Claims for Patent: 6,287,539

Summary for Patent: 6,287,539

Title:	Methods of imaging using osmotically stabilized microbubble preparations
Abstract:	A microbubble preparation formed of a plurality of microbubbles comprising a first gas and a second gas surrounded by a membrane such as a surfactant, wherein the first gas and the second gas are present in a molar ratio of from about 1:100 to about 1000:1, and wherein the first gas has a vapor pressure of at least about (760-x) mm Hg at 37.degree. C., where x is the vapor pressure of the second gas at 37.degree. C., and wherein the vapor pressure of each of the first and second gases is greater than about 75 mm Hg at 37.degree. C.; also disclosed are methods for preparing microbubble compositions, including compositions that rapidly shrink from a first average diameter to a second average diameter less than about 75% of the first average diameter and are stabilized at the second average diameter; methods and kits for preparing microbubbles; and methods for using such microbubbles as contrast agents.
Inventor(s):	Schutt; Ernest G. (San Diego, CA), Anderson; Charles David (Lebanon, NJ), Evitts; David P. (La Jolla, CA)
Assignee:	Alliance Pharmaceuticals Corp. (San Diego, CA)
Application Number:	08/841,846
Patent Claims:	1. A method of imaging an object, a body part or a body cavity comprising the steps of: introducing into said object, body part or body cavity a microbubble preparation comprising an aqueous medium having dispersed therein a plurality of osmotically stabilized microbubbles, said microbubbles comprising a generally spherical microbubble membrane containing at least one modifier gas and at least one gas osmotic agent, wherein said modifier gas and said gas osmotic agent are present in a molar ratio from about 1:100 to about 1,000:1, wherein said ratio is effective to stabilize said microbubble preparation, with the proviso that said modifier gas is not water vapor, wherein said gas osmotic agent is selected from the group consisting of perfluoropentane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclobutane, perfluorohexane, perfluorocyclohexane, perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane, perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane, perfluorotriethylamine and combinations thereof; and imaging at least a portion of said object, body part or body cavity by ultrasound or magnetic resonance. 2. The method of claim 1 wherein said modifier gas is a fluorocabon gas. 3. The method of claim 2 wherein said fluorocarbon modifier gas is selected from the group comprising perfluoropropane, perfluorobutane, perfluorocyclobutaxe, perfluoromethylcyclobutane, perfluoropentane and perfluorocyclopentane. 4. The method of claim 1 wherein said modifier gas is a nonfluorocarbon gas. 5. The method of claim 4 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixtures thereof. 6. The method of claim 1, wherein said gas osmotic agent has a water solubility of not more than about 0.5 mM at 25.degree. C. and one atmosphere. 7. The method of claim 1 wherein the microbubble membrane comprises a surfactant. 8. The method of claim 7 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 9. The method of claim 7 wherein said surfactant is a non-Newtonian surfactant. 10. The method of claim 7 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextran fatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, phospholipids, polyoxyethylene fat acids esters polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol, oleic acid, sodium oleate and combinations thereof. 11. The method of claim 1 wherein the microbubble membrane comprises a liposome. 12. The method of claim 1 wherein the microbubble membrane comprises a proteinaceous material. 13. The method of claim 12 wherein said proteinaceous material is albumin. 14. The method of claim 1 wherein said gas osmotic agent is perfluoropentane. 15. The method of claim 14 wherein said modifier gas is a fluorocabon modifier gas. 16. The method of claim 14 wherein said modifier gas is a nonfluorocarbon modifier gas. 17. The method of claim 16 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixtures thereof. 18. The method of claim 16 wherein said nonfluorocarbon modifier gas is air. 19. The method of claim 14 wherein the microbubble membrane comprises a surfactant. 20. The method of claim 19 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 21. The method of claim 19 wherein said surfactant is a fluorinated surfactant. 22. The method of claim 14 wherein the microbubble membrane comprises a proteinaceous material. 23. The method of claim 1 wherein said gas osmotic agent is perfluoropentane and said modifier gas is nitrogen. 24. The method of claim 23 wherein the microbubble membrane comprises a surfactant. 25. The method of claim 24 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 26. The method of claim 24 wherein said surfactant is a fluorinated surfactant. 27. The method of claim 24 wherein said surfactant is a non-Newtonian surfactant. 28. The method of claim 24 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextrans fatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol oleic acid, sodium oleate and combinations thereof. 29. The method of claim 1 wherein said gas osmotic agent is perfluorohexane. 30. The method of claim 29 wherein said modifier gas is a fluorocarbon modifier gas. 31. The method of claim 29 wherein said modifier gas is a nonfluorocarbon modifier gas. 32. The method of claim 31 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixtures thereof. 33. The method of claim 31 wherein said nonfluorocarbon modifier gas is air. 34. The method of claim 29 wherein the microbubble membrane comprises a surfactant. 35. The method of claim 34 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 36. The method of claim 34 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextran fatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol oleic acid, sodium oleate and combinations thereof. 37. The method of claim 34 wherein said surfactant comprises a mixture of a phospholipid and a polyoxyethylene-polyoxypropylene copolymer. 38. The method of claim 37 wherein said microbubble membrane further comprises a hydroxy ethyl starch. 39. The method of claim 29 wherein the microbubble membrane comprises a proteinaceous material. 40. The method of claim 39 wherein said proteinaceous material is albumin. 41. The method of claim 1 wherein said gas osmotic agent is perfluorohexane and said modifier gas is nitrogen. 42. The method of claim 41 wherein the microbubble membrane comprises a surfactant. 43. The method of claim 42 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 44. The method of claim 42 wherein said surfactant is a non-Newtonian surfactant. 45. The method of claim 42 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxyethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextran fatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxysmate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol, oleic acid, sodium oleate and combinations thereof. 46. The method of claim 42 wherein said surfactant comprises at least one phospholipid. 47. The method of claim 42 wherein said surfactant comprises a mixture of a phospholipid and a polyoxyethylene-polyoxypropylene copolymer. 48. The method of claim 42 wherein said microbubble membrane further comprises a hydroxy ethyl starch. 49. The method of claim 1 wherein said administrating step comprises intravenous administration of said microbubble preparation. 50. The method of claim 1 wherein said object, body part or body cavity to be imaged comprises a vascular system. 51. The method of claim 1 wherein said object, body part or body cavity to be imaged comprises a perfusion defect. 52. The method of claim 1 wherein said object, body part or body cavity to be imaged comprises myocardial tissue. 53. A method of imaging an object, a body part or a body cavity comprising the steps of: introducing into said object, body part or body cavity a microbubble preparation comprising an aqueous medium having dispersed therein a plurality of osmotically stabilized microbubbles, said microbubbles comprising a generally spherical microbubble membrane containing at least one modifier gas and at least one gas osmotic agent, wherein said modifier gas and said gas osmotic agent are present in a molar ratio from about 1:100 to about 1,000:1, wherein said ratio is effective to stabilize said microbubble preparation and wherein said gas osmotic agent comprises the vapor of a compound which is a liquid at 37.degree. C. and 760 Torr, and imaging at least a portion of said object, body part or body cavity by ultrasound or magnetic resonance. 54. The method of claim 53 wherein said gas osmotic agent is selected from the group consisting of perfluorohexane, perfluorocyclohexane, perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane, perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane, perfluorotriethylamine and combinations thereof. 55. The method of claim 53 wherein said modifier gas is a fluorocarbon gas. 56. The method of claim 55 wherein said fluorocabon modifier gas is selected from the group comprising perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane and perfluorocyclopentane. 57. The method of claim 53 wherein said modifier gas is a nonfluorocarbon gas. 58. The method of claim 57 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixtures thereof. 59. The method of claim 57 wherein said nonfluorocarbon modifier gas is air. 60. The method of claim 53 wherein the microbubble membrane comprises a surfactant. 61. The method of claim 60 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 62. The method of claim 60 wherein said surfactant is a non-Newtonian surfactant. 63. The method of claim 60 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextran fatty acid esters, sorbitol, sorbitol fatty acid ester gelatin, serum albumins, phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol, oleic acid, sodium oleate and combinations thereof. 64. The method of claim 53 wherein the microbubble membrane comprises a liposome. 65. The method of claim 53 wherein the microbubble membrane comprises a proteinaceous material. 66. The method of claim 53 wherein said proteinaceous material is albumin. 67. The method of claim 53 wherein said object, body cavity or body part to be imaged comprises a vascular systems. 68. The method of claim 53 wherein said object, body cavity or body part to be imaged comprises a perfusion defect. 69. The method of claim 53 wherein said object body cavity or body part to be imaged comprises myocardial tissue. 70. A method of imaging an object, a body part or a body cavity comprising the steps of: providing a container having therein microbubble precursor components comprising an aqueous medium, a surfactant, at least one modifier gas that is relatively soluble in the aqueous medium, and at least one gas osmotic agent that is relatively insoluble in the aqueous medium, wherein said modifier gas and said gas osmotic agent are present in a molar ratio from about 1:100 to about 1.000:1, wherein said ratio is effective to stabilize a resulting microbubble, with the proviso that said modifier gas is not water vapor, and wherein said microbubble precursor components are adapted to form microbubbles upon the application of energy thereto; applying energy to said microbubble precursor components to form a microbubble preparation comprising a plurality of microbubbles dispersed in said aqueous medium that are osmotically stabilized when introduced into a physiological liquid, in that the gas osmotic agent is present in an amount that dilutes the modifier gas sufficiently that gases ordinarily dissolved in the physiological liquid in vivo seek to diffuse into the bubble with an osmotic pressure sufficient to counteract the Laplace pressure of the microbubble, said microbubbles comprising a generally spherical microbubble membrane containing said gas osmotic agent and said modifier gas; introducing at least a portion of said microbubble preparation into said object, body part or body cavity; and imaging at least a portion of said object, body part or body cavity by ultrasound or magnetic resonance. 71. The method of claim 70 wherein said gas osmotic agent comprises a fluorocarbon. 72. The method of claim 71 wherein said fluorocarbon gas osmotic agent is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclobutanes, perfluorohexane, perfluorocyclohexane, perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane, perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane, perfluorotriethylamine and combinations thereof. 73. The method of claim 70 wherein said modifier gas is a nonfluorocarbon gas. 74. The method of claim 73 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixture thereof. 75. The method of claim 70 wherein said modifier gas is a fluorocarbon gas. 76. The method of claim 70 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 77. The method of claim 70 wherein said gas osmotic agent is perfluoropentane. 78. The method of claim 77 wherein said microbubble precursor components comprise perfluoropentane in a liquid state. 79. The method of claim 78 wherein said perfluoropentane is emulsified in said aqueous medium. 80. The method of claim 79 wherein the application of energy reduces pressure in said container. 81. The method of claim 80 wherein the application of energy boils said emulsified liquid perfluoropentane to form said microbubble preparation. 82. The method of claim 77 wherein said container is a syringe. 83. The method of claim 78 wherein said modifier gas is nitrogen. 84. The method of claim 83 wherein said perfluoropentane is emulsified in said aqueous medium. 85. The method of claim 84 wherein the application of energy reduces pressure in said container. 86. The method of claim 85 wherein the application of energy boils sad emulsified liquid perfluoropentane to form said microbubble preparation. 87. The method of claim 86 wherein said container is a syringe. 88. The method of claim 83 wherein said object, body cavity or body part to be imaged comprises a vascular system. 89. The method of claim 83 wherein said object body cavity or body part to be imaged comprises a perfusion defect. 90. The method of claim 83 wherein said object, body cavity or body part to be imaged comprises myocardial tissue. 91. The method of claim 83 wherein said administrating step comprises intravenous administration of said microbubble preparation. 92. A method of imaging an object, a body part or a body cavity comprising the steps of: introducing into said object, body part or body cavity a microbubble preparation, comprising an aqueous medium having dispersed therein a plurality of microbubbles, said microbubbles comprising a generally spherical microbubble membrane comprising proteinaceous material containing at least one relatively water-soluble modifier gas and at least one relatively water-insoluble gas osmotic agent in a molar ratio from about 1:100 to about 1.000:1 such that the microbubbles are osmotically stabilized when introduced into a physiological liquid, with the proviso that said modifier gas is not water vapor, and whereby said gas osmotic agent dilutes the modifier gas sufficiently that gases ordinarily dissolved in the physiological liquid in vivo seek to diffuse into the bubble with an osmotic pressure sufficient to counteract the Laplace pressure of the microbubble; and imaging at least a portion of said object, body part or body cavity by ultrasound or magnetic resonance. 93. The method of claim 92 wherein said gas osmotic agent comprises a fluorocarbon. 94. The method of claim 93 wherein said fluorocarbon gas osmotic agent is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclobutanes, perfluorohexane, perfluorocyclohexane, perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane, perfluorodimethylcyclopentane, perfluoromethyl cyclobutane, perfluorotriethylamine and combinations thereof. 95. The method of claim 92 wherein said modifier gas is a nonfluorocarbon gas. 96. The method of claim 95 wherein said nonfluorocarbon modifier gas is selected from the group consisting of nitrogen, oxygen, carbon dioxide and mixtures thereof. 97. The method of claim 92 wherein said modifier gas is a fluorocarbon gas. 98. The method of claim 92 wherein the microbubble membrane further comprises a surfactant. 99. The method of claim 98 wherein said surfactant is selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof. 100. The method of claim 98 wherein said surfactant is a fluorinated surfactant. 101. The method of claim 98 wherein said surfactant is selected from the group consisting of polyoxypropylene polyoxyethylene copolymers, sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxy ethyl starch fatty acid esters, dextrans, dextran fatty acid esters, sorbitol, sorbitol fatty acid esters, gelatin, phospholipids, polyoxyethylene fatty acids esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, cholesterol, oleic acid, sodium oleate and combinations thereof. 102. The method of claim 92 wherein said proteinaceous material is albumin. 103. The method of claim 102 wherein said administrating step comprises intravenous administration of said microbubble preparation. 104. The method of claim 102 wherein said gas osmotic agent comprises perfluoropropane. 105. The method of claim 104 wherein said modifier gas comprises nitrogen. 106. The method of claim 104 wherein said modifier gas comprises oxygen. 107. The method of claim 104 wherein said modifier gas comprises air. 108. The method of claim 104 wherein said modifier gas comprises carbon dioxide. 109. The method of claim 102 wherein said gas osmotic agent comprises perfluoropropane and said modifier gas comprises nitrogen. 110. The method of claim 102 wherein said gas osmotic agent comprises perfluoropropane and said modifier gas comprises oxygen. 111. The method of claim 102 wherein said gas osmotic agent comprises perfluoropropane and said modifier gas comprises a mixture of oxygen and nitrogen. 112. The method of claim 102 wherein said gas osmotic agent comprises perfluoropropane and said modifier gas comprises air. 113. The method of claim 102 wherein said gas osmotic agent comprises perfluorohexane. 114. The method of claim 113 wherein said modifier gas comprises nitrogen. 115. The method of claim 113 wherein said modifier gas comprises oxygen. 116. The method of claim 113 wherein said modifier gas comprises air. 117. The method of claim 113 wherein said modifier gas comprises carbon dioxide. 118. The method of claim 113 wherein said gas osmotic agent comprises perfluorohexane and said modifier gas comprises nitrogen. 119. The method of claim 102 wherein said gas osmotic agent comprises perfluorohexane and said modifier gas comprises oxygen. 120. The method of claim 112 wherein said gas osmotic agent comprises perfluorohexane and said modifier gas comprises a mixture of oxygen and nitrogen. 121. The method of claim 102 wherein said gas osmotic agent comprises perfluorohexane and said modifier gas comprises air. 122. The method of claim 1, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:100 and 1:1. 123. The method of claim 1, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:10 and 1:1. 124. The method of claim 124, in which the molar ratio of said modifier gas and said gas osmotic agent is greater than 1:1. 125. The method of claim 125, wherein the modifier gas is a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 126. The method of claim 126, wherein said plurality of osmotically stabilized microbubbles have a diameter from about 1 to 10 .mu.m. 127. The method of claim 70, wherein the diameter of said plurality of osmotically stabilized microbubbles is about 6 .mu.m. 128. The method of claim 120, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:100 and 1:1. 129. The method of claim 70, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:10 and 1:1. 130. The method of claim 70, in which the molar ratio of said modifier gas and said gas osmotic agent is greater than 1:1. 131. The method of claim 130, wherein the modifier gas is a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 132. The method of claim 131, wherein said plurality of osmotically stabilized microbubbles have a diameter from about 1 to 10 .mu.m. 133. The method of claim 132, wherein said plurality of osmotically stabilized microbubbles have a diameter from about 1 to 10 .mu.m. 134. The method of claim 133, wherein the diameter of said plurality of osmotically stabilized microbubbles is about 6 .mu.m. 135. The method of claim 92, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:100 and 1:1. 136. The method of claim 92, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:1 0 and 1:1. 137. The method of claim 82, in which the molar ratio of said modifier gas and said gas osmotic agent is greater than 1:1. 138. The method of claim 137, wherein the modifier gas is a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 139. The method of claim 138, wherein said plurality of osmotically stabilized microbubbles have a diameter from about 1 to 10 .mu.m. 140. The method of claim 139, wherein the diameter of said plurality of osmotically stabilized microbubbles is about 6 .mu.m. 141. The method of claim 53, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:100 and 1:1. 142. The method of claim 53, wherein the molar ratio of said modifier gas and said gas osmotic agent is between about 1:10 and 1:1. 143. The method of claim 53, in which the molar ratio of said modifier gas and said gas osmotic agent is greater than 1:1. 144. The method of claim 143, wherein the modifier gas is a non-fluorocarbon and the gas osmotic agent is a fluorocarbon. 145. The method of claim 144, wherein said plurality of osmotically stabilized microbubbles have a diameter from about 1 to 10 .mu.m. 146. The method of claim 145, wherein the diameter of said plurality of osmotically stabilized microbubbles is about 6 .mu.m.