Evaluation of a new liposome preparation technique, the freeze-thawing method, using L-asparaginase as a model drug.

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Abstract
A new and simple technique for the preparation of liposomes, the freeze-thawing method (FT method), was developed and evaluated for encapsulation efficiency by using L-asparaginase (A-ase) as a model drug. The encapsulation percentage of A-ase in liposomes (EN%) was determined by 3 methods, i.e., the measurement of free and total activities of A-ase, the gel filtration method and the trypsin treatment method. A-ase was encapsulated in FT liposomes with a higher efficiency that in liposomes prepared by the hydration method (HY liposomes), although the shape, the particle size distribution and the appearance of multilamellar structure of FT and HY liposomes were similar of each other. A-ase encapsulated in FT liposomes was resistant to proteolysis by trypsin treatment and to leakage by osmotic shrinkage. The FT method has the following advantages over the HY method for the practical preparation of liposomes as drug carriers: the use of an organic solvent or a detergent is unnecessary; sterilization can be carried out with a membrane filter; and the preparation technique is simple.