Actinomycin D(AMD), a potent antitumor agent, was incorporated into egg phosphatidylcholine vesicles mixed with various molar ratio of cholesterol, to yield information about effective loading method. Also, the influences of cholesterol and temperature on the molecular transport of actinomycin D across large unilamellar lipid bilayer were studied.
The two types of loading methods for liposomal formulation of AMD were introduced and each efficacy was investigated in terms of entrapment efficiency and stability of drug. In physical binding method, the drug formed complexes with lipid mixtures and the entrapment efficiency was reduced with the addition of cholesterol because AMD served as membrane constituents. In transmembrane loading method, the drug transported through the bilayer by pH gradient with high entrapment efficiency. The size distribution of liposome suspensions was not significantly affected by the addition of cholesterol in transmembrane loading method, whereas in the physical binding method, the increase of effective diameter was observed, originated from the aggregates of cholesterol. In the natural release experiment, the liposome formulation was more stable when prepared by the physical binding method than that of the transmembrane loading method was.
In the transmembrane loading method, the effect of transbilayer pH gradient was studied on the rate of uptake and entrapment efficiency. Liposomes prepared with a transbilayer pH gradient of 1.4 units (inner pH is 7.4, outer pH is 8.8), showed the higher entrapment efficiency and the faster rates of translocation of drug than vesicles without pH gradient did. In order to increase the rate of uptake, various temperatures of loading were employed. A First order kinetic analysis was employed to determine the activation energy and to compare the rates of uptake quantitatively. The activation energy was 2.1 fold larger in liposomes without ΔpH than that of vesicles with ΔpH 1.4. The effects of cholesterol and temperature on the transport kinetics of AMD on lipid bilayer, of which pH gradient was 1.4, were investigated. Liposomes were prepared with transbilayer ΔpH 1.4, and the temperature dependent parameter, uptake rate constant and activation energy were experimentally determined. The transport kinetics of AMD across bilayer observed with time. The results showed that cholesterol retarded the rate of transport of AMD across liposome bilayer. At fixed content of cholesterol, the elevation of loading temperature also, enhanced the rate of uptake. The activation energy of transbilayer movement of AMD was varied with the content of cholesterol.
The reason why the rate of AMD transport across EPC liposomes was sensitive to the content of cholesterol and loading temperature was assumed due to change of membrane fluidity. To verify the effects of temperature and cholesterol on the fluidity of EPC liposomes, membrane polarization was measured using fluorescence probe. The addition of cholesterol decreases the membrane fluidity and elevation of temperature increases the membrane fluidity. Therefore, it can be proposed that, in EPC bilayers, addition of cholesterol and a decrease of temperature are ultimately equivalent. These results were well accordance with the experiments of kinetic analysis.
