Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH₂-ATP required the inclusion of Cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH₂-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.