pH-Controlled Microbubble Shell Formation and Stabilization

May 12, 2014 - Loess, 67034 Strasbourg, France. •S Supporting Information. ABSTRACT: We report on microbubbles with a shell self-assembled from an ...
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pH-Controlled Microbubble Shell Formation and Stabilization Artem Kovalenko,† Prasad Polavarapu,† Geneviève Pourroy,‡ Gilles Waton,† and Marie Pierre Krafft*,† †

Institut Charles Sadron (ICS, UPR CNRS 22), University of Strasbourg, 23 rue du Loess, 67034 Strasbourg, France Institut de Physique et de Chimie des Matériaux de Strasbourg (IPCMS, UMR CNRS 7504), University of Strasbourg, 23 rue du Loess, 67034 Strasbourg, France

‡

S Supporting Information *

ABSTRACT: We report on microbubbles with a shell self-assembled from an anionic perfluoroalkylated surfactant, perfluorooctyl(ethyl)phosphate (F8H2Phos). Microbubbles were formed and effectively stabilized from aqueous solutions of F8H2Phos at pH 5.6−8.5. This range overlaps the domains of existence of the monosodic and disodic salts. The shell morphology of microbubbles formed spontaneously by heating aqueous solutions of F8H2Phos was monitored during cooling, directly on the microscope’s stage. At pH 5.6, the shell collapses through nucleation of folds, as typical for insoluble surfactants. At pH 8.5, no folds were seen during shrinking. At higher pH, the microbubbles rapidly adsorb on the glass. The effect of pH (from 5.6 to 9.7) on adsorption kinetics of F8H2Phos at the air/water interface, and on the elasticity of its Gibbs films, was determined. At low pH, F8H2Phos is highly surface active. The interfacial film undergoes a dilute-to-condensed phase transition and a dramatic increase of elastic module, leading to extremely high values (up to 500 mN m−1). At high pH, the surfactant’s adsorption is quasi-instantaneous, but interfacial tension lowering is limited, leading to very low elastic module (∼5 mN m−1). At pH 5.6 and 8.5, the interfacial tension of F8H2Phos adsorbed on millimetric bubbles and compressed at a rate similar to that exerted on micrometric bubbles during deflation is lower than the equilibrium interfacial tension. Langmuir monolayers of F8H2Phos are highly stable at low pH and feature a liquid expanded/ liquid condensed transition; at high pH, they do not withstand compression. Both mono- and disodic F8H2Phos salts are needed to effectively stabilize microbubbles: the rapidly adsorbed disodic salt stabilizes a newly created air/water interface; the more surface active monosodic salt then replaces the more water-soluble disodic salt at the interface. During deflation, the surfactant shell undergoes a transition toward a highly elastic phase, which further contributes to bubble stabilization.

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INTRODUCTION Microbubbles have raised interest in the past decade, owing to their potential as contrast agents for ultrasound diagnosis and as drug/gene delivery systems.1−7 In consequence, the physical chemistry of such bubbles, and in particular the properties of their shells with regards to microbubbles’ basic attributes such as size and stability control,8−11 viscosity,12 resonance frequency,13 capacity to generate sound harmonics,14−16 to enable gene transfer,7,17 and to undergo ultrasound-triggered disruption,18 have been investigated. As uncoated gaseous microbubbles in water disappear after a few seconds as a result of gas transfer and Laplace pressure effects,19 proper shell engineering is required in order to stabilize microbubbles against both dissolution and coalescence.1,7 Various types of shells, made of biodegradable block copolymers, such as poly(DL-lactide-co-glycolide),20 polyelectrolyte multilayers,21 proteins,22 phospholipids,4,23 and neutral24 and charged surfactants,25 have been investigated. Each type of bubble has its own advantages and can be tailored for specific functions. Most of the injectable soft-shell microbubble contrast agents commercially available for diagnosis or under development use phospholipids as the main bubble wall component. A series of studies aiming at determining the © 2014 American Chemical Society

role of the shell of phospholipids on microbubble dissolution have been conducted.26−29 Microbubble deflation was shown to be accompanied by the expulsion of phospholipids from the bubble’s surface in the form of bilayer fragments through complex collapse and shedding mechanisms. The existence of a critical radius below which bubbles are quasi-stable has been established.26−29 Here, we report on the capacity for perfluorooctyl(ethyl)phosphate C8F17(CH2)2OP(O)(OH)2 (F8H2Phos) to generate and stabilize aqueous dispersions of air microbubbles that are sensible to pH. Negatively charged carboxylic acid salts have been previously used in microbubble compositions.25 However, the influence of pH was not studied. The use of a surfactant that can exist as two differently charged mono- and disodic forms in aqueous solutions has never been investigated to the best of our knowledge. In addition, perfluoroalkylated surfactants have specific properties that distinguish them markedly from their alkyl analogues.30−32 Fluorocarbon chains, CnF2n+1, have larger cross-section areas and hence greater Received: February 23, 2014 Revised: May 12, 2014 Published: May 12, 2014 6339

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A stirred solution of phosphorus oxychloride (1.65 g, 98 μL, 10.8 mM, 2.5 equiv) in dry diethyl ether (13 mL) was cooled to 0 °C in an ice bath under argon. A solution of 2-(perfluorooctyl)ethanol (2.0 g, 4.31 mM) in diethyl ether (40 mL) was added dropwise over 1 h at 0 °C. The solution was allowed to return to room temperature and was kept under stirring for 48 h. Water (5 mL) was added, and the solution was stirred at room temperature for 24 h in order to hydrolyze the phosphorus dichloride intermediate. The aqueous phase was extracted 3 times with diethyl ether, and the combined ether phases were extracted 3 times with a 3% NaOH solution. The pH of the aqueous phase was adjusted to ∼1 with a diluted HCl solution and extracted 3 times with ethyl acetate. The organic phase was washed with water and dried over MgSO4, and the solvent evaporated to yield 1.91 g (81%) of F8H2Phos as a white solid. 1H NMR (CD3OD, 400 MHz) δ (ppm): 2.62 (m, 2H), 4.27 (dt, J = 7.6, 6.6 Hz, 2H). 13C NMR (CD3OD, 100 MHz) δ (ppm): 33.1 (m), 59.6 (m), 107−125 (m). 19F NMR (CD3OD, 282 MHz) δ (ppm): −82.4 (t, J = 10.2 Hz, 3F), −114.7 (m, 2F), −122.7 (m, 2F), −122.9 (m, 4F), −123.7 (m, 2F), −124.7 (m, 2F), −127.3 (m, 2F). 31P NMR (CD3OD, 162 MHz) δ: 0.4 ppm. MS (MALDI-TOF): m/z = 566.9 [M + Na]+. Anal. Calcd for C10H6F17O4P: C, 22.07; H, 1.11. Found C, 22.09; H, 1.25. F8H2Phos Titration. F8H2Phos (27.2 mg) was dispersed in 10 mL of NaCl 0.1 M. The solutions were allowed to equilibrate for 15 min before measuring each pH point during titration with NaOH (0.01 M) at room temperature. Microbubble Preparation. F8H2Phos (6.8 mg) was first dispersed under stirring in 10 mL of NaOH at the following concentrations: 1, 2, 2.5, 3, and 4 mM (in order to adjust the pH to 5.6, 7.5, 8.5, 9.3, and 9.7) and then diluted by NaCl aqueous solutions (0.1 M). The F8H2Phos concentration in the resulting solutions was 1.25 mM, and NaCl concentration was 0.025 M. Microbubbles were prepared by manual shaking of the above solutions (10 mL in 30 mL glass vials) for 10 s. They were allowed to float for 1 min (unless otherwise mentioned) in order to eliminate large bubbles before observation by optical microscopy. Optical Microscopy. Three to four droplets of bubble dispersion were placed into a concave glass slide, covered with a glass slide, and observed with a Nikon Eclipse 90i microscope, 1 min after preparation. Temperature-Induced Microbubble Formation, Growth, and Deflation Monitored by Optical Microscopy. The F8H2Phos solutions were deposited on concave glass slides and covered by a glass slide, the latter being sealed by Scotch tape in order to avoid evaporation. They were heated up to 50 °C using a temperaturecontrolled Pelletier stage (Linkham Scientific Instruments PE 94). They were then allowed to cool back to room temperature at the cooling rate of 10 °C min−1. Bubble Profile Analysis Tensiometry. Axisymmetric bubble shape analysis was applied to a rising air bubble formed in the F8H2Phos solution (1.25 mM in 0.025 M NaCl). Time dependence of the interfacial tension during surfactant adsorption at the gas/liquid interface was measured using a Tracker tensiometer (Teclis, Longessaigne, France).43 A lid fitted on the measuring glass cell (10 mL) prevented water evaporation during long equilibration times. The bubble (typically 1−2 μL) was formed at the tip of a stainless steel capillary with a tip diameter of 0.25 mm. In some experiments, the surface area of the bubble was kept constant. In other experiments, the bubble was submitted to compression of its surface at a relative rate of 0.01−0.02 s−1. The experimental error on the interfacial tension data was ±1 mN m−1. Oscillating Bubble Measurements. The oscillations were produced by a position-encoded motor and transmitted to the bubble through a piston coupled to the syringe carrying the capillary. The bubble was submitted to sinusoidal oscillations with a period T of 20 s and a relative variation of the bubble’s surface area ΔA of 1−3% at 25 ± 0.5 °C.44 Depending on the experiments, the oscillatory regime was applied immediately after formation of the bubble or after the equilibrium tension has been reached. Compression Isotherms. Surface pressure versus molecular area (π/A) isotherms were recorded on a Langmuir minitrough (KSV Nima, Finland) equipped with two movable barriers. The surface

sterical requirements than their hydrocarbon analogues.33,34 They are also more rigid and have lesser conformational freedom. The strength of the C−F and C−C bonds and dense repellent electron sheath that covers fluorocarbon chains ensure outstanding chemical and biological inertness. Interactions among fluorinated chains are weaker, due to the lower polarizability of fluorine as compared to hydrogen (and hence lower van der Waals forces), resulting in higher vapor pressures relative to molecular weight, higher gas solubilities, and extremely low water solubility. These attributes are used to osmotically stabilize microbubbles for diagnostic use.1 Perfluoroalkyl chains are much more hydrophobic than alkyl chains; they are also lipophobic. Fluorinated surfactants can reduce air/water interfacial tension more effectively than their hydrogenated analogues, reaching values that cannot be obtained with the latter. Their capacity for segregation and self-association is also much more pronounced, fostering higher organization. A major objective of this work is to examine the relations between the properties of microbubbles with those of the Falkyl phosphate that forms their shell, in particular its adsorption kinetics at the air/water interface and the viscoelasticity of the interfacial film. Although we are aware that methods based on microfluidic devices6,35−39 can produce monodisperse microbubble populations down to 2 μm in size, we prepared our microbubbles by manual shaking followed by a short flotation step. This very simple preparation method was efficient when using fluorinated surfactants, which lower the interfacial tension and therefore require minimal energy for bubble formation. Consequent to low energy use, any interference with the adsorption of the surfactant at the newly formed air/water interface is minimized. This is expected to reduce formation of defects within the surfactant shell. Such defects often occur when using high-energy methods such as sonication40 and can alter microbubble behavior. Microbubble preparations obtained by low-energy mechanical agitation are notoriously highly polydisperse. However, narrowly sized bubble populations can be obtained from these preparations by fractionation using flotation or centrifugation.8,24,41,42 The paper is organized as follows: A first section focuses on the influence of pH on the formation and stability of micrometric F8H2Phos-based bubbles, as observed by optical microscopy. This section also includes observations of surfactant shell morphology conducted during the shrinking of the microbubbles. Section 2 presents the dynamic interfacial tension profiles of F8H2Phos at the air/water interface of millimetric bubbles having constant or variable surface areas. In the latter case, a compression rate similar to that exerted on microbubbles during their deflation was used. Section 3 describes the viscoelastic properties of F8H2Phos Gibbs films as a function of pH, as investigated by submitting the tensiometric bubbles to sinusoidal oscillations. Section 4 reports on the effect of pH on F8H2Phos spread as Langmuir monolayers on water.

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MATERIALS AND METHODS

Materials. Water was obtained from a Milli-Q (Millipore) system (γ = 71.7 ± 0.2 mN m−1 at 20 °C; resistivity 18.2 MΩ cm). 2(Perfluorooctyl)ethanol (C8F17(CH2)2OH) was from Atochem. Synthesis of Perfluorooctyl(ethyl)phosphate (F8H2Phos). F8H2Phos was synthesized by phosphorylation with POCl3 of the perfluorooctyl alcohol precursor followed by hydrolysis, according to the following protocol: 6340

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pressure was measured using the Wilhelmy plate method. 40−100 μL of a solution of F8H2Phos (2 mM) in NaCl (0.1 M) was spread on aqueous NaCl (0.1 M) subphases adjusted to different pH values (7.0, 8.5, and 9.7) using NaOH (0.1 M). Compression speed was kept at 50 cm2 min−1 for all experiments. Temperature was regulated at 25 ± 0.5 °C. Each experiment was run at least three times. Experimental errors were ±0.5 mN m−1 on the surface pressure values, and reproducibility on the molecular area values was ±2.5 Å2.

5.6 < pH < 8.5, the dispersions were opalescent, indicating that microbubbles had formed under these conditions. In this pH range, the opalescence of the dispersions and the thickness of the foam layer that overlays them increased with pH. Optical micrographs show that microbubbles are formed under these conditions and that their number increases with pH (Figure 2b,c). In contrast, for pH lower than 5.6 or higher than 9.2, the dispersions were transparent or only slightly opalescent, showing that none or only few bubbles were present. It is noteworthy that the pH domain for which bubbles can be obtained (5.6−8.5) overlaps the pH domain of coexistence of the monosodic salt F8H2Phos.1Na and disodic salt F8H2Phos.2Na. The fact that only few bubbles are seen at pH > 9.2 strongly suggests that the monosodic salt is indispensable for the bubble stabilization. Conversely, the increase in the number of bubbles for 5.6 ≤ pH ≤ 8.5 indicates that the disodic salt helps their formation. These results suggest already that both the monosodic and the disodic salts are needed in order to form and stabilize microbubbles, each one playing a specific role. b. Microbubble Deflation Rate Determination. The deflation of microbubbles prepared at pH 5.6 and 8.5 was monitored by optical microscopy. The small bubbles were seen to progressively shrink to the benefit of the larger ones, likely by an Ostwald ripening mechanism (see Supporting Information, Figure S1). The variations of microbubble diameters D as a function of time are also presented in the Supporting Information (Figure S2). Microbubbles with diameters of 2−5 μm progressively shrink with an absolute deflation rate dD/dt ∼ 0.005−0.01 μm s−1. It is noteworthy that the deflation rate of microbubbles stabilized by these water-soluble surfactants is lower than that reported for microbubbles made of phospholipids (about 0.03 μm s−1).29 The relative rate of microbubble deflation was also calculated by using the following equation: (1/V)(∂V/∂t) = ∂(ln V)/∂t, where V is the microbubble volume. The values of the relative deflation rate were obtained by fitting the variation of ln V as a function of t and found to be in the 0.01−0.02 s−1 range. c. Shell Morphology Monitoring during Microbubble Deflation. The mechanical properties of their shell play an important role in the stability of microbubbles. Monitoring the deflation of small, fast moving bubbles in a microbubble dispersion using optical microscopy is difficult, and no information can be gained on the surfactant shell morphology. We devised a simple method for visualizing the surfactant shells of bubbles of ∼20−100 μm in diameter during deflation under the effect of temperature. Therefore, bubbles were generated directly on the observation stage of the microscope by heating F8H2Phos solutions (1.25 mM). At room temperature, no bubbles are present in the F8H2Phos solutions. When temperature increases, the solubility of the gases dissolved in water decreases, leading to generation of bubbles. At any given temperature, the gas dissolved in the solution is in equilibrium with the gas inside the bubble. At equilibrium, the total pressure within a bubble, Pint, can be written as Pint = Patm + Phyd + PL = PH2O(v) + Pair, where Patm is the atmospheric pressure, Phyd the hydrostatic pressure of water, PL the Laplace pressure, PH2O(v) the vapor pressure of water, and Pair the partial pressure of air.45 When a dispersion of bubbles is heated, two processes take place that lead to bubble growth: (1) water evaporation increases, increasing PH2O(v) and (2) the solubility

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RESULTS AND DISCUSSION 1. Coexistence of Mono- and Disodic F8H2Phos Salts as a Function of pH. Depending on pH, the monosodic (F8H2Phos.1Na) and/or disodic (F8H2Phos.2Na) salts of the surfactant are present in aqueous solutions. The titration curve of F8H2Phos comprises several regions (Figure 1). The acidic form is present for pH < 2, the monosodic salt predominates between pH 2 and pH 8, and the disodic salt is present for pH > 8.

Figure 1. Titration curve of F8H2Phos with NaOH (0.01 M). The volumes of 5 and 10 mL correspond to 1 and 2 equiv of the fluorinated phosphate surfactant, respectively.

The pH domain of coexistence of the two salts ranges roughly between pH 5 and pH 9 (Scheme 1). This is mere qualitative information, however, because determination of domain boundaries lacks precision, owing to the difficulty to determine the pKa. Scheme 1. Domains of Coexistence of the Mono- and Disodic Salts of F8H2Phos as a Function of pH

Also important to keep in mind is that, whatever the relative concentration of the two salts in solution, it is the outcome of their competitive adsorption at the air/water interface that matters where microbubble stabilization is concerned. 2. F8H2Phos-Based Microbubbles. a. Formation and Stability: pH Dependency. Bubble formation from F8H2Phos solutions was observed visually and by optical microscopy (1.25 mM in NaCl 0.025 M; 5.6 < pH < 9.7) (Figure 2a). Although bubbles were prepared by manual shaking, their size was quite small (1−10 μm in diameter) after application of the 1 min flotation period that eliminated most of the larger bubbles. For 6341

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Figure 2. (a) Turbidity of F8H2Phos-based microbubble dispersions prepared at different pH. Optical micrographs (×40) of the dispersions prepared at (b) pH 5.6 and (c) pH 8.5. F8H2Phos concentration was 1.25 mM in NaCl 0.025 M. Insets: bubble size histograms (∼70 and ∼100 microbubbles measured for parts b and c, respectively).

very large microbubbles (50−100 μm) at pH 5.6 (Figure 4a), whereas they were detected in phospholipid bubbles of much lower size. This reflects other differences such as the fact that F8H2Phos salts are water-soluble and thus adsorb much faster than phospholipids at the air/water interface. These observations show that the monosodic salt of F8H2Phos is able to decrease the interfacial tension to sufficiently low values so that the energy penalty resulting from maintaining the surfactant at the bubble’s surface is lower than that involved in its expulsion toward the aqueous phase. During shrinking, the only possibility for the surfactant molecules that remain confined at the surface is to form folds. At pH 8.5, on the other hand, no folds were ever seen to form during the deflation process, whatever their initial size (Figures 3b and 4b). The bubbles deflate monotonously and keep their spherical shape throughout the dissolution process until they become very small (Figure 3b). This indicates that the more water-soluble disodic salt is progressively and continuously expelled in the water phase, thus preventing the formation of folds. At a certain time point (60 s in the conditions used), the bubble is no longer spherical. Its size, however, is then too small to determine whether folds are present or not on its surface. At pH 9.3, the bubbles adhere to the glass plate/water interface within seconds. They are therefore no longer spherical, and their shape is primarily governed by their interaction with the substrate (Figures 3c and 4c). No folds were seen on their surface. These observations support the data obtained in the bubble stability study and shed light on the shrinking mechanism of F8H2Phos microbubbles. When the monosodic salt is predominant, the bubble’s shell behaves like an insoluble shell and forms folds upon compression. As pH increases, the concentration of monosodic salt decreases at the bubble’s surface and that of the disodic salt increases. Being more soluble in water, the latter is expelled into the aqueous solution during shrinking. This prevents fold formation until a critical diameter is reached at which the microbubbles are predominantly stabilized by a shell of monosodic salt. At high pH, the more water-soluble disodic salt that forms the shell provides no elasticity and cannot oppose bubble deflation. 3. Influence of pH on Gibbs Films of F8H2Phos. a. Adsorption Kinetics and Equilibrium Interfacial Tension. In order to further investigate the effect of pH on the bubble stabilization mechanism, the kinetics of adsorption of F8H2Phos solutions at the air/water interface was determined as a function of pH using bubble profile analysis tensiometry. This dynamic interfacial tension study was expected to provide information on how fast the surfactant adsorbs at the interface

of air in water decreases, resulting in increased diffusion of air from the supersaturated solution into the bubble, which increases Pair. Conversely, cooling the bubble suspension results in bubble deflation. Perfectly spherical bubbles appear spontaneously in F8H2Phos solutions when temperature reaches ∼50 °C. 1− 20 min was then allowed until these bubbles reached a diameter of ∼10−20 μm. The bubble-containing dispersions were then slowly cooled back to room temperature. Depending on pH, the behavior of the bubbles was very different during the deflation process (Figure 3). At pH 5.6, bubble deflation is

Figure 3. Optical micrographs (×40) of microbubbles submitted to cooling-induced deflation. The microbubbles were formed spontaneously by heating at 50 °C aqueous solutions of F8H2Phos (1.25 mM in NaCl 0.025 M) with pH adjusted to (a) pH 5.6, (b) pH 8.5, and (c) pH 9.3. Their deflation process was induced by cooling at a rate of 10 °C min−1.

characterized by alternate episodes during which the surfactant shell visibly develops folds and creases, wrinkles, and pleats (Figure 3a). The process involves successive steps of formation of folds, followed by re-formation of a spherical, but smaller bubble, a sequence that is repeated several times until the bubble disappears. This process is reminiscent of that described for bubbles stabilized by phospholipids.27−29 With substantial differences, however, for F8H2Phos the folds are observed for 6342

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Figure 4. Optical micrographs (×40) of large microbubbles (∼50−100 μm) obtained by heating aqueous solutions of F8H2Phos under the same conditions as described in Figure 3. The pH was adjusted to (a) 5.6, (b) 8.5, and (c) 9.3.

and hence is able to stabilize microbubbles at the very first moments of their formation. Therefore, F8H2Phos concentration was set to 1.25 mM (in NaCl 0.025 M), the same as above for microbubble preparation. The study also aimed at determining the equilibrium interfacial tension γeq. The interfacial adsorption behavior was found to vary drastically with pH. At pH 9.3 (not shown) and 9.7 (Figure 5, curve a), the interfacial tension γ already reaches its

For pH ranging from 8.5 to 5.6 (Figure 5, curves b−d), the adsorption profiles are substantially different. First, the adsorption rates are higher than for pH 9.3, and they increase when pH decreases. Second, the γeq values become much lower (down to ∼18 mN m−1). This shows that, at high pH (≥9.3), F8H2Phos adsorption is governed by the characteristics of the disodic salt (high water solubility, bulky charged polar head, critical micelle concentration about 25 mM in water at 25 °C, as compared to 1 mM for the monosodic salt),46 translating in weak (but quasi-instantaneous) adsorption. When pH decreases, the surfactant behavior progressively reflects the properties of the monosodic salt (lower water solubility, reduced head repulsion). b. Interfacial Tension of Overcompressed Bubbles. In the case of phospholipids, the most common shell component of medical microbubbles, it is assumed that the microbubbles are stabilized by a monolayer of surfactant maximally compressed due to the Laplace “overpressure”.47 According to Epstein and Plesset,19 the variation of the surface area of a shrinking microbubble remains constant upon time. Hence, the relative surface variation increases when microbubble size decreases. Consequently, the microbubble is expected to disappear irremediably once deflation has started. However, there are reports showing that phospholipid-based microbubbles actually stabilize at small diameters of 1−2 μm. Examples of such behavior include microbubbles of various shell compositions dissolving either naturally8,39,42 or while submitted to acoustic pulsing.18 A recent critical review on monolayer collapse did

Figure 5. Adsorption kinetics at 25 °C of aqueous solutions of F8H2Phos (1.25 mM) in NaCl 0.025 M when pH is adjusted to (a) 9.7, (b) 8.5, (c) 7.5, and (d) 5.6.

equilibrium value (γeq ∼ 54 mN m−1) within the first few seconds of the experiment and remains unchanged thereafter.

Figure 6. (a) Variation of the interfacial tension γ and bubble surface area A at pH 5.6, 8.5, and 9.7 as a function of time. The time point when compression starts is indicated by an arrow. (b) The bubble is compressed at the beginning of surfactant adsorption (green). The pH is 8.5. The adsorption at constant bubble surface area (red) is shown for comparison. 6343

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Figure 7. Variation at 25 °C of the interfacial tension γ of aqueous solutions of F8H2Phos (1.25 mM in NaCl 0.025 M) as the function of the bubble’s normalized surface area A/A0. The pH was adjusted to (a) 5.6, (b) 8.5, and (c) 9.7. The bubbles were submitted to sinusoidal oscillations (T = 20 s, ΔA = 1−3%) when the equilibrium interfacial tension was reached. In (a) and (c) the values of E can be directly determined from the slope of the linear regressions (red lines).

not provide obvious reasons for the stabilization of microbubbles at such small diameters.29 The interfacial tension that determines Laplace pressure according to PL = 2γ/r (r being the microbubble’s radius) is a key element of microbubble stability. The interfacial tension of microbubbles has, however, never been measured. Having determined the rate of deflation of microbubbles, we were able to set the experimental conditions for the adsorption of F8H2Phos solutions (1.25 mM) so that, after having reached the equilibrium interfacial tension, the millimetric bubble used for bubble profile analysis was submitted to a compression regime similar to that experienced by 2−5 μm microbubbles during their “natural” deflation (the microbubble deflation rate is reported in section 2b). Figure 6a shows the variation of γ, along with that of the bubble’s surface area A, as a function of time for pH 5.6, 8.5, and 9.7. The results show that, for pH 5.6 and 8.5, the reduction in the bubble’s surface is accompanied by a rapid and strong reduction of γ to values equal to or lower than 10 mN m−1. It is noteworthy that γ rapidly increases back to its equilibrium value when the bubble’s surface area is restored to its initial value, showing some reversibility of the surfactant packing at the bubble’s surface. In contrast, at pH 9.7, compression of the microbubble was not accompanied by a decrease of γ, which remained above 40 mN m−1. A comparable behavior was also observed; that is, γ decreases down to a few mN m−1, when the bubble is compressed during the first stages of surfactant adsorption (Figure 6b). These results strongly support the idea that the actual interfacial tension of microbubbles is very low at pH 5.6−8.5, which explains the observed stabilization of the microbubbles in this pH range. c. Rheological Behavior. Elasticity is a crucial parameter of interfacial surfactant films that largely conditions a surfactant’s

suitability for applications. Since microbubbles are submitted to Laplace pressure, their volume tends to decrease or increase, depending on conditions (temperature, aqueous and gas phase compositions). The effect of surface dilation (or compression) on interfacial tension is characterized by the surface elasticity module E according to Δγ = E × ΔA, where Δγ is the interfacial tension variation and ΔA is the relative bubble surface area variation. In order to determine E, experiments were conducted on F8H2Phos solutions (1.25 mM) at pH 5.6, 8.5, and 9.7 adsorbed at the surface of a millimetric bubble submitted to sinusoidal oscillations (T = 20 s; ΔA = 1−3%). Figure 7 features the variation of γ as a function of the normalized bubble’s surface area A/A0. At pH 5.6, γ is seen to vary linearly with A/A0, with no hysteresis during dilation/compression cycles (Figure 7a). This reveals a strong elastic behavior, as illustrated by the high value of E ∼ 300 mN m−1. It means that, under these conditions, the monosodic salt remains at the interface during the oscillations (or that its desorption rate is slower than the oscillation period). At pH 8.5, the behavior is typical of a monolayer with viscoelastic properties and features a large hysteresis of γ during inflation and subsequent deflation of the bubble (Figure 7b). The hysteresis reflects the effect of relaxation of the two salts on the interfacial tension, with the disodic form being expelled from, and rapidly readsorbed at, the interface, while the monosodic form remains adsorbed at this interface. At pH 9.7, E is very small, ∼5 mN m−1; the behavior of the monolayer is dominated by the rapid adsorption/desorption of the disodic salt and has therefore no elastic properties (Figure 7c). d. Gibbs Film Elasticity as a Function of Phase Density. Reducing F8H2Phos concentration to 0.1 mM revealed that the adsorption profile comprises a first regime that reflects a dilute 6344

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phase, followed by a second regime that corresponds to a condensed phase (Figure 8, curve a). A dilute-to-condensed phase transition occurs after 40 s and is clearly visible at 42 mN m−1.

Figure 9. Compression isotherms of F8H2Phos solutions spread on aqueous subphases of NaCl (0.1 M) adjusted to (a) pH 7.0, (b) pH 8.5, and (c) pH 9.7. Two different volumes (40 and 100 μL) were spread at pH 8.5. Inset: the subphase was NaCl 0.025 M adjusted to pH 7.0.

Figure 8. (a) Adsorption kinetics at 25 °C of aqueous solutions of F8H2Phos (0.1 mM) in NaCl 0.1 M at the surface of a static bubble. (b) The bubble was submitted to sinusoidal oscillations (T = 20 s; ΔA = 3%) immediately after its formation.

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When the bubble was submitted to oscillations (Figure 8, curve b), it was found that, immediately after the dilute-tocondensed phase transition, γ strongly varied with surface variations, reflecting the sudden, strong increase in E value. E then attained the extremely high value of 500 mN m−1 at equilibrium. These results are supported by a study on the adsorption kinetics of either the mono- or disodic salts of F8H2Phos, when stoichiometrically synthesized.46 4. Influence of pH on Langmuir Monolayers of F8H2Phos. The ability of a surfactant monolayer to withstand compression during bubble shrinking is another crucial parameter that determines the suitability of the surfactant for microbubble stabilization. We therefore investigated the nature (liquid expanded LE or liquid condensed LC) of Langmuir films of F8H2Phos at the air/water interface. Owing to the low solubility of F8H2Phos in common organic solvents (hexane, chloroform, THF), we have spread aqueous solutions of this surfactant (10 mM in NaCl 0.1 M) on aqueous NaCl subphases (0.1 M) adjusted to different pH values using NaOH (0.1 M). At pH 7.0 and 8.5 (Figure 9ab), that is, at pH values for which the monosodic salt is predominant, the isotherms were found to exhibit a transition between a LE phase and a LC phase. For surface pressures π higher than 30 mN m−1, the compression isotherms are particularly steep, reflecting a condensed phase with very low compressibility. The transition occurs at π ∼30 mN m−1, that is, at γ ∼42 mN m−1, the same value as seen for the Gibbs films. For pH 8.5, we spread two different volumes (40 and 100 μL) of the F8H2Phos solution in order to span a larger range of molecular areas (Figure 9b). By contrast, at pH 9.7 (Figure 9c), when the disodic salt is predominant, it was not possible to compress the monolayer, owing to rapid desorption of the more water-soluble disodic salt from the interface and loss to the subphase. It is noteworthy that decreasing the NaCl concentration to 0.025 M (in order to allow comparison with the microbubble experiment conditions) (inset in Figure 9) did not change the shape of the isotherm or the plateau pressure value significantly, but for a slight decrease of the latter.

CONCLUSIONS

Investigation of the interfacial film adsorption and compression behavior of the short, yet microbubble-forming (perfluorooctyl)ethyl phosphate surfactant F8H2Phos as a function of pH revealed a marked contrast between its monoand disodic salts. Adsorption of the disodic salt is very fast (less than 5 s), but its interfacial tension reduction capacity is low (∼18 mN m−1). On the other hand, the monosodic salt is considerably more effective in terms of interfacial tension reduction (∼50 mN m−1). Yet, these divergent characteristics cooperate to ensure efficacious bubble formation and stabilization: the disodic salt rapidly colonizes the nascent bubble’s surface; then, the monosodic salt progressively replaces the disodic salt at the interface and provides bubble stability, in particular by reducing Laplace pressure. It is noteworthy that a liquid expanded to liquid condensed (or dilute to condensed) film transition was observed in both Langmuir and Gibbs films. The extremely high film elasticity (E 300−500 mN m−1) observed for F8H2Phos films at pH 5.6 after its transition to the condensed phase further increases bubble resilience and hence stability. The optimal pH range for microbubble formation and stabilization was determined to be 5.6−8.5. In this range, the interfacial tension of overcompressed monolayers of F8H2Phos is significantly lower than the equilibrium tension, showing that the main contribution to bubble stabilization is the interfacial tension reduction induced by the monosodic salt. The mechanism of bubble deflation was also found to be pH dependent: at low pH, deflation proceeds by repeated episodes comprising a surfactant film wrinkling phase, followed by stabilization of a smaller size spherical microbubble. At pH around 8.5, smooth monotonous size decrease is observed while the bubbles remain spherical throughout the deflation process. At the higher pH, the bubbles adsorb rapidly at the glass surface, and their shape is governed by its interaction with the substrate. 6345

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(16) Sprague, M. R.; Chérin, E.; Goertz, D. E.; Foster, F. S. Nonlinear emission from individual bound microbubbles at high frequencies. Ultrasound Med. Biol. 2010, 36, 313−324. (17) Alter, J.; Sennoga, C. A.; Lopes, D. M.; Eckersley, R. J.; Wells, D. J. Microbubble stability is a major determinant of the efficiency of ultrasound and microbubble mediated in vivo gene transfer. Ultrasound Med. Biol. 2009, 35, 976−984. (18) Borden, M. A.; Kruse, D. E.; Caskey, C. F.; Zhao, S.; Dayton, P. A.; Ferrara, K. W. Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction. IEEE Trans. Ultrasound Ferroelectr. Freq. Control 2005, 52, 1992−2002. (19) Epstein, P. S.; Plesset, M. S. On the stability of gas bubbles in liquid-gas solutions. J. Chem. Phys. 1950, 18, 1505−1509. (20) Forsberg, F.; Lathia, J. D.; Merton, D. A.; Liu, J.-B.; Le, N. T.; Goldberg, B. B.; Wheatley, M. A. Effect of shell type on the in vivo backscatter from polymer-encapsulated microbubbles. Ultrasound Med. Biol. 2004, 30, 1281−1287. (21) Shchukin, D. G.; Köhler, K.; Möhwald, H.; Sukhorukov, G. B. Gas-filled polyelectrolyte capsules. Angew. Chem., Int. Ed. 2005, 44, 3310−3314. (22) Cox, A. R.; Cagnol, F.; Russell, A. B.; Izzard, M. J. Surface properties of class II hydrophobins from Trichoderma reesei and influence on bubble stability. Langmuir 2007, 23, 7995−8002. (23) Kim, D. H.; Costello, M. J.; Duncan, P. B.; Needham, D. Mechanical properties and microstructure of polycrystalline phospholipid monolayer shells: novel solid microparticles. Langmuir 2003, 19, 8455−8466. (24) Wheatley, M. A.; Forsberg, F.; Dube, N.; Patel, M.; Oeffinger, B. E. Surfactant-stabilized contrast agent on the nanoscale for diagnostic ultrasound imaging. Ultrasound Med. Biol. 2006, 32, 83−93. (25) Soetanto, K.; Chan, M. Study of the lifetime and attenuation properties of microbubbles coated with carboxylic acid salts. Ultrasonics 2000, 38, 969−977. (26) Borden, M.; Longo, M. Dissolution behavior of lipid monolayercoated, air-filled microbubbles: Effect of lipid hydrophobic chain length. Langmuir 2002, 18, 9225−9233. (27) May, D. J.; Allen, J. S.; Ferrara, K. W. Dynamics and fragmentation of thick-shelled microbubbles. IEEE Trans. Ultrasound Ferroelect. Freq. Control 2002, 49, 1400−1410. (28) Pu, G.; Borden, M. A.; Longo, M. L. Collapse and shedding transitions in binary lipid monolayers coating microbubbles. Langmuir 2006, 22, 2993−2999. (29) Kwan, J. J.; Borden, M. A. Lipid monolayer collapse and microbubble stability. Adv. Colloid Interface Sci. 2012, 183−184, 82− 99. (30) Riess, J. G.; Krafft, M. P. Fluorocarbons and fluorosurfactants for in vivo oxygen transport (blood substitutes), imaging and drug delivery. Mater. Res. Soc. Bull. 1999, 24, 42−48. (31) Kissa, E. Fluorinated Surfactants and Repellents; Surfactant Science Series; Marcel Dekker: New York, 2001; Vol. 97. (32) Krafft, M. P. Highly fluorinated compounds induce phase separation in, and nanostructuration of liquid media. Possible impact on, and use in chemical reactivity control. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 4251−4258. (33) Krafft, M. P. Large organized surface domains self-assembled from non-polar amphiphiles. Acc. Chem. Res. 2012, 45, 514−524. (34) Krafft, M. P.; Riess, J. G. Chemistry, physical chemistry and uses of molecular fluorocarbon-hydrocarbon diblocks, triblocks and related compounds - Unique apolar components for self-assembled colloid and interface engineering. Chem. Rev. 2009, 109, 1714−1792. (35) Xu, J. H.; Li, S. W.; Wang, Y. J.; Luo, G. S. Controllable gasliquid phase flow patterns and monodisperse microbubbles in a microfluidic T-junction device. Appl. Phys. Lett. 2006, 88, 133506−1− 3. (36) Stride, E.; Edirisinghe, M. J. Soft Matter 2008, 4, 2350−2359. (37) Pancholi, K. P.; Farook, U.; Moaleji, R.; Stride, E.; Edirisinghe, M. J. Eur. Biophys. J. 2008, 37, 515−520. (38) Wan, J.; Stone, H. A. Microfluidic generation of a high volume fraction of bubbles in droplets. Soft Matter 2010, 6, 4677−4680.

ASSOCIATED CONTENT

S Supporting Information *

Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail kraff[email protected] (M.P.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the French National Research Agency (ANR, grant 2010-BLAN-0816-01). A.K. acknowledges ANR for research fellowship. We thank Teclis (Longessaigne, France) for technical help.

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REFERENCES

(1) Schutt, E. S.; Klein, D. H.; Mattrey, R. M.; Riess, J. G. Injectable microbubbles as contrast agents for diagnostic ultrasound imaging: The key role of perfluorochemicals. Angew. Chem., Int. Ed. 2003, 42, 3218−3235. (2) Lindner, J. R. Microbubbles in medical imaging: Current applications and future directions. Nat. Rev. Drug Discuss. 2004, 3, 527−532. (3) Ferrara, K.; Pollard, R.; Borden, M. Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery. Annu. Rev. Biomed. Eng. 2007, 9, 415−447. (4) Ferrara, K. W.; Borden, M. A.; Zhang, H. Lipid-shelled vehicles: Engineering for ultrasound molecular imaging and drug delivery. Acc. Chem. Res. 2009, 42, 881−892. (5) Hernot, S.; Klibanov, A. L. Microbubbles in ultrasound-triggered drug and gene delivery. Adv. Drug Delivery Rev. 2008, 60, 1153−1166. (6) Stride, E.; Edirisinghe, M. J. Special issue on microbubbles: From contrast enhancement to cancer therapy. Med. Biol. Eng. Comput. 2009, 47, 809−811. (7) Sirsi, S. R.; Borden, M. A. Advances in ultrasound mediated gene therapy using microbubble contrast agents. Theranostics 2012, 2, 1208−1222. (8) Rossi, S.; Waton, G.; Krafft, M. P. Small phospholipid-coated gas bubbles can last longer than larger ones. ChemPhysChem 2008, 9, 1982−1985. (9) Sirsi, S.; Borden, M. Microbubble compositions, properties and biomedical applications. Bubble Sci. Eng. Technol. 2009, 1, 3−17. (10) Rossi, S.; Waton, G.; Krafft, M. P. Phospholipid-coated gas bubble engineering - Key parameters for size and stability control as determined by an acoustic method. Langmuir 2010, 26, 1649−1655. (11) Dicker, S.; Mleczko, M.; Schmitz, G.; Wrenn, S. P. Size distribution of microbubbles as a function of shell composition. Ultrasonics 2013, 53, 1363−1367. (12) Hosny, N. A.; Mohamedi, G.; Rademeyer, P.; Owen, J.; Wu, Y.; Tang, M.-X.; Eckersley, R. J.; Stride, E.; Kuimova, M. K. Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 9225−9230. (13) Dicker, S.; Mleczko, M.; Siepmann, M.; Wallace, N.; Sunny, Y.; Bawiec, C. R.; Schmitz, G.; Lewin, P.; Wrenn, S. P. Influence of shell composition on the resonance frequency of microbubble contrast agents. Ultrasound Med. Biol. 2013, 39, 1292−1302. (14) De Jong, N.; Bouakaz, A.; Frinking, P. Basic acoustic properties of microbubbles. Echocardiography 2002, 19, 229−240. (15) Kimmel, E.; Krasovitski, B.; Hoogi, A.; Razansky, D.; Adam, D. Subharmonic response of encapsulated microbubbles: conditions for existence and amplification. Ultrasound Med. Biol. 2007, 33, 1767− 1776. 6346

dx.doi.org/10.1021/la5007023 | Langmuir 2014, 30, 6339−6347

Langmuir

Article

(39) Talu, E.; Hettiarachchi, K.; Powell, R. L.; Lee, A. P.; Dayton, P. A.; Longo, M. L. Maintaining monodispersity in a microbubble population formed by flow-focusing. Langmuir 2008, 24, 1745−1749. (40) Zasadzinski, J. A. N. Transmission electron microscopy observation of sonication-induced changes in liposome structure. Biophys. J. 1986, 49, 1119−1130. (41) Kvåle, S.; Jacobsen, H. A.; Asbjørnsen, O. A.; Omtveit, T. Size fractionation of gas-filled microspheres by flotation. Sep. Technol. 1996, 6, 219−226. (42) Feshitan, J. A.; Chen, C. C.; Kwan, J. J.; Borden, M. A. Microbubble size isolation by differential centrifugation. J. Colloid Interface Sci. 2009, 329, 316−324. (43) Benjamins, J.; Cagna, A.; Lucassen Reynders, E. H. Viscoelastic properties of triacylglycerol/water interfaces covered by proteins. Colloids Surf., A 1996, 114, 245−254. (44) Nguyen, P. N.; Waton, G.; Vandamme, T.; Krafft, M. P. Behavior of an adsorbed phospholipid monolayer submitted to prolonged periodical surface density variations. Angew. Chem., Int. Ed. 2013, 52, 6404−6408. (45) Leighton, T. G. The Acoustic Bubble; Academic Press: San Diego, 1994. (46) Kovalenko, A.; Polavarapu, P.; Gallani, J.-L.; Pourroy, G.; Waton, G.; Krafft, M. P. ChemPhysChem 2013, DOI: 10.1002/ cphc.201402248R1. (47) Lozano, M. M.; Longo, M. L. Complex formation and other phase transformations mapped in saturated phosphatidylcholine/ DSPE-PEG2000 monolayers. Soft Matter 2009, 5, 1822−1834.

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