An Experimental Model for Interpreting Percutaneous Penetration of Oligonucleotides that Incorporates the Role of Keratinoctyes1  Rhonda M. Brand, Kristina Haase, Tracy L. Hannah, Patrick L. Iversen  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1166-1171 (December 1998) DOI: 10.1046/j.1523-1747.1998.00453.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The impact of size on keratinocyte uptake of oligonucleotides is sequence dependent. Cultured human keratinocytes were incubated for 4 h with 5 μM fluorescein-labeled phosphorothioate oligonucleotides. Uptake levels were assayed fluorometrically. Results from the telomerase (TAG) sequence 5′-d(TTAGGG)n-3′ and its complement (CTA) 5′-d(CCCTAA)n-3′ are shown in (a) as T and C, respectively. Then ontelomeric sequences are shown in (b). Data are plotted as mean ± SEM. Journal of Investigative Dermatology 1998 111, 1166-1171DOI: (10.1046/j.1523-1747.1998.00453.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Photomicrographs and flow cytometry give results consistent with the fluorometric assay. Photomicrographs and flow cytometry were performed on cells that had been incubated with 5 μM fluorescein-labeled phosphorothioate oligonucleotides for 4 h. Sections (a), (d), and (g) were photographed under phase contrast, whereas (b), (e), and (h) are the same field using florescence. Exposure times and magnifications were the same for all photographs. Sections (c), (f), and (i) are the flow cytometry data revealing the percentage of cells with associated oligonucleotides. Sections (a), (b), and (c) are controls in which the procedure was followed in the absence of any oligonucleotide. Sections (d), (e), and (f) and (g), (h), and (i) represent the oligonucleotides CTA-6 and P53, respectively. Journal of Investigative Dermatology 1998 111, 1166-1171DOI: (10.1046/j.1523-1747.1998.00453.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Keratinocyte uptake influences iontophoretically enhanced transdermal delivery of phosphorothioate oligonucleotides. The relationship between log transdermal flux (pmole per cm2 h) and log average cellular uptake (fmole per cell h) for phosphorothioate oligonucleotides is presented. Data include all oligonucleotides tested, the best fit curve with the equation log transdermal flux = –0.795 × log(uptake – 0.287), r2 = 0.52 and 95% confidence intervals (dashed lines). TAG and CTA sequences are abbreviated as T and C, respectively. Journal of Investigative Dermatology 1998 111, 1166-1171DOI: (10.1046/j.1523-1747.1998.00453.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Model describing transport of oligonucleotides across skin. The three rate constants k1, k2, and k3 represent transdermal transport, uptake, and release by keratinocytes, respectively. The relative sizes of k1, k2, and k3 can be used to help predict potential clinical uses of particular oligonucleotides. Journal of Investigative Dermatology 1998 111, 1166-1171DOI: (10.1046/j.1523-1747.1998.00453.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions