title: Oligonucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell type.
abstract: The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation.
authors.affiliation: Center for Hormone Research, Royal Children's Hosptial, Parkville, Victoria, Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: White
authors.forename: R D
authors.initials: RD
authors.lastname: Fogarty
authors.forename: S C
authors.initials: SC
authors.lastname: McKean
authors.forename: D J
authors.initials: DJ
authors.lastname: Venables
authors.forename: G A
authors.initials: GA
authors.lastname: Werther
authors.forename: C J
authors.initials: CJ
authors.lastname: Wraight
mesh_terms: D000328:Adult; D002411:Cation Exchange Resins / Q000494:pharmacology; D002452:Cell Count; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008055:Lipids / Q000494:pharmacology; D008081:Liposomes / Q000494:pharmacology; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002411:Cation Exchange Resins; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine