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

Anti-sense oligonucleotide uptake by keratinocytes in human skin grafts on athymic mice was examined using live confocal microscopy, Fluorescein isoth iocyanate-labeled 15-mer C-5 propyne modified phosphorothioate anti-sense o ligonucleotide (10-50 mu M) was intradermally injected into normal human sk in grafts on athymic mice, and the localization of the anti-sense oligonucl eotide was assessed after 1-24 h postinjection. Anti-sense oligonucleotide was found to localize in the nuclei of basal and suprabasal keratinocytes a fter 1-2 h, and this localization was still observed after 24 h. This live in vivo observation of anti-sense oligonucleotide uptake in basal keratinoc ytes was confirmed using conventional fluorescence microscopy of fixed sect ions of skin grafts. Neither single nucleotides which were fluorescein isot hiocyanate-labeled nor fluorescein isothiocyanate alone was able to penetra te into the nuclei of human skin graft keratinocytes after intradermal inje ction, and hence it is likely that the anti-sense oligonucleotide was not d egraded prior to intracellular localization. Topical administration of anti -sense oligonucleotide and anti-sense oligonucleotide-liposome complexes re sulted primarily in localization in the stratum corneum of human skin graft s. When grafts were tape stripped prior to anti-sense oligonucleotide admin istration, however, as little as 5 mu M anti-sense oligonucleotide was requ ired to observe nuclear antisense oligonucleotide accumulation. These resul ts suggest that cutaneous anti-sense strategies can be tested using deliver y via intradermal anti-sense oligonucleotide injection in human skin grafts on athymic mice, and that agents providing penetration of antisense oligon ucleotide across the stratum corneum are likely to be required for successf ul topical therapies.