Increased oligonucleotide permeability in keratinocytes of artificial skin correlates with differentiation and...

We tested the permeability of fluorescent oligonucleotides in cultured human epidermal keratinocyte monolayers and keratinocytes grown in a 3-dimensional skin model. Oligonucleotide permeability in living cells was determined by confocal microscopy after either simple addition to the culture medium or topical application via oligonucleotide-saturated filters placed atop the artificial skin. In cultured monolayers, few keratinocytes (9%) were found to acquire intracellular oligonucleotides that were primarily localized to the nucleus. In contrast, keratinocytes grown in an artificial, 3-dimensional skin matrix acquired extensive oligonucleotide permeability as differentiation progressed. About 95% of the granular cells showed nuclear accumulation of oligonucleotides. About 70% of the oligonucleotide-permeable granular cells were viable as verified by a mitochondria-specific, potential-sensitive dye, tetramethyl rhodamine ethyl ester. A marker used to study apoptotic cells with altered membrane potential, merocyanine 540, was found elevated in the cytoplasm of granular cells. In contrast, cultured keratinocyte monolayers or basal keratinocytes of skin showed a membrane staining pattern typical of undifferentiated cells. Few cells (<3%) of the basal layer had nuclear oligonucleotides, but none of the labeled cells were viable. These results suggest that the development of oligonucleotide and merocyanine 540 permeability in differentiated granular cells parallels the changes in membrane permeability found in other apoptotic systems.