theory has also been performed. Almost all examined properties were found to depend on which of the two forms of lidocaine, charged or uncharged, is studied. The overall picture is a rather static behavior determined by the lipids for the charged molecules and more mobile situation of the uncharged form with higher diffusion and lower orientational and positional order.     

Large divalent cations and electrostatic potentials adjacent to membranes. Experimental results with hexamethonium.

A simple extension of the Gouy-Chapman theory predicts that the ability of a divalent cation to screen charges at a membrane-solution interface decreases significantly if the distance between the charges on the cation is comparable with the Debye length. We tested this prediction by investigating the effect of hexamethonium on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes. The distance between the two charges of an extended hexamethonium molecule is approximately 1 nm, which is the Debye length in the 0.1 M monovalent salt solutions used in these experiments. Six different experimental approaches were utilized. We measured the electrophoretic mobility of multilamellar vesicles to determine the zeta potential, the line width of the 31P nuclear magnetic resonance (NMR) signal from sonicated vesicles to calculate the change in potential at the phosphodiester moiety of the lipid, and the conductance of planar bilayer membranes exposed to either carriers (nonactin) or pore formers (gramicidin) to estimate the change in potential within the membrane. We also measured directly the effect of hexamethonium on the potential above a monolayer formed from negative lipids, and attempted to calculate the change in the surface potential of a bilayer membrane from capacitance measurements. With the exception of the capacitance calculations, each of the techniques gave comparable results: hexamethonium exerts a smaller effect on the potential than that predicted by the classic screening theory. The results are consistent with the predictions of the extended Gouy-Chapman theory and are relevant to the interpretation of physiological and pharmacological experiments that utilize hexamethonium and other large divalent cations.     

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.     

Membrane packing geometry of diphytanoylphosphatidylcholine is highly sensitive to hydration: phospholipid polymorphism induced by molecular rearrangement in the headgroup region.

Diphytanoylphosphatidylcholine (DPhPC) has often been used in the study of protein-lipid interaction and membrane channel activity, because of the general belief that it has high bilayer stability, low ion leakage, and fatty acyl packing comparable to that of phospholipid bilayers in the liquid-crystalline state. In this solid-state 31P and 2H NMR study, we find that the membrane packing geometry and headgroup orientation of DPhPC are highly sensitive to the temperature studied and its water content. The phosphocholine headgroup of DPhPC starts to change its orientation at a water content as high as approximately 16 water molecules per lipid, as evidenced by hydration-dependent 2H NMR study at room temperature. In addition, a temperature-induced structural transition in the headgroup orientation is detected in the temperature range of approximately 20-60 degrees C for lipids with approximately 8-11 water molecules per DPhPC. Dehydration of the lipid by one more water molecule leads to a nonlamellar, presumably cubic, phase formation. The lipid packing becomes a hexagonal phase at approximately 6 water molecules per lipid. A phase diagram of DPhPC in the temperature range of -40 degrees C to 80 degrees C is thus constructed on the basis of NMR results. The newly observed hydration-dependent DPhPC lipid polymorphism emphasizes the importance of molecular packing in the headgroup region in modulating membrane structure and protein-induced pore formation of the DPhPC bilayer.     

A mean-field model of the alkane-saturated lipid bilayer above its phase transition. I. Development of the model

A statistical mechanical model of a bilayer of dipalmitoyl-3-sn-phosphatidylcholine molecules in equilibrium with an aqueous phase saturated with an n-alkane is presented. A mean-field approach developed in previous work on a solventless bilayer (Gruen, Biochim. Biophys. Acta. 595:161--183, 1980) is extended to allow alkane chains to exist in the hydrophobic core of the membrane. As the alkane chains are chemically similar to the lipid chains, much of the analysis follows directly from the solventless model. Novel features of the present model are the inclusion of (a) a term which models the free energy cost of creating space for alkane conformations, (b) a term which constrains the chains to pack evenly in the hydrophobic region of the membrane, and (c) a term which estimates the free energy of mixing of the lipid and alkane molecules in the plane of the bilayer. On uptake of alkane, the dimensions of the bilayer increase. Allowance is made for an increase in thickness and/or an increase in area per lipid. A thermodynamic framework is established which allows evaluation of the free energy of a bilayer of arbitrary dimensions with a view to predicting the equilibrium structure.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

A glycophospholipid covalently attached to the C-terminus of the Thy-1 glycoprotein

The Thy-1 glycoprotein is a very abundant cell surface molecule of rat thymocytes and neuronal cells with the properties of a molecule that inserts into the lipid bilayer. The hydrophobicity is due to a glycophospholipid component covalently attached to the carboxy group of the C-terminal cysteine residue. The mature glycoprotein does not contain a stretch of hydrophobic amino acids that could traverse the membrane bilayer. These findings present a new mode of membrane attachment for a cell surface molecule that can mediate lymphokine release and cell division after cross-linking by antibodies.     

Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation

Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcumin–liposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). The computed free energy profile exhibits a global minimum at the solvent–headgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (?8.39 vs. ?1.69 k_BT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin–lipid bilayer.     

Alcohols reduce lateral membrane pressures: predictions from molecular theory

We explore the effects of alcohols on fluid lipid bilayers using a molecular theory with a coarse-grained model. We show that the trends predicted from the theory in the changes in area per lipid, alcohol concentration in the bilayer, and area compressibility modulus, as a function of alcohol chain length and of the alcohol concentration in the solvent far from the bilayer, follow those found experimentally. We then use the theory to study the effect of added alcohol on the lateral pressure profile across the membrane, and find that added alcohol reduces the surface tensions at both the headgroup/solvent and headgroup/tailgroup interfaces, as well as the lateral pressures in the headgroup and tailgroup regions. These changes in lateral pressures could affect the conformations of membrane proteins, providing a nonspecific mechanism for the biological effects of alcohols on cells.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Molecular dynamics simulation of a palmitoyl-oleoyl phosphatidylserine bilayer with Na+ counterions and NaCl

Two 40 ns molecular dynamics simulations of a palmitoyl-oleoyl phosphatidylserine (POPS) lipid bilayer in the liquid crystalline phase with Na(+) counterions and NaCl were carried out to investigate the structure of the negatively charged lipid bilayer and the effect of salt (NaCl) on the lipid bilayer structure. Na(+) ions were found to penetrate deep into the ester region of the water/lipid interface of the bilayer. Interaction of the Na(+) ions with the lipid bilayer is accompanied by a loss of water molecules around the ion and a simultaneous increase in the number of ester carbonyl oxygen atoms binding the ion, which define an octahedral and square pyramidal geometry. The amine group of the lipid molecule is involved in the formation of inter- and intramolecular hydrogen bonds with the carboxylate and the phosphodiester groups of the lipid molecule. The area per lipid of the POPS bilayer is unaffected by the presence of 0.15M NaCl. There is a small increase in the order parameter of carbon atoms in the beginning of the alkyl chain in the presence of NaCl. This is due to a greater number of Na(+) ions being coordinated by the ester carbonyl oxygen atoms in the water/lipid interface region of the POPS bilayer.     

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.     

Effect of ion-binding and chemical phospholipid structure on the nanomechanics of lipid bilayers studied by force spectroscopy

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.