PREPARATION OF LIPOSOMES ENCAPSULATING WATER-SOLUBLE COMPOUNDS USING SUPERCRITICAL CARBON-DIOXIDE

In this paper the development of a new preparation method of liposomes containing a water soluble marker (fluorescein isothiocyanate-dextran (FITC-dextran) or zinc phthalocyanine tetrasulfonic acid (TSZnPc) usi ng supercritical carbon dioxide (called ''the supercritical liposome m ethod'') is described. The apparatus used consisted of two main parts: the high-pressure part, in which the lipid components 1-palmitoyl-2-o leoylphosphatidylcholine (POPC) and cholesterol (Chol) (7:3 molar rati o) were dissolved under pressure in supercritical carbon dioxide, and a low-pressure part, in which the homogeneous supercritical solution i s expanded and simultaneously mixed with the aqueous phase to yield li posomes encapsulating the water soluble marker. Addition of 7% absolut e ethanol to carbon dioxide at 25 MPa and 60 degrees C and the use of a high-pressure recycling system during 30 min form the homogeneous so lution with high reproducibility of both lipid components and resulted in an equal expansion profile (recovery after expansion versus time) of POPC and Chol, incubation of the lipid components during 60 min at the above mentioned conditions generated only 3% degradation. The aver age size of the liposomes was about 200 nm and could not be influenced by the experimental conditions used. Optimal values for encapsulated Volume (1.25 L/mol) and efficiency (20%); of the liposomes were obtain ed using statistical experimental design by using the water soluble ma rker TSZnPc and an encapsulation capillary with 5.0 cm length and 0.5 mm inner diameter. The total amount of ethanol used to obtain an encap sulation efficiency of 20% was 15-fold reduced compared to the ethanol injection method of Batzri and Korn.