Live confocal microscopy of oligonucleotide uptake by keratinocytes in human skin grafts on nude mice

Paul J. White, Rhys Fogarty, Ingrid J. Liepe, Peter M. Delaney, George A. Werther, Christopher J. Wraight

Research output: Contribution to journalArticle

38 Citations (Scopus)

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 μM) 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 μM 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.

Original languageEnglish
Pages (from-to)887-892
Number of pages6
JournalJournal of Investigative Dermatology
Volume112
Issue number6
DOIs
Publication statusPublished - 1 Jan 1999

Keywords

  • Anti-sense
  • DNA
  • Localization

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

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