Decafluorobutane Microbubble Formulation

Decafluorobutane microbubbles were formulated by the dissolution of 1,2-dipalmitoyl-sn-glycero 3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-polyethyleneglycol-2000 (DPPE-PEG-2000), and 1,2-dipalmitoyl-3-trimethylammonium propane (chloride salt; 16:0 TAP) in a molar ratio of 65:5:30 and a total lipid concentration of 0.75 mg/mL, 1.5 mg/mL, and 3 mg/mL. The excipient liquid was comprised of propylene glycol, glycerol, and normal saline (15:5:80). After adding 1.5 mL of the resulting solution to a 2 mL vial, microbubbles were formed via agitation using a Vialmix™ shaker (Bristol-Myers-Squibb, New York, NY) for 45 seconds. The 2 mL vial containing the formed microbubbles was then immersed in a CO₂/isopropanol bath controlled to a temperature of approximately −5° C. A 25 G syringe needle containing 30 mL of room air was then inserted into the vial septum and the plunger depressed slowly until the headspace of the vial was pressurized to between 600 – 750 kPa (approximately 85–110 psi). Lipid freezing was avoided by observing the contents of the vial as well as the temperature of the CO₂/isopropanol solution periodically. The syringe needle was removed from the vial after pressurizing, leaving a pressure head on the solution.