title: Efficient cationic lipid-mediated delivery of antisense oligonucleotides into eukaryotic cells: down-regulation of the corticotropin-releasing factor receptor.
abstract: Oligonucleotides (ODNs) can be employed as effective gene-specific regulators. However, before ODNs can reach their targets, several physical barriers have to be overcome, as although ODNs may pass cell membranes, most become sequestered in endocytic compartments. Accordingly, sophisticated strategies are required for efficient delivery. Here we have employed a pyridinium-based synthetic amphiphile, called SAINT-2, which carries ODNs into cells in a highly efficient, essentially non-toxic and serum-insensitive manner. Intracellular delivery was examined by monitoring the trafficking of fluorescent ODNs and lipid, and by measuring the effect of specific antisense ODNs on target mRNA and protein levels of the receptor for the neuropeptide corticotropin-releasing factor (CRF-R), expressed in Chinese hamster ovary cells. ODN delivery is independent of lipoplex size, and fluorescently tagged ODNs readily acquire access to the nucleus, whereas the carrier itself remains sequestered in the endosomal-lysosomal pathway. While the release is independent of the presence of serum, it is not observed when serum proteins gain access within the lipoplex, and which likely stabilizes the lipoplex membrane. We propose that the amphiphile-dependent aggregate structure governs complex dissociation, and hence, the biological efficiency of ODNS: We demonstrate an essentially non-toxic and effective antisense-specific down-regulation of the CRF-R, both at the mRNA and protein level.
authors.affiliation: Department of Membrane Cell Biology, University of Groningen, Faculty of Medical Sciences, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Shi
authors.forename: A
authors.initials: A
authors.lastname: Nomden
authors.forename: V
authors.initials: V
authors.lastname: Oberle
authors.forename: J B
authors.initials: JB
authors.lastname: Engberts
authors.forename: D
authors.initials: D
authors.lastname: Hoekstra
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001798:Blood Proteins / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002412:Cations / Q000008:administration & dosage / Q000378:metabolism / Q000633:toxicity; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism*; D005562:Formazans; D020404:Glycerophospholipids / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D008247:Lysosomes / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism*; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds / Q000008:administration & dosage / Q000378:metabolism* / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D013778:Tetrazolium Salts
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D001798:Blood Proteins; D002412:Cations; D005562:Formazans; D020404:Glycerophospholipids; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; C414306:SAINT 2; D013778:Tetrazolium Salts; C079782:MTT formazan