A Lipid-based Delivery System for Antisense Oligonucleotides Derived from a Hydrophobic Complex

Antisense oligodeoxynucleotides (ASOs) prevent expression of proteins by binding to specific regions of mRNA. This report investigates a potential lipid-based delivery system for ASO. A hydrophobic complex was recovered following addition of cationic lipids to ASOs in a Bligh and Dyer monophase [chloroform/methanol/water (1:2.1:1, v/v/v)]. The addition of monovalent cationic lipids (dioleyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dioleoyltrimethylammonium propane), resulted in >95% recovery of the ASOs from the organic phase when ASO phosphate charge was neutralized. Cholesteryldimethylaminoethylcarbamate mediated efficient extraction at a charge ratio (+/ −) >5.2. ASOs could not be extracted into the organic phase by the polyvalent lipids, dioctadecylamidoglycyl spermine and 2,3-dioleyloxy- N -[2(sperminecarboxamido)ethyl]- N, N -dimethyl-1-propaminium trifluoroacetate, even at a charge ratio (+/ −) >5. Dioleoylphosphatidylethanolamine, but not dioleoylphosphatidylcholine, prevented formation and destabilized the hydrophobic complexes. The characterization of the hydrophobic complex led to the development of lipid-ASO particles containing dioleyldimethylammonium chloride, dioleoylphosphatidylethanolamine and poly(ethylene glycol)-conjugated phosphatidylethanolamine (LAPs). When FITC-labeled ASOs in LAPs were added to B-cell lymphoma cells (DoHH2) in vitro, cell-associated ASO decreased as poly(ethylene glycol)-conjugated phosphatidylethanolamine incorporation increased. Western Blot analysis demonstrated that no significant downregulation of Bcl-2 protein was observed when using LAPs. The results suggest that the use of stabilized PEG-conjugated lipids may be detrimental for cationic lipid-based ASO delivery.