Full Length Research Paper

Cholesteryl cytofectin-oligodeoxyribonucleotide lipoplexes: Protection against serum nuclease digestion and interaction with mammalian cells in vitro

Sharda Balram¹, Mario Ariatti^2*, Moganavelli Singh² and Virendra Rambiritch¹

¹Department of Pharmacology, Westville Campus, University of KwaZulu-Natal, P Bag X54001, Durban 4000, South Africa.

²Department of Biochemistry, Westville Campus, University of KwaZulu-Natal, P Bag X54001, Durban 4000, South Africa.

*Corresponding author. E-mail: ariattim@ukzn.ac.za. Tel.: +27 (0)31 2607981.

Fax: +27 (0)31 2607942.

Abbreviations: Chol-T, 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol; dT₁₈, octadecylthymidylate; [³H]dT₁₈A_7, tritiated octadecylthymidylylheptosyldeoxyadenylate; DOPE, dioleoylphosphatidylethanolamine; FBS, foetal bovine serum; HBS, HEPES buffered saline; HEPES, 2-[4-(2-hydroxyethyl)-piperazinyl]-ethanesulfonic acid; MEM, minimum essential medium; ODN, oligodeoxyribonucleotide; PO-ODN, phosphor-diester oligodeoxyribonucleotide; PS-ODN, phosphorothioate oligodeoxyribonucleotide, PBS, phosphate buffered saline.

Accepted 24 July, 2009

Submicron unilamellar cationic liposomes constructed from equimolar amounts of a cholesteryl cytofectin 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and dioleoylphosphati-dylethanolamine (DOPE) were shown to bind a model antisense oligodeoxyribonucleotide (ODN) dT₁₈ in a saturable manner to afford electropositive lipoplexes. Liposomes imparted to the ODN [³H]dT₁₈A₇ full protection against nuclease catalyzed degradation in 10% foetal calf serum at 37^oC for 3 h, while under the same conditions the naked ODN underwent approximately 50% degradation. Liposomes also promoted association of ODNs to Chinese hamster ovary (CHO) cells while at a liposome (+ve) : ODN (-ve) charge ratio of 1.3:1 lipoplexes were well tolerated by the human hepatoma cell line HepG2 at levels of ODN ordinarily employed  in  antisense ODN-mediated gene knockdown experiments. The findings of this study suggest that cationic liposomes based on the cationic cholesterol derivative Chol-T and the neutral co-lipid DOPE may be used in antisense ODN applications in mammalian cell lines grown in culture.

Key words: Cationic liposome, antisense oligodeoxyribonucleotide, CHO cells, HepG2 cells.