Molecular Study of Ultrasound-Triggered Release of Fluorescein from Liposomes.

Several investigations have suggested that ultrasound triggers the release of drugs encapsulated into liposomes at acoustic pressures low enough to avoid cavitation or high hyperthermia. However, the mechanism leading to this triggered release as well as the adequate composition of the liposome membrane remains unknown. Here, we investigate the ultrasound-triggered release of fluorescein disodium salt encapsulated into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-distearoylphosphatidyl-ethanolamine (DSPC) lipids with various concentrations of cholesterol (from 0 to 44 mol %). The passive release of encapsulated fluorescein was first characterized. It was observed to be higher when the membrane is in a fluid phase and increased with temperature but decreased upon addition of cholesterol. Next, the release of fluorescein was measured at different acoustic frequencies (0.8, 1.1, and 3.3 MHz) and peak-to-peak pressures (0, 2, 2.5, 5, and 8 MPa). Measurements were performed at temperatures where DOPC and DSPC liposomes were, respectively, in the fluid or gel phase. We found that the release rate did not depend on the ultrasound frequency. For DOPC liposomes, the ultrasound-triggered release of fluorescein decreased with increasing concentration of cholesterol in liposomes, while the behavior was more complex for DSPC liposomes. Overall, the triggered release from DSPC liposomes was up to ten times less than DOPC liposomes. Molecular dynamics simulations performed on a pure DOPC membrane showed that a membrane experiences, under a directional pressure of ±2.4 MPa, various changes in properties such as the area per lipid (APL). An increase in the APL was notably observed when the simulation box was laterally stretched or perpendicularly compressed, which was accompanied by an increase in the number of water molecules crossing the membrane. This suggests that ultrasound most probably enhances the diffusion of encapsulated molecules at small acoustic pressures by increasing the distance between lipids.

Simulating Bilayers of Nonionic Surfactants with the GROMOS-Compatible 2016H66 Force Field.

Polyoxyethylene glycol alkyl ether amphiphiles (CiEj) are important nonionic surfactants, often used for biophysical and membrane protein studies. In this work, we extensively test the GROMOS-compatible 2016H66 force field in molecular dynamics simulations involving the lamellar phase of a series of CiEj surfactants, namely C12E2, C12E3, C12E4, C12E5, and C14E4. The simulations reproduce qualitatively well the monitored structural properties and their experimental trends along the surfactant series, although some discrepancies remain, in particular in terms of the area per surfactant, the equilibrium phase of C12E5, and the order parameters of C12E3, C12E4, and C12E5. The polar head of the CiEj surfactants is highly hydrated, almost like a single polyethyleneoxide (PEO) molecule at full hydration, resulting in very compact conformations. Within the bilayer, all CiEj surfactants flip-flop spontaneously within tens of nanoseconds. Water-permeation is facilitated, and the bending rigidity is 4 to 5 times lower than that of typical phospholipid bilayers. In line with another recent theoretical study, the simulations show that the lamellar phase of CiEj contains large hydrophilic pores. These pores should be abundant in order to reproduce the comparatively low NMR order parameters. We show that their contour length is directly correlated to the order parameters, and we estimate that they should occupy approximately 7-10% of the total membrane area. Due to their highly dynamic nature (rapid flip-flops, high water permeability, observed pore formation), CiEj surfactant bilayers are found to represent surprisingly challenging systems in terms of modeling. Given this difficulty, the results presented here show that the 2016H66 parameters, optimized independently considering pure-liquid as well as polar and nonpolar solvation properties of small organic molecules, represent a good starting point for simulating these systems.

AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity.

Elasticity of polymer microcapsules (MCs) filled with a liquid fluorinated core is studied by atomic force microscopy (AFM). Accurately characterized spherical tips are employed to obtain the Young's moduli of MCs having four different shell thicknesses. We show that those moduli are effective ones because the samples are composites. The strong decrease of the effective MC elasticity (from 3.0 to 0.1 GPa) as the shell thickness decreases (from 200 to 10 nm) is analyzed using a novel numerical approach. This model describes the evolution of the elasticity of a coated half-space according to the contact radius, the thickness of the film, and the elastic moduli of bulk materials. This numerical model is consistent with the experimental data and allows simulating the elastic behavior of MCs at high frequencies (5 MHz). While the quasi-static elasticity of the MCs is found to be very dependent on the shell thickness, the high frequency (5 MHz) elastic behavior of the core leads to a stable behavior of the MCs (from 2.5 to 3 GPa according to the shell thickness). Finally, the effect of thermal annealing on the MCs elasticity is investigated. The Young's modulus is found to decrease because of the reduction of the shell thickness due to the loss of the polymer.

Stability of C(12)E(j) Bilayers Probed with Adhesive Droplets.

The stability of model surfactant bilayers from the poly(ethylene glycol) mono-n-dodecyl ether (C12Ej) family was probed. The surfactant bilayers were formed by the adhesion of emulsion droplets. We generated C12Ej bilayers by forming water-in-oil (w/o) emulsions with saline water droplets, covered by the surfactant, in a silicone and octane oil mixture. Using microfluidics, we studied the stability of those bilayers. C12E1 allowed only short-lived bilayers whereas C12E2 bilayers were stable over a wide range of oil mixtures. At high C12E2 concentration, a two-phase region was displayed in the phase diagram: bilayers formed by the adhesion of two water droplets and Janus-like particles consisting of adhering aqueous and amphiphilic droplets. C12E8 and C12E25 did not mediate bilayer formation and caused phase inversion leading to o/w emulsion. With intermediate C12E4 and C12E5 surfactants, both w/o and o/w emulsions were unstable. We provided the titration of the C12E2 bilayer with C12E4 and C12E5 to study and predict their stability behavior.

Properties of theranostic nanoparticles determined in suspension by ultrasonic spectroscopy.

In the context of growing use of nanoparticles, it is important to be able to characterize all their physical properties in order to understand their behavior, to optimize them, and to control their quality. We showed that ultrasonic spectroscopy provides many of the desired properties. To do so, we used as an example nanocapsules made of a polymer shell encaspulating a liquid perfluorocarbon core and designed them for theranostic applications. Frequency-dependent measurements of both ultrasound velocity and attenuation were performed on nanocapsule suspensions. Then the desired properties were extracted by analyzing the experimental data using a recently developed model that relates the speed of sound and attenuation of a suspension to the geometrical and viscoelastic properties of the nanocapsules.

Surfactant bilayers maintain transmembrane protein activity.

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca(2+)ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase.

Kinetic study of membrane protein interactions: from three to two dimensions.

Molecular interactions are contingent upon the system's dimensionality. Notably, comprehending the impact of dimensionality on protein-protein interactions holds paramount importance in foreseeing protein behaviour across diverse scenarios, encompassing both solution and membrane environments. Here, we unravel interactions among membrane proteins across various dimensionalities by quantifying their binding rates through fluorescence recovery experiments. Our findings are presented through the examination of two protein systems: streptavidin-biotin and a protein complex constituting a bacterial efflux pump. We present here an original approach for gauging a two-dimensional binding constant between membrane proteins embedded in two opposite membranes. The quotient of protein binding rates in solution and on the membrane represents a metric denoting the exploration distance of the interacting sites-a novel interpretation.

A model for ultrasound absorption and dispersion in dilute suspensions of nanometric contrast agents.

Ultrasound dispersion and absorption are examined in dilute suspensions of contrast agents of nanometric size, with a typical radius around 100 nm. These kinds of contrast agents are designed for targeted delivery of drugs for cancer treatment. Compared to standard contrast agents used for imaging, particles are of smaller size to pass through the endothelial barrier, their shell, made up of biocompatible polymer, is stiffer to undergo a longer lifetime, and they have a liquid core instead of a gaseous one. Ultrasound propagation in dilute suspension is modeled by combining two modes for particle oscillations. The first one is a dilatational mode assuming an incompressible shell with a rheological behavior of Kelvin-Voigt or Maxwell type. The second one is a translational mode induced by visco-inertial interaction with the ambient fluid. The relative importance of these two modes of interaction on both dispersion and absorption is quantified and analyzed for a model system and for two radii (75 and 150 nm) and the two rheological models. The influence of shell parameters (Young modulus, viscosity, and relative thickness) is finally discussed.

How to best estimate the viscosity of lipid bilayers.

The viscosity of lipid bilayers is a property relevant to biological function, as it affects the diffusion of membrane macromolecules. To determine its value, and hence portray the membrane, various literature-reported techniques lead to significantly different results. Herein we compare the results issuing from two widely used techniques to determine the viscosity of membranes: the Fluorescence Lifetime Imaging Microscopy (FLIM), and Fluorescence Recovery After Photobleaching (FRAP). FLIM relates the time of rotation of a molecular rotor inserted into the membrane to the macroscopic viscosity of a fluid. Whereas FRAP measures molecular diffusion coefficients. This approach is based on a hydrodynamic model connecting the mobility of a membrane inclusion to the viscosity of the membrane. We show that: This article emphasizes the pitfalls to be avoided and the rules to be observed in order to obtain a value of the bilayer viscosity that characterizes the bilayer instead of interactions between the bilayer and the embedded probe.

Two-dimensional simulation of linear wave propagation in a suspension of polymeric microcapsules used as ultrasound contrast agents.

A generation of tissue-specific stable ultrasound contrast agent (UCA) composed of a polymeric capsule with a perfluorocarbone liquid core has become available. Despite promising uses in clinical practice, the acoustical behavior of such UCA suspensions remains unclear. A simulation code (2-D finite-difference time domain, FDTD) already validated for homogeneous particles [Galaz Haiat, Berti, Taulier, Amman and Urbach, (2010). J. Acoust. Soc. Am. 127, 148-154] is used to model the ultrasound propagation in such UCA suspensions at 50 MHz to investigate the sensitivity of the ultrasonic parameters to physical parameters of UCA. The FDTD simulation code is validated by comparison with results obtained using a shell scatterer model. The attenuation coefficient (respectively, the sound velocity) increases (respectively, decreases) from 4.1 to 58.4 dB/cm (respectively, 1495 to 1428 m/s) when the concentration varies between 1.37 and 79.4 mg/ml, while the backscattered intensity increases non-linearly, showing that a concentration of around 30 mg/ml is sufficient to obtain optimal backscattering intensity. The acoustical parameters vary significantly as a function of the membrane thickness, longitudinal and transverse velocity, indicating that mode conversions in the membrane play an important role in the ultrasonic propagation. The results may be used to help manufacturers to conceive optimal liquid-filled UCA suspensions.

Experimental validation of a time domain simulation of high frequency ultrasonic propagation in a suspension of rigid particles.

Ultrasonic propagation in suspensions of particles is a difficult problem due to the random spatial distribution of the particles. Two-dimensional finite-difference time domain simulations of ultrasonic propagation in suspensions of polystyrene 5.3 mum diameter microdisks are performed at about 50 MHz. The numerical results are compared with the Faran model, considering an isolated microdisk, leading to a maximum difference of 15% between the scattering cross-section values obtained analytically and numerically. Experiments are performed with suspensions in through transmission and backscattering modes. The attenuation coefficient at 50 MHz (alpha), the ultrasonic velocity (V), and the relative backscattered intensity (I(B)) are measured for concentrations from 2 to 25 mg/ml, obtained by modifying the number of particles. Each experimental ultrasonic parameter is compared to numerical results obtained by averaging the results derived from 15 spatial distributions of microdisks. alpha increases with the concentration from 1 to 17 dB/cm. I(B) increases with concentration from 2 to 16 dB. The variation of V versus concentration is compared with the numerical results, as well as with an effective medium model. A good agreement is found between experimental and numerical results (the larger discrepancy is found for alpha with a difference lower than 2.1 dB/cm).

Recent applications of fluorescence recovery after photobleaching (FRAP) to membrane bio-macromolecules.

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods.

Effect of Dimethyl Sulfoxide on the Binding of 1-Adamantane Carboxylic Acid to ß- and γ-Cyclodextrins.

Most therapeutic targets are proteins whose binding sites are hydrophobic cavities. For this reason, the majority of drugs under development are hydrophobic molecules exhibiting low solubility in water. To tackle this issue, a few percent of cosolvent, such as dimethyl sulfoxide (DMSO), is usually employed to increase drug solubility during the drug screening process. However, the few published studies dealing with the effect of adding DMSO showed that the affinity of hydrophobic ligands is systematically underestimated. To better understand the effect of DMSO, there is a need of studying its effect on a large range of systems. In this work, we used ß- and γ-cyclodextrins (made of 6 and 7 α-d-glucopyranoside units, respectively) as models of hydrophobic cavities to investigate the effect of the addition 5% DMSO on the affinity of 1-adamantane carboxylic acid (ADA) to these cyclodextrins. The two systems differ by the size of the cyclodextrin cavity. The evaluation of binding constants was performed using ultrasound velocimetry, nuclear magnetic resonance spectroscopy, and molecular simulations. All techniques show that the presence of 5% DMSO does not significantly modify the affinity of ADA for γ-cyclodextrin, while the affinity is dramatically reduced for ß-cyclodextrin. The bias induced by the presence of DMSO is thus more important when the ligand volume better fits the cyclodextrin cavity. Our work also suggests that free energy calculations provide a sound alternative to experimental techniques when dealing with poorly water-soluble drugs.

Effect of a neutral water-soluble polymer on the lamellar phase of a zwitterionic surfactant system.

We have studied the effect of adding a water-soluble polymers (PEG) to the lamellar phases of the ternary system tetradecyldimethylaminoxide (C14DMAO)-hexanol-water. The results of Freeze-Fracture Electron Microscopy (FFEM) and Small Angle X-ray Scattering (SAXS) experiments show that the addition of the polymer induces the spontaneous formation of highly monodisperse multilayered vesicles above a threshold polymer concentration.

FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.

Self-diffusion and collective diffusion in a model viscoelastic system.

We use dynamic light scattering (DLS) and fluorescence recovery after pattern photobleaching (FRAPP) to investigate the dynamics of a model transient network made of an oil-in-water droplet microemulsion to which small amounts of a telechelic polymer are added. The DLS correlation functions exhibit three relaxation modes. The two first modes can be interpreted quantitatively in the frame of the classical De Gennes-Brochard theory of DLS in viscoelastic system. The third, slower mode is diffusive and arises from the ternary character (droplets, polymers, and water) of the system. By contrast, the pattern relaxation in FRAPP exhibits a single-, slow-exponential decay with a characteristic time proportional to the squared inverse scattering vector: the corresponding self-diffusion coefficient of the droplets is found to be close to the diffusion coefficient characterizing the third mode in DLS. We interpret these results in terms of the coupled relaxation of the concentration fluctuations of the polymers and the droplets.

Compressibility of nano inclusions in complex fluids by ultrasound velocity measurements.

We present a high precision ultrasonic velocimeter for a small volume sample (1 cm3) for a path length of 1 cm achieved. The method used is based on the time of flight measurement with an original signal processing technique: the barycenter method. With our system, we have measured the sound velocity with an accuracy of 10(-5). The detection of a difference in velocity between two liquids of about 2 cm/s is achieved. The compressibility of the reference liquid can then be deduced with an accuracy better than 0.2%. Using this custom-made system, we have studied and characterized complex fluids, systems biomimetic of biological membranes, as well as proteins included in nanometric water droplets. Under these experimental conditions, we have reached the value of protein compressibilities with an accuracy better than 10%.

Variation of the lateral mobility of transmembrane peptides with hydrophobic mismatch.

A hydrophobic mismatch between protein length and membrane thickness can lead to a modification of protein conformation, function, and oligomerization. To study the role of hydrophobic mismatch, we have measured the change in mobility of transmembrane peptides possessing a hydrophobic helix of various length d(pi) in lipid membranes of giant vesicles. We also used a model system where the hydrophobic thickness of the bilayers, h, can be tuned at will. We precisely measured the diffusion coefficient of the embedded peptides and gained access to the apparent size of diffusing objects. For bilayers thinner than d(pi), the diffusion coefficient decreases, and the derived characteristic sizes are larger than the peptide radii. Previous studies suggest that peptides accommodate by tilting. This scenario was confirmed by ATR-FTIR spectroscopy. As the membrane thickness increases, the value of the diffusion coefficient increases to reach a maximum at h approximately = d(pi). We show that this variation in diffusion coefficient is consistent with a decrease in peptide tilt. To do so, we have derived a relation between the diffusion coefficient and the tilt angle, and we used this relation to derive the peptide tilt from our diffusion measurements. As the membrane thickness increases, the peptides raise (i.e., their tilt is reduced) and reach an upright position and a maximal mobility for h approximately = d(pi). Using accessibility measurements, we show that when the membrane becomes too thick, the peptide polar heads sink into the interfacial region. Surprisingly, this "pinching" behavior does not hinder the lateral diffusion of the transmembrane peptides. Ultimately, a break in the peptide transmembrane anchorage is observed and is revealed by a "jump" in the D values.

Tracking membrane protein association in model membranes.

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5.Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.