Optimization and modeling of the remote loading of luciferin into liposomes.

We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na(+), K(+), Ca(+2), Mg(+2)), and the initial extra-liposomal amount of luciferin (nL(add)) on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and nL(add) was established, and under our conditions the maximum LC was attained using an nL(add) of around 2.6µmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between nL(add) and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1×10(-8)cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.

Slow Relaxation of Shape and Orientational Texture in Membrane Gel Domains.

Gel domains in lipid bilayers are structurally more complex than fluid domains. Growth dynamics can lead to noncircular domains with a heterogeneous orientational texture. Most model membrane studies involving gel domain morphology and lateral organization assume the domains to be static. Here we show that rosette shaped gel domains, with heterogeneous orientational texture and a central topological defect, after early stage growth, undergo slow relaxation. On a time scale of days to weeks domains converge to circular shapes and approach uniform texture. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) enriched gel domains are grown by cooling 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DPPC bilayers into the solid-liquid phase coexistence region and are visualized with fluorescence microscopy. The relaxation of individual domains is quantified through image analysis of time-lapse image series. We find a shape relaxation mechanism which is inconsistent with Ostwald ripening and coalescence as observed in membrane systems with coexisting liquid phases. Moreover, domain texture changes in parallel with the changes in domain shape, and selective melting and growth of particular subdomains cause the texture to become more uniform. We propose a relaxation mechanism based on relocation of lipids from high-energy lattice positions, through evaporation-condensation and edge diffusion, to low-energy positions. The relaxation process is modified significantly by binding Shiga toxin, a bacterial toxin from Shigella dysenteriae, to the membrane surface. Binding alters the equilibrium shape of the gel domains from circular to eroded rosettes with disjointed subdomains. This observation may be explained by edge diffusion while evaporation-condensation is restricted, and it provides further support for the proposed overall relaxation mechanism.

Tight coupling of metabolic oscillations and intracellular water dynamics in Saccharomyces cerevisiae.

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D2O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process.

Propofol modulates the lipid phase transition and localizes near the headgroup of membranes.

The compound 2,6-diisopropylphenol (Propofol, PRF) is widely used for inducing general anesthesia, but the mechanism of PRF action remains relatively poorly understood at the molecular level. This work examines the possibility that a potential mode of action of PRF is to modulate the lipid order in target membranes. The effect on monolayers and bilayers of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) was probed using Langmuir monolayer isotherms, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations. Increasing amounts of PRF in a DPPC monolayer causes a decrease in isothermal compressibility modulus at the phase transition. A partition constant for PRF in DPPC liposomes on the order of K≈1500 M(-1) was found, and the partitioning was found to be enthalpy-driven above the melting temperature (Tm). A decrease in Tm with PRF content was found whereas the bilayer melting enthalpy ΔHm remains almost constant. The last finding indicates that PRF incorporates into the membrane at a depth near the phosphatidylcholine headgroup, in agreement with our MD-simulations. The simulations also reveal that PRF partitions into the membrane on a timescale of 0.5 µs and has a cholesterol-like ordering effect on DPPC in the fluid phase. The vertical location of the PRF binding site in a bacterial ligand-gated ion channel coincides with the location found in our MD-simulations. Our results suggest that multiple physicochemical mechanisms may determine anesthetic potency of PRF, including effects on proteins that are mediated through the bilayer.

Electromechanics of a membrane with spatially distributed fixed charges: flexoelectricity and elastic parameters.

We investigate the electrostatic contribution to the lipid membrane mechanical parameters: tension, bending rigidity, spontaneous curvature, and flexocoefficient, using an approach where stress in the membrane is explicitly balanced. Our model includes an applied electrostatic potential as well as a charge distribution in the membrane. We apply our theory to membranes having surface charges and electric dipoles at the surface.

Applying a potential across a biomembrane: electrostatic contribution to the bending rigidity and membrane instability.

We investigate the effect on biomembrane mechanical properties due to the presence an external potential for a nonconductive incompressible membrane surrounded by different electrolytes. By solving the Debye-Hückel and Laplace equations for the electrostatic potential and using the relevant stress-tensor we find (1) in the small screening length limit, where the Debye screening length is smaller than the distance between the electrodes, the screening certifies that all electrostatic interactions are short range and the major effect of the applied potential is to decrease the membrane tension and increase the bending rigidity; explicit expressions for electrostatic contribution to the tension and bending rigidity are derived as a function of the applied potential, the Debye screening lengths, and the dielectric constants of the membrane and the solvents. For sufficiently large voltages the negative contribution to the tension is expected to cause a membrane stretching instability. (2) For the dielectric limit, i.e., no salt (and small wave vectors compared to the distance between the electrodes), when the dielectric constant on the two sides are different the applied potential induces an effective (unscreened) membrane charge density, whose long-range interaction is expected to lead to a membrane undulation instability.

Dynamic force spectroscopy on soft molecular systems: improved analysis of unbinding spectra with varying linker compliance.

Dynamic force spectroscopy makes it possible to measure the breaking of single molecular bonds or the unfolding of single proteins subjected to a time-dependent pulling force. The force needed to break a single bond or to unfold a domain in a protein depends critically on the time dependence of the applied force. In this way the elastic response couples to the unbinding force. We have performed an experimental and theoretical examination of this coupling by studying the well-known biotin-streptavidin bond in systems incorporating two common types of linkers. In the first case biotin is linked by bovine serum albumin (BSA) and it is observed that this linker has a linear elastic response. More surprisingly we find that its force constant varies significantly between repeated force curves. It is demonstrated that by sorting the force curves according to the force constant of the linker we can improve the data analysis and obtain a better agreement between experimental data and theory. In the second case biotin is linked by poly(ethylene glycol) (PEG), which has a soft nonlinear elastic response. A numerical calculation of the unbinding statistics for the polymer system agrees quantitatively with experiments. It demonstrates a clear decrease in unbinding forces resulting from the polymer linker.

Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis.

The activity of phospholipase A(2) (PLA(2)) which catalyzes the hydrolysis of phospholipids into free fatty acids and lysolipids, depends on the structure and thermodynamic state of the membrane. To further understand how the substrate conformation correlates with enzyme activity, model systems that are based on time-resolved membrane microscopy are needed. We demonstrate a methodology for preparing and investigating the dynamics of fluid supported phospholipid membranes hydrolyzed by snake venom PLA(2). The method uses quantitative analysis of time-lapse fluorescence images recording the evolution of fluid bilayer islands during hydrolysis. In order to minimize interactions with the support surface, we use double bilayer islands situated on top of a complete primary supported membrane prepared by hydration of spincoated lipid films. Our minimal kinetic analysis describes adsorption of enzyme to the membrane in terms of the Langmuir isotherm as well as enzyme kinetics. We use two related models assuming hydrolysis to occur either at the perimeter or at the surface of the membrane island. We find that the adsorption constant is similar for the two cases, while the estimated turnover rate is markedly different. The PLA(2) concentration series is measured in the absence and presence of beta-cyclodextrin which forms water soluble complexes with the reaction products. The results demonstrate the versatility of double bilayer islands as a membrane model system and introduces a new method for quantifying the kinetics of lipase activity on membranes by directly monitoring the evolution in substrate morphology.

Activation of interfacial enzymes at membrane surfaces.

A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A(2) (sPLA(2)), are only activated at the interface between water and membrane surfaces, where they lead to a break-down of the lipid molecules into lysolipids and free fatty acids. The activation is critically dependent on the physical properties of the lipid-membrane substrate. A topical review is given of our current understanding of the physical mechanisms responsible for activation of sPLA(2) as derived from a range of different experimental and theoretical investigations.

DNA overstretching transition: ionic strength effects.

As double-stranded DNA is stretched to its B-form contour length, models of polymer elasticity can describe the dramatic increase in measured force. When the molecule is stretched beyond the contour length, it further shows a highly cooperative overstretching transition. We have developed a theoretical description for this transition by coupling the two-state model and the elasticity theory proposed earlier by others. Furthermore, we have extended this model to account for monovalent salt effects on elastic moduli during the transition. We find that this theoretical description is in very good agreement with recent measurements for the salt dependence of the overstretching transition, allowing us to gain insight into the mechanisms that govern the transition. In double-stranded DNA, the effective length per unit charge varies with salt in agreement with the Manning and Poisson-Boltzmann models for thin polyelectrolyte rods, whereas the other model parameters describing structural features have barely any salt dependence. The results thus suggest that the electrostatic component of force-induced overstretching is mediated mesoscopically via elasticity.

Nanobubbles give evidence of incomplete wetting at a hydrophobic interface.

The appearance of a hydrophobic surface, namely a crystalline (111) Si wafer coated with a thick soft polystyrene film, and the morphological changes along this interface depending on the polarity of an adjoining liquid phase were studied with magnetic tapping mode atomic force microscopy. Interfacially associated nanobubbles of decreasing size and number are observed as the hydrophobicity of the subphase increases. The disturbance of the water structure in the contact region induces the formation of nanobubbles. The topology of the interface is visualized, starting with the dry polymer under normal atmosphere conditions and observing the changes as air is replaced by a series of liquids. With water, the surface coverage of the substrate with bubbles is almost a close-packed configuration. The bubble shape is well approximated by spherical caps of a rather low aspect ratio. The Gaussian size distributions of bubble shape parameters are discussed. The contact angle of the nanobubbles is substantially smaller than the corresponding number measured for a macroscopic droplet. This apparent discrepancy might be resolved if the nanobubbles were assumed to exist along the interface as a connecting sublayer between a depleted water film at the hydrophobic polymer surface and an adsorbed macrodroplet.

Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws.

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.