STRUCTURAL REQUIREMENTS FOR CATIONIC LIPID-MEDIATED PHOSPHOROTHIOATE OLIGONUCLEOTIDES DELIVERY TO CELLS IN CULTURE

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formula ted with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule I (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes p repared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligo nucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cati onic lipids synthesized with dimyristyl (C-14:0) and dioleyl (C-18:1) alkyl chains were equally effective. Activity of cationic lipids conta ining saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C-14:0) was more effective than dipalmityl (C-16: 0) lipid, which was more effective than distearyl (C-18:0). The phase transition temperature of cationic lipids containing saturated aliphat ic chains was 56 degrees C for the distearyl lipid, 42 degrees C for t he dipalmityl lipid and 24 degrees C for the dimyristyl lipid, Cationi c lipids with dioleyl alkyl chains required DOPE for activity, with op timal activity occurring at 50 mole%. In contrast, a dimyristyl contai ning cationic lipid did not require DOPE to enhance the activity of IS IS 1570. Formulation with different phosphatidylethanolamine derivativ es, revealed that optimal activity was obtained with DOPE. These studi es demonstrate that several cationic lipid species enhance the activit y of phosphorothioate antisense oligonucleotides and provide further i nformation on the mechanism by which cationic lipids enhance the activ ity of phosphorothioate oligodeoxynucleotides.