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Abstract
G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3β[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms — either large unilamellar vesicles (∼100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.
Acknowledgments
This study was supported in part by ISF 30/98-16.1 to J.S.C. and Y.B., ISF grant 62/03 to YB, and by the Chief Scientist of the Israeli Ministry of Health, grant to J.C. We would like to acknowledge Dr. Robert Clarke of the Lombardi Cancer Research Center, Georgetown University Medical Center for providing us with MCF-7 cells. We thank Dr. Leaf Huang of the Department of Pharmacology and Pharmaceutical sciences, University of Pittsburgh, for providing us with DC-CHOL, Dr. Eli Rochlin of BioLab Ltd., Jerusalem, Israel, for the gift of CCS triacetate salt, Dmitri Simberg of Prof. Y. Barenholz’s lab for characterization of CCS triacetate salt and Genta Inc. for the supplies of G3139 and the reverse sequence G3622.