University of Ulsan College of Medicine, Seoul 05505, Republic of Korea.

Transcription

1 Nanoparticle-Assisted Transcutaneous Delivery of a Signal Transducer and Activator of Transcription 3-Inhibiting Peptide Ameliorates Psoriasis-like Skin Inflammation Jin Yong Kim 1,5, Jinhyo Ahn 3,4, Jinjoo Kim 2, Minsuk Choi 2, Hyungsu Jeon 2, Kibaek Choe 3,4, Dong Yun Lee 1,6, Pilhan Kim 3,4, Sangyong Jon 1,2,* 1 Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea. 2 KAIST Institute for the BioCentury, Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea. 3 Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141, Republic of Korea. 4 KAIST Institute for Health Science and Technology, KAIST, Daejeon 34141, Republic of Korea. 5 Division of Gastroenterology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea. 6 Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea. * Corresponding author Sangyong Jon, PhD, Graduate School of Medical Science and Engineering, and KAIST Institute for the BioCentury, Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea. Phone: ; Fax: ; syjon@kaist.ac.kr Supplementary Figures

2 Supplementary Figure 1. Viability of NIH3T3 cells following exposure to APTstat3-9R. NIH3T3 cells were incubated with PBS, APTscr-9R (20 µm), or different concentrations (1, 5, 10, and 20 µm) of APTstat3-9R for 24 h. Cell viability was measured by WST-1 assay. Viability of aptide-treated cells was expressed relative to that of control cells as a percentage. Data are means ± SEM of three independent experiments. Supplementary Figure 2. Topical imiquimod causes adverse systemic effects. Imiquimod or Vaseline cream (control) was topically applied to the shaved back skin and ears of mice for 6 consecutive days. During imiquimod application, 100 µg of APTscr-9R or APTstat3-9R or an equivalent volume of PBS was intradermally injected into the indicated area of the back skin. Mice were weighed daily. On day 7, mice were sacrificed and their spleens were isolated and weighed separately. (A) Change in body weight during exposure to applied imiquimod, expressed as a percentage of that for the Vaseline-applied control mice. (B) Spleen weight at day 7. Data are presented as means ± SEM (n = 5 6 mice/group; **P <.01, and ***P <.001; one-way ANOVA with post hoc Tukey test). Ctrl, Vaseline-applied mice; IMQ, imiquimod-applied mice.

3 Supplementary Figure 3. IL-17 expression in imiquimod-induced psoriatic skin lesions. Representative immunohistochemical staining for IL-17 in psoriatic skin lesions induced by imiquimod after 6 days of treatment with PBS, APTscr-9R or APTstat3-9R. Scale bar, 100 µm. Supplementary Figure 4. Transcutaneous administration of APTstat3-9R to imiquimodinduced psoriasis-like ear skin. (A) Experimental protocol. During the course of imiquimod-induced psoriasis-like ear skin inflammation, 50 µg of APTstat3-9R was applied transcutaneously twice per day at a 4-h interval beginning on day 2 of imiquimod application and continuing to day 6. (B) Ear thickness during the course of treatment. (C) Clinical score of skin erythema, scaling, and induration at day 7. Each score is summed and expressed as a cumulative score. In B and C, clinical parameters were assessed as described in Materials and Methods. (D) Ear skin punch biopsy weights. (E) Representative histological images of H&E-stained mouse ear skin after 6 days of transcutaneous application of distilled water (DW) or APTstat3-9R. Scale bar, 100 µm. (F) Quantification of epidermal thickness. Data are presented as means ± SEM (n = 5 mice/group). IMQ, imiquimod-applied mice.

4 Supplementary Figure 5. Graphic representation of the scheme for preparing aptideencased discoid-shaped lipid nanoparticles. DMPC and DHPC were dissolved in chloroform. After mixing appropriate ratios of DMPC and DHPC, the lipid mixture was vortexed and evaporated under a stream of nitrogen gas to yield a thin lipid film. The lipid film was then hydrated with an aqueous solution of APTstat3-9R and sonicated at ambient temperature until a transparent solution was obtained.

5 Supplementary Figure 6. TEM images of lipid nanoparticles at different DMPC/DHPC molar ratios in the absence or presence of APTstat3-9R. Scale bar, 100 nm.

6 Supplementary Figure 7. Analysis of hydrodynamic size and zeta potential. (A, B) Analysis of the hydrodynamic size of lipid nanoparticles with different DMPC/DHPC molar ratios in the absence or presence of APTstat3-9R. Bar graph shows the average hydrodynamic size of lipid nanoparticles, as measured by dynamic light scattering. (C) Zeta potential analysis showing the surface charge distribution of lipid nanoparticles with different DMPC/DHPC molar ratios in the absence or presence of APTstat3-9R. Data are presented as means ± SD.

7 Supplementary Figure 8. The stability of [APTstat3-9R]-DLNPs in various solutions. The stability of [APTstat3-9R]-DLNPs upon incubation in DW, PBS, or 5% FBS containing DW at ambient temperature over time was monitored by dynamic light scattering. Data are presented as means ± SD.

8 Supplementary Figure 9. Optical sectioning by multiphoton microscopy. FITC-APT (20 µg) or [FITC-APT]-DLNPs (20 µg aptide) was transcutaneously applied to the inside ear skin of a disease-free mouse. Images were acquired with a two-photon microscope 6 h after the application of each preparation. (A) Representative serial images of disease-free ear skin at 6- µm intervals below the skin surface. Scale bar, 100 µm. (B) Representative x-z axis orthogonal images of disease-free ear skin. Horizontal scale bar, 100 µm; vertical scale bar, 25 µm. (C) Quantitative analysis of relative fluorescence signals in disease-free ear skin. Average signals were plotted as a function of depth by collecting fluorescence signals for each specimen, subtracting values for untreated specimens from each treated specimen, and normalizing to the signal at z = 0 µm. Data are presented as means ± SEM (n = 4 mice/group).