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.

Perfluorocarbon Nanodroplets as Potential Nanocarriers for Brain Delivery Assisted by Focused Ultrasound-Mediated Blood-Brain Barrier Disruption.

The management of brain diseases remains a challenge, particularly because of the difficulty for drugs to cross the blood-brain barrier. Among strategies developed to improve drug delivery, nano-sized emulsions (i.e., nanoemulsions), employed as nanocarriers, have been described. Moreover, focused ultrasound-mediated blood-brain barrier disruption using microbubbles is an attractive method to overcome this barrier, showing promising results in clinical trials. Therefore, nanoemulsions combined with this technology represent a real opportunity to bypass the constraints imposed by the blood-brain barrier and improve the treatment of brain diseases. In this work, a stable freeze-dried emulsion of perfluorooctyl bromide nanodroplets stabilized with home-made fluorinated surfactants able to carry hydrophobic agents is developed. This formulation is biocompatible and droplets composing the emulsion are internalized in multiple cell lines. After intravenous administration in mice, droplets are eliminated from the bloodstream in 24 h (blood half-life (t1/2) = 3.11 h) and no long-term toxicity is expected since they are completely excreted from mice' bodies after 72 h. In addition, intracerebral accumulation of tagged droplets is safely and significantly increased after focused ultrasound-mediated blood-brain barrier disruption. Thus, the proposed nanoemulsion appears as a promising nanocarrier for a successful focused ultrasound-mediated brain delivery of hydrophobic agents.

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.

Micron-sized PFOB liquid core droplets stabilized with tailored-made perfluorinated surfactants as a new class of endovascular sono-sensitizers for focused ultrasound thermotherapy.

The purpose of this study was to develop micron-sized droplet emulsions able to increase the heat deposition of high intensity focused ultrasound (HIFU), aiming to accelerate the tumour ablation in highly perfused organs with reduced side effects. The investigated droplets consisted of a perfluorooctyl bromide (PFOB) core coated with a biocompatible fluorinated surfactant called F-TAC. The novelty of this work relies on the use, for this application, of a high boiling point perfluorocarbon core (142 °C), combined with an in-house fluorinated surfactant to formulate the emulsion, yielding quasi-reversible strong interactions between the HIFU beam and the droplets. In order to fine-tune the emulsion size, surfactants with different hydrophobic/hydrophilic ratios were screened. Different concentrations of PFOB droplets were homogeneously embedded in two different MRI compatible materials, exhibiting either ultrasound (US) absorbing or non-absorbing properties. For the US absorbing TMM, the speed of sound at each droplet concentration was also assessed. These TMM were sonicated by 1 MHz HIFU with acoustical power of 94 W at two different duty cycles. The temperature elevation was monitored accurately by MRI proton shift resonance frequency in near real-time. The presence of sono-sensitive droplets induced a significant increase of the HIFU thermal effect that persisted under repeated sonication of the same locus. Optimal enhancement was observed at the lowest concentration tested (0.1%) with an additional temperature rise at the focal point of approximately 4 °C per applied kJ of acoustic energy corresponding to one order of magnitude augmentation of the thermal dose. Furthermore, no deformation of the heating pattern pre- or post-focal was observed.

Gamma-cyclodextrin forms a highly compressible complex with 1-adamantanecarboxylic acid.

We report temperature-dependent ultrasonic velocimetric and densimetric data on changes in volume, expansibility, and adiabatic compressibility associated with the binding of 1-adamantanecarboxylic acid (AD) to gamma-cyclodextrin (gamma-CD). We compare these results with our previous data on the binding of AD to beta-cyclodextrin (beta-CD) [Taulier, N.; Chalikian, T.V.J. Phys. Chem. B 2006, 110, 12222-12224]. The comparison reveals that, in contrast to the tight AD-beta-CD complex with little void space left inside the cavity, AD forms a loose complex with gamma-CD with approximately 30 A3 of void space between the guest molecule and the inner walls of the cavity. The presence of the void renders the AD-gamma-CD complex highly compressible; the intrinsic coefficient of compressibility of the AD-gamma-CD complex is 37x10(-6) bar(-1) at 18 degrees C and decreases to 23x10(-6) bar(-1) at 55 degrees C. Such large compressibility is suggestive of only weak contacts between the interacting AD and gamma-CD atomic groups in the cavity. Our results are consistent with the notion that the AD-gamma-CD complex is predominantly stabilized by the hydrophobic effect with only modest contribution from intermolecular van der Waals interactions. This notion is in contrast to the AD-beta-CD complex which is stabilized by strong host-guest van der Waals interactions in addition to the hydrophobic effect.

A calmodulin-binding site on cyclin E mediates Ca2+-sensitive G1/s transitions in vascular smooth muscle cells.

Calcium transients are known to control several transition points in the eukaryotic cell cycle. For example, we have previously shown that a coordinate elevation in the intracellular free calcium ion concentration is required for G1- to S-phase cell cycle progression in vascular smooth muscle cells (VSMC). However, the molecular basis for this Ca2+ sensitivity was not known. Using buffers with differing [Ca2+], we found that the kinase activity of mouse and human cyclin E/CDK2, but not other G1/S-associated cell cycle complexes, was responsive to physiological changes in [Ca2+]. We next determined that this Ca2+-responsive kinase activity was dependent on a direct interaction between calmodulin (CaM), one of the major Ca2+-signal transducers of eukaryotic cells, and cyclin E. Pharmacological inhibition of CaM abrogated the Ca2+ sensitivity of cyclin E/CDK2 and retarded mouse VSMC proliferation by causing G1 arrest. We next defined the presence of a highly conserved 22 amino acid N-terminal CaM-binding motif in mammalian cyclin E genes (dissociation constant, 1.5+/-0.1 micromol/L) and showed its essential role in mediating Ca2+-sensitive kinase activity of cyclin E/CDK2. Mutant human cyclin E protein, lacking this CaM-binding motif, was incapable of binding CaM or responding to [Ca2+]. Taken together, these findings reveal CaM-dependent cyclin E/CDK2 activity as a mediator of the known Ca2+ sensitivity of the G1/S transition of VSMC.

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.

Molecular oxygen loading in candidate theranostic droplets stabilized with biocompatible fluorinated surfactants: Particle size effect and application to in situ19F MRI mapping of oxygen partial pressure.

OBJECTIVE: Perfluorocarbon nano- and micron-sized emulsions are a new field of investigation in cancer treatment due to their ability to be used as imaging contrast agents, or as delivery vectors for pharmaceuticals. They also demonstrated capability to enhance the efficiency of high intensity focused ultrasound thermo-therapy. In the context of new biomedical applications we investigated perfluorooctyl bromide (PFOB) theranostic droplets using 19F NMR. Each droplet contains biocompatible fluorinated surfactants composed of a polar Tris(hydroxymethyl)aminomethane head unit and hydrophobic perfluorinated tail (abbreviated as F-TAC). The influence of the droplet size on the oxygen loading capacity was determined from longitudinal relaxation (T1) data of 19F NMR signal. MATERIAL AND METHODS: Liquid PFOB and five samples of PFOB droplets of average diameter 0.177, 0.259, 1.43, 3.12 and 4.53 µm were tested with different oxygen levels. A dedicated gas exchange system was validated to maintain steady state oxygen concentrations, including a spatial gradient of oxygen concentration. A prototyped transmit-receive switchable 19F/1H quadrature coil was integrated on a 3 T clinical scanner. The coil is compatible with focused ultrasound sonication for future application. A spectroscopy FID inversion-recovery (IR) sequence was used to measure the T1 value per sample and per value of equilibrium oxygen pressure. Pixel wise, spatial T1 mapping was performed with magnetization prepared 2D gradient echo sequences in tissue mimicking gels doped with theranostic droplets. RESULTS: Experimental data indicated that the longitudinal relaxation rate of 19F signal of the investigated theranostic droplets depended approximately linearly on the oxygen level and its slope decreased with the particle size according to a second order polynomial over the investigated range. This semi-empirical model was derived from general thermodynamics and weak electrostatic forces theory and fitted the experimental data within 0.75% precision. The capacity of oxygen transportation for the described theranostic droplets tended to that of pure PFOB, while micron-sized droplets lost up to 50% of this capacity. In a specific setup producing a steady state gradient of oxygen concentration, we demonstrated spatial mapping of oxygen pressure gradient of 6 kPa/mm with 1 mm in-plane resolution. CONCLUSION: The size-tunable PFOB theranostic droplets stabilized with F-TAC surfactants could be characterized by 19F MRI in a clinical setup readily compatible with interventional in vivo studies under MR guidance. Current precision and spatial resolution of T1 mapping are promising. A potential challenge for further in vivo studies is the reduction of the imaging time.

Hydrophobic hydration in cyclodextrin complexation.

We report temperature-dependent acoustic and densimetric data on changes in volume, expansibility, and adiabatic compressibility accompanying the binding of 1-adamantanecarboxylic acid (AD) to beta-cyclodextrin (beta-CD). We interpret our volumetric results in terms of hydration. Based on our compressibility and expansibility data, we estimate that, at 25 degrees C, the binding of AD to beta-CD is accompanied by displacement of 20 to 25 water molecules from the hydration shells of the two interacting species. Comparison of the temperature-dependent compressibility changes accompanying the binding of AD to beta-CD with the compressibility contribution of aliphatic groups suggests that displaced water molecules predominantly come from the hydrophobic loci of AD and beta-CD. Thus, we conclude that hydrophobic interactions play a major role in stabilizing the AD-beta-CD complex. Our estimate of the number of water molecules released to the bulk is consistent with structural considerations. There is also good agreement between our volumetric data and osmotic stress results reported by Harries et al. (Harries, D.; Rau, D. C.; Parsegian, V. A. J. Am. Chem. Soc. 2005, 127, 2184). This observation is consistent with the picture in which the two techniques probe the same population of water molecules solvating AD and beta-CD.