Preparation and characterization of echogenic liposome as an ultrasound contrast agent: size-dependency and stabilizing effect of cholesterol on the echogenicity of gas-entrapping liposome

The liposome entrapping CO2 gas inside the vesicle, which is called the echogenic liposome, has been made and characterized in vitro as an ultrasound contrast agent. The small unilamellar vesicle (SUV), large unilamellar vesicle (LUV) and multilamellar vesicle (MLV) as echogenic liposomes were compared in their echogenic efficiency and stability, and the effect of size and acoustic property was tested. The acoustic reflectivity increased with the increase in size of the vesicle, largest for the gas filled MLV among the three liposome suspensions. The acoustic reflectivity obtained with the echogenic MLV was larger than that of the gas bubbles enclosed within a surfactant mixture. A half-lifetime of 39 min was observed for the MLV prepared from egg-yolk phosphatidylcholine liposomes. The duration of reflectivity was prolonged drastically to a half-lifetime of 866 min by incorporating cholesterol into the MLV, although the echogenicity was decreased by such incorporation. The stabilizing effect of cholesterol for the ordinary liposomal membrane was thus ascertained in the present case of the gas-entrapping liposome. Our findings encourage the future development of improved gas-entrapping liposomes for the clinical trials of ultrasound contrast agents.