Langmuir Trough Study of Surfactant Mixtures Used in the Production

Publication Date (Web): August 8, 1996 ... Medical ultrasound imaging is based on the pulse-echo principle. ..... initial area, the large degree of hy...
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J. Phys. Chem. 1996, 100, 13815-13821

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Langmuir Trough Study of Surfactant Mixtures Used in the Production of a New Ultrasound Contrast Agent Consisting of Stabilized Microbubbles Wenhua Wang,† Chris C. Moser,‡ and Margaret A. Wheatley*,† Department of Chemical Engineering, Drexel UniVersity, Philadelphia, PennsylVania 19104, and Department of Biophysics and Biochemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104 ReceiVed: May 9, 1996X

Span-type surfactants (sorbitan fatty acid esters) and Tween-type surfactants (sorbitan polyoxyethylene fatty acid esters) are employed by us to generate stabilized microbubbles for use in diagnostic ultrasound. After sonication of an aqueous surfactant solution, only some mixtures of Span-type and Tween-type surfactants at certain conditions can form stable microbubbles. This work investigated the stability of the surfactant-stabilized microbubbles by using a Langmuir trough to measure the π-A isotherms of the surfactant monolayer. The experimental results, which agreed with a theoretical analysis of the microbubble stability, indicate that the surfactant-stabilized microbubbles have a solid-condensed monolayer “skin” which functions to reduce the surface tension, prevent coalescence between microbubbles, and increase their aqueous compatibility. The higher surface pressure obtained for the case of a microbubble preparation, compared with that of unsonicated mixtures, indicates that sonication enhances the structure of surfactant monolayer and makes the microbubbles extremely stable.

a

Introduction Medical ultrasound imaging is based on the pulse-echo principle. With the introduction of grayscale ultrasound imaging, it has become possible to visualize differences between and within tissue structures. However, the ultrasound differentiation of normal from abnormal tissue is sometimes difficult. In 1968 Gramiak and Shah1 found that the injection of indocyanine into the ascending aorta enhanced the ultrasound image. Later, the causative agent was found to be microbubbles of gas, and since that time the development of ultrasound contrast agents to increase the reflectivity of tissue as well as to study blood flow has aroused the interest of researchers throughout the world.2-4 The underlying physical principles4 indicate that the most significant enhancement of ultrasound imaging occurs for gasbased constrast agents through the increased backscattered signal intensity. An ideal contrast agent, in addition to being highly echogenic, should be (1) small in size (10 µm). This investigation was conducted to understand, on a molecular level, why a mixture of Span 40 and Tween 40 can form © 1996 American Chemical Society

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Figure 2. Phase behavior of the mixture of Span 40 and Tween 40 after sonication.

a

Wang et al. the coalescence among gas bubbles, an energy barrier could be built to prevent the system from reaching the primary minimum state, as shown in Figure 3b. This could be achieved by the addition of an anticoalescence agent. The secondary minimum should be deep enough to prevent the system from jumping over the energy barrier. Practically, the anticoalescence agents should be surface-active materials which mainly participate in the interfacial region of microbubbles, and they should have bulky hydrophillic groups which remain in the aqueous environment outside the microbubbles to stop the direct interbubble contact. Several effective anticoalescence agents have been used to stabilize microbubbles for use as ultrasound contrast agents: (i) globular protein,7,8 (ii) lipid,9,10 and (iii) surface-active polymer.11,12 The bulky hydrophillic headgroup of Tween 40 (Figure 1c), causes the surfactant to act as an anticoalescence agent. If the stability of a single microbubble is investigated from the perspective of intrabubble interaction, a thermodynamic equation can be written for the air-liquid interface (monolayer) of the microbubble at constant temperature and pressure:

dGs ) γ dA + ∑µi dnis

b

Figure 3. Interbubble interaction energy profile between two gas bubbles: (a) the case for free gas bubbles and (b) the case for the gas bubbles with an anticoalescence agent.

stable microbubbles, while the individual components, as well as a mixture of Span 80 and Tween 40, cannot. To answer this question, we investigated the interaction of Span and Tween from the perspective of an air-liquid interface of a surfactant monolayer. The answers should not only provide an explanation for the stability of Span/Tween combinations but also help in the development of improved microbubble contrast agents for diagnostic ultrasound. Theoretical Background of the Microbubble Stability. The stability of a microbubble depends on both inter- and intrabubble interactions. According to the general principle of colloidal stability,6 Figure 3 qualitatively represents microbubble stability from the perspective of interbubble interaction. When a solution traps gas bubbles after agitation, the free gas bubbles quickly approach one another as they concentrate on the top of the solution. Figure 3a shows that the interaction energy of the two bubbles monotonically decreases to a minimum as two free gas bubbles almost contact each other. At this energy minimum, coalescence of the two free gas bubbles is inevitable due to flocculation and a continuous process of coalescence would lead to breakup of all of the free gas bubbles. To prevent

(1)

where Gs represents surface free energy, γ and A are the surface tension and the surface area of a microbubble, respectively, µi stands for the chemical potential of component i in the monolayer, and nsi represents the number of moles of component i in the monolayer. There are three possible avenues to reduce the surface free energy, which either stabilize the microbubbles or destroy them: (i) The surface tension is reduced. When a surfactant is present at the gas-liquid interface of a microbubble, the surface tension of the microbubble will decrease. The surface tension is mainly a function of the surfactant structure and the concentration of the surfactant at the surface of the microbubble. Hence, the addition of a surfactant could stabilize microbubbles,5 especially if the surfactant reduces the surface tension to zero at high surface concentrations. (ii) The surface of the microbubble decreases. The Laplace equation relates the difference in pressure on either side of the microbubble surface, ∆P, to the uniform surface tension:

∆P ) 2γ/R

(2)

where R is the radius of the microbubble. If the microbubble surface is not rigid but is compressible, the size of the microbubble will continue to decrease in order to satisfy the principle of the energy minimization, as required by eq 1. According to eq 2, a decrease in the microbubble size will increase ∆P. If the encapsulated gas pressure is higher than the collapse pressure of the microbubble surface, the microbubble will be destroyed. Hence, the hardening of the microbubble surface by the addition of a globular protein7,8 or a coating polymer11 will contribute to the stabilization of the microbubbles. (iii) The encapsulated gas diffuses through the surface monolayer of microbubbles. Due to higher chemical potential, the encapsulated gas tends to diffuse through the surface monolayer of microbubbles into the suspending medium. This will decrease the second term on the right-hand side of eq 1. As a result, the microbubbles will continue to shrink in order to satisfy the principle of the energy minimization. If the monolayer is closely packed, the encapsulated gas encounters resistance to diffusion into the aqueous environment, and the microbubbles are more stable. As indicated from the theoretical analysis above, the surface tension and the molecular arrangement of a monolayer have an

Langmuir Trough Study of Surfactant Mixtures

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important influence on the microbubble stability. Hence, to follow up on preliminary work13 we used a Langmuir-trough measurement as an experimental tool to investigate the stability of monolayers which represent a two-dimensional “skin” of the surfactant-stabilized microbubbles. Experimental Materials and Methods The surfactants Span 40 (sorbitan monopalmitate), Span 80 (sorbitan monooleate), and Tween 40 (polyoxyethylene 20 sorbitan monopalmitate), were obtained from Aldrich (Milwaukee, WI) and used without further purification. Methylene chloride and ethanol, both HPLC grade, were purchased from Pfaltz & Bauer Inc. (Waterbury, CT). All salts with certified A.C.S. grade were obtained from Fisher Scientific (Pittsburgh, PA). Deionized water, with an electrical conductivity below 1.0 µS/cm, was used for all experiments. The PBS (phosphatebuffered saline, pH 7.4) buffer solution was prepared by mixing 24.0180 g of sodium chloride, 0.5817 g of potassium chloride, 2.7255 g of sodium phosphate, and 0.5715 g of potassium phosphate monobasic and then adding deionized water to 3.0 L. This PBS buffer solution was used immediately after preparation to avoid problems with bacterial contamination. The surface pressure-area isotherms were measured using a computerized Langmuir film balance (MGW Lauda Filmwaage, Germany) with a Teflon-coated trough at an initial area of 927.0 cm2 and a constant temperature of 23.0 °C. The experimental procedure was as follows: About 1.2 L of a phosphate-buffered saline, pH 7.4, was poured into the Langmuir trough as a subphase. Thirty microliters of a methylene chloride solution containing 1 mM surfactants with various molar ratios of Span and Tween was carefully applied on the subphase to form a monolayer by using the glass rod method.14 Seven minutes after spreading, the monolayer was compressed by a movable barrier at a rate of 0.342 Å2/(molecule‚s). The compression was continued until the monolayer collapsed. The surface pressure and surface area of the monolayer were measured by a positionsensitive transducer coupled to the movable barrier. The reversibility of the isotherms was checked by periodic expansion, followed by recompression. Results and Discussion The surface pressure is defined as

π ) γ0 - γ

(3)

where γ0 and γ are the surface tension of the subphase and the surfactant monolayer, respectively. The value of γ0 for PBS buffer is around 73 mN/m. The surface tension of the surfactant monolayer γ approaches zero while the surface pressure π is close to its maximum value of 73 mN/m. The properties of monolayers at high surface pressure can be summarized as (i) the surface resists motion, and there is a high surface viscosity, and (ii) the monolayer itself is a resistance to gas transport. Strong evidence exists that four phase phenomena in the monolayers of long-chain fatty acids and their esters might be found with an increase of surface pressure, π: gas phase (G), liquid-expanded phase (LE), liquid-condensed phase (LC), and solid-condensed phase (SC).15-20 The location of phase boundaries and the characterization of the phases are sometimes ambiguous since the nature of the phase transition in Langmuir monolayers is not clearly understood. While the surface pressure of the monolayer is very low (typically below several mN/m), the hydrocarbon chains in a monolayer in the gas phase are widely separated and lie horizontally on the surface with both conformational and orientational disorder. As the phase

Figure 4. (a) Surface pressure π-molecular area A isotherm and (b) ∆π/∆A ∼ A curve for Span 40 at 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase.

changes from gas to LE, the disordered hydrocarbon chains begin to be raised off the surface and the surface pressure starts to increase. When the LE monolayer is compressed, a transition to an LC phase is often observed. For the isotherms of longchain fatty acids and their esters between 20 and 30 °C, the compressed monolayer starts the LE to LC transition (molecular area of about 32 Å2/molecule) as the hydrocarbon chains are orienting to a 45° angle with respect to the surface normal, and finally the polar angle reaches 30° at the end of the transition region (molecular area of about 25 Å2/molecule).20 The isotherm measurements on fatty acid/ester mixtures suggested the existence of several LC phases in which there is quasi-longrange orientational order but different tilt order.21 At molecular areas below about 22 Å2/molecule, the monolayer appears to be solid-condensed.16,20 When oriented normal to the surface in a SC monolayer, the hydrocarbon chains are close-packed or hexagonal-packed. The close-packed chains have a crosssectional area of about 18.2 Å2, whereas the hexagonal-packed chains, which possess oscillational or rotational disorder have a cross-sectional area of about 19.5 Å2.15 Figure 4a shows a π-A isotherm which we obtained for a sample of Span 40. In order to determine the inflection point accurately, Figure 4b shows the curve of the first derivative of the surface pressure to molecular area with respect to molecular area. To obtain the curve in Figure 4b a 5- to 10-point curvesmoothing was performed on the first derivative using the software Kaleidagraph. The values of the surface pressure and the molecular area at the inflection point were taken at the point where the maximum dip of the derivative curve occurred. As shown in Figure 4b, only one inflection point at the molecular area of about 22 Å2/molecule, which corresponds to the monolayer collapse, exists for the case of Span 40. The value of molecular area indicates that the Span 40 begins to form a SC monolayer at the collapse point. The absence of an inflection point representing the LE-LC phase transition may suggest a second-order transition by changes in slope or even

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Figure 5. (a) Surface pressure π-molecular area A isotherm and (b) ∆π/∆A ∼ A curve for Span 80 at 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase.

a direct LE-SC phase transition.20 Figure 5 shows the π-A isotherm and the first-derivative curve for Span 80. The collapsed molecular area of about 44 Å2/molecule indicates that, unlike Span 40, Span 80 forms an LE monolayer at the collapse point. Although the hydrophillic headgroups of Span 40 and Span 80 are the same, as shown in Figure 1, an ethylene group, -CHdCH-, in the hydrocarbon chain makes Span 80 less flexible, with lower surface activity. Hence, Span 80 cannot pack closely on the air-liquid interface. Figure 6 shows the π-A isotherm and the first-derivative curve for Tween 40. There is only one inflection point which corresponds to the collapse point at the molecular area of about 50 Å2/molecule. This means that the Tween 40 monolayer remains liquid-expanded at the monolayer collapse. This phenomenon may be caused by several factors which prevent the hydrophobic tail groups from approaching closely to one another at the air-liquid interface: Tween 40 has a bigger hydrophillic headgroup than does Span 40, and the polyoxyethylene groups make Tween 40 more hydrophillic, allowing it to migrate more easily into the PBS subphase at relatively low pressure. Figure 7 is an example of the π-A isotherm and the firstderivative curve for the mixture of Span 80 and Tween 40 with a mole fraction of Span 80 of 0.50. It should be recalled that while a mixture of Span 40 and Tween 40 can produce stable microbubbles, mixtures of Span 80 and Tween 40 can not. The single inflection point at a molecular area of about 40 Å2/ molecule indicates that the monolayer for the mixture of Span 80 and Tween 40 at the collapse point is liquid-expanded. An interesting phenomenon, which is illustrated in Figure 8, is that for the case of a Span 40 and Tween 40 mixture (0.50 mole fraction of Span 40) two inflection points exist. A similar situation has been observed in Langmuir monolayers of heneicosanol16 and C18-ferrocenecarboxamide19 as would be expected in a monolayer that has a phase diagram that consists of more than three phases. At the inflection point a in Figure 8, the molecular area of about 34 Å2/molecule indicates the start

Wang et al.

Figure 6. (a) Surface pressure π-molecular area A isotherm and (b) ∆π/∆A ∼ A curve for Tween 40 at 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase.

Figure 7. (a) Surface pressure π-molecular area A isotherm and (b) ∆π/∆A ∼ A curve for the mixtures of Span 80 and Tween 40 at a mole fraction of Span 80 of 0.50 and 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase.

of the LE to LC phase transition. The short plateau of the isotherm between inflection points a and b corresponds to the LE-LC two phase region with a first-order transition. The molecular area at b (about 16.5 Å2/molecule), which is below 22 Å2/molecule, shows that this inflection point refers to the

Langmuir Trough Study of Surfactant Mixtures

Figure 8. (a) Surface pressure π-molecular area A isotherm and (b) ∆π/∆A ∼ A curve for the mixtures of Span 40 and Tween 40 at a mole fraction of Span 40 of 0.50 and 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase.

monolayer collapse, and the monolayer is solid-condensed at the collapse. The value 16.5 Å2/molecule, which was calculated with the assumption that all the surfactant molecules remain in the monolayer, is less than 18.2 Å2/molecule (the value for the close-packed hydrocarbon chains). This is physically impossible and may indicate that some molecules of Tween 40, which possesses bigger hydrophillic headgroups, migrate out of the monolayer into the PBS subphase at high surface pressure. The possibility of migration of Tween 40 into the PBS subphase at high surface pressure was investigated using the data of isotherm reversibility. Figure 9 shows the π-A isotherms in compression and expansion. It can be seen in Figure 9a that when the monolayer formed by Tween 40 alone was compressed to a pressure of about 34 mN/m (point 1 on the graph), and then completely expanded to the initial position, little hysteresis was observed. The small decrease in surface pressure which was observed could be attributed to the fact that the surfactant molecules were forced into an ordered arrangement during the initial compression, and upon expansion intermolecular attraction forces between the arranged molecules are still present, resulting in a decreased surface pressure. The isotherm of the second compression fell on the same curve as the continually compressed isotherm (same as Figure 6(a)). Both of these results indicate that very few molecules of Tween 40 leave the monolayer to migrate into the PBS subphase before the collapse of the monolayer. However, when compression was continued to a pressure of about 43 mN/m (point 2, beyond the collapse point) followed by complete expansion to the initial area, the large degree of hysteresis indicates that some molecules of Tween 40 moved into the PBS subphase after collapse. It was interesting to observe that some molecules of Tween 40 appear to return to the surface from the PBS subphase after full expansion. The recompressed isotherm was below, but close to the first compression isotherm, indicating that only a

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Figure 9. Investigation of the reversibility of π-A isotherms by the compression-expansion technique for the cases of (a) Tween 40 and (b) mixture of Span 40 and Tween 40 with a mole fraction of Span 40 of 0.50 at 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase: (O) continually compressed isotherm; (s) compressed part of isotherm; (- - -) expanded part of isotherm). Points 1, 2, and 3 indicate the points at which compression was stopped and expansion was started.

small number of Tween 40 molecules was permanently lost. Likewise, the monolayer of Span 40 was expanded to 22 Å2/ molecule and 49 mN/m (just before its collapse point), and no Span 40 molecules were found to leave the monolayer (data not shown). For the monolayer of a Span 40 and Tween 40 mixture (mole fraction of Span 40 of 0.50), Figure 9b shows when the monolayer was compressed to 49 Å2/molecule and 31 mN/m (point 1, below the collapse pressure of Tween 40), and then partly expanded to 105 Å2/molecule and 13 mN/m, little hysteresis was observed. Upon recompression, the isotherm closely followed that of the first compression, indicating that few molecules of Tween 40 passed into the PBS subphase. The monolayer was next compressed past the first inflection point, to 25 Å2/molecule and 45 mN/m (point 2, above the collapse pressure of Tween 40 at 38.5 mN/m). Upon expansion to 88 Å2/molecule and 13 mN/m, the isotherm was below the first and second compression isotherms, indicating that some of the Tween 40 molecules have left the monolayer to migrate into the PBS subphase. Molecules of Tween 40 that are surrounded by other molecules of Tween 40 have a strong potential to leave the monolayer when the surface pressure is higher than the collapse pressure of Tween 40. Hence, the actual molecular areas of the monolayer, formed by a mixture of Span 40 and Tween 40, between the first and second inflection points should be larger than our reported values which were calculated on the basis of all the surfactant molecules staying in the monolayer. Upon a final expansion from a point beyond the collapse point (point 3) to the point with about 66 Å2/molecule and 13 mN/m, a great deal of hysteresis was observed since a

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Wang et al.

Figure 11. Surface pressure π-total monolayer area AT isotherm for the microbubbles generated from a sonicated mixture of Span 40 and Tween 40 at a mole fraction of Span 40 of 0.50 and 23.0 °C, a compression rate of 0.342 Å2/(molecule‚s), and a PBS buffer solution (see Experimental Materials and Methods) as the subphase. Dried sample was dissolved in ethanol before application to the trough.

Figure 10. Effect of the composition of the surfactant mixtures on (a) molecular area and (b) surface pressure at inflection points (0, Span 80 and Tween 40; [, Span 40 and Tween 40 at inflection point a; ] Span 40 and Tween 40 at inflection point b). The shadow area represents the mole fraction range in which a mixture of Span 40 and Tween 40 can form stable microbubbles when sonicated.

large number of surfactant molecules (both Tween 40 and Span 40) passed into the PBS subphase after the collapse point. Figure 10 collects the Langmuir trough data for measurements made with increasing mole fraction of Span. It is to be noted that for these mixtures of Span 40 and Tween 40, two inflection points appear on the isotherms only in a range of mole fraction of Span 40 between 0.3 to 0.9. It is exactly the same range of mole fraction from which we have found that stable microbubbles can be produced.13 The molecular areas below 22 Å2/molecule for the inflection point b indicate that the monolayer is solid-condensed, and the decrease of molecular areas from 40 to 20 Å2/molecule at the inflection point a demonstrates that the nature of the monolayer changes from liquid-expanded to solid-condensed with the increase in mole fraction of Span 40. The molecular area at collapse of the mixture of Span 40 and Tween 40 is smaller than that of Tween 40 (about 50 Å2/molecule) and even of Span 40 (about 22 Å2/molecule) alone. This may be explained by various factors: (i) The headgroups of Tween 40 are squeezed out of the headgroup plane of Span 40, and the hydrocarbon chains of Span 40 and Tween 40 are well-packed and oriented normal to the trough surface in an extended zigzag conformation. (ii) The polyoxyethylene group in the hydrophobic tail of Tween 40 adjacent to the fatty acid carboxylate group (Figure 1c) causes stronger van der Waals attractive forces between hydrocarbon chains of Tween 40 and of Span 40. (iii) Some Tween 40 molecules migrate completely out of the monolayer into the subphase after the isotherm has passed the first inflection point, especially at low Span 40 mole fraction, resulting in some “values” of area at collapse even less than 18.2 Å2/molecule since they are calculated with the assumption that all the surfactant molecules remain in the monolayer. Figure 10a also shows that the molecular areas for the mixture of Span 40 and Tween 40 at both inflection points a and b are

less than those for the mixture of Span 80 and Tween 40 at the collapse point. Span 80, with a less flexible hydrocarbon chain with an ethylene group, cannot pack as closely together on the air-liquid interface as can Span 40. The molecular areas of above 35 Å2/molecule for mixtures of Span 80 and Tween 40 indicate that the monolayers are liquid-expanded at the monolayer collapse. Also the average molecular areas of the surfactant mixtures of Span 80 and Tween 40 are considerably lower than those of the “ideal surfactant mixtures” (i.e., algebraic average molecular areas as indicated by the straight dashedline in Figure 10a), since the ethylene group in the hydrocarbon chain of Span 80 enhances the van der Waals attractive forces holding the headgroups together. However, this lowering is not sufficient to stabilize microbubbles. Figure 10b shows that, for a mixture of Span 40 and Tween 40 in the range of mole fraction of Span 40 between 0.3 to 0.9, the surface pressures derived from the inflection point b associated with a SC monolayer are higher than those derived from the inflection point a. This suggests that the gas bubbles with a solid-condensed “skin” have lower surface tension which reduces the surface free energy to stabilize the gas bubbles as indicated by eq 1. Figure 10b also shows that the surface collapse pressure of Span 80 alone is much lower than that of Span 40 alone due to an ethylene group -CHdCH- in the hydrocarbon chain. The mixture of Span 80 and Tween 40 cannot form stable microbubbles because the surface activity of Span 80 is too weak to reduce the surface tension. Figure 10 suggests that the microbubbles in the middle phase shown in Figure 2 have a solid-condensed skin with the zigzag conformation of the mixture of Span 40 and Tween 40. The solid-condensed zigzag monolayer contributes to the stability of the microbubbles by (i) creating an energy barrier, due to the big hydrophilic headgroups of Tween 40 surrounding the microbubbles, to prevent the coalescence of the microbubbles; (ii) reducing the surface free energy due to low surface tension; (iii) resisting diffusion of the encapsulated gas through the surface of the microbubbles due to a well-packed monolayer; and (iv) making the microbubbles compatible with the aqueous environment due to the hydrophillic headgroup of Tween 40. Finally, Figure 11 shows an isotherm for a sample of microbubbles prepared by sonicating a mixture (0.50 mole fraction of Span 40) of Span 40 and Tween 40 (preparation method; see ref 5). Since the composition of the microbubble phase (the middle phase shown in Figure 2) is unknown, Figure 11 can only show the information of total monolayer area instead of molecular area. Before being applied on the Langmuir trough, the dried microbubbles were dissolved in ethanol since they were not soluble in methylene chloride. This change in

Langmuir Trough Study of Surfactant Mixtures solubility characteristics indicates that the sonication process may cause a strong association between the molecules of Span 40 and Tween 40.22 The polyoxyethylene groups of the Tween 40 have been shown to be chemically active.23 In Figure 11 there are more than two inflection points, which indicates that sonication causes a structure change of the surfactant monolayer. It is also interesting to observe that the collapse pressure of the microbubble monolayer is above 70 mN/m, which (eq 3) means that the surface tension of the microbubble monolayer is near zero! Compared with the collapse pressure below 65 mN/m in Figure 10b for the monolayer of the Span 40 and Tween 40 mixtures without sonication, Figure 11 indicates that sonication enhances the structure of surfactant monolayer and makes the microbubbles extremely stable. These findings are the focus of future investigation. Conclusions The stability of the microbubbles depends on both inter- and intrabubble interactions. Span 40 alone cannot form stable microbubbles without an anticoalescence agent and a component to enhance their aqueous compatibility. Tween 40 alone cannot form stable microbubbles, due to insufficient surface activity to reduce surface tension. The mixture of Span 80 and Tween 40 cannot form the stable microbubbles since Span 80 has insufficient surface activity and the hydrocarbon chain presents large steric effects due to an ethylene group -CHdCH- in the hydrocarbon chain. On the basis of the information from the Langmuir trough measurements, the mixture of Span 40 and Tween 40 can form stable microbubbles by the following methods: (i) The skin of the microbubbles is a solid-condensed zigzag monolayer in which the headgroups of Tween 40 are squeezed out of the headgroup plane of Span 40. (ii) The well-packed monolayer inhibits the encapsulated gas diffusion through the surface of the microbubbles. (iii) Span 40 functions as a surface-active component to reduce surface tension. (iv) Tween 40 functions by creating an energy barrier to prevent the coalescence of microbubbles and by increasing the compatibility of the microbubbles with the aqueous environment. (v) Sonication enhances the structure of the surfactant monolayer by reducing surface tension to near zero. Acknowledgment. The authors are grateful to Mallinckrodt Medical Co. and the National Institutes of Health (NIH

J. Phys. Chem., Vol. 100, No. 32, 1996 13821 HL52901, NIH CA52823) for financial support and to Dr. Leslie P. Dutton, Department of Biophysics and Biochemistry, University of Pennsylvania, for generously allowing us to use the Langmuir trough. References and Notes (1) Gramiak, R.; Shah, P. M. InVest. Radiol. 1968, 3, 356. (2) Reisner, S. A.; Shapiro, J. R.; Schwarz, K. Q.; Meltzer, R. S. J. CardioVasc. Ultrasonog. 1988, 7, 273. (3) Balen, F. G.; Allen, C. M.; Lees, W. R. Clinical Radiol. 1994, 49, 77. (4) Goldberg, B. B.; Liu, J. B.; Forsberg, F. Ultrasound Med. Biol. 1994, 20, 319. (5) Wheatley, M. A.; Peng, S.; Singhal, S.; Goldberg, B. B. United States Patent, 5352436, 1994. (6) Evans, D. F.; Wennerstro¨m, H. The colloidal domain where Physics, Chemistry, Biology, and Technology meet. VCH Publishers, Inc.: New York, 1994; Chapters 8 and 11. (7) Hilpert, P. L.; Mattrey, R. F.; Mitten, R. M.; Peterson, T. Am. J. Roentgenol. 1989, 153, 613. (8) Christiansen, C.; Kryvi, H.; Sontum, P. C.; Skotland, T. Biotechnol. Applied Biochem. 1994, 19, 307. (9) Darrigo, J. S.; Imae, T. J. Colloid Interface Sci. 1992, 149, 592. (10) Simon, R. H.; Ho, Sy; Lange, S. C.; Uphoff, D. F.; D’Arrigo, J. S. Ultrasound Med. Biol. 1993, 19, 123. (11) Wheatley, M. A.; Schrope, B.; Shen, P. Biomaterials 1990, 11, 713. (12) Wheatley, M. A.; Singhal, S. React. Polym. 1995, 25, 157. (13) Singhal, S.; Moser, C. C.; Wheatley, M. A. Langmuir 1993, 9, 2426. (14) Trurnit, H. J. J. Colloid Sci. 1960, 15, 1. (15) Larsson, K. Surf. Colloid Sci. 1973, 6, 261. (16) Buontempo, J. T.; Rice, S. A. J. Chem. Phys. 1993, 98, 5835. (17) Schwartz, D. K.; Knobler, C. M. J. Phys. Chem. 1993, 97, 8849. (18) Fischer, B.; Tsao, M.; Ruiz-Garcia, J.; Fischer, T. M.; Schwartz, D. K.; Knobler, C. M. J. Phys. Chem. 1994, 98, 7430. (19) Lindholm-Sethson, B.; Aberg, S. Langmuir 1995, 11, 1244. (20) Andelman, D.; Brochard, F.; Knobler, C.; Rondelez, F. Structures and phase transitions in Langmuir monolayers. In Micelles, Membranes, and Monolayers; Gelbart, W. M., Ben-Shaul, A., Roux, D., Eds.; SpringerVerlag: New York, 1994. (21) Bibo, A. M.; Knobler, C. M.; Peterson, I. R. J. Phys. Chem. 1991, 95, 5591. (22) Fendler, J. H.; Tundo, P. In Eighteen years of colloid and surface chemistry; Fort, T., Mysels, K. J., Eds.; American Chemical Society: Washington, DC, 1991; pp 207-212. (23) Donbrow, M. In Nonionic surfactants: physical chemistry; Schick, M. J., Ed.; Surfactant Science Series, Vol. 23; Marcel Dekker: New York, 1987; Chapter 18.

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