All the liposome preparation protocols, which involve drug encapsulation are multi-step processes, i.e. they consist of one or several steps of preparation and homogenization. The conditions of converting all lipids into vesicles smaller than 200 nm were determined by replacing ultrasonication with mechanical stirring of the buffer and solution of lipids in a low-boiling point organic solvent or solvents in a simple preparator. Preferably, the process should be carried out at a temperature higher than the temperature of the gel/fluid phase transition (Tm), and higher than the boiling point of the organic solvent(s) used to obtain the lipid solution. For many lipid membrane compositions, the products of preparation are as follows: a dominant fraction of unilamellar vesicles (vesicle of diameter smaller than 200 nm) and a fraction of much larger multivesicular or multilamellar vesicles, easily separated by simple centrifugation at 15000´g. If PEG-phosphatidylethanolamine or cholesteryl palmitate are additional membrane components, multivescular or multilamellar vescicles are virtually absent in the final product, of a single-step process and all the used lipids were quantitatively converted into vesicles smaller than 200 nm in diameter.
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