Development of inherently echogenic liposomes as an ultrasonic contrast agent

Ultrasonic contrast agents have been developed for improved assessment of blood flow and tissue perfusion. Many of these agents are not inherently acoustically reflective (echogenic), and nearly all are not suitable for tissue specific targeting. The purpose of this study was to develop acoustically reflective liposomes, which are suitable for antibody conjugation, without using gas or any other agent entrapment. Echogenic liposomes were prepared from phosphatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH), using a dehydration/rehydration method. The formulation was optimized for higher acoustic reflectivity by varying the lipid composition. Liposomes were imaged with a 20 MHz intravascular ultrasonic imaging catheter. Echogenicity levels were expressed using pixel gray scale. The presence of PE and PG at specific concentrations improved echogenicity due to their effects on liposomal morphology as confirmed by freeze-etch electron microscopy. The acoustic reflectivity of liposomes was retained when liposomes were treated with blood at room temperature and 37 degrees C under in vitro conditions. It was demonstrated that the liposomes were also acoustically reflective in vivo after they were injected into a miniswine model. We have developed echogenic liposomes that are stable and suitable for tissue specific targeting as a novel contrast agent. This new contrast agent can be used for ultrasonic image enhancement and/or treatment of targeted pathologic sites.