1 2 dipalmitoyl sn glycero 3 phosphatidylcholine

Soy phosphatidylcholine ［ (2R) -2,3-di (tetradecanoyloxy) propyl］ -2-(trimethylazaniumyl) ethyl phosphate, (SPC) ］ was purchased from Calbiochem (Billerica, MA, USA) . 1, 2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) , 1,2-dipalmitoyl-sn-glycero-3-phospho-(1 -rac-glycerol) (DPPG) , (3β ) -cholest-5-en-3-ol (cholesterol) , and curcumin ［1,7-bis (4-hydroxy-3-methoxyphenyl) -1,6-hepatadiene-3,5-dione］ were from Sigma-Aldrich (St. Louis, MO, USA) . Analytical reagent-grade disodium hydrogen phosphate (Na 2 HPO 4 .2H 2 O) , sodium dihydrogen phosphate (NaH 2 PO 4 .2H 2 O) , sodium chloride (NaCl) , HPLC-grade chloroform, and methanol were from Merck Specialities Pvt. Ltd. (Karnataka, India) . The nonionic surfactant polyoxyethylenesorbitanmonooleate (Tween 80) was purchased from Sisco Research Laboratory (Mumbai, India) . Doubledistilled water with specific conductance 2-4 μS (at 25℃) was used to prepare the solutions. All chemicals were ≥ 99％ pure and used as received.

1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) with molecular weight 734 (99% pure), 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) with molecular weight 790 (99% pure), Ethanol 99%, Triton X100, and Trizma buffer with a molecular weight of 121.1 were purchased from Sigma-Aldrich company (St. Louis, Mo, USA). Doxorubicin hydrochloride with mean molecular weight 579 (Adriblastina ®) was purchased from pharmacia Italia S.P.A, Italy. All other chemicals used in this work were of research grade.

Lipid-stabilized microbubbles were prepared as described previously [60] . Briefly, a stock solution is prepared containing 270 mg of 1,2-dipalmitoyl-Sn-glycero-3-phosphatidylcholine, (Sigma, St. Louis, MO), 30 mg of 1,2dipalmitoyl-Sn-glycero-3-phosphatidylethanolamine, (Sigma, St. Louis, MO), and 1 g of glucose. These ingredients were dissolved in a boiling water bath for 20-30 min, with pipetting of contents up and down until no visible particles remain. This stock solution was stored at 4-8 ˚C. Plasmid-containing microbubbles were prepared by mixing 2 mg of dried plasmid with 50 µL of lipofectamine 2000 (Invitrogen, Carlsbad, CA) and incubating at room temperature for 15 min. This liposome/plasmid DNA mixture was added to 400 µL of lipid stock solution, 50 µL of pure glycerol, and 5 µL of 10% albumin solution, mixed well with a pipette, and then placed in ice. Aliquots of 0.5 mL of this phospholipid-plasmid solution were placed in 1.5 mL clear vials; the air in the headspace of the vials was replaced with perfluoropropane gas (Air Products, Inc., Allentown, PA). Each vial was incubated at 4-8˚C for 30 min and then mechanically shaken for 30 s by a dental amalgamator (VialmixTM, Bristol-Myers Squibb Medical Imaging, N. Billerica, MA). The mean diameter and concentration of the microbubbles in the upper layer were measured by a particle counter (Beckman Coulter Multisizer III).