Microbubbles with a Self-Assembled Poloxamer Shell and a

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Microbubbles with a Self-Assembled Poloxamer Shell and a Fluorocarbon Inner Gas Yu ANDO, Hiraku Tabata, Michaël Sanchez, Alain Cagna, Daisuke Koyama, and Marie Pierre Krafft Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01883 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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Microbubbles with a Self-Assembled Poloxamer Shell and a Fluorocarbon Inner Gas

Yu Ando1,2, Hiraku Tabata1,2, Michaël Sanchez3, Alain Cagna3, Daisuke Koyama1*, and Marie Pierre Krafft2* 1

Faculty of Life and Medical Sciences, Doshisha University, Kyoto 610-0321, Japan

2

Institut Charles Sadron (CNRS), University of Strasbourg, 23 rue du Loess. Strasbourg

67034, France 3

TECLIS Instruments, Tassin 69160, Lyon Métropole, France

Keywords: Fluorocarbon; Poloxamer 188; Pluronic F-68; Ultrasound; Diagnostic; Phase transition; Film elasticity; Bubble shape analysis tensiometry; Laser Doppler vibratometry; Gibbs film; Self-assembly; Soft matter

ACS Paragon Plus Environment

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ABSTRACT: The numerous applications of microbubbles in food science and medicine call for a better understanding and control of the effects of the properties of their shells on stability and ability to resonate at chosen frequencies when submitted to an ultrasound field. We have investigated both millimetric and micrometric bubbles stabilized by an amphiphilic block copolymer, Poloxamer 188 (e.g. Pluronic F-68). Although Pluronic F-68 is routinely being used as a dispersing and foaming agent to facilitate phospholipid-based microbubble preparation, it has never been studied as a shell component per se. First, we have investigated the adsorption kinetics of Pluronic F-68 at the interface between water and air, or air saturated with vapors of perfluorohexane (F-hexane), using bubble profile tensiometry analysis. F-hexane was found to strongly accelerate the adsorption of Pluronic F-68 (at low concentrations) and decrease the interfacial tension values at equilibrium (at all concentrations). We also found that relatively stable microbubbles could unexpectedly be prepared from Pluronic F-68 in the absence of any other surfactant, but only when F-hexane was present. These bubbles showed only limited volume increase for ~3 h, while a tenfold size growth occurred within 200 s in the absence of fluorocarbon. Remarkably, their deflation rate decreased when Pluronic F-68 concentration decreased, suggesting that bubbles with semi-dilute copolymer coverage are more stable than those more densely covered by copolymer brushes. Single bubble experiments using laser Doppler vibratometry showed that, by contrast with other surfactant-coated microbubbles, the resonance radius of the Pluronic F-68-coated microbubbles was lower than that of naked microbubbles, meaning that they are less elastic. It was also found that the bubble’s vibrational displacement amplitude decreased substantially when the microbubbles are covered with Pluronic F-68, an effect that was further amplified by F-hexane.


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I. INTRODUCTION Microbubbles (MBs) have found multiple applications, in particular in food science, microfluidic technologies and medicine. In medical diagnosis, MBs are used as contrast agents for ultrasound imaging.1-9 Recent ultrasound molecular imaging relies on MB agents for detecting intravascular targets.10-13 Therapeutic agents and genes can be incorporated into MBs for the purpose of drug delivery and therapy.14-21 Moreover, the sonoporation phenomenon that results from MB resonance creates transient pores in cell membranes allowing efficient transportation of therapeutic agents into tissues.15-16, 19, 22-24 Microbubbles are commonly classified in two categories depending on whether they have hard or soft shells.21 Hard shell MBs are usually made from polymers, whilst soft shell MBs consist of thin molecular films of self-assembled surfactants, generally phospholipids. MBs have a viscoelastic behavior, but the relative contribution of viscosity and elasticity can differ critically. On one side, viscosity has a strong damping effect on the vibrational amplitude of the microbubble when insonated. On the other side, elasticity acts on the MB resonance frequency radius; the higher the elasticity, the higher the resonance radius. As a consequence, MBs with hard, elastic shells are highly echogenic, but the downside is that they resonate at high frequencies. This strongly limits their use to the imaging of superficial organs and tissues, as the penetration of ultrasound waves in the body is inversely proportional to their frequency. Obtaining stable MBs from water-soluble components is desirable as they are easier to implement than the commonly used quasi-insoluble, oxidizable phospholipids. Among polymers, Poloxamers, which are triblock copolymers consisting of a hydrophobic poly(propylene oxide) (PPO) segment flanked by two hydrophilic poly(oxyethylene oxide) (PEO) chains, are candidates of choice because some of them, including Poloxamer 188 (Pluronic F-68) are well accepted and routinely used in applications related to bioprocesses (e.g. for protecting cell cultures against shear in reactors), pharmaceuticals (for sensitizing resistant cancers to chemotherapy and personal care products).25-26 Pluronic F-68 is commonly used in MB preparation as dispersant and foaming agent.2 However, to the best of our knowledge, Pluronic F-68 has never been investigated as a sole MB shell component. The adsorption kinetics of Pluronic F-68 at the air/water interface and its


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properties at equilibrium have been investigated in details, providing information on the copolymer’s conformation during spontaneous adsorption.27-28 Pluronic F-68 films successively undergo two first-order transitions, first from a dilute to a semi-dilute state (π1), then from a semidilute to a dense state (π2), after which the film progressively forms a dense polymer brush. Recent work from our group demonstrated that the structure and properties of self-assembled interfacial surfactant films and colloids could be drastically modified when contacted with a fluorocarbon (FC) gas. FC gases can reduce osmotic pressure,2, 29-30 lower interfacial tensions,31 control phospholipid versus protein competitive adsorption,32 stabilize microbubbles,33-34 and foster transmembrane recognition.35 FC gases have been identified as promising components of novel compositions of lung surfactant substitutes able to counteract the deleterious inhibiting effect of serum proteins.36 It is noteworthy that all phospholipid-based developed MB contrast agents contain a FC gas.37 The unique physicochemical characteristics of the latter, including high vapor pressures with respect to molecular weight and low surface tensions, along with biological inertness, underlie their medical applications.2 Here, we have first studied the kinetics of adsorption of Pluronic F-68 solutions at the air/water interface using bubble profile analysis tensiometry with and without perfluorohexane (F-hexane) gas-saturated inner bubble phase in order to gain information on the surfactant’s ability to adsorb at the interface. Next, we have evaluated the feasibility of obtaining MBs coated with Pluronic F-68 as the only surfactant and to determine whether their stability could be increased by saturating the gaseous phase with a FC gas. Another goal was to investigate the echogenicity of such Pluronic F68-only MBs by measuring the attenuation coefficient of an ultrasound wave that propagates in a dispersion of such microbubbles. Finally, we have determined the vibrational displacement amplitude of isolated large (tens of micrometers in diameter) Pluronic F-68 microbubbles by using laser Doppler vibratometry (LDV). This technique allowed successful determination of the elasticity of hard shell capsules submitted to ultrasound,38 but had never been applied to soft shell MBs. The combined use of bubble profile analysis tensiometry and LDV was expected to allow assessment of the relative contributions of viscosity and elasticity to microbubble echogenicity. Our data support the notion that a surfactant coating with a high viscous character, such as a


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Pluronic F-68 coating when in contact with F-hexane, actually allows obtaining of microbubbles with a resonance radius smaller than that of naked microbubbles.

II. EXPERIMENTAL SECTION A. Materials Pluronic F-68 was purchased from Aldrich ((PEO)A-(PPO)B(PEO)A; CAS: 9003-11-6; Mw ∼8400 g mol-1 according to the supplier; ∼9050 g mol-1 according to our light scattering experiments). The degree of polymerization of each block, NA = 2 x 76 and NB = 29, was calculated from the nominal value of Mw. The polydispersity index (Mw/Mn 1.15; Mn: number average molecular weight) was obtained from GPC analysis using THF as solvent. Perfluorohexane (Fhexane, purity >99%) was from Sigma. Water was obtained from a MilliQ (Millipore) system (surface tension: 71.7 ± 0.2 mN m-1 at 20°C; resistivity 18.2 MΩ cm). All measurements were made at 22 ± 1°C and repeated three to five times. B. Bubble Profile Analysis Tensiometry Axisymmetric bubble shape analysis was applied to a rising bubble of air formed in Pluronic F68 solutions in Milli-Q water. The time dependence of the surface tension during adsorption of the block copolymer at the gas/liquid interface was measured using a Tracker® tensiometer (TECLIS Instrument, France). The bubble (5 µL) was formed at the end of a steel capillary with a tip diameter of 1 mm. Since the experiments lasted for up to 2 h, a lid was fitted on the measuring glass cell (10 mL) to prevent evaporation of water during the long equilibration times. It was carefully determined that the systems had reached equilibrium at the end of each experiment. For the experiments achieved at the F-hexane-saturated air/water interface, a 1 mL syringe was purged three times with the F-hexane-saturated air that surmounts liquid F-hexane at ambient temperature (F-hexane saturated vapor pressure and concentration at 25°C are 2.9 104 Pa and 11.66 mol m-3, respectively; water solubility at 25°C: 2.7 10-4 mol m-3). The syringe was mounted on the injection cell of the tensiometer in order to form the rising bubble. C. Microbubble Preparation


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The microbubble samples were prepared by submitting the aqueous solutions of Pluronic F-68 to brief sonication. The sonicator (Vibracell sonicator, Bioblock Scientific, Illkirch, France) was equipped with a 3 mm titanium probe and operated at 20 kHz. 5 mL of the solution were placed into a glass tube (diameter 2 cm). The sonicator probe was positioned slightly above the bottom of the tube, which was maintained at 25°C in a water bath. Dry N2 was saturated with F-hexane by bubbling through three vials containing liquid F-hexane, and the atmosphere above the dispersion in the sample tubes was flushed with F-hexane-saturated N2 for 5 min. Pure N2 was used for the controls. The samples were then sonicated for 15 s (power setting 5, duty cycle 40%). Each sample was immediately diluted with 10 mL of deionized water to obtain the aqueous MB test dispersion. D. Optical Microscopy The samples were observed using a Nikon Eclipse 90i microscope. Three to four droplets of bubble dispersion were placed into a concave slide and covered with a glass slide. Rapid image acquisition was achieved using a Lumenera Infinity 2 CCD camera (Lumenera, Ottawa, Canada). Bubble morphology was investigated and their mean diameter measured using the ImageJ software on 5 to 10 slides. These experiments were not achieved under ultrasound irradiation. E. Echogenicity and Acoustical Determination of Microbubble Size Distribution Our method is based on the attenuation, that is, the reduction in amplitude, of an acoustical wave that propagates through an aqueous dispersion of bubbles. A method has been developed that fits standard simple-harmonic resonator curves to measured attenuations in order to infer the size of the bubbles.39-41 Determination of radii distribution is based on the fact that the attenuation is maximal when the frequency of the acoustic wave is the same as the resonance frequency of the bubble. The half-life of the bubbles is obtained directly, since the number of bubbles is directly proportional to the attenuation coefficient. A low-power emitter was used in order to avoid altering bubble characteristics and stability (acoustical power: 0.1 W cm-2; peak-to-peak acoustical pressure 3 10-7 mol L-1,


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a concentration at which F-hexane does not play its co-surfactant role anymore, likely because it is ejected from the interface, an hypothesis also supported by previous observations.31 Surface Tension Properties The equilibrium surface tension data σeq for Pluronic F-68 at the air/water and F-hexanesaturated air/water interfaces were obtained from the corresponding dynamic curves σ(t) at 22 ± 1°C (Figure 5). The variations of the surface pressure π = σ0 - σ are represented as a function of the Pluronic F-68 concentration c.

Figure 5. Variation of the surface pressure π at 22 ± 1°C as a function of Pluronic F-68 concentration c under air (empty symbols) and under F-hexane-saturated air (filled symbols). π1 and π2 are the phase transitions occurring in the Pluronic F-68 Gibbs film (π1 and π2). π3 corresponds to the cmc. Schematical representation of Pluronic F-68 adsorption as a function of π. At the air/water interface, our results qualitatively agree with an earlier report.27 In that study, both adsorbed and spread monolayers of Pluronic F-68 were studied using surface tension and ellipsometric measurements.27 An analysis of the experimental data in terms of the scaling theory of adsorption of polymer brushes showed that the copolymer undergoes at least two successive first-order phase transitions (Figure 5). At the early times of the process, adsorption is weak (dilute regime). The copolymer is water-soluble and only few molecules are loosely adsorbed flat on the interface. Subsequently, a reorientation occurs that leads to a differentiated anchoring of the central


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hydrophobic segment, which remains horizontal at the interface, while the hydrophilic segments are loosely oriented towards the aqueous phase (initial first-order transition π1, semi-dilute regime) forming a dilute polymer brush. This reorientation enables the adsorption of additional Poloxamer molecules, until a second first-order transition is reached (π2). At this stage, the hydrophilic segments start adopting a stretched more ordered conformation. The polymer then forms dense brushes. When compared to the earlier report,27 our results show that, under an atmosphere of air, the π(c) curve is shifted towards the larger concentrations. This discrepancy may be explained by the difference in method used (Wilhelmy plate versus bubble profile analysis tensiometry). Nevertheless, the transitions occur at approximately the same pressure in both studies, that is, ~10 mN m-127 and ~12 mN m-1 (us) for the first transition, and ~18 mN m-127 and ~20 mN m-1 (us) for the second one. We show that F-hexane increases markedly the equilibrium interfacial pressure πeq, by ~5 to 10 mN m-1 and this throughout the concentration range (Figure 5). Even at the lowest Pluronic F-68 concentration investigated (1 10-8 M), the surface pressure is still ~3 mN m-1, which corresponds to the surface pressure exerted by the FC gas at the naked air/water interface. This suggests that the FC molecules add to the lateral pressure experienced by the Pluronic F-68 molecules, or that more copolymer molecules are adsorbed. The adsorption profile also displays three plateaus that reflect the occurrence of first-order transitions. The fact that the transitions exist both in the presence or in the absence of the FC gas - and for comparable concentrations - suggests that the FC molecules do not hinder the changes in Poloxamer conformation during adsorption, and hence, the adsorption of additional Poloxamer molecules. The reason for this being that F-hexane is probably expelled from the interface during Poloxamer conformational changes.

B. Microbubbles With a Shell Made of Pluronic F-68 Only Size and Stability Characteristics


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Air microbubbles stabilized by water-soluble surfactants are notoriously unstable.44 Pluronic F68-coated air microbubbles are indeed highly instable, as shown on Figure 6, irrespective of the Poloxamer’s concentration, and undergo a rapid and large increase in volume within a few minutes. This growth is mainly assigned to the fast coalescence of the bubbles owing to the poor stabilizing properties of the water-soluble Poloxamer. On the other hand, when F-hexane is present, the MBs are strongly stabilized, with a much slower increase of their mean volume (Figure 6).

Figure 6. Average change in volume of microbubbles ∆V as a function of time for various Pluronic F-68 concentrations in the absence (empty symbols) and presence of F-hexane (filled symbols). Squares: 10-2 M; up triangles: 10-3 M; circles: 10-5 M; left triangles: 10-7 M. This regime lasts about ~3h, after which, the MBs were observed to coalesce. This suggests that the major stabilization effect is due to the osmotic effect of the highly hydrophobic poorly watersoluble F-hexane that therefore remains in the bubbles’ inner gas phase and does not diffuse in the aqueous phase. A second contribution to MB stabilization arises from the co-surfactant effect of the F-hexane with respect to the Poloxamer, in agreement with previous observations obtained with Fhexane-stabilized phospholipid-shelled MBs.34 However, our present results show that, in the case of F-hexane stabilized microbubbles, stability also depends on Poloxamer concentration. Counterintuitively, the microbubbles prepared with the lowest Pluronic F-68 concentrations (1 10-5 and 1 10-7 mol L-1) are the most stable. This likely means that bubble stability is related to polymer


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conformation. Stabilization by the more resilient, semi-dilute copolymer brushes that are formed at low concentrations may be more effective than that by the dense brushes obtained at higher concentrations. Echogenicity of Pluronic F-68 Shelled Microbubbles The acoustic behavior of the Poloxamer-shelled MBs was studied by measuring the attenuation coefficient α of ultrasound waves that propagate through the bubble dispersion as a function of time for a range of excitation frequencies f. The MBs were found echogenic, and their mean diameter, ~5 µm, as inferred from the attenuation coefficient measured immediately after injection of the bubble dispersion in the cell, was supported by optical microscopy and static light scattering measurements (Figure 7). No signal was detected for the Pluronic F-68 MBs prepared in the absence of F-hexane, likely reflecting insufficient bubble stability.

Figure 7. Diameter distributions of Pluronic F-68-shelled microbubbles determined by optical microscopy (bars), static light scattering (dashed line) and the acoustical attenuation method (solid line). C. Single Pluronic F-68 Microbubbles Subjected to Ultrasound Isolated large microbubbles were submitted to ultrasound and investigated using a laser Doppler vibratometer (LDV). Figure 8 shows the relationships between bubble radius and the normalized radial vibration amplitude of the bubble (amplitude divided by initial bubble radius, ∆R/R0), which is also known as the bubble resonance curves.


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Figure 8. Relationship between the vibrational amplitude displacement of the bubble and the bubble radius (red: naked F-hexane-saturated air; blue: naked air; green: air/Pluronic F-68 (1 10-3 mol L-1); blue, purple and magenta: F-hexane-saturated air/Pluronic F-68 (1 10-4, 1 10-3, 1 10-2 mol L-1, respectively)); acoustic power: 1.5 W m-2. Inset: magnification of the resonance curves for the Pluronic F-68-coated bubbles. Air bubbles covered with Pluronic F-68 (1 10-3 mol L-1) and F-hexane-saturated air bubbles covered with Pluronic F-68 (1 10-4, 1 10-3, 1 10-2 mol L-1) were investigated, as well as, for comparison, air bubbles and F-hexane-saturated air naked bubbles. First, it can be seen that the naked bubbles display larger vibrational amplitude displacements than Pluronic F-68 coated bubbles. The strong reduction of the vibrational amplitude observed for all Pluronic F68-coated MBs, whatever the surfactant concentration and whether F-hexane is present or not, is assigned to the increased shell viscosity caused by the Poloxamer coating. Second, it is important to note that all Poloxamer-coated bubbles show a shift of their resonance radii towards the smaller values, revealing a decrease in bubble elasticity. Whilst the effect of the surfactant on the shell viscosity is well-known, its impact on the resonance radius was unexpected, as it has been repeatedly observed that microbubbles with a surfactant coating have a resonance radius shifted towards the larger values, owing to an increase in elasticity.45-46 This is the case, for example, of commercial ultrasonic contrast agents such as Levovist (stabilized by palmitic acid) and Sonazoid (stabilized by


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phospholipids in the presence of a FC gas). For F-hexane-stabilized Pluronic F-68-coated bubbles, it is seen that the higher the Pluronic F-68 concentration, the higher the damping effect, due to increased viscosity. These data support that our Pluronic F-68-shelled microbubbles have smaller resonance diameter at the same driving frequency than naked microbubbles, that is, they have lower resonance frequency as compared to similarly sized naked MBs. Furthermore, we found that introducing F-hexane gas in the MB’s gas phase further enhances this effect by increasing the “softness” of the Pluronic F-68 interfacial film. We also see that the increase of viscosity also results in a decrease in echogenicity, which, of course, is less desirable. Altogether, this study shows that controlling viscosity and elasticity of MB shells may be useful for the design of microbubbles specially tailored for specific applications, and in particular for producing microbubbles of biologically compatible sizes that resonate at practical frequencies for medical uses. The use of a fluorocarbon gas that contributes to decreasing MB elasticity may be valuable for ensuring effective echogenicity control. In particular, this finding may help enhancing contrast in nonlinear ultrasound imaging methods because MBs with softer shells generate harmonic and subharmonic components more efficiently than bubbles with hard shells. IV. CONCLUSIONS The capacity of Poloxamer 188 (Pluronic F-68) to stabilize microbubbles in the presence, and only in the presence, of F-hexane, was unexpected. Shell film viscosity and elasticity, as determined by single bubble laser Doppler vibratometry in an ultrasound field indicates that the Poloxamer shell has a low elasticity, allowing the MBs to resonate at a frequency comparable to that of similarly sized naked MBs, in contrast to the standard surfactant and phospholipid-coated MBs that resonate at higher frequency. The bubble stabilization data provide a further example of the outstanding capacity for fluorocarbon gases to promote self-assembly at interfaces and in the shell of dispersed colloids and control over adsorption of different classes of molecules at the air/water interface. The most important lesson from this study is probably that, contrary to common belief and practice, Pluronic F-68 (and other Poloxamers) can definitely not be considered as the


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“surface passive” excipient it is generally considered to be. This Poloxamer’s definite capacity for self-assembly in films at interfaces cannot be dismissed.

AUTHOR INFORMATION Corresponding Authors *DK Tel: +81 0774-65-6327 E-mail: [email protected] *MPK Tel: +33 3 88414060. Fax: +33 3 88414099. E-mail: [email protected] ACKNOWLEDGEMENTS We thank the French National Research Agency (ANR-14-CE35-0028-01), the Centre National de la Recherche Scientifique (CNRS) and the Japan Society for the promotion of Science (JSPS) for financial support (PRC 2015-2016). We also acknowledge Goho Life Science (Japan) for a travel grant.

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TOC Graphic


Microbubbles with a Self-Assembled Poloxamer Shell and a

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