Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design.

The effect of different formulation factors (lipid type, cholesterol, charge, internal buffer capacity, drug-to-lipid incubation ratio) on the encapsulation efficiency and size of primaquine liposomes (SUV's) in response to a pH gradient was investigated by a fractional factorial screen ing design. Three of the factors (charge, internal buffer capacity, drug -to-lipid incubation ratio) were further studied in a Box--Behnken optimisation design. The lipid type was the most important parameter followed by the drug-to-lipid incubation ratio, buffer capacity, cholesterol and charge. Several of the interactions wer e important. In the optimisation design a robust region with high encapsulation efficiency (>95%) was obtained for DSPC: 33.33 mol% cholesterol-liposomes at high internal citrate concentration (200 mM) by maintaining the drug-to-lipid incubation ratio below 0.15:1 (mol:mol) and varying the charge incorporation between 2 and 10%. In order to achieve long-term stability and sterility, the liposomes were lyophilised followed by gamma irradiation. The pH gradient was maintained during this treatment with little chemical degradation of the substances. The final preparation consisted of three separate vials with lyophilised liposomes, solid state primaquine and hydration medium.