Volume 22, Issue 2, Pages 397-408 (February 2014) TNF-α Gene Silencing Using Polymerized siRNA/Thiolated Glycol Chitosan Nanoparticles for Rheumatoid Arthritis  So Jin Lee, Aeju Lee, Seung Rim Hwang, Jong-Sung Park, Jiyeon Jang, Myung Sook Huh, Dong-Gyu Jo, Soo-Young Yoon, Youngro Byun, Sun Hwa Kim, Ick Chan Kwon, Inchan Youn, Kwangmeyung Kim  Molecular Therapy  Volume 22, Issue 2, Pages 397-408 (February 2014) DOI: 10.1038/mt.2013.245 Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Schematic diagrams. (a) Uptake of psi-tGC-NPs into macrophage cells leading to tumor necrosis factor (TNF)-α gene knockdown. (b) Formation of poly-siRNA. (c) Synthesis of tGC polymers. (d) Complexation of poly-siRNA with tGC polymers. Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Characterization of psi-tGC-NPs. (a) Confirmation of poly-siRNA formation by gel electrophoresis and gel retardation of mono- or poly-siRNA and complex of tGC with poly-siRNA at different weight ratios (1:2, 1:5, and 1:10). Lane 7 shows reduction of poly-siRNA by adding 1,4-dithiothreitol (DTT). (b) Size and morphology of psi-tGC-NPs by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 In vitro cellular uptake and gene silencing effect of psi-tGC-NPs in murine macrophage RAW 264.7 cells. (a) Confocal microscopic images of RAW 264.7 cells treated with FPR-675–labeled psi-tGC-NPs. Scale bar = 20 µm. (b) Reverse transcription PCR (RT-PCR) images for tumor necrosis factor (TNF)-α mRNA levels obtained from lipopolysaccharide (LPS)-activated RAW 264.7 cells treated with free poly-siRNA, psi(scramble)-tGC-NPs, psi-LF and psi-tGC-NPs targeting TNF-α (100 nmol/l of poly-siRNA) for 4 hours. (c) Semiquantitative analysis for TNF-α mRNA expression. Graph was plotted after normalizing with the gene expression intensity of β-actin. Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 In vivo imaging of psi-tGC-NPs accumulated in the arthritic joints of collagen-induced arthritis (CIA) mice. (a) The paw swelling in CIA model by naked eyes. (b) Arthritic score in CIA model. (c) In vivo near-infrared fluorescence (NIRF) tomographic images of arthritic joints after intravenous injection of psi-tGC-NPs or free poly-siRNA. (d) Fluorescence intensity of FPR-675–labeled poly-siRNA at hind limbs was recorded at each time point (n = 3 mice per group).(e) NIRF reflectance images of CIA mice 24 hours after intravenous injection of psi-tGC-NPs or free poly-siRNA.(f) Ex vivo NIRF images of dissected organs after 24 hours. Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Monitoring of the disease progression in collagen-induced arthritis (CIA) mice. (a) In vivo near-infrared fluorescence (NIRF) images of normal mice (left, top) and CIA mice in each group (untreated, methotrexate (MTX)-treated, and psi-tGC-NP–treated) after intravenous injection of MMP-3 nanoprobe (n = 8) at different stages of disease (at 3, 4, 5, and 7 weeks after the primary immunization). (b) Enlarged paws in the untreated and psi-tGC-NP–treated CIA mice. (c) Mice were scored according to the disease progression. Arthritic score of untreated CIA mice increased rapidly and reached the highest score at 5–7 weeks after the primary immunization. On the other hand, arthritic scores of MTX- and psi-tGC-NP–treated groups were sustained like normal state. (d) The severity of arthritis assessed by measuring hind paw thickness over time (n = 5 mice per group). Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Three-dimensional reconstruction of paws and knee joints and bone volume analysis using Micro-CT. (a) Paw damage was visualized using micro-CT. Computed tomographic images of the paws from untreated, methotrexate (MTX)-treated, and psi-tGC-NP–treated mice with collagen-induced arthritis (CIA) at 4, 5, 6, and 7 weeks (w) after the first immunization are shown (n = 3 per each week). Bone volume of MTX-treated (10.59 ± 1.76) and psi(TNF-α)-tGC-NP–treated (10.76 ± 1.83) knee joint was sustained like normal one (data not shown, 12.10 ± 0.94). (b) Knee joint damage was visualized using micro-CT. Bone volume of MTX-treated (17.95 ± 2.73) and psi(TNF-α)-tGC-NP–treated (16.51 ± 2.91) knee joint was sustained like normal one (data not shown, 19.06 ± 0.73). Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Analysis of tumor necrosis factor (TNF)-α expression in serum and arthritic joints and histological examination. (a) Safranin O staining (upper; red: cartilage, gray green: cytoplasm) and anti-TNF-α staining (lower; brown: TNF-α, purple: counter stain) of an arthritic knee joint at 7 weeks after the first immunization (n = 3 per each group). Scale bar = 100 µm. (b) Each group of collagen-induced arthritis (CIA) mice blood serum was obtained at 4, 5, and 7 weeks after the first immunization, and serum concentrations of TNF-α were measured. Values are the mean ± SEM. *P < 0.05, **P < 0.01 by one-way analysis of variance. (c) Immunoblot results show the expression of TNF-α and MMP-3 in knee joints at 4, 5, and 7 weeks after the first immunization. The band of TNF-α was detected approximately at 52 kDa and the band of MMP-3 was detected at 57 kDa (pro) and 28 kDa (active). β-actin was used as loading control (n = 3 per each group). Lane 1 (N), lane 2 (U4), lane 3 (U5), lane 4 (U7), lane 5 (M4), lane 6 (M5), lane 7 (M7), lane 8 (S4), lane 9 (S5) and lane 10 (S7) indicates normal control group, untreated saline control groups at 4, 5, and 7 weeks, methotrexate (MTX)-treated groups at 4, 5, and 7 weeks, and psi-tGC-NP–treated groups at 4, 5, and 7 weeks, respectively. (d) Representative histological images of transferase dUTP nick end labeling (TUNEL)–stained sections of small intestinal tissues from the normal and CIA mice treated with saline, MTX and psi-tGC-NPs (n = 3 per each group). Scale bar = 100 µm. Molecular Therapy 2014 22, 397-408DOI: (10.1038/mt.2013.245) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions