An Experimental Model for Interpreting Percutaneous Penetration of Oligonucleotides that Incorporates the Role of Keratinoctyes1

Oligonucleotides have been extensively studied for their potential as therapeutic agents. Phosphorothioate oligonucleotides have been demonstrated to be particularly useful due to their stability against nucleases, their ability to be internalized by many cell types, and the ease with which they hybridize with target mRNA. These compounds have previously been delivered across the skin with the aid of iontophoresis. During transdermal delivery, the first viable cells exposed to the oligonucleotides are the keratinocytes. The purpose of this study was to determine the relationship between internalization of these compounds by keratinocytes and their transport across the skin. The in vitro uptake of 15 different fluorescently labeled phosphorothioate oligonucleotides into human keratinocytes was quantitatively measured with a fluorometer. Photomicrographs of keratinocytes indicate diffuse cytoplasmic and nuclear localization. The ability of these molecules to enter cells was linearly related to size. Cellular uptake data were inversely correlated with previously reported steady-state transport levels of oligonucleotides that had been transdermally delivered by iontophoresis across hairless mouse skin. Oligonucleotides that readily entered keratinocytes had a decreased ability to penetrate skin under iontophoretic conditions. The results indicate that oligonucleotide sequences may be designed for treating skin diseases (high uptake, low transport) or systemic disorders (low uptake, high transport).