<p>[Supplementary Information]</p><p>Chitosan-Based Hybrid Nanocomplex for siRNA Delivery and Its Application for</p><p>Cancer Therapy</p><p>Running title: Chitosan-Based Hybrid Nanocomplex for siRNA Delivery</p><p>Min-Hyo Ki · Ji-Eon Kim · Young-Nam Lee · Sang Myoung Noh · Sung-Won An · Hyun-</p><p>Jong Cho · Dae-Duk Kim</p><p>M. H. Ki · Y. N. Lee · S. M. Noh · S. W. An</p><p>Chong Kun Dang Research Institute </p><p>CKD Pharmaceuticals Inc. </p><p>Yongin 446-916, Republic of Korea</p><p>M. H. Ki · J. E. Kim · D. D. Kim ()</p><p>College of Pharmacy and Research Institute of Pharmaceutical Sciences </p><p>Seoul National University </p><p>Seoul 151-742, Republic of Korea e-mail: [email protected]</p><p>H. J. Cho College of Pharmacy </p><p>Kangwon National University </p><p>Chuncheon 200-701, Republic of Korea Table SI Specifications of Chitosan Used in this Study.</p><p>Chitosan Molecular weight (MW; kDa) Deacetylation degrees (%)</p><p>A Chitosan acetate 100 84 B Chitosan acetate 300 84 C Chitosan HCl 50-150 83 D Chitosan HCl 150-400 95 FIGURE LEGENDS</p><p>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.</p><p>Fig. S2 Gel retardation assays of siRNA with different chitosans. (A) chitosan acetate (MW:</p><p>100 kDa), (B) chitosan acetate (MW: 300 kDa), (C) chitosan HCl (MW: 50-150 kDa), and</p><p>(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. </p><p>Fig. S3 In vitro gene transfection efficiency of different chitosans. (A) chitosan acetate (MW:</p><p>100 kDa), (B) chitosan acetate (MW: 300 kDa), (C) chitosan HCl (MW: 50-150 kDa), and</p><p>(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).</p><p>Fig. S4 Gel retardation assays of siRNA from nanocomplexes. siRNA-binding capacities were investigated with (A) naked siRNA (Naked), (B) siRNA/Lipofectamine 2000 (L2K),</p><p>(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.</p><p>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-</p><p>CT).</p><p>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),</p><p>(B) siRNA/Lipofectamine 2000 (L2K), and (C) siRNA/protamine/lecithin/chitosan/TPP (GP-</p><p>L-CT) are presented. 1600</p><p> d 1400 n a r t y = 13.896x - 8.5689 s</p><p>1200</p><p> e 2</p><p> s R = 0.9998 n</p><p> e 1000 s - i t</p><p> n 800 a</p><p> f o 600 a e r a 400 k a e</p><p>P 200</p><p>0 0 20 40 60 80 100 120 Standard concentration (ug/ml)</p><p>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</p><p>SVN expression rate (%) 3 Figure S Figure (A) (B) (C) (D) (E)</p><p>Figure S4 (A) (B)</p><p>(C) (D)</p><p>(E)</p><p>Figure S5 (A) (B) (C)</p><p>Fluorescence</p><p>Optical</p><p>Figure S6</p>