Antibody Conjugation of Acoustically Reflective Liposomes for Tissue Targeting

We have previously demonstrated the formulation of acoustic multilamellar liposomes that does not require gas entrapment. Successful antibody conjugation of liposomes, while retaining acoustic properties, could provide an ultrasonic contrast agent for the early detection of atherosclerosis, thrombi, and vegetations. We prepared liposomes from (55 : 10 : 5 : 30) molar mixture of egg phosphatidylcholine, 4-(p-maleimidophenyl)butyrylphosphatidylethanolamine, dipalmitoyalphosphatidylglycerol, and cholesterol by hydration of the lipid film and sonication followed by freeze drying and resuspension. Human gamm globulin was thiolated for conjugation to the liposomes with N-succinimidyl 3-(2-pyridyldithio)propionate. Following reaction, the unbound antibody was removed by separation on Sepharose 4B column. To determine acoustic properties, the conjugated preparation was imaged with 20 mHz intravascular ultrasound catheter (Cardiovascular Imaging System, Sunnyvale, CA) and compared to water and our previously unconjugated liposomes. Results: This method allowed near complete conjugation of liposomes. The average liposomal size following antibody conjugation was 0.5 μm. Magnitude of brightness Δ vs. water in dB (20log[lipo/water]) was 14.5 for unconjugated and 12.7 for conjugated liposomes. We have demonstrated method of antibody conjugation to liposomes that allows the liposomes to retain their acoustic reflectivity. This novel technique allows for target-specific in-vivo acoustic enhancement of pathologic vascular structures.