Running Title: Chitosan-Based Hybrid Nanocomplex for Sirna Delivery

[Supplementary Information]

Chitosan-Based Hybrid Nanocomplex for siRNA Delivery and Its Application for

Cancer Therapy

Running title: Chitosan-Based Hybrid Nanocomplex for siRNA Delivery

Min-Hyo Ki · Ji-Eon Kim · Young-Nam Lee · Sang Myoung Noh · Sung-Won An · Hyun-

Jong Cho · Dae-Duk Kim

M. H. Ki · Y. N. Lee · S. M. Noh · S. W. An

Chong Kun Dang Research Institute

CKD Pharmaceuticals Inc.

Yongin 446-916, Republic of Korea

M. H. Ki · J. E. Kim · D. D. Kim ()

College of Pharmacy and Research Institute of Pharmaceutical Sciences

Seoul National University

Seoul 151-742, Republic of Korea e-mail: [email protected]

H. J. Cho College of Pharmacy

Kangwon National University

Chuncheon 200-701, Republic of Korea Table SI Specifications of Chitosan Used in this Study.

Chitosan Molecular weight (MW; kDa) Deacetylation degrees (%)

A Chitosan acetate 100 84 B Chitosan acetate 300 84 C Chitosan HCl 50-150 83 D Chitosan HCl 150-400 95 FIGURE LEGENDS

Fig. S1. Calibration curve for the quantification of SVN siRNA. Peak areas of anti-sense strand of SVN siRNA according to standard concentrations are presented.

Fig. S2 Gel retardation assays of siRNA with different chitosans. (A) chitosan acetate (MW:

100 kDa), (B) chitosan acetate (MW: 300 kDa), (C) chitosan HCl (MW: 50-150 kDa), and

(D) chitosan HCl (MW: 150-400 kDa). Complex formation according to the weight ratios between chitosan and siRNA was investigated by gel electrophoresis in 2.5% agarose gel.

Fig. S3 In vitro gene transfection efficiency of different chitosans. (A) chitosan acetate (MW:

100 kDa), (B) chitosan acetate (MW: 300 kDa), (C) chitosan HCl (MW: 50-150 kDa), and

(D) chitosan HCl (MW: 150-400 kDa). SVN expression rate (%) was shown after incubating for 48 h with various weight ratios between chitosan and SVN siRNA in PC-3 cells. Each value was presented as the mean ± SD (n = 3).

Fig. S4 Gel retardation assays of siRNA from nanocomplexes. siRNA-binding capacities were investigated with (A) naked siRNA (Naked), (B) siRNA/Lipofectamine 2000 (L2K),

(C) siRNA/protamine (GP), (D) siRNA/protamine/lecithin (GP-L), and (E) siRNA/protamine/lecithin/chitosan/TPP (GP-L-CT) by gel electrophoresis in 2.5% agarose gel.

Fig. S5 In vitro cellular uptake efficiency of the nanocomplexes in PC-3 cells. Fluorescent siRNA was loaded into various nanocomplex formulations and fluorescence intensity measured by flow cytometry after incubating for 24 h. Plots are presented of side-scattered light (SSC) and fluorescence intensity from (A) naked siRNA (Naked), (B) siRNA/Lipofectamine 2000 (L2K), (C) siRNA/protamine (GP), (D) siRNA/protamine/lecithin (GP-L), and (E) siRNA/protamine/lecithin/chitosan/TPP (GP-L-

Fig. S6 Morphology of cells observed by fluorescence and optical microscopy after cellular uptake study using fluorescent siRNA. The images treated with (A) naked siRNA (Naked),

(B) siRNA/Lipofectamine 2000 (L2K), and (C) siRNA/protamine/lecithin/chitosan/TPP (GP-

L-CT) are presented. 1600

d 1400 n a r t y = 13.896x - 8.5689 s

s R = 0.9998 n

e 1000 s - i t

n 800 a

f o 600 a e r a 400 k a e

0 0 20 40 60 80 100 120 Standard concentration (ug/ml)

Figure S1 Figure S2 80 60 (D) 40 20 100 80 (C) 60 40 100 80 (B) 60 40 120 100 (A) 80 60 UT 0 20 80 60 40 100 120

SVN expression rate (%) 3 Figure S Figure (A) (B) (C) (D) (E)

Figure S4 (A) (B)

(C) (D)

Figure S5 (A) (B) (C)

Fluorescence

Optical

Figure S6