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Supplementary Data

Bioreducible branched poly(modified nona-arginine) cell-penetrating

peptide as a novel gene delivery platform

Jisang Yoo1*, DaeYong Lee1*, Vipul Gujrati2, N. Sanoj rejinold1, Kamali Manickavasagam Lekshmi3, Saji Uthaman3, Chanuk Jeong1, In-Kyu Park3, Sangyong Jon2, and Yeu-Chun Kim1†

1 Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and

Technology (KAIST), Daejeon 305-701, Republic of Korea.

2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST),

Daejeon 305-701, Republic of Korea

3 Department of Biomedical Science and BK21 PLUS Centre for Creative Biomedical Scientists,

Chonnam National University Medical School, 160 Baekseo-ro, Gwangju 501-746, Republic of Korea

* These authors contributed equally to this work

† To whom correspondence should be addressed

E-mail: dohnanyi@kaist.ac.kr (Y.C.K.);

Tel (Y.C.K.): +82-42-350-3939; Fax (Y.C.K.): +82-42-350-3910

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Fig. S1. Determination of the absolute molecular weight of B-mR9 by a static light scattering

(SLS) method at different concentrations (1, 0.5, 0.25, 0.125, and 0.0625 mg/mL) of B-mR9.

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Fig. S2. Size change measurement of the B-mR9/siVEGF polyplex at an N/P ratio of 10 in the

presence or absence of 10 mM Glutathione (GSH) for 25 min. siVEGF (5 μg) was mixed with B-

mR9 in 10 mM HEPES buffer (pH7.4). After 30 and 90 min, the size was measured by DLS.

After the GSH treatment, the polyplex was incubated at 37oC and the size change was checked at

10, 15, 20, and 25 min.

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Fig. S3. siVEGF cellular uptake and biocompatibility. (A) An amount of 75 pmol of FITC-

siVEGF with B-mR9 polyplexes (N/P ratio 1, 3, 5, 10, and 15) was delivered in each case into

Hela, SKOV3, and NCI-H460 cells and the cellular uptake was then analyzed by flow cytometry.

(B) Cell viability test by a MTT assay for siVEGF with R9, mR9, B-mR9, and PEI 25k

polyplexes at an N/P ratio of 10 in Hela, SKOV3, and NCI-H460 cells. Phosphate-buffered

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saline (PBS) was used as a positive control (n=3, error bars represent the standard deviation). (C)

Hemolytic activity test using horse blood agar plate, in which 20 μg of siVEGF with R9, mR9,

PEI 25k, and B-mR9 diluted in 0.9% saline (total volume 200 μL) was treated and incubated for

24 h. Triton X-100 diluted in saline was used as a negative control.

.

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Fig. S4. Cell penetration mechanism experiment of the B-mR9/pDNA polyplex. Various

endocytosis inhibitors were added to SKOV3 cells. After incubation for 1 h, cells were treated

with the B-mR9/pDNA polyplex and incubated for 3 h. After 2 days, the pDNA transfection

efficiency was measured by a spectrofluorophotometer (n=3, error bars represent the standard

deviation, ***P < 0.001 versus 37oC). The fluorescence intensities of cells treated only with B-

mR9/pDNA (the 37oC group) without inhibitors were used as a control.

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Fig. S5. Tumor images of each group after extraction at the end of the in vivo experiments (n=5).

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Fig. S6. Intratumoral VEGF contents. After the 22nd day of the in vivo tumor inhibition study,

each tumor was dissected. The tumor tissues (20 mg) were homogenized in lysis buffer and then

centrifuged. The amount of VEGF was measured using a human VEGF ELISA kit (n=3, error

bars represent the standard deviation, **P < 0.01 versus the control).