Gilles Waton

Wall slip, shear banding, and instability in the flow of a triblock copolymer micellar solution

The shear flow of a triblock copolymer micellar solution (PEO-PPO-PEO Pluronic P84 in brine) is investigated using simultaneous rheological and velocity profile measurements in the concentric cylinder geometry. We focus on two different temperatures below and above the transition temperature T{c} which was previously associated with the apparition of a stress plateau in the flow curve. (i) At T=37.0 degrees C<T{c}, the bulk flow remains homogeneous and Newtonian-like, although significant wall slip is measured at the rotor that can be linked to an inflexion point in the flow curve. (ii) At T=39.4 degrees C>T{c}, the stress plateau is shown to correspond to stationary shear-banded states characterized by two high shear rate bands close to the walls and a very weakly sheared central band, together with large slip velocities at the rotor. In both cases, the high shear branch of the flow curve is characterized by flow instability. Interpretations of wall slip, three-band structure, and instability are proposed in light of recent theoretical models and experiments.

Small Phospholipid-Coated Gas Bubbles Can Last Longer than Larger Ones

Microbubbles are actively being investigated as contrast agents for ultrasound imaging and as ultrasound-mediated drug delivery vehicles. Gas-filled microcapsules stabilized by multilayers of oppositely charged polyelectrolytes [poly(allylamine hydrochloride) and poly(styrene sulfonate)] have recently been reported. These rigid microbubbles are promising owing to their high stability, tunable surface charge and easy functionalization of the polyelectrolyte surface. Bubbles stabilized by polystyrene beads have also been studied. However, rigid shells, which are made either from polymers or crystallized lipids, increase the resonance frequency of the microbubbles dramatically. As maximum echogenicity is obtained for an ultrasound frequency close to the bubble resonance frequency (which varies as the inverse of bubble size), high-intensity ultrasound is required to obtain successful echo imaging of hard-shell bubbles. High-intensity ultrasound pulses can compromise shell integrity and may limit the use of hard-shell microbubbles with small diameters (1–5 mm) for drug delivery. Because they are supple, monolayers of self-assembled phospholipids in the fluid state allow production of small bubbles that resonate at much lower frequency. Their stability, however, remains a critical issue. Under the action of Laplace pressure, which increases as the inverse of their radii, bubbles dissolve in aqueous media all the more rapidly when they are smaller. Naked gas bubbles smaller than 10 mm persist only for a few seconds in an aqueous environment. Enclosing the bubbles within a phospholipid shell allows an increase in bubble persistence by a factor of 10 or more, provided the aqueous phase is saturated with the gas. Micron-size bubbles with half-lives sufficiently long for practical diagnostic ultrasound examination have been obtained using phospholipids and a perfluorocarbon filling gas. 12] Perfluorocarbons retard bubble dissolution very effectively, due to very low solubility in water. The half-life of the commercially available soft-shell bubbles is limited, however, to a few minutes. We found that, against expectations, and contrary to all published experimental evidence, small (~1.55 0.20 mm in radius) bubbles covered with a shell of fluid phospholipid and dispersed in an aqueous phase can last longer, by an order of magnitude, than larger bubbles (~5.40 0.50 mm) made of the same components. This was made possible because we were able to prepare monodisperse populations of bubbles and determine the size and stability characteristics of the bubbles accurately using a new acoustic method (Supporting Information). Initial bubble size distributions and stability over time were determined by measuring the attenuation coefficient of ultrasound waves, that is, the reduction in amplitude of a wave package that propagates through the bubble dispersion. The technique is based on the fact that, as a bubble is a resonator, the product of the bubble’s radius, r, and its resonance frequency, f0, is about 3. [9] The attenuation spectra of ultrasound by the microbubbles were monitored using a multi-frequency analysis set-up (Supporting information). The microbubbles investigated contained N2 saturated with perfluorohexane (C6F14, PFH) as filling gas. [12] The bubble wall consisted of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). Two DMPC concentrations, 10 or 50 mm, were investigated. Bubbles were obtained by sonicating DMPC (10 or 50 mm) in an isotonic solution (Isoton II), the volume above the dispersion being filled with PFH-saturated N2. The bubble dispersion was diluted with 14 mL of Isoton and the bubbles were allowed to float. 300 mL (for the 10 mm-concentrated dispersion) or 1 mL (for the 50 mm-concentrated dispersion) were pipeted after 3 min and 20 min, respectively, and transferred to the ultrasonic cell. The ratio of the partial pressure of oxygen in the aqueous phase to that at saturation was approximately 1. We checked that the ultrasound field did not modify the size characteristics or stability of the bubbles (Experimental Section). The low DMPC concentrations (10 mm), after 3 min of flotation of the dispersions at 25 8C, lead consistently to a monomodal population of bubbles with an average radius of 5.40 0.50 mm (Figure 1). The bubble radius and radius distribution are confirmed both by static light scattering (5.90 0.50 mm) and by optical microscopy (6.3 1.2 mm) [Figure 1].

A Nonpolar, Nonamphiphilic Molecule Can Accelerate Adsorption of Phospholipids and Lower Their Surface Tension at the Air/Water Interface

The adsorption dynamics of a series of phospholipids (PLs) at the interface between an aqueous solution or dispersion of the PL and a gas phase containing the nonpolar, nonamphiphilic linear perfluorocarbon perfluorohexane (PFH) was studied by bubble profile analysis tensiometry. The PLs investigated were dioctanoylphosphatidylcholine (DiC(8)-PC), dilaurylphosphatidylcholine, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine. The gas phase consisted of air or air saturated with PFH. The perfluorocarbon gas was found to have an unexpected, strong effect on both the adsorption rate and the equilibrium interfacial tension (γ(eq)) of the PLs. First, for all of the PLs, and at all concentrations investigated, the γ(eq) values were significantly lower (by up to 10 mN m(-1)) when PFH was present in the gas phase. The efficacy of PFH in decreasing γ(eq) depends on the ability of PLs to form micelles or vesicles in water. For vesicles, it also depends on the gel or fluid state of the membranes. Second, the adsorption rates of all the PLs at the interface (as assessed by the time required for the initial interfacial tension to be reduced by 30%) are significantly accelerated (by up to fivefold) by the presence of PFH for the lower PL concentrations. Both the surface-tension reducing effect and the adsorption rate increasing effect establish that PFH has a strong interaction with the PL monolayer and acts as a cosurfactant at the interface, despite the absence of any amphiphilic character. Fitting the adsorption profiles of DiC(8)-PC at the PFH-saturated air/aqueous solution interface with the modified Frumkin model indicated that the PFH molecule lay horizontally at the interface.

Effects of perfluorocarbon gases on the size and stability characteristics of phospholipid-coated microbubbles: osmotic effect versus interfacial film stabilization.

Micrometer-sized bubbles coated with phospholipids are used as contrast agents for ultrasound imaging and have potential for oxygen, drug, and gene delivery and as therapeutic devices. An internal perfluorocarbon (FC) gas is generally used to stabilize them osmotically. We report here on the effects of three relatively heavy FCs, perfluorohexane (F-hexane), perfluorodiglyme (F-diglyme ), and perfluorotriglyme (F-triglyme), on the size and stability characteristics of microbubbles coated with a soft shell of dimyristoylphosphatidylcholine (DMPC) and on the surface tension and compressibility of DMPC monolayers. Monomodal populations of small bubbles (~1.3 ± 0.2 μm in radius, polydispersivity index ~8%) were prepared by sonication, followed by centrifugal fractionation. The mean microbubble size, size distribution, and stability were determined by acoustical attenuation measurements, static light scattering, and optical microscopy. The half-lives of F-hexane- and F-diglyme-stabilized bubbles (149 ± 8 and 134 ± 3 min, respectively) were about 2 times longer than with the heavier F-triglyme (76 ± 7 min) and 4-5 times longer than with air (34 ± 3 min). Remarkably, the bubbles are smaller than the minimal size values calculated assuming that the bubbles are stabilized osmotically by the insoluble FC gases. Particularly striking is that bubbles 2 orders of magnitude smaller than the calculated collapse radius can be prepared with F-triglyme, while its very low vapor pressure prohibits any osmotic effect. The interface between an aqueous DMPC dispersion and air, or air (or N(2)) saturated with the FCs, was investigated by tensiometry and by Langmuir monolayer compressions. Remarkably, after 3 h, the tensions at the interface between an aqueous DMPC dispersion (0.5 mmol L(-1)) and air were lowered from ~50 ± 1 to ~37 ± 1 mN m(-1) when F-hexane and F-diglyme were present and to ~40 ± 1 mN m(-1) for F-triglyme. Also noteworthy, the adsorption kinetics of DMPC at the interface, as obtained by dynamic tensiometry, were accelerated up to 3-fold when the FC gases were present. The compression isotherms show that all these FC gases significantly increase the surface pressure (from ~0 to ~10 mN m(-1)) at large molecular areas (70 Å(2)), implying their incorporation into the DMPC monolayer. All three FC gases increase the monolayers collapse pressures significantly (~61 ± 2 mN m(-1)) as compared to air (~54 ± 2 mN m(-1)), providing for interfacial tensions as low as ~11 mN m(-1) (vs ~18 mN m(-1) in their absence). The FC gases increase the compressibility of the DMPC monolayer by 20-50%. These results establish that, besides their osmotic effect, FC gases contribute to bubble stabilization by decreasing the DMPC interfacial tension, hence reducing the Laplace pressure. This contribution, although significant, still does not suffice to explain the large discrepancy observed between calculated and experimental bubble half-lives. The case of F-triglyme, which has no osmotic effect, indicates that its effects on the DMPC shell (increased collapse pressure, decreased interfacial tension, and increased compressibility) contribute to bubble stabilization. F-hexane and F-diglyme provided both the smallest mean bubble sizes and the longest bubble half-lives.

Microbubbles with exceptionally long life- : synergy between shell and internal phase components

Highly effective stabilization of microbubbles has been obtained by using a fluorocarbon as part of their filling gas and a fluorinated phospholipid, instead of a standard phospholipid, as a shell component. An unexpected strong synergistic effect between the fluorocarbon gas and the fluorinated phospholipid has been discovered.

Long-Range Nanometer-Scale Organization of Semifluorinated Alkane Monolayers at the Air/Water Interface

We have determined the structure formed at the air-water interface by semifluorinated alkanes (C(8)F(17)C(m)H(2m+1) diblocks, F8Hm for short) for different lengths of the molecule (m = 14, 16, 18, 20) by using surface pressure versus area per molecule isotherms, Brewster angle microscopy (BAM), and grazing incidence x-ray experiments (GISAXS and GIXD). The behavior of the monolayers of diblocks under compression is mainly characterized by a phase transition from a low-density phase to a condensed phase. The nonzero surface pressure phase is crystalline and exhibits two hexagonal lattices at two different scales: a long-range-order lattice of a few tens of nanometers lateral parameter and a molecular array of about 0.6 nm parameter. The extent of this organization is sufficiently large to impact larger scale behavior. Analysis of the various compressibilities evidences the presence of non organized molecules in the monolayer for all 2D pressures. At room temperature, the self-assembled structure appears generic for all the F8Hm investigated.

Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking

A UV-cross-linkable agent was incorporated and polymerized in Pluronic micelle core to create an interpenetrating polymer network (IPN) of poly(pentaerythritol tetraacrylate). This stabilization prevented micelle disruption below the critical micelle temperature (CMT) and concentration (CMC), while maintaining the integrity of the PEO corona and the hydrophobic properties of the PPO core. The prepared stabilized spherical micelles of Pluronic P94 and F127 presented hydrodynamic diameters ranging from 40 to 50 nm. The stability of cross-linked Pluronic micelles at 37 °C in the presence of serum proteins was studied and no aggregation of the micelles was observed, revealing the colloidal stability of the system. Cytotoxicity experiments in NIH/3T3 mouse fibroblasts revealed that the presence of the cross-linking agent did not induce any further toxicity in comparison to the respective pure polymer solutions. Furthermore, stabilized micelles of Pluronic P94 were shown to be less toxic than the polymer itself. A hydrophobic fluorescent probe (Nile red) was absorbed in the cross-linked core of pre-stabilized micelles to mimic the incorporation of a poorly water-soluble drug, and the internalization and intracellular localization of Nile red was studied by confocal microscopy at different incubation times. Overall, the results indicate that Pluronic micelles stabilized by core cross-linking are capable of delivering hydrophobic components physically entrapped in the micelles, thus making them a potential candidate as a delivery platform for imaging or therapy of cancer.

Long Lived Microbubbles for Oxygen Delivery

Exceptionally long lived microbubbles containing a fluorocarbon as part of their filling gas have been obtained by using a fluorinated phospholipid instead of a standard phospholipid as shell component. An unexpected, strong synergistic effect between the fluorocarbon gas and the fluorinated phospholipid has been discovered. Such bubbles could be used for in vivo oxygen delivery, ultrasound contrast imaging and drug delivery.

Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state

This work reveals how the physicochemical properties of Pluronic block copolymers influence significantly their interactions with cancer cells, whether in monolayer or spheroid cultures, and how different clinical applications can be foreseen. Two-dimensional (2D) and three-dimensional (3D) cell culture models were used to investigate the interactions of Pluronic carriers with different PEO block length and aggregation state (unimers versus cross-linked micelles) in HeLa and U87 cancer cells. Stabilized micelles of Pluronic P94 or F127 were obtained by polymerization of a crosslinking agent in the micelles hydrophobic core. Nanocarriers were functionalized with a fluorescent probe for visualization, and with a chelator for radiolabeling with Indium-111 and gamma-quantification. The 2D cell models revealed that the internalization pathways and ultimate cellular localization of the Pluronic nanocarriers depended largely on both the PEO block size and aggregation state of the copolymers. The smaller P94 unimers with an average radius of 2.1nm and the shortest PEO block mass (1100gmol(-1)) displayed the highest cellular uptake and retention. 3D tumor spheroids were used to assess the penetration capacity and toxicity potential of the nanocarriers. Results showed that cross-linked F127 micelles were more efficiently delivered across the tumor spheroids, and the penetration depth depends mostly on the transcellular transport of the carriers. The Pluronic P94-based carriers with the shortest PEO block length induced spheroid toxicity, which was significantly influenced by the spheroid cellular type.

Compressible multi-scale magnetic constructs: decorating the outer surface of self-assembled microbubbles with iron oxide nanoparticles

This paper reports the elaboration of size-controlled resilient echogenic micron-size bubbles that have a soft shell made from self-assembled perfluoroalkylated phosphates and iron oxide nanoparticles grafted on their external surface. The high echogenicity, characteristic of compressible gas bubbles, is retained. These hybrid constructs have potential as bimodal contrast agents for ultrasound and magnetic resonance imaging.