title: Live confocal microscopy of oligonucleotide uptake by keratinocytes in human skin grafts on nude mice.
abstract: Anti-sense oligonucleotide uptake by keratinocytes in human skin grafts on athymic mice was examined using live confocal microscopy. Fluorescein isothiocyanate-labeled 15-mer C-5 propyne modified phosphorothioate anti-sense oligonucleotide (10-50 microM) was intradermally injected into normal human skin grafts on athymic mice, and the localization of the anti-sense oligonucleotide was assessed after 1-24 h postinjection. Anti-sense oligonucleotide was found to localize in the nuclei of basal and suprabasal keratinocytes after 1-2 h, and this localization was still observed after 24 h. This live in vivo observation of anti-sense oligonucleotide uptake in basal keratinocytes was confirmed using conventional fluorescence microscopy of fixed sections of skin grafts. Neither single nucleotides which were fluorescein isothiocyanate-labeled nor fluorescein isothiocyanate alone was able to penetrate into the nuclei of human skin graft keratinocytes after intradermal injection, and hence it is likely that the anti-sense oligonucleotide was not degraded prior to intracellular localization. Topical administration of anti-sense oligonucleotide and anti-sense oligonucleotide-liposome complexes resulted primarily in localization in the stratum corneum of human skin grafts. When grafts were tape stripped prior to anti-sense oligonucleotide administration, however, as little as 5 microM anti-sense oligonucleotide was required to observe nuclear anti-sense oligonucleotide accumulation. These results suggest that cutaneous anti-sense strategies can be tested using delivery via intradermal anti-sense oligonucleotide injection in human skin grafts on athymic mice, and that agents providing penetration of anti-sense oligonucleotide across the stratum corneum are likely to be required for successful topical therapies.
authors.affiliation: Center for Hormone Research, Royal Children's Hospital, 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: I J
authors.initials: IJ
authors.lastname: Liepe
authors.forename: P M
authors.initials: PM
authors.lastname: Delaney
authors.forename: G A
authors.initials: GA
authors.lastname: Werther
authors.forename: C J
authors.initials: CJ
authors.lastname: Wraight
mesh_terms: D000287:Administration, Topical; D000818:Animals; D004355:Drug Stability; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008808:Mice, Inbred CBA; D008819:Mice, Nude; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000493:pharmacokinetics*; D016038:Skin Transplantation; D014183:Transplantation, Heterologous
chemical_list: D016376:Oligonucleotides, Antisense