Perfluorobutane Microbubbles for siRNA Delivery

Perfluorobutane gas-filled lipid MBs carrying either Silencer Select siRNA (Ambion) against luciferase or a negative control sequence were prepared as previously described [16 (link), 17 (link)]. Briefly, chloroform solutions of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (Avanti Lipids), and polyethylene glycol-40 stearate (Sigma) at a mole ratio 100:43:1:4.5 were dried with argon gas. An aqueous micellar solution of lipid was then prepared by adding phosphate buffered saline (PBS) containing 1 mmol/L EDTA and the solution was sonicated (Misonix) to disperse lipids. The resulting lipid solution was diluted 1:4 in PBS/EDTA, and 8 μg of Silencer Select siRNA against luciferase or a negative control sequence was added. The solution was placed in a sealed glass vial and the head space was filled with perfluorobutane gas (FlouroMed), followed by amalgamation (Bristol Myers-Squibb) to form perfluorobutane gas-filled MBs [17 (link)]. The resulting siRNA-loaded MB solution contained approximately 7×10⁸ MB/mL with a mean diameter of 2.1±1.1 μm as measured by a Coulter Multisizer 3 (Beckman Coulter).