Solid-in-oil peptide nanocarriers for transcutaneous cancer vaccine delivery. against melanoma
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1 Supplementary Information for Solid-in-oil peptide nanocarriers for transcutaneous cancer vaccine delivery against melanoma Rie Wakabayashi,,a,b Masato Sakuragi,,a Shuto Kozaka, a Yoshiro Tahara, a Noriho Kamiya, a,b and Masahiro Goto, *,a,b,c a Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Motooka 744, Nishi-ku, Fukuoka , Japan. b Advanced Transdermal Drug Delivery System Center, Kyushu University, Motooka 744, Nishiku, Fukuoka , Japan. c Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka , Japan. These authors contributed equally. *Corresponding author. m-goto@mail.cstm.kyushu-u.ac.jp Table of Contents 1. Characterization of K-TRP-2 peptides 2. Morphological analysis of S/O nanodispersions carrying K-TRP-2 peptide 3. Stability of S/O nanodispersions carrying K-TRP-2 peptide 4. Skin permeation of FITC-K-TRP-2 peptide 5. Skin permeation route of FITC-K-TRP-2 peptide 6. Size distribution of L-195 S/O co-loading with R Characterization of L-195 S/O co-loading with R Distribution of CD8 + cells in tumors 9. Tumour growth in individual mouse 10.Delivery of K-TRP-2 peptide using patch 11.Transcutaneous delivery of K-TRP-2 peptide to Langerhans cells S2 S3 S3 S4 S4 S5 S5 S6 S7 S8 S9 S1
2 1. Characterization of K-TRP-2 peptides Synthesized K-TRP-2 peptide was analyzed by HPLC and MALDI TOF MS as follows: Shimadzu HPLC system (pump: LC-20AT, UV detector: SPD-20A, degasser: DGU-20A5, control unit: GL science START CONTROL UNIT) was equipped with a GL science Inertsil ODS-3 column ( mm). K-TRP-2 peptide was dissolved in water at a concentration of 1 mg/ ml and 20 μl of the solution was injected for the analysis. Gradient elution of 80%A 20%B to 50%A 50%B in 60 min was used for the analysis, where A is water and B is acetonitrile, both containing 0.1% TFA. For MALDI TOF MS, above K-TRP-2 peptide solution was diluted ca. 100-fold using water and mixed with CHCA solution, then spotted on a sample stage. The mass analysis was conducted using linear positive mode on a Bruker Autoflex-III. Figure S1. Characterization of K-TRP-2 peptide by HPLC (a) and MALDI TOF MS (b). Conditions: (a) Inertsil ODS-3 (GL science, mm), 20%B to 50%B in 60 min (A: water, B: acetonitrile, both containing 0.1% TFA), 1 ml/min, 220 nm detection and (b) linear positive mode, matrix: α-chca. S2
3 2. Morphological analysis of S/O nanodispersions carrying K-TRP-2 peptide Figure S2. Transmission electron microscope images of L-195 S/O (a) and ER-290 S/O (b) stained with uranyl acetate. Bars: 200 nm. 3. Stability of S/O nanodispersions carrying K-TRP-2 peptide Figure S3. Particle stability of S/O nanodispersions carrying K-TRP-2 peptide. n = 3. S3
4 4. Skin permeation of FITC-K-TRP-2 peptide Figure S4. Fluorescence intensity profile of treated mice skin with FITC-K-TRP-2 PBS solution, L-195 S/O and ER-290 S/O. 5. Skin permeation route of FITC-K-TRP-2 peptide Figure S5. Z-axis projection view of confocal fluorescence microscopic images of mouse ear skin (acquired through 60 μm) treated with FITC-K-TRP-2 L-195 S/O for 1 h. Bar: 100 μm. S4
5 6. Size distribution of L-195 S/O co-loading with R-848 Figure S6. Size distribution of L-195 S/O co-loading with R-848 in the oil phase. 7. Skin permeation and release of FITC-K-TRP-2 using L-195 S/O co-loading with R-848 Release rate [%] Time [h] L-195 S/O L-195 S/O (+R-848) Figure S7. Skin permeation of FITC-K-TRP-2 using Yucatan micro pig skin (a) and release of FITC-K-TRP-2 from L-195 S/O with and without R-848 (b). S5
6 8. Distribution of CD8+ cells in tumors Figure S8. Distribution of CTLs in tumors of unvaccinated (top) or vaccinated (bottom) mice. Harvested tumors were frozen sectioned with 20 µm thickness and the sections were stained with APC anti-mouse/human CD8 and DAPI. Bars: 100 µm. S6
7 9. Tumour growth in individual mouse Figure S9. Tumor growth of individual mice vaccinated with K-TRP-2 peptide. S7
8 10.Delivery of K-TRP-2 peptide using patch Figure S10. Skin permeation of FITC-K-TRP-2 peptide using a patch. S8
9 11. Transcutaneous delivery of K-TRP-2 peptide to Langerhans cells K-TRP-2 peptide was labeled with Cy5 using Cy5 monofunctional reactive dye (GE healthcare UK Ltd.). S/O nanodispersion containing Cy5-labeled K-TRP-2 was prepared as reported in the main manuscript and was administered transcutaneously to back of C57BL/6N mice. After 6 h, the skin of the administration site was harvested and washed with ethanol/pbs. The skin segments were embedded in O.C.T. compound and frozen in liquid nitrogen. Sections of 20 μm thickness were prepared with a Leica CM1860UV cryostat. For the detection of Langerhans cells, PE-conjugated anti-mouse/human CD207 (BioLegend) was diluted 20-fold using 0.1% Tween 20/PBS containing 5% Blocking One (nacalai tesque) and skin sections were immersed overnight at 4 C. Lymph node was harvested after 24 h of administration and stained sections were prepared by the same procedure. Stained sections were imaged on a Zeiss LSM700 with diode lasers (555 nm for PE, 639nm for Cy5). Figure S11. Transcutaneous delivery of Cy5-labeled antigen peptide in the skin. Encircled regions indicate the uptake of Cy5-K-TRP-2 by Langerhans cells. Bars: 100 μm. S9
10 Figure S12. Migration of Langerhans cells internalizing Cy5-labeled antigen peptide in lymph node. Bars: 100 μm. S10
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