1. Volume 17, Issue 1, Pages 162-168 (January 2009)
Chitosan/siRNA Nanoparticle–mediated TNF-α Knockdown in Peritoneal Macrophages for Anti-inflammatory Treatment in a Murine Arthritis Model 
Kenneth A Howard, Søren R Paludan, Mark A Behlke, Flemming Besenbacher, Bent Deleuran, Jørgen Kjems 
Molecular Therapy 
Volume 17, Issue 1, Pages (January 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Nanoparticle-mediated TNF-α silencing in murine primary macrophages in vitro. Isolated peritoneal macrophages from C57BL/6J mice were transfected with chitosan/TNF-α-specific siRNA or chitosan/TNF-α control for 4 hours (50 nmol/l siRNA/well). The level of TNF-α in the cellular supernantant, measured 5 hours after LPS stimulation 24 or 48 hours after transfection, was used as a measure of TNF-α knockdown (normalized to untreated macrophages (cells only), arbitrarily set to 100). Error bars represent ±SD. Significant differences between NP/TNF-α and NP/Control siRNA are indicated (*P < 0.01; **P < 0.02; two-sided t-test). Data from single experiment with triplicate samples.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Nanoparticle-mediated transfection and TNF-α knockdown in murine peritoneal macrophages in vivo. The ability of chitosan/siRNA nanoparticles to transfect and silence TNF-α production in peritoneal macrophages after i.p. administration. (a) Fluorescence micrograph showing chitosan nanoparticle uptake in macrophages extracted 4 hours after i.p. administration of 200 μl chitosan/Cy3-siRNA nanoparticles (panel A, light image; panel B, Cy3-labeled siRNA (red); panel C, fluorescence overlay of Cy3-labeled siRNA (red) and cells). (b) Macrophage TNF-α production measured by enzyme-linked immunosorbent assay (ELISA) (normalized to untreated LPS-stimulated macrophages (cells only), set to 100; error bars represent ±SD) in LPS-stimulated peritoneal macrophages extracted 2 hours after i.p. administration of 200 μl of chitosan nanoparticles containing ~5 μg of 2′-O-methyl modified anti-TNF-α DsiRNA (NP/TNF-α modified siRNA) or unmodified anti-TNF-α DsiRNA (NP/TNF-α siRNA) or TNF-α Dicer-substrate control siRNA (NP/Control siRNA) in mice (N = 3). Two-sided t-test statistics relative to chitosan/TNFα-control are given (**P < 0.003). (c) ELISA determination of type I IFN expression in macrophages shown in b (normalized to control untreated LPS-stimulated macrophages (cells only), set to 100; error bars represent ± SD). Two-sided t-test statistics for unmodified versus modified siRNA is shown (*P < 0.03).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Therapeutic effects in collagen-induced arthritic (CIA) mice after i.p. administration of chitosan/siRNA nanoparticles. (a) The therapeutic effect of i.p. administration of chitosan/siRNA-TNF-α nanoparticles on the clinical progression of established collagen-induced arthritis. Animals were scored for clinical symptoms using an arthritic scoring method for level of joint inflammation (scale: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling of entire paw, 4 = maximal inflammation in multiple joints; sum total for 4 limbs = 16). Day 1 corresponds to onset of arthritis and assignments to treatment groups (N = 5). Animals were dosed i.p. on days 1, 3, 5, 7, and 9 with 200 μl nanoparticles containing either unmodified (closed circles) or 2′-O-Me modified (open circles) anti-TNF-α DsiRNA or control (closed squares) DsiRNA siRNA, or 0.2 mol/l sodium acetate buffer (open squares). Dexamethasone control group were dosed (400 μg/kg) daily (days 1–14) subcutaneously (open diamonds). Kruskal–Wallis one-way analysis of variance on ranks, followed by pairwise multiple comparison procedures (Dunnett's method) for 2′-O-Me modified versus control siRNA is shown (*P = 0.028), NS, non significant (b) The effect of nanoparticle treatment on CIA mouse survival in groups shown in a. Data from single experiment.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Histopathological evaluation of joints from CIA mice dosed i.p. with chitosan/siRNA nanoparticles. (a) Histological sections from nanoparticle-treated mice. Paws were taken 5 days after treatment with dexamethasone (Panel A), 0.2 mol/l sodium acetate buffer (panel B), chitosan nanoparticles containing unmodified (panel C), 2′-O-Me modified (panel D) anti-TNF-α DsiRNA or control siRNA (panel E). Formalin fixed, decalcified, paraffin-embedded, and hematoxylin and eosin–stained 2-μm sections were assessed for cartilage and bone erosion, and cellular infiltration as evidence of joint inflammation. Representative images of the different groups are shown. Intact cartilage (c) and Panus-forming cellular infiltrates (p) are marked in panels A and B. (b) Histological scoring. Histological scores (scale: 0 = normal, 1 = mild, 2 = moderate, 3 = severe, 4 = maximal) used for cartilage damage (closed triangles) and cellular infiltration (open triangles) evaluation are shown for the different groups (N = 5). Dex, Dexamethasone. Counting was performed using a doublet scoring method.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Prophylactic effects in CIA mice after i.p. administration of chitosan/siRNA nanoparticles. The effect of i.p. administration of chitosan/siRNA-TNF-α nanoparticles on the onset of collagen-induced arthritis. Animals were scored for clinical symptoms using an arthritic scoring method for level of joint inflammation indicated in Figure 3. Animals (N = 8) were dosed i.p. with 200 μl chitosan nanoparticles containing 5 μg of 2′-O-Me modified (open circles) anti-TNF-α DsiRNA or 2′-O-Me modified control siRNA (closed circles) 2 days before collagen immunization and after immunization on days 1, 5, 9, 13, 17, and 21. Untreated group is shown (open squares). Data from single experiment.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions