An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes

Topotecan can be encapsulated in liposomes, however little is known about the role encapsulated counter ions play in drug loading efficiency and drug release. Using 1,2-distearoyl-sn-glycero-3 phosphatidylcholine and cholesterol liposomes (55:45 mote ratio), encapsulation was achieved using manganese ion gradients (MnSO4 or MnCl2) with the addition of A23187, a divalent cation/proton exchanger, to maintain a pH gradient. This methodology was compared to procedures where the pH gradient was generated by use of encapsulated (NH4)(2)SO4 or citrate (300 mM, pH 3.5). All methods facilitated topotecan encapsulation. Liposomes prepared in the presence of the citrate and MnCl2 (+A23187) exhibited reduced loading capacities. Liposomes prepared in the presence of (NH4)(2)SO4 and MnSO4 (+A23187) could be used to generate liposomes exhibiting a drug-to-lipid ratio of 0.3 (wt/wt) with an encapsulation efficiency of >90%. In vitro drug release data suggested that the (NH4)(2)SO4 and MnSO4 (+A23187) formulations released drug at a reduced rate. For these formulations, the drug release rates decreased as the drug-to-lipid ratio (wt/wt) increased from 0.1 to 0.2. Cryo-electron micrographs indicated that encapsulated topotecan precipitated as linear particles within liposomes. The stability of topotecan loaded liposomes appeared to be dependent on the presence of both a pH gradient and encapsulated sulfate. (C) 2004 Elsevier B.V. All rights reserved.