title: A lipid-based delivery system for antisense oligonucleotides derived from a hydrophobic complex.
abstract: 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.
authors.affiliation: Department of Advanced Therapeutics, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, B.C, Canada VSZ L3.
authors.forename: F M P
authors.initials: FM
authors.lastname: Wong
authors.forename: S A
authors.initials: SA
authors.lastname: MacAdam
authors.forename: A
authors.initials: A
authors.lastname: Kim
authors.forename: C
authors.initials: C
authors.lastname: Oja
authors.forename: E C
authors.initials: EC
authors.lastname: Ramsay
authors.forename: M B
authors.initials: MB
authors.lastname: Bally
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000737:chemistry; D004337:Drug Carriers; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D016393:Lymphoma, B-Cell / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D004337:Drug Carriers; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D011092:Polyethylene Glycols; D004247:DNA; D016650:Fluorescein-5-isothiocyanate