Langmuir 2008, 24, 10035-10040
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Influence of the Dissolution Rate on the Collapse and Shedding Behavior of Monostearin/Monopalmitin-rich Coated Microbubbles Yuyi Shen, Robert L. Powell, and Marjorie L. Longo* Department of Chemical Engineering and Materials Science, UniVersity of California, DaVis, California 95616 ReceiVed May 30, 2008. ReVised Manuscript ReceiVed July 7, 2008 A low cost food grade emulsifier (a mixture of monoglycerides, diglycerides, and sodium stearoyl lactylate) in combination with polyethylene glycol-40 stearate (PEG-40S) was used as an alternative to pure saturated phospholipids to form the thin shell of a microbubble. To investigate the stability of these microbubbles in a water system over time, their dissolution behavior was studied at various degas factors and at two percentages of PEG-40S. It was found that the favored shell collapse/shedding mechanism switched, as the dissolution rate increased (degas factor decreased), from folding with a smooth surface contour to buckling accompanied by surface folding/expulsion with a cyclic buckled-smooth surface contour. The compositional change that we made played a more minor role, mainly controlling the resistance to mass transfer of the microbubble shell and again modifying the mechanism-determinant dissolution rate. The shell resistance behavior for these microbubbles varied from that of previous lipid/PEG-40S-coated microbubbles by the presence of a maximum in shell resistance during dissolution. We hypothesize that the dominance of one collapse mechanism over another for these compositions is related to the time scales of two competing processes, fold nucleation and area compression. For these mixtures at room temperature, we estimate that the maximum area compression rate for folding as the major collapse mechanism is ∼0.2 s-1, a rate unattainable in a traditional Langmuir trough but achievable by the use of a dissolving microbubble.
Introduction Bubbles are ubiquitous in the environment, the chemical and biomedical industries, and food. Bubbles in the sea are produced by breaking waves and are further stabilized by the presence of organic surfactants, particulates, and inorganic salts.1-5 Injected bubbles are important for chemical engineering processes such as scrubbers, strippers, fermentation, sewage treatment plants, and bio-oxidizers. Medical applications of micrometer-scale bubbles (microbubbles) include ultrasound contrast agents (UCAs), blood substitutes, and targeted drug and gene delivery vehicles.6-9 Recently, we investigated food emulsifiers, for the first time, for the sole purpose of stabilizing a microbubble population.10 The study was carried out to enhance and understand the presence of microbubbles in foods. We found that the commercially available emulsifier MYVATEX p14k (MYVATEX) mixed with 4 mol % PEG-40S stabilized a visible microbubble layer. By monolayer and microbubble studies, we showed that the monoglyceride (rich in monostearin and monopalmitin) and diglyceride components were enriched in condensed domains that appear to be phase separated from the two expanded phase components, sodium stearoyl-2-lactylate (SSL2) and PEG-40S. * Corresponding author. E-mail:
[email protected]. (1) Johnson, B. D.; Cooke, R. C. Science 1981, 213, 209–211. (2) D’Arrigo, J. S. Stable Gas-in-Liquid Emulsions: Production in Natural Waters and Artificial Media; Elsevier Science: New York, 1986; Vol. 40. (3) Lozano, M. M.; Talu, E.; Longo, M. L. J. Geophys. Res., [Oceans] 2007, 112, C12001. (4) Tsai, W.; Liu, K.-K. J. Geophys. Res. 2003, 108, 3127. (5) Fox, F. E.; Herzfeld, K. F. J. Acoust. Soc. Am. 1954, 26, 984–989. (6) Ferrara, K.; Pollard, R.; Bordeni, M. Annu. ReV. Biomed. Eng. 2007, 9, 415–447. (7) Klibanov, A. L. J. Nucl. Cardiol. 2007, 14, 876–884. (8) Talu, E.; Lozano, M. M.; Powell, R. L.; Dayton, P. A.; Longo, M. L. Langmuir 2006, 22, 9487–9490. (9) Pancholi, K. P.; Farook, U.; Moaleji, R.; Stride, E.; Edirisinghe, M. J. Eur. Biophys. J. Biophys. Lett. 2008, 37, 515–520. (10) Shen, Y.; Powell, R. L.; Longo, M. L. J. Colloid Interface Sci. 2008, 321, 186–194.
Bubble dissolution is of general importance in medicine, the environment, and food applications. For UCAs, preformed microbubbles are injected intravenously, and circulation time is on the order of tens of minutes.7,11 The general consensus is that circulation time is limited by dissolution of the gas core into the blood pool.12 Of additional interest is the collapse and shedding behavior of the shells of microbubbles formed in the environment.3 Such bubbles profoundly impact the transport, mechanical, and acoustical properties of seawater.2 When microbubbles are closedpacked, such as in a food application, a portion of the bubbles will dissolve because of interaction with the surrounding bubbles through Ostwald ripening.13-15 The presence of a surfactant monolayer shell can have a significant impact on the dissolution behavior of a microbubble in a degassed medium.16 Previously, we demonstrated that the relative stability of diacyl phosphatidylcholine/PEG-40S-coated microbubbles in degassed media is primarily due to the resistance of the lipid monolayer shell to air permeation, which increases by as much as 2 orders of magnitude with increasing hydrophobic chain length.17-19 During gas-core dissolution, the monolayer shell of the microbubble undergoes compression and ultimately collapses. We showed that the morphology of microbubble shell collapse structures and shed particles at a single degassed fraction (11) Fritz, T. A.; Unger, E. C.; Sutherland, G.; Sahn, D. InVest. Radiol. 1997, 32, 735–740. (12) Kabalnov, A.; Bradley, J.; Flaim, S.; Klein, D.; Pelura, T.; Peters, B.; Otto, S.; Reynolds, J.; Schutt, E.; Weers, J. Ultrasound Med. Biol. 1998, 24, 751–760. (13) Shen, Y.; Powell, R. L.; Longo, M. L. J. Colloid Interface Sci. 2008, 321, 186–194. (14) Kloek, W.; van Vliet, T.; Meinders, M. J. Colloid Interface Sci. 2001, 237, 158–166. (15) Lau, C. K.; Dickinson, E. Food Hydrocolloids 2005, 19, 111–121. (16) Yount, D. E. J. Acoust. Soc. Am. 1979, 65, 1429–1439. (17) Borden, M. A.; Longo, M. L. Langmuir 2002, 18, 9225–9233. (18) Borden, M. A.; Longo, M. L. J. Phys. Chem. B 2004, 108, 6009–6016. (19) Pu, G.; Longo, M. L.; Borden, M. A. J. Am. Chem. Soc. 2005, 127, 6524–6525.
10.1021/la801668h CCC: $40.75 2008 American Chemical Society Published on Web 08/22/2008
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Table 1. Composition and Chemical Structure of Materials
depended upon shell composition17,20 with expanded phase components collapsing via suboptical particles, vesicles, and tubes as the chain length increased. Primarily condensed-phase shells collapsed via the rapid propagation of monolayer folds, and for longer, more rigid chain lengths, substantial surface buckling with the simultaneous nucleation and growth of surface folds was involved. The sequence is in remarkable agreement with the temperature-dependent sequence of collapse in Langmuir monolayers by Gopal and Lee21 with the new observation of surface buckling accompanied by folding at higher chain lengths in comparison to their study. The microbubble shell collapse mechanism could significantly impact food texture or drug targeting in food or biomedical applications, respectively. Herein, we report on the dissolution of microbubbles, formed by tip sonication and stabilized primarily by a food-grade emulsifier, MYVATEX p14k (MYVATEX) with PEG-40S, for the first time. Different from our previous studies that maintained one degas factor (f, the ratio of the actual partial pressure of oxygen in water to that at saturation),17,20 here we vary f. Thereby, we investigate the dependence of the microbubble shell collapse and shedding mechanism upon the air-water interfacial area compression rate. In addition, we varied the PEG-40S mole fraction and determined its impact on both the collapse and shedding mechanism and the overall resistance to gas transport across the shell. This work provides new insights with regard to the impact of the area compression rate and composition on monolayer collapse and permeability. (20) Pu, G.; Borden, M. A.; Longo, M. L. Langmuir 2006, 22, 2993–2999. (21) Gopal, A.; Lee, K. Y. C. J. Phys. Chem. B 2001, 105, 10348–10354.
Materials and Methods Materials. Two mixtures composed of two commercially available surfactants were studied in this work as listed in Table 1. Foodgrade emulsifier MYVATEX p14k (MYVATEX) in the form of an ivory-colored solid powder was obtained from the Kerry Bio-Science Company (Brantford, Ontario, Canada). It is a mixture of five components, 77 ( 2 mol % monoglycerides (rich in monostearin and monopalmitin), 2.5 ( 2 mol % diglycerides (rich in distearin and dipalmitin) and 20.5 ( 2 mol % sodium stearoyl-2-lactylate (SSL2). The same production batch of MYVATEX was used in all experiments to ensure consistency. The MYVATEX mixture alone cannot be used to make microbubbles. Another commercial emulsifier, PEG-40 stearate (PEG40S, product No. Myrj 52), from Sigma (St. Louis, MO), was added. Two mixtures of MYVATEX/PEG-40S were used. The mole ratios for MYVATEX and PEG-40S in mixture A and mixture B are 96:4 and 9:1, respectively. For fluorescent imaging, 3H-Indolium, 2-[3(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol- 2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-perchorate (DiI) was added before probe sonication at 1 µL DiI solution as supplied by Molecular Probes (Eugene, OR) per 1 mg of MYVATEX/PEG-40s. The solvent for all experiments was ultrapure water (resistivity ∼ 18 MΩ cm) purified with a NANOpure ultrapure water system (Barnstead| Thermolyne Corporation, Dubuque, IA). Air was used as the filling gas for the microbubbles. All the experiments were conducted at room temperature. Microbubble Formation Methods. A multilamellar vesicle dispersion of MYVATEX plus PEG-40S was prepared by dissolving the materials in powder form with ultrapure water in a glass vial. See Table 1 for concentrations and compositions. An ultrasonic cleaner (Branson Ultrasonics Corporation, Danbury, CT) was used to heat and sonicate the solution in a bath for 10 min to ensure good
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Figure 1. Typical microbubble surface contour during dissolution: (A) smooth, (B) buckle-smooth, and (C) severe buckle-smooth. Scale bars represent 10 µm.
Figure 2. Fluorescence videomicrographs of MYVATEX/PEG-40S microbubble shell collapse and shedding during dissolution in degassed media. (A) Smooth dissolution with white arrows pointing to locations of surface folds forming between 11 and 19 s and black arrows pointing to location of surface fold forming between 19 and 22 s. (B) Buckle-smooth with arrows at 1 and 8 s pointing to the meeting of surface folds and arrows at 2 and 10 s pointing to the rounding associated with the shedding of excess area and returning to sphericity. Scale bars represent 10 µm. Table 2. Microbubble Surface Contour Statistics with Bracketed Numbers Indicating the Total Number of Bubbles degas factor (mixture A, bubble radius 5-45 µm)
degas factor (mixture B, bubble radius 5-45 µm)
bubble radius (mixture A, degas factor ∼ave 0.85)
surface contour
0.87 [95] %b
0.87 [52]%b
5-7 µm [90]%
7-30 µm [274]%
30-45 µm [16]%
smooth buckle-smooth severe buckle-smooth
24.7 30.9 44.4
54.9 27.5 17.6
90.5 8.4 1.1
8.6 22.9 68.6
44.9 34.6 20.5
67.3 21.2 11.5
21.1 35.6 43.3
51.5 23.7 24.8
81.2 18.8 0
a
Minimum, 0.78.
b
Maximum, 0.91.
mixing. The method of probe sonication was used to generate polydisperse microbubbles ranging from 0.1 to 100 µm.22 In this method, the tip of a probe sonicator (model 250, Branson Ultrasonics Corporation, Danbury, CT) was first plunged below the air-water interface in a 6 mL vial at low power for 1 to 2 min to ensure (22) Borden, M. A. Surface Morphology and Transport Properties of Lipid/ Emulsifier Monolayer-Coated Microbubbles. Ph.D Dissertation, University of California, Davis, CA, 2003.
hydration. Then the solution was sonicated with the tip at the air-water interface at high power for 10 to 30 s. Three phases of separated layers were observed after the solution was sonicated: an upper foam layer with large bubbles, a middle microbubble layer, and a lower layer containing excess surfactants aggregates. The microbubbles, ranging from 0.1 to 100 µm, were obtained from the middle layer using an airtight microsyringe (Hamilton Company, Reno, NV).
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Shen et al. atmospheric pressure and is equal to 105 Pa. C ) 0.693 when the effect of the impermeable wall is included.26 The unknown σ is assumed to be negligible, as is typical for monolayers that collapse by folding mechanisms,21 as observed here and in agreement with the lowest surface tensions that we measured for this mixture in a previous study.10 On the basis of this assumption, eq 1 can be simplified to
-
Figure 3. Radius-time plots for MYVATEX/PEG-40S-coated microbubbles dissolved in degassed water. The degas factor, f, is the ratio of the actual partial pressure of oxygen in water to that at saturation. Solid curves represent fits from the theoretical model varying Rshell.
Dissolution Experiments and Fluorescence Microscopy. The dissolution experiments tracked the size change over time of an individual microbubble in degassed water. Degassed water was prepared in two steps: first, oxygen-saturated nanopure water was obtained by exposure to the atmosphere under continuous stirring at 2 to 3 Hz overnight; second, the saturated water was placed in a controlled vacuum for 2 to 3 h, and the final f value was measured by a multiprobe system (model YSI 556 MPS, multiprobe system; YSI Incorporated, Yellow Spring, OH). The microbubbles were then injected via a syringe into a closed laminar flow perfusion chamber (series 20 chamber platform P-2 and cell culture/imaging chamber RC-21BR, Warner Instruments, Hamden, CT) between two coverglass slides pretreated with SurfaSil (Pierce, Rochford, IL). This closed chamber was connected to a programmed controlled pump (Kd Scientific, Holliston, MA) to inject the degassed water into the system. Images of the entire dissolution of a chosen microbubble were viewed and captured using a Hamamatsu OrcaER digital camera (Hamamatsu Photonics, K.K, Japan) mounted on a Nikon microscope (Fryer Company Inc., Scientific Instruments, Cincinnati, OH). For fluorescence imaging, a fluorescent lamp (Nikon Inc., Hawthorne, NY) was used to excite the fluorescent probe. The whole setup was secured on a vibration isolation table (Newport Corporation, Baldy Circle Fountain Valley, CA). The criterion for choosing a microbubble for observation was that the chosen microbubble should be a reasonable distance (more than 100 µm was preferred) away from microbubble aggregates and it should not move during the whole dissolution process. Data Analysis and Model Fitting. The area and perimeter of each individual microbubble were analyzed using ScionImage software (Frederick, Maryland). The model to fit the behavior of “clean” bubble dissolution was first derived by Epstein and Plesset23 and verified by others.24,25 The model of shell resistance (Rshell) to gas permeation (dissolution model) was further developed by Borden.17,18 In this model, the change in microbubble radius (R) with respect to dissolution time (t) can be derived as17
-
dR H ) dt R + Rshell CDw
(
)
2σ -f PaR 4σ 1+ 3PaR
1+
(1)
where H is the ratio of gas concentration in the aqueous phase to that in the gas phase (i.e., 0.019), f is the degas factor, and σ is the surface tension. The value of Dw is 2 × 10-5 cm2/s. Pa is the (23) Epstein, P. S.; Plesset, M. S. J. Chem. Phys. 1950, 18, 1505–1509. (24) Duncan, P. B.; Needham, D. Langmuir 2004, 20, 2567–2578. (25) Hanwright, J.; Zhou, J.; Evans, G. M.; Galvin, K. P. Langmuir 2005, 21, 4912–4920.
dR H (1 - f ) ) dt R + Rshell CDw
(2)
The previous study of lipid-coated microbubbles by Borden17,18 found that Rshell could be treated as a constant. However, the assumption of constant Rshell for this system gives an unacceptably high average sum of squares error, >10-1 µm. Therefore, to predict the change in Rshell as a function of time and radius, the following algorithm was used. Equation 2 was integrated to obtain
R(t) ) - RshellCDw +
√Rshell2C2Dw2 - 2tCDwH + 2tCDwHf + R02 + 2R0RshellCDw (3) In eq 3, Rshell values are obtained at each given time and radius by using the fsolVe optimization function from MATLAB (MathWorks.Inc., Natick, MA).27 At the end of optimization, Rshell values are obtained for each given experimental data point of radius/time. Termination tolerance on the function value is taken as 10-8. By the use of this algorithm, the fitting of the theoretical microbubble radius data and experimental microbubble radius data is controlled with an average sum of squares error under 10-4 µm.
Results and Discussion Microbubble Surface Contours during Dissolution. Airfilled microbubbles coated with the two MYVATEX and PEG40S compositions given in Table 1 were dissolved by the introduction of degassed water. Three qualitative bubble surface contours were observed during microbubble dissolution in degassed water: smooth (Figure 1A), buckle-smooth (Figure 1B), and severe buckle-smooth (Figure 1C). For the smooth contour, as shown in Figure 1A, the surface remained smooth; however, the shape deviated from perfectly spherical much of the time, and there was no observed skin roughness. The buckle-smooth contour (Figure 1B) involved a cycle of increasing skin roughness and a shape deviating from perfectly spherical, followed by a discrete event (less than 1/32 s) in which the surface contour became smooth and spherical. The buckling shape of the severe buckle-smooth contour (Figure 1C) deviated significantly from sphericity, and there was pronounced skin roughness during many of the cycles. Similar behaviors were observed previously in (Di16PC-Di22PC)/PEG-40S-shelled microbubbles while investigating the effects of phosphatidyl choline (PC) lipid hydrophobic chain length on dissolution behavior at room temperature.17 We observed the dissolution behavior by fluorescence microcopy to determine if the same kind of collapse and shedding transitions were occurring in comparison to previous observations of (Di16PC-Di20PC)/PEG-40S at room temperature.20 A fluorescent probe was included that partitioned into the expanded phase; condensed phases appeared dark. Indeed, in agreement with previous results, we find that the smooth contour during dissolution is associated with the formation of micrometer-scale surface folds as shown in Figure 2A and Movie 1A, also explaining (26) Wise, D. L.; Houghton, G. Chem. Eng. Sci. 1968, 23, 1502–1503. (27) Optimisation Toolbox for Use with Matlab, User’s Guide, version 3.1; The MathWorks Inc.: 2006, Vol. R2006b.
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Figure 4. Comparison of microbubble morphology using fluorescence microscopy: (A, B) microbubble coated with mixture A and (C, D) microbubble coated with mixture B. The scale bars represent 10 µm.
Figure 5. Change in shell resistance with microbubble radius during dissolution. Insets at the bottom show the microbubbles used to measure these two varying Rshell values and the microbubble size (dashed circles) expected if Rshell remains constant. The scale bar is 10 µm.
the deviation of the shape from perfectly spherical. We recommend viewing Movie 1A where approximately 30 surface folding events can be observed. Very bright spots, evident in Figure 2A and the beginning of Movie 1A, are an indication of the “squeeze out”/collapse of the expanded-phase components (SSL2 and PEG-40S) of the MYVATEX/PEG-40S mixture, studied previously in Langmuir monolayers,10 before the surface began folding. Similar observations have been interpreted as the squeeze-out of expanded-phase PEG-40S from DinPC/PEG-40Scoated microbubbles.20 Also in agreement with previous results, we find that the buckle-smooth contour during dissolution is associated with the formation of folds that would often come to a point or a region (top arrows in Figure 2B) and then would disappear from the surface (Figure 2B at 2 and 10 s and Movie 1B). This process occurred in less than 1/32 s and was accompanied by stepping down in size and rounding of the microbubble, as evidenced by circular outlines (bottom arrows in Figure 2B). As before, it appeared that excess material was being ejected from the surface, as was evidenced by fluorescent material remaining while the core of the microbubble stepped down in size. This behavior was cyclical, with folds reappearing in nearly the same pattern and place, followed by ejection. Crossing surface folds are regions of high curvature where largescale shedding occurs in a cyclic pattern, removing the surface buckles temporarily.
Statistical Analysis of Dissolution Surface Contours: Degas Factor and Size. Earlier results employing phospholipids indicated that buckling is associated with the lateral cohesiveness and stiffness of longer-chain-length saturated components.17 In our case, the major components of MYVATEX, monostearin and monopalmitin (carbon numbers 16 and 18), are also saturated and have long chain lengths. However, given that several behaviors were observed within the same surface composition in previous studies17,20 and here, we decided to perform statistical analysis comparing experimental variables to functional behavior. The results in Table 2 show that initial surface contours during dissolution were influenced by two factors that we purposely varied for this study: the degas factor (f ) and shell composition as well as a factor outside of our controlsthe initial size of microbubbles. As shown in Table 2, the influence of f is significant. For example, 90.5% of 95 microbubbles coated with mixture A in the more saturated media (f > 0.87) displayed smooth dissolution behavior, whereas in media with f < 0.83 only 24.7% of 81 microbubbles with the same coating displayed smooth dissolution. For mixture A, the dissolution times (for R ) 10 µm (area ) 1300 µm2) to R ) 1 µm (area ) 13 µm2)) increased with f: 0.87 ) 66.29 ( 15.35 s, where all populations are statistically distinct by the t test. Therefore, the collapse mechanism is strongly influenced by the monolayer area compression rate, -d(A/Ao)/ dt, that is slower in higher-f media. The influence of f on the dissolution rate can be also seen in Figure 3, where dissolution times can be doubled by increasing f by 5%. Collapse structures such as folds are created through a nucleation process with a nucleation rate that is dependent on the change in enthalpy per molecule and line tension.21 Our results suggest that excess area created at a given monolayer area compression rate, -d(A/Ao)/dt, during dissolution can be eliminated by the nucleation and rapid propagation of each fold when area compression rates are below some critical value. However, when the monolayer area compression rate exceeds a critical value, the folding process is not rapid enough to shed the excess area and instead surface buckling occurs, accompanied by the continued formation of folds. We have estimated the critical value of the area compression rate for the transition from folding to buckling/folding by measuring -d(A/Ao)/dt for microbubbles that switch from smooth contour to buckling/smooth contour during dissolution (i.e., -d(A/Ao)/dtcrit. On the basis of eight microbubbles, -d(A/Ao)/dtcrit is 0.2 ( 0.05 s-1 or ∼1/5 s-1. Therefore, the folding process can eliminate as much as 1/5 of the surface area every second! It should be noted that such area compression rates are not accessible in traditional Langmuir troughs, with a maximum -d(A/Ao)/dt of 0.01 s-1. The initial radius is another major influencing factor. As shown in Table 2, 81.2% of 16 microbubbles with an initial radius ranging from 30 to 45 µm exhibited initially smooth dissolution
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behavior, whereas only 21.1% of 90 small microbubbles (initial radius of 5-7 µm) showed similar behavior. This behavior can be explained by the faster monolayer area compression of smaller microbubbles, as a result of larger area/volume ratios for gas permeation,17 favoring the buckling mechanism. In the future, the radius could be controlled more precisely for this experiment by the production of coated microbubbles using a microfluidics technique.8,9 These results and past results17,20,21 taken as a whole are consistent with time-temperature superposition in viscoelastic systems.28 Hence, compression at a lower reduced temperature has the same effect as increasing the area compression rate. Reduced temperature is defined as T/Tm with the main phasetransition temperature of Tm. Here, increasing the compression rate favored buckling, whereas in the past increasing the chain length favored buckling; a longer chain length has a lowered reduced temperature. Statistical Analysis of the Dissolution Surface Contours: Composition. With different f values, two different mole percentages of MYVATEX and PEG-40S (4 and 8%) led to similar initial dissolution behavior with f between 0.83 and 0.87, as can be seen in Table 2. However, at low (0.87) f values the differences became evident. Specifically, mixture A favored smooth dissolution in comparison to mixture B. For example, at f > 0.87, 90.5% of 95 microbubbles coated with mixture A showed smooth dissolution behavior, whereas 67.3% of 52 microbubbles coated with mixture B showed similar behavior. In the simplistic but functional view given above, this result would indicate a longer dissolution time (slower area compression rates) and/or a higher fold nucleation rate (or both) for mixture A compared to those for mixture B. Indeed, for >0.87f, the dissolution time (for R ) 10 µm (area ) 1300 µm2) to R ) 1 µm (area ) 13 µm2)) was longer with mixture A, 66.29 ( 15.35 s, compared to that for mixture B, 43.31 ( 9.8 s, and the two populations are statistically distinct by the t test. It is important to note here that because MYVATEX is a mixture of expanded- and condensed-phase components the more gaspermeable18 expanded phase (SSL2 and PEG-40S) mole percentages of mixtures A and B were 16.3 and 28.5%, respectively, in the mixtures used to stabilize the microbubbles. As we showed in our previous work, these components are squeezed out of the monolayer to a large extent, but pressure-area Langmuir isotherms indicated that perhaps as much as 50% of the PEG40S is retained in the case of mixture B.10 Therefore, we should expect the gas permeability, microstructure, and mechanical properties to vary between these two microbubble coatings, with mixture A being the less permeable coating. This is also demonstrated in Figure 3 by the longer dissolution times of mixture A. A lower permeability of the mixture A coating would explain our results; therefore, we investigate the differences in permeability (mass-transfer resistance) between these two mixtures in the section below. We compared microbubbles coated with mixture A to mixture B by fluorescence microscopy in air-saturated water as demonstrated in Figure 4. In all cases, the coexistence of the majority condensed phase (dark domains) and minority expanded phase (homogeneous fluorescence with occasionally bright spots) was evident.29 The condensed phase consists primarily of monostearin and monopalmintin and SSL2 and PEG-40S for the expanded phase.10 The observed morphology differences include (1) for microbubbles coated with mixture B, the domains formed networks (28) Deng, J.; Hottle, J. R.; Polidan, J. T.; Kim, H.-J.; Farmer-Creely, C. E.; Viers, B. D.; Esker, A. R. Langmuir 2004, 20, 109–115. (29) Borden, M. A.; Pu, G.; Runner, G. J.; Longo, M. L. Colloids Surf., B 2004, 35, 209–223.
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(a complex pattern of associated connection), whereas in mixture A the domains are isolated by the expanded phase with weaker interconnection and (2) for mixture A, most domains had smaller sizes (average 3-5 µm) compared to the domain sizes of mixture B. Therefore, mixture A contained more of the domain boundary, known to be the location of fold nucleation.21,19 The domain boundary density β (L/Ad; ratio of the boundary length of bright areas and total dark domain area) is 0.13 ( 0.03 µm-1 for mixture A and 0.08 ( 0.06 µm-1 for mixture B, the two populations are statistically distinct by the t test. It may follow, then, that the fold nucleation rate in mixture A may be higher than that in mixture B, kinetically favoring the folding mechanism and smooth dissolution contour as observed in Table 2. Change in Shell Resistance during Dissolution. The modified Epstein and Plesset model (eq 1)17,18 to determine the gas permeation resistance of the microbubble shells did not fit our radius versus time dissolution data well enough to yield a constant shell resistance. By applying a fitting routine (eq 3), the shell resistance (Rshell) at each radius and time was obtained. Example fits are shown in Figure 3. As demonstrated in Figure 5, Rshell initially increased as excess expanded-phase components were squeezed out of the shell monolayer,13 in agreement with previous results.29 However, we then observe a significant difference from our previous studies that used DinPC/PEG-40S. Here, we observe a maximum in shell resistance followed by a decrease in shell resistance, where previous results indicated a plateau to a constant shell resistance. As a control, we ran the same experiments using DSPC/PEG-40S-coated microbubbles, the results agreed with the previous investigation,17 (i.e., we obtained a constant/plateau Rshell), and the average sum of squares error of the fitting data for microbubble radius was
