Targeted Microbubble-Liposome Complexes for Cancer

Microbubble-liposome complexes were prepared as previously described (7 (link)). Lipid stocks (Avanti Polar Lipids, Albaster, AL, USA) comprising 15.4 mg of 1,2-dipalmitory-sn-glycero-3-phosphatidylcholine, 3.5 mg of cholesterol, 1 mg of dicetyl phosphate, 1.2 mg of 1,2-dipalmitory-sn-glycero-3-phosphoethanolamine, and 5 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(PDP[polyethylene glycol]-2000) were dissolved in 5 mL of 99.9% chloroform (Sigma-Aldrich). The mixture was lyophilized for 24 hours to remove chloroform, and mixed with 2 mL of solvent comprising glycerin, propylene glycol, and H₂O (1:2:7). Cores of synthesized microbubbles were filled with sulfur hexafluoride gas (SF₆). Liposomes were formed by freezing and thawing the mixture 5 times using liquid nitrogen, followed by agitation in a sonicator at 60℃ for 5 minutes with 2 mL of H₂O. The resultant multilamellar vesicles were extruded using polycarbonate filters (filter size, 200 nm) to obtain liposomes smaller than 200 nm. Microbubbles and liposomes were shaken together at 25℃ for 2 hours to form MLC. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (5 mg; Sigma-Aldrich) was added to MLCs, followed by conjugation with anti-Her2 antibodies (Herceptin®; Roche, Berlin, Germany) by shaking at 4℃ for 24 hours to allow MLCs to target Her2-expressing prostate cancer cells (12 (link)).