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Chapter 9 Concentrated gas-in-liquid emulsions in artificial media. I. Demonstration by laser-light scattering
151
Chapter 9
CONCENTRATED FICIAL MEDIA. SCATTERING
GAS-IN-LIQUID EMULSIONS IN ARTII. DEMONSTRATION BY LASER-LIGHT
9.1 PHYSIOLOGICAL HINTS FOR THE PRODUCTION OF ARTIFICIAL MICROBUBBLES The overall impression one obtains from the various studies and competing hypotheses described in Chapter 8 is that stable gas microbubbles more likely do not occur naturally in physiological fluids; on the other hand, injected artificial surfactant-stabilized microbubbles are likely to have useful clinical applications. Interestingly, these proposed long-lived, artificial microbubbles would resemble in many ways (including their having a lowdielectric material in their core) the biochemical assemblies, i.e., oil-in-water emulsions, already formed in the human body during the process of digestion and absorption of fats from inside the small intestine. For example, once digested fat is absorbed into the intestinal epithelium, the fat is processed further and organized into protein-coated lipid droplets called "chylomicrons" (ref. 447-449). The 0.1-3.5 ~tm diameter sizes of these structures (ref. 270,448) closely resemble the size distribution, usually < 5 ~tm, of long-lived gas microbubbles (see Sections 3.2 and 4.1.1). Moreover, the protein content of chylomicrons even though low (about 2% by weight) is a very consistent feature (ref. 447-450), as is also true for natural microbubble surfactant (see Chapters 3-5) where the protein content is less than 5% by weight (see Chapter 6). The protein coat of chylomicrons, even though incomplete and mixed with various lipids in the interfacial film, helps keep them suspended and prevents them from sticking to each other or to the walls of the lymphatics or blood vessels (ref. 449,450). (Similarly, one might expect that the protein content of natural microbubble surfactant (see Section 1.3.1 and Chapters 3-5) helps prevent the
152 coalescence and/or destruction of the stable gas microbubbles known to be present in natural waters (see Chapters 1, 3, 4, and 5).) However, the presence of a partial protein coat (ref. 194,451,452) is not, of course, an indispensable requirement for all (or even most) stable, spontaneously forming oil-in-water emulsions (ref. 194,453-463). Returning to another related physiological example from the intestinal processing of fats in humans, undigested fat enters the intestine in the form of large lipid globules which undergo emulsification through the detergentlike action of various cholesterol derivatives, i.e., the bile salts (ref. 447-449). This emulsification facilitates subsequent enzymatic degradation (by pancreatic lipase) of the triglycerides to monoglycerides. The monoglycerides, cholesterol derivatives (bile salts), cholesterol, and other lipid components then spontaneously form stable "mixed micelles" (ref. 447,448) in the intestinal lumen. Parallel consideration of results from related artificial oil-in-water emulsion studies offer the suggestion, however, that if the monoglycerides had sufficiently long chain lengths and the rest of the interfacial film contained only cholesterol and/or cholesterol derivatives, one could expect larger, spontaneously forming structures (containing low-dielectric material in their core) to be formed. For example, Schulman et al. (ref. 454) reported that "it has been shown that the chain length of a normal alcohol, when benzene was the oil phase and potassium oleate the micelle forming compound, was a governing factor in controlling the size of the droplets. Increasing the chain length greatly diminished the range of the phase diagram such that above decyl alcohol, no microemulsions could be formed with this system. Only coarse emulsions could be formed of 0.5 ~tm diameter droplets. This occurred also with systems stabilized with cetyl alcohol or cholesterol ... and thus, although the emulsions were spontaneously formed, no microemulsions could be made. It was considered that the interfacial mixed monolayer was too highly condensed with the strong associating components, and thus no great degree of curvature necessary for the very small droplets could be produced, a vapor condensed film being essential" (ref. 454). Accordingly, a detailed series of tests in this laboratory revealed that aqueous solutions of saturated monoglycerides (with acyl chain lengths greater than 10 carbons) combined with
153 cholesterol and cholesterol derivatives did, in fact, form stable zasin-water emulsions when shaken vigorously (in an air atmosphere). Evidence for the formation of these significantly stable (hours to days) emulsions is presented below. 9.2 LASER-BASED FLOW CYTOMETRY A N D FORWARDANGLE LIGHT SCATTERING
All experimental measurements were carried out with a Coulter EPICS V System. This instrument is a laser-based flow cytometer which, as one of its simpler analytical functions, utilizes light scatter measurements to accurately size cells or similar particles (e.g., artificial surfactant-stabilized microbubbles) suspended in aqueous media. The light scatter measurements are sensitive to particle sizes as small as 0.3 ~tm in diameter. To make the measurements, particles in liquid suspension are presented under mild pressure to a flow cell where they are surrounded by a laminar sheath of particle-free liquid. This coaxial stream then exits through a flow chamber as a (76-~tm diameter) jet. This hydrodynamic focusing, which is ordinarily bubble-free when only solid particles are measured, insures that all particles follow the same path through the detection zone. Sample pressure, sheath pressure, and particle concentration are controlled so that particles are presented one-at-a-time to the beam of a UV-enhanced argon-ion laser. As the particles pass through the laser beam, the scatter emissions are collected by detectors which convert the signals to voltage pulses proportional to the amount of light scattered. The pulses are amplified, converted to digital form, and displayed in numeric or histogram formats. In this study, light scattered in specifically the forward direction was monitored, since it is generally proportional to the cross-sectional area (radius 2) of large-size particles (i.e., on the order of a micron) (ref. 129). 9.3 SYNTHETIC MICROBUBBLE COUNTS VERSUS THE CONTROL
A mixture of saturated monoglycerides (with acyl chain lengths greater than 10 carbons) combined with cholesterol and (nordonic) choiesterol derivatives, all initially in powdered form, was used to form high concentrations of artificial gas microbubbles
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Fig. 9.1. Synthetic microbubble histograms determined by laser-light scattering in aqueous media (at 21 ~ (A) saturated solution of Filmix 3 surfactant mixture; (B) distilled water alone; (C) computed difference in above two histograms. (See text for fitrther discussion.)
155 in distilled water. This proprietary surfactant mixture, CAV-CON Filmix 3, contains approximately equal parts of monoglycerides and sterol products. A saturated aqueous solution of the surfactant mixture was simply shaken vigorously for a few seconds, in an air atmosphere at room temperature, to form the concentrated gas-inwater emulsions. Evidence for the formation of synthetic microbubbles was obtained by comparing the particle histograms (Fig. 9.1) for identically preshaken, equivolumetric samples of the artificialmicrobubble-surfactant solution (Fig. 9.1(A)) versus distilled water alone (Fig. 9.1(B)). Collection of the scatter emissions, for a total period of 400 see, commenced one minute after shaking ended in both cases; this one-minute delay allowed essentially all macroscopic bubbles formed in both samples to rise to the surface. It can be seen from the histograms (of identical scale), shown in Fig. 9.1, that the amount of microscopic particles remaining over several minutes in the artificial-microbubble-surfactant solution (Fig. 9.1(A)) was enormously greater than in an equal volume of the distilled water alone (Fig. 9.1(B)); this finding is further confirmed by the total particle counts (over the 400-sec collection period) shown in the upper right corner of both Figs. 9.1(A) and 9.1(B), which differed by more than two orders of magnitude, as well as by the computed difference in the two histograms shown in Fig. 9.1(C ). Additional evidence that the large number of microscopic particles detected in Fig. 9.1(A) are, in fact, artificial surfactantstabilized microbubbles includes the following" 1) Prior to shaking and microbubble formation, the surfactant solution was passed through a membrane filter which removed all solid debris greater than 6.0 ~tm in diameter; 2) The detection limit of the instrument, i.e., particle sizes down to 0.3 ~tm in diameter, strongly argues against the particle count being a representation of a micelle population (which will also be present, but undetected in these experiments). This is especially true since forward-angle light scattering was used, which favors the detection of larger particles in the detection range (0.3-40 ~tm) of the instrument; 3) Since none of the surfactants used are liquids, oil-in-water microemulsions/emulsions could not be the basis of the particle histogram obtained; 4) In accompanying decompression tests, it was found, in the course of developing Filmix 3, that those
156 surfactant solutions which produced the higher concentrations of growing bubbles upon decompression (i.e., below 1 atm) similarly produced the greater degree of light scatter in the absence of decompression. An estimate of the actual concentration of synthetic microbubbles present in the (shaken) artificial-microbubble-surfactant solution, represented by Fig. 9.1(A), is given by the fact that 360400 particles/see were consistently detected at a flow rate of approximately 1 ml/30 min in order to produce the histogram shown. Therefore, the calculated approximate concentration of synthetic microbubbles in the sample is 7 x 105 microbubbles/ml. (A similar calculation for the distilled water sample shown in Fig. 9.1(B) results in an estimated concentration of only 5 x 10 3 microbubbles/ml.) 9.4
MICROBUBBLE FLOTATION WITH TIME
In the next experiment, the relative size distribution of the artificial microbubbles formed with the Filmix 3 surfactant mixture (described in the preceding section) was monitored over a 1,000sec period (Fig. 9.2). While the Coulter EPICS V System used did not record the absolute sizes of microbubbles detected, relative sizes were indicated by the relative intensity of scattered light (increasing toward the right on the abscissa of Figs. 9.1-9.3). Furthermore, during the 1,000-sec collection period, the instrument accumulated microbubble counts over successive 4-sec intervals and, in each case, determined the separate scattered-light intensity levels above which no more than 50 and 5 microbubbles reached during the interval (see Fig. 9.2(B)). The averaged results for the 1,000-see collection period are plotted in both three and two dimensions in Fig. 9.2 (with the total counts given by the 6-digit number). It can be seen that the relative proportions of microbubbles falling below the "50-microbubble" and "5-microbubble" scatteredlight intensity levels (Fig. 9.2) changed little over a period of more than 15 min (i.e., 1,000 see); this finding indicates that little, if any, loss of microbubbles due to (rapid) flotation occurred during this time period. Close inspection of part B of Fig. 9.2, however, does reveal a slight skewing of the microbubble population toward smaller sizes during the 1,000-see period.
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SIZE Fig. 9.2. Relative size distribution of artificial microbubbles in an unstirred medium. Light-scatter signals were collected over a total period of 1,000 see, and plotted in three (part A) and two (part B) dimensions. (See text for further discussion.)
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SIZE Fig. 9.3. Relative size distribution of artificial microbubbles in a stirred medium. Light-scatter signals were collected over a total period of 1,500 sec, and plotted in three (part A) and two (part B) dimensions. (See text for further discussion.)
159 9.5
MICROBUBBLE PERSISTENCE WITH TIME
An effort was made to evaluate the possibility that the slight skewing of the artificial microbubble population (in Fig. 9.2) with time might be due to a continuing, even though very slow, dissolution of the newly formed, surfactant-eoated microbubbles. Unlike the experiment referred to in Fig. 9.2, gentle automatic swirling of the preshaken (Filmix 3) surfactant stock solution was now performed as it was fed into the flow cell (see Section 9.2) of the Coulter EPICS V System. Data collection and analysis were the same as in Section 9.4, except that the total collection period was now extended to 1,500 see and each collection interval was 6 see long. The averaged results for the 1,500-see collection period used in this experiment are plotted in both three and two dimensions in Fig. 9.3 (with the total counts given by the 6-digit number). It can be seen that with the constant, gentle swirling of the surfactant solution in this experiment, the relative size of microbubbles falling below either the "50-microbubble" or the "5microbubble" scattered-light intensity levels (Fig. 9.3) decreased more noticeably during the entire collection period (i.e., 25 min). This finding indicates that very slow dissolution of the newly formed, surfactant-coated microbubbles does continue, at least during the first half hour of their existence. Moreover, this gradual rate of dissolution of the artificial microbubbles can apparently be increased somewhat by circulation of the liquid.
Chapter 9
CONCENTRATED FICIAL MEDIA. SCATTERING
GAS-IN-LIQUID EMULSIONS IN ARTII. DEMONSTRATION BY LASER-LIGHT
9.1 PHYSIOLOGICAL HINTS FOR THE PRODUCTION OF ARTIFICIAL MICROBUBBLES The overall impression one obtains from the various studies and competing hypotheses described in Chapter 8 is that stable gas microbubbles more likely do not occur naturally in physiological fluids; on the other hand, injected artificial surfactant-stabilized microbubbles are likely to have useful clinical applications. Interestingly, these proposed long-lived, artificial microbubbles would resemble in many ways (including their having a lowdielectric material in their core) the biochemical assemblies, i.e., oil-in-water emulsions, already formed in the human body during the process of digestion and absorption of fats from inside the small intestine. For example, once digested fat is absorbed into the intestinal epithelium, the fat is processed further and organized into protein-coated lipid droplets called "chylomicrons" (ref. 447-449). The 0.1-3.5 ~tm diameter sizes of these structures (ref. 270,448) closely resemble the size distribution, usually < 5 ~tm, of long-lived gas microbubbles (see Sections 3.2 and 4.1.1). Moreover, the protein content of chylomicrons even though low (about 2% by weight) is a very consistent feature (ref. 447-450), as is also true for natural microbubble surfactant (see Chapters 3-5) where the protein content is less than 5% by weight (see Chapter 6). The protein coat of chylomicrons, even though incomplete and mixed with various lipids in the interfacial film, helps keep them suspended and prevents them from sticking to each other or to the walls of the lymphatics or blood vessels (ref. 449,450). (Similarly, one might expect that the protein content of natural microbubble surfactant (see Section 1.3.1 and Chapters 3-5) helps prevent the
152 coalescence and/or destruction of the stable gas microbubbles known to be present in natural waters (see Chapters 1, 3, 4, and 5).) However, the presence of a partial protein coat (ref. 194,451,452) is not, of course, an indispensable requirement for all (or even most) stable, spontaneously forming oil-in-water emulsions (ref. 194,453-463). Returning to another related physiological example from the intestinal processing of fats in humans, undigested fat enters the intestine in the form of large lipid globules which undergo emulsification through the detergentlike action of various cholesterol derivatives, i.e., the bile salts (ref. 447-449). This emulsification facilitates subsequent enzymatic degradation (by pancreatic lipase) of the triglycerides to monoglycerides. The monoglycerides, cholesterol derivatives (bile salts), cholesterol, and other lipid components then spontaneously form stable "mixed micelles" (ref. 447,448) in the intestinal lumen. Parallel consideration of results from related artificial oil-in-water emulsion studies offer the suggestion, however, that if the monoglycerides had sufficiently long chain lengths and the rest of the interfacial film contained only cholesterol and/or cholesterol derivatives, one could expect larger, spontaneously forming structures (containing low-dielectric material in their core) to be formed. For example, Schulman et al. (ref. 454) reported that "it has been shown that the chain length of a normal alcohol, when benzene was the oil phase and potassium oleate the micelle forming compound, was a governing factor in controlling the size of the droplets. Increasing the chain length greatly diminished the range of the phase diagram such that above decyl alcohol, no microemulsions could be formed with this system. Only coarse emulsions could be formed of 0.5 ~tm diameter droplets. This occurred also with systems stabilized with cetyl alcohol or cholesterol ... and thus, although the emulsions were spontaneously formed, no microemulsions could be made. It was considered that the interfacial mixed monolayer was too highly condensed with the strong associating components, and thus no great degree of curvature necessary for the very small droplets could be produced, a vapor condensed film being essential" (ref. 454). Accordingly, a detailed series of tests in this laboratory revealed that aqueous solutions of saturated monoglycerides (with acyl chain lengths greater than 10 carbons) combined with
153 cholesterol and cholesterol derivatives did, in fact, form stable zasin-water emulsions when shaken vigorously (in an air atmosphere). Evidence for the formation of these significantly stable (hours to days) emulsions is presented below. 9.2 LASER-BASED FLOW CYTOMETRY A N D FORWARDANGLE LIGHT SCATTERING
All experimental measurements were carried out with a Coulter EPICS V System. This instrument is a laser-based flow cytometer which, as one of its simpler analytical functions, utilizes light scatter measurements to accurately size cells or similar particles (e.g., artificial surfactant-stabilized microbubbles) suspended in aqueous media. The light scatter measurements are sensitive to particle sizes as small as 0.3 ~tm in diameter. To make the measurements, particles in liquid suspension are presented under mild pressure to a flow cell where they are surrounded by a laminar sheath of particle-free liquid. This coaxial stream then exits through a flow chamber as a (76-~tm diameter) jet. This hydrodynamic focusing, which is ordinarily bubble-free when only solid particles are measured, insures that all particles follow the same path through the detection zone. Sample pressure, sheath pressure, and particle concentration are controlled so that particles are presented one-at-a-time to the beam of a UV-enhanced argon-ion laser. As the particles pass through the laser beam, the scatter emissions are collected by detectors which convert the signals to voltage pulses proportional to the amount of light scattered. The pulses are amplified, converted to digital form, and displayed in numeric or histogram formats. In this study, light scattered in specifically the forward direction was monitored, since it is generally proportional to the cross-sectional area (radius 2) of large-size particles (i.e., on the order of a micron) (ref. 129). 9.3 SYNTHETIC MICROBUBBLE COUNTS VERSUS THE CONTROL
A mixture of saturated monoglycerides (with acyl chain lengths greater than 10 carbons) combined with cholesterol and (nordonic) choiesterol derivatives, all initially in powdered form, was used to form high concentrations of artificial gas microbubbles
154 155522
A
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C
SIZE
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Fig. 9.1. Synthetic microbubble histograms determined by laser-light scattering in aqueous media (at 21 ~ (A) saturated solution of Filmix 3 surfactant mixture; (B) distilled water alone; (C) computed difference in above two histograms. (See text for fitrther discussion.)
155 in distilled water. This proprietary surfactant mixture, CAV-CON Filmix 3, contains approximately equal parts of monoglycerides and sterol products. A saturated aqueous solution of the surfactant mixture was simply shaken vigorously for a few seconds, in an air atmosphere at room temperature, to form the concentrated gas-inwater emulsions. Evidence for the formation of synthetic microbubbles was obtained by comparing the particle histograms (Fig. 9.1) for identically preshaken, equivolumetric samples of the artificialmicrobubble-surfactant solution (Fig. 9.1(A)) versus distilled water alone (Fig. 9.1(B)). Collection of the scatter emissions, for a total period of 400 see, commenced one minute after shaking ended in both cases; this one-minute delay allowed essentially all macroscopic bubbles formed in both samples to rise to the surface. It can be seen from the histograms (of identical scale), shown in Fig. 9.1, that the amount of microscopic particles remaining over several minutes in the artificial-microbubble-surfactant solution (Fig. 9.1(A)) was enormously greater than in an equal volume of the distilled water alone (Fig. 9.1(B)); this finding is further confirmed by the total particle counts (over the 400-sec collection period) shown in the upper right corner of both Figs. 9.1(A) and 9.1(B), which differed by more than two orders of magnitude, as well as by the computed difference in the two histograms shown in Fig. 9.1(C ). Additional evidence that the large number of microscopic particles detected in Fig. 9.1(A) are, in fact, artificial surfactantstabilized microbubbles includes the following" 1) Prior to shaking and microbubble formation, the surfactant solution was passed through a membrane filter which removed all solid debris greater than 6.0 ~tm in diameter; 2) The detection limit of the instrument, i.e., particle sizes down to 0.3 ~tm in diameter, strongly argues against the particle count being a representation of a micelle population (which will also be present, but undetected in these experiments). This is especially true since forward-angle light scattering was used, which favors the detection of larger particles in the detection range (0.3-40 ~tm) of the instrument; 3) Since none of the surfactants used are liquids, oil-in-water microemulsions/emulsions could not be the basis of the particle histogram obtained; 4) In accompanying decompression tests, it was found, in the course of developing Filmix 3, that those
156 surfactant solutions which produced the higher concentrations of growing bubbles upon decompression (i.e., below 1 atm) similarly produced the greater degree of light scatter in the absence of decompression. An estimate of the actual concentration of synthetic microbubbles present in the (shaken) artificial-microbubble-surfactant solution, represented by Fig. 9.1(A), is given by the fact that 360400 particles/see were consistently detected at a flow rate of approximately 1 ml/30 min in order to produce the histogram shown. Therefore, the calculated approximate concentration of synthetic microbubbles in the sample is 7 x 105 microbubbles/ml. (A similar calculation for the distilled water sample shown in Fig. 9.1(B) results in an estimated concentration of only 5 x 10 3 microbubbles/ml.) 9.4
MICROBUBBLE FLOTATION WITH TIME
In the next experiment, the relative size distribution of the artificial microbubbles formed with the Filmix 3 surfactant mixture (described in the preceding section) was monitored over a 1,000sec period (Fig. 9.2). While the Coulter EPICS V System used did not record the absolute sizes of microbubbles detected, relative sizes were indicated by the relative intensity of scattered light (increasing toward the right on the abscissa of Figs. 9.1-9.3). Furthermore, during the 1,000-sec collection period, the instrument accumulated microbubble counts over successive 4-sec intervals and, in each case, determined the separate scattered-light intensity levels above which no more than 50 and 5 microbubbles reached during the interval (see Fig. 9.2(B)). The averaged results for the 1,000-see collection period are plotted in both three and two dimensions in Fig. 9.2 (with the total counts given by the 6-digit number). It can be seen that the relative proportions of microbubbles falling below the "50-microbubble" and "5-microbubble" scatteredlight intensity levels (Fig. 9.2) changed little over a period of more than 15 min (i.e., 1,000 see); this finding indicates that little, if any, loss of microbubbles due to (rapid) flotation occurred during this time period. Close inspection of part B of Fig. 9.2, however, does reveal a slight skewing of the microbubble population toward smaller sizes during the 1,000-see period.
157
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SIZE Fig. 9.2. Relative size distribution of artificial microbubbles in an unstirred medium. Light-scatter signals were collected over a total period of 1,000 see, and plotted in three (part A) and two (part B) dimensions. (See text for further discussion.)
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SIZE Fig. 9.3. Relative size distribution of artificial microbubbles in a stirred medium. Light-scatter signals were collected over a total period of 1,500 sec, and plotted in three (part A) and two (part B) dimensions. (See text for further discussion.)
159 9.5
MICROBUBBLE PERSISTENCE WITH TIME
An effort was made to evaluate the possibility that the slight skewing of the artificial microbubble population (in Fig. 9.2) with time might be due to a continuing, even though very slow, dissolution of the newly formed, surfactant-eoated microbubbles. Unlike the experiment referred to in Fig. 9.2, gentle automatic swirling of the preshaken (Filmix 3) surfactant stock solution was now performed as it was fed into the flow cell (see Section 9.2) of the Coulter EPICS V System. Data collection and analysis were the same as in Section 9.4, except that the total collection period was now extended to 1,500 see and each collection interval was 6 see long. The averaged results for the 1,500-see collection period used in this experiment are plotted in both three and two dimensions in Fig. 9.3 (with the total counts given by the 6-digit number). It can be seen that with the constant, gentle swirling of the surfactant solution in this experiment, the relative size of microbubbles falling below either the "50-microbubble" or the "5microbubble" scattered-light intensity levels (Fig. 9.3) decreased more noticeably during the entire collection period (i.e., 25 min). This finding indicates that very slow dissolution of the newly formed, surfactant-coated microbubbles does continue, at least during the first half hour of their existence. Moreover, this gradual rate of dissolution of the artificial microbubbles can apparently be increased somewhat by circulation of the liquid.