In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement

Tissue-specific ultrasonic enhancement can be used for the detection and characterization of atherosclerosis. We have previously demonstrated the generation of inherently echogenic (acoustically reflective) liposomes solely by varying lipid composition and controlling the method of production. In this study, echogenic liposomes composed of phosphatidylcholine (PC), 4-(p-maleimidophenyl) butyryl phosphatidylethanolamine (MPB-PE), phosphatidylglycerol (PG), and cholesterol were conjugated to human gamma globulin to determine the effect of antibody conjugation on liposomal acoustic reflectivity. The liposomes remained highly echogenic following antibody conjugation. Echogenic liposomes were also conjugated to rabbit antihuman fibrinogen to study their ability to target fibrin. Antibody-conjugated liposomes were targeted to fibrin-coated filter paper and slides, thrombi made in vitro, and segments of atheroma in an animal model of atherosclerosis. Liposomes were detected by scanning electron microscopy, radiolabeling, and imaging with intravascular ultrasound. Electron microscopy revealed attachment of antibody-conjugated liposomes to fibrin on slides and to the fibrous plaques of the arterial segments, whereas unconjugated liposomes did not attach. Similarly, conjugated liposomes did not attach to normal arteries, indicating their binding to the arterial segment is directed towards a component of the fibrous plaque. Ultrasound imaging of the thrombi demonstrated surface attachment of the acoustic conjugated liposomes. I-125-Labeled liposomes conjugated to rabbit anti-human were targeted to fibrin-coated paper. Counting specifically bound radioactivity showed that >84% of applied liposomes remained attached to the fibrin after washing with saline. These results demonstrate the potential of acoustically reflective liposomes for site-specific targeting and acoustic enhancement.