Generation of contrast-carrying liposomes of defined size with a new continuous high pressure extrusion method

Abstract

A novel continuous high pressure extrusion method was evaluated for the generation of radiopaque and paramagnetic liposomes. The magnetic resonance contrast agent gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) as well as the X-ray contrast agent iopromide were used as water-soluble model substances for liposomal encapsulation. The continuous process, which is introduced here, allows the fast and efficient extrusion of large batches of liposomal preparations with maximum flow rates of 500 ml/min. Applying high pressures up to 10.5 MPa, MLV prepared by the film method were sequentially extruded through polycarbonate membranes of decreasing pore size. Encapsulation efficiency was found to be dependent on lipid composition and concentration, amount of contrast agent in the preparation as well as choice of final pore size for extrusion. Application of freeze-thaw cycles markedly improved the entrapment of iopromide, whereas for Gd-DTPA freeze-thaw surprisingly turned out to have only minor effects. Entrapment values rose with increasing lipid concentration and fell sharply with increasing solute concentration. Mean liposome diameters could be varied using polycarbonate membranes of differing pore sizes. Smaller final pore sizes led to vesicle populations with smaller mean diameters and lower encapsulation efficiencies. Excellent maximum encapsulation efficiencies of more than 50% for iopromide and over 60% for Gd-DTPA were obtained for vesicles with mean diameters of around 100 nm, as determined by photon correlation spectroscopy (PCS) and confirmed by negative-staining electron microscopy. Employing medium contrast (100 mg/g iodine and 180μmol/g Gd) and lipid concentrations (150 mg/g), entrapment values as high as 40% for the X-ray and 50% for the paramagnetic contrast agent could still be achieved. Best results were obtained using a lipid mixture of soy phosphatidylcholine (SPC), cholesterol (Chol) and soy phosphatidylglycerol (SPG) in a molar ratio of 6:3:1 for iopromide and SPC, Chol 7:3 for Gd-DTPA. Liposomal preparations remained stable upon storage at 2–8°C for 6 months. The new continuous high pressure extrusion method proved to be suitable for the generation of large volumes of stable, contrast-carrying liposomes with outstanding encapsulation efficiencies.