Liposomes as ultrasound imaging contrast agents and as ultrasound-sensitive drug delivery agents.

Ultrasound imaging contrast agents that exhibit an affinity for specific tissue types, and especially, disease sites, would have considerable clinical value. Liposomal dispersions can be prepared such that they both reflect diagnostic ultrasound [1] and, when conjugated to an appropriate antibody, target themselves to thrombi in the vascular circulation [2]. The procedure for preparation of ultrasound-reflective liposomes, consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH), requires sonication to hydrate the lipid thoroughly, addition of an excipient, lyophilization, and reconstitution. These steps were examined to generate an optimal preparation [3]. Ultrasound reflectivity was assessed using a 20 MHz intravascular ultrasound catheter and computer-assisted videodensitometry. Ultrasound reflectivity was found to be maximal at a CH concentration of 10 mol %. Variation in PG had little effect, although in the total absence of PG, aggregation was undesirably high. Optimal acoustic stability (resistance to loss of reflectivity upon standing following the reconstitution step) was observed with CH concentrations of 10-15 mol % and with PG concentrations greater than 4 mol %. Preparations made with 0.2 M mannitol present during lyophilization were much more ultrasound reflective than those made with similar concentrations of lactose, trehalose, or sucrose. The preparations were stable when stored in the lyophilized state, but became acoustically inactive a few hours after reconstitution at room temperature. Such preparations could be rejuvenated by lyophilizing a second time. Careful attention to formulation conditions produced preparations that could be diluted to 10-50 :g/ml and still produce strong ultrasound reflection. An indication of the basis of the acoustic activity came from measurements of effects of variations in ambient pressure; echogenicity was greatly reduced by exposure to 0.5 atm vacuum or 1.5 atm pressure for 10 s [4]. (Pressure changes of the magnitude that are present in the arterial circulation had little effect on echogenicity.) Such a response suggested that the active lipid preparations contained small amounts of highly dispersed air. Indeed, application of vacuum resulted in the release of approximately 100 :L of air from a standard preparation of 10 mg lipid in 1 mL of buffer.