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

Abstract

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.

Keywords::

• 8-Cl-ATP
• 8-NH₂-ATP
• EDOPC
• EDPPC
• encapsulation

Acknowledgments

The authors are very grateful to Nancy L. Krett and Steven T. Rosen (Northwestern University) for MM cells, 8-NH₂-ATP, and scientific discussions.

The authors thank Theodore Jardetzky (Northwestern University) for access to the microplate reader.

The authors dedicate this contribution to the memory of Alec Bangham, an unusually thoughtful and considerate person, who proclaimed, “Let there be liposomes”….and there were!

Declaration of interest

This work was supported by a grant from the Mazza Foundation administered by the Cancer Center of Northwestern University.

Cationic phospholipids of the type used in this investigation were invented by Northwestern University researchers, including one of the authors (R.C.M.). Avanti Polar Lipids holds a license to market these compounds, for which Northwestern University receives royalties.