Chitosan/siRNA Nanoparticle–mediated TNF-α Knockdown in Peritoneal Macrophages for Anti-inflammatory Treatment in a Murine Arthritis Model
Authors: Kenneth A Howard, Søren R Paludan, Mark A Behlke, Flemming Besenbacher, Bent Deleuran, Jørgen Kjems
Journal: Molecular Therapy (2008)

Secretion of tumor necrosis factor-α (TNF-α) by macrophages plays a predominant role in the development and progression of rheumatoid arthritis. We demonstrate that knockdown of TNF-α expression in systemic macrophages by intraperitoneal (i.p.) administration of chitosan/small interfering RNA (siRNA) nanoparticles in mice downregulates systemic and local inflammation. Chitosan nanoparticles containing an unmodified anti-TNF-α Dicer-substrate siRNA (DsiRNA) mediated TNF-α knockdown (~66%) in primary peritoneal macrophages in vitro. The presence of Cy3-labeled nanoparticles within peritoneal macrophages and specific TNF-α knockdown (~44%) with TNF-α siRNA after i.p. injection supports our therapeutic approach. Downregulation of TNF-α-induced inflammatory responses arrested joint swelling in collagen-induced arthritic (CIA) mice dosed i.p. with anti-TNF-α DsiRNA nanoparticles. The use of 2′-O-Me-modified DsiRNA resulted in the lowest arthritic scores and correlated with reduced type I interferon (IFN) activation in macrophages in vivo compared with unmodified DsiRNA. Histological analysis of joints revealed minimal cartilage destruction and inflammatory cell infiltration in anti-TNF-α-treated mice. The onset of arthritis could be delayed using a prophylactic dosing regime. This work demonstrates nanoparticle-mediated TNF-α knockdown in peritoneal macrophages as a method to reduce both local and systemic inflammation, thereby presenting a novel strategy for arthritis treatment. Secretion of tumor necrosis factor-α (TNF-α) by macrophages plays a predominant role in the development and progression of rheumatoid arthritis. We demonstrate that knockdown of TNF-α expression in systemic macrophages by intraperitoneal (i.p.) administration of chitosan/small interfering RNA (siRNA) nanoparticles in mice downregulates systemic and local inflammation. Chitosan nanoparticles containing an unmodified anti-TNF-α Dicer-substrate siRNA (DsiRNA) mediated TNF-α knockdown (~66%) in primary peritoneal macrophages in vitro. The presence of Cy3-labeled nanoparticles within peritoneal macrophages and specific TNF-α knockdown (~44%) with TNF-α siRNA after i.p. injection supports our therapeutic approach. Downregulation of TNF-α-induced inflammatory responses arrested joint swelling in collagen-induced arthritic (CIA) mice dosed i.p. with anti-TNF-α DsiRNA nanoparticles. The use of 2′-O-Me-modified DsiRNA resulted in the lowest arthritic scores and correlated with reduced type I interferon (IFN) activation in macrophages in vivo compared with unmodified DsiRNA. Histological analysis of joints revealed minimal cartilage destruction and inflammatory cell infiltration in anti-TNF-α-treated mice. The onset of arthritis could be delayed using a prophylactic dosing regime. This work demonstrates nanoparticle-mediated TNF-α knockdown in peritoneal macrophages as a method to reduce both local and systemic inflammation, thereby presenting a novel strategy for arthritis treatment. IntroductionRheumatoid arthritis is a chronic inflammatory disease affecting ~1% of the population. Joint destruction associated with this autoimmune condition results from synovial infiltration by helper T cells, and mono-polymorphonuclear leukocytes eliciting local proinflammatory and regulatory cytokine effects,1Arend WP Physiology of cytokine pathways in rheumatoid arthritis.Arthritis Rheum. 2001; 45: 101-106Crossref PubMed Google Scholar modulated predominantly by tumor necrosis factor-α (TNF-α) secreted by systemic-derived macrophages. Intervention of the inflammation cascade in mice with anti-TNF-α,2Williams RO Feldmann M Maini RN Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis.Proc Natl Acad Sci USA. 1992; 89: 9784-9788Crossref PubMed Scopus (941) Google Scholar CD4+ T-cell receptor,3Williams RO Mason LJ Feldmann M Maini RN Synergy between anti-CD4 and anti-tumor necrosis factor in the amelioration of established collagen-induced arthritis.Proc Natl Acad Sci USA. 1994; 91: 2762-2766Crossref PubMed Scopus (194) Google Scholar and interleukin (IL)-1 (ref. 4Williams RO Marinova-Mutafchieva L Feldmann M Maini RN Evaluation of TNF-α and IL-1 blockade in collagen-induced arthritis and comparison with combined anti-TNF-α/anti-CD4 therapy.J Immunol. 2000; 165: 7240-7245Crossref PubMed Scopus (97) Google Scholar) monoclonal antibodies provides the basis for present clinical immunotherapy treatments;5Taylor PC Williams RO Maini RN Immunotherapy for rheumatoid arthritis.Curr Opin Immunol. 2001; 13: 611-616Crossref PubMed Scopus (61) Google Scholar however, cost and possible auto-immunity to antibodies and side-effects associated with standard methotrexate and corticosteroid combinational therapy necessitates different approaches.Small interfering RNA (siRNA)-mediated knockdown of proinflammatory cytokines at the messenger RNA (mRNA) level (termed RNA interference),6Lieberman J Song E Lee SK Shankar P Interfering with disease: opportunities and roadblocks to harnessing RNA interference.Trends Mol Med. 2003; 9: 397-403Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar offers an alternative therapeutic strategy to overcome inflammatory conditions. In this process, double-stranded RNAs are cleaved by the cellular nuclease Dicer into short 21–22mer fragments referred to as siRNA, which enter a ribonuclear protein complex termed the RNA-induced silencing complex. Guided by the antisense strand of the siRNA, this complex mediates a specific degradation of the corresponding mRNA.7Elbashir SM Harborth J Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8079) Google ScholarDirect intra-articular injection of TNF-α-specific siRNA has been reported to reduce joint inflammation in murine collagen-induced arthritis (CIA) and supports the use of siRNA-based therapies.8Schiffelers RM Xu J Storm G Woodle MC Scaria PV Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis.Arthritis Rheum. 2005; 52: 1314-1318Crossref PubMed Scopus (132) Google Scholar Furthermore, systemic delivery of TNF-α-specific siRNA using cationic lipid-based nanoparticles show effective anti-inflammatory effects in arthritic mice.9Khoury M Louis-Plence P Escriou V Noel D Largeau C Cantos C et al.Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor α in experimental arthritis.Arthritis Rheum. 2006; 54: 1867-1877Crossref PubMed Scopus (171) Google Scholar Delivery is a key determinant as to whether or not RNAi-based therapeutics will have clinical relevance. In this regard, delivery describes extracellular transport to target cells, intracellular RNA trafficking, and processing. The recruitment of systemic macrophages to local sites and pivotal role during inflammation10Simon J Surber R Kleinstauber G Petrow PK Henzgen S Kinne RW et al.Systemic macrophage activation in locally-induced experimental arthritis.J Autoimmun. 2001; 17: 127-136Crossref PubMed Scopus (39) Google Scholar,11Kinne RW Brauer R Stuhlmuller B Palombo-Kinne E Burmester GR Macrophages in rheumatoid arthritis.Arthritis Res. 2000; 2: 189-202Crossref PubMed Scopus (590) Google Scholar makes these an ideal target for RNAi-based TNF-α therapies. We have previously reported knockdown of endogenous genes in primary macrophages using a chitosan/siRNA nanoparticle (or polyplex) system;12Howard KA Rahbek UL Liu X Damgaard CK Glud SZ Andersen MO et al.RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.Mol Ther. 2006; 14: 476-484Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar however, systemic delivery of polyplexes by the intravenous route is restricted by serum protein–induced aggregation and clearance to the liver.13Dash PR Read ML Fisher KD Howard KA Wolfert M Oupicky D et al.Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.J Biol Chem. 2000; 275: 3793-3802Crossref PubMed Scopus (167) Google Scholar,14Howard KA Dash PR Read ML Ward K Tomkins LM Nazarova O et al.Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA.Biochim Biophys Acta. 2000; 1475: 245-255Crossref PubMed Scopus (68) Google Scholar As an alternative, i.p. injection allows delivery into a blood-free macrophage-rich environment. This has been exploited as a strategy to silence systemic TNF-α production in mice using liposomal/siRNA formulations for the treatment of murine sepsis15Sorensen DR Leirdal M Sioud M Gene silencing by systemic delivery of synthetic siRNAs in adult mice.J Mol Biol. 2003; 327: 761-766Crossref PubMed Scopus (432) Google Scholar and investigating antiviral host immunity in herpes simplex virus infection in mice.16Lundberg P Welander PV Edwards 3rd, CK van Rooijen N Cantin E Tumor necrosis factor (TNF) protects resistant C57BL/6 mice against herpes simplex virus-induced encephalitis independently of signaling via TNF receptor 1 or 2.J Virol. 2007; 81: 1451-1460Crossref PubMed Scopus (59) Google Scholar RNA duplexes, which are ~27 nucleotides in length, termed Dicer-substrate siRNA (DsiRNA),17Rose SD Kim DH Amarzguioui M Heidel JD Collingwood MA Davis ME et al.Functional polarity is introduced by Dicer processing of short substrate RNAs.Nucleic Acids Res. 2005; 33: 4140-4156Crossref PubMed Scopus (258) Google Scholar,18Kim DH Behlke MA Rose SD Chang MS Choi S Rossi JJ Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.Nat Biotechnol. 2005; 23: 222-226Crossref PubMed Scopus (727) Google Scholar have been shown to exhibit higher silencing activity due to processing by Dicer before RNA-induced silencing complex incorporation. Moreover, incorporation of 2′-O-methyl modified (2′-O-Me) nucleotides,19Judge AD Bola G Lee AC Maclachlan I Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.Mol Ther. 2006; 13: 494-505Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar recently in Dicer-substrate duplexes20Collingwood MA Rose SD Huang L Hillier C Amarzguioui M Wiiger MT et al.Chemical modification patterns compatible with high potency Dicer-substrate small interfering RNAs.Oligonucleotides. 2008; 18: 187-200Crossref PubMed Scopus (76) Google Scholar and asymmetric strand design17Rose SD Kim DH Amarzguioui M Heidel JD Collingwood MA Davis ME et al.Functional polarity is introduced by Dicer processing of short substrate RNAs.Nucleic Acids Res. 2005; 33: 4140-4156Crossref PubMed Scopus (258) Google Scholar eliminate nonspecific innate immune effects, e.g., type I interferon (IFN), induced by RNA duplexes21Marques JT Devosse T Wang D Zamanian-Daryoush M Serbinowski P Hartmann R et al.A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.Nat Biotechnol. 2006; 24: 559-565Crossref PubMed Scopus (321) Google Scholar and toll-like receptor (TLR)-rich endosomal localization that is potentiated with nanoparticle delivery.This work presents a novel treatment for arthritis using polymeric nanoparticles, based on systemic knockdown of TNF-α production for reduction in inflammation at local sites by i.p. targeting of macrophages. Investigation into chitosan/DsiRNA nanoparticle knockdown of TNF-α production in stimulated peritoneal macrophages is followed by determination of TNF-α silencing and type I IFN induction on macrophages harvested after i.p. administration using modified and unmodified DsiRNA formulations. The approach was then evaluated as a therapeutic and prophylactic treatment strategy in a type II collagen murine arthritis model by arthritic scoring and histological joint analysis.ResultsPeritoneal macrophage studiesThe fundamental requirement for the strategy presented in this work is an ability to silence TNF-α in difficult-to-silence peritoneal macrophages. We have previously demonstrated knockdown of endogenous enhanced green fluorescent protein in peritoneal macrophages harvested from a transgenic enhanced green fluorescent protein mouse with the chitosan-based nanoparticle system.12Howard KA Rahbek UL Liu X Damgaard CK Glud SZ Andersen MO et al.RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.Mol Ther. 2006; 14: 476-484Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar Self-assembly-driven nanoparticles formed between 27mer DsiRNA TNF-α or control siRNA and chitosan (N:P 63) ranging from ~350 to 450 nm in size were used for in vitro and in vivo silencing studies. The decrease in TNF-α secretion, detected by enzyme-linked immunosorbent assay, in the supernatants of lipopolysaccharide (LPS)-stimulated peritoneal macrophages was used as the determinant for RNA interference in vitro. We accomplished similar levels of TNF-α knockdown at 24 (61.3%) and 48 hours (66.0%) after transfection with chitosan nanoparticles containing unmodified anti-TNF-α-siRNA (at 50 nmol/l concentration per well) compared to nontreated LPS-stimulated control (Figure 1). A slight increase in TNF-α production was seen in the control siRNA nanoparticles (~11%) after 48 hours.Investigation into the ability of i.p.-administered nanoparticles to deliver siRNA into peritoneal macrophage targets was carried out in female C57BL/6J mice. Nanoparticles containing Cy3-labeled DsiRNA were clearly evident within peritoneal macrophages harvested 4 hours after injection with a 200 μl nanoparticle volume (Figure 2a). Punctate Cy3 fluorescence restricted to cytosolic but not nuclear (DAPI blue stain; data not shown) localization in the majority of cells correlated with previous intracellular studies using this nanoparticle system.12Howard KA Rahbek UL Liu X Damgaard CK Glud SZ Andersen MO et al.RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.Mol Ther. 2006; 14: 476-484Abstract Full Text Full Text PDF PubMed Scopus (508) Google ScholarFigure 2Nanoparticle-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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nanoparticle-mediated in vivo silencing of peritoneal macrophage TNF-α production was determined with unmodified and 2′-O-Me modified TNF-α DsiRNA at ~5 μg per dose. Two hours after i.p. injection of nanoparticles, the peritoneal macrophages were harvested and incubated for 24–48 hours ex vivo to allow macrophage adherence and RNAi to function. Significant reduction in the TNF-α level in the supernatants of LPS-stimulated peritoneal macrophages was revealed for both unmodified (36% and 44.3%) and modified (23% and 33%) compared to the untreated and control siRNA nanoparticles, respectively (Figure 2b). The relatively low knockdown may be a consequence of sampling freshly recruited nontransfected macrophages or loss of migratory silenced macrophages from the peritoneum during the 2-hour incubation period. This is supported by lower knockdown in peritoneal macrophages isolated 24 hours after nanoparticle incubation (data not shown). The induction of innate immune responses such as type I IFN by the RNA duplexes and nanoparticle-mediated delivery into a TLR environment was also evaluated in the harvested macrophages. Macrophages taken from mice treated with nanoparticles containing modified DsiRNA revealed lower levels of type I IFN in comparison with both control siRNA (54.8% reduction) and unmodified DsiRNA (58.8% reduction) formulations (Figure 2c). Cells treated with the modified DsiRNA formulation also showed reduced amounts of type I IFN in comparison with untreated, which suggests noninduction of innate immunity. The same physicochemical characteristics such as particle size and charge and amount of siRNA in both nanoparticle systems suggest that the reduced type I IFN induction is indeed due to nucleotide modification.Murine type II CIA treatmentThe therapeutic potential of silencing TNF-α production in systemic macrophages with polymeric nanoparticles was investigated in a collagen type II DBA/I arthritic model.22Moore AR Collagen-induced arthritis.Methods Mol Biol. 2003; 225: 175-179PubMed Google Scholar Onset of arthritis was evident as joint inflammation at ~28 days after collagen immunization and presented on an arthritic scoring scale in Figure 3a (scale: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling of entire paw, 4 = maximal inflammation in multiple joints; sum total for all four limbs 0–16). Rapid and aggressive arthritis progression, characteristic of this arthritic model, was observed in animals treated i.p. with sodium acetate buffer (0.2 mol/l, pH 5.5) that resulted in reduced animal numbers over the course of the study because of the termination of animals exhibiting extreme inflammation. The arthritic scores were carried over if mice were killed to counteract the dropout. The animals were dosed five times (days 1, 3, 5, 7, and 9) i.p. with 200 μl of nanoparticle formulations containing 5 μg unmodified or 2.5 μg 2′-O-Me modified TNF-α DsiRNA or control DsiRNA starting at an arthritic score ~3 (day 1 in Figure 3). Accelerated increase in inflammation during the acute arthritic phase at 1–3 days was observed with all DsiRNA formulations compared to the dexamethasone positive control which stayed at ~3 over the dosing period. Arrested development of inflammation was observed with the modified formulation after the second dose (day 3), while the unmodified and control siRNA continued to rise. The trend with the modified siRNA formulation continued during the dosing period showing a slight rise from the initial 3.5 score to 5. After termination of dosing, control siRNA nanoparticles and buffer-treated mice rose with accelerated progression. The reduced arthritic score of the modified anti-TNF-α siRNA nanoparticle–treated mice compared to those with control siRNA nanoparticles was statistically significant (P = 0.028), whereas the difference between control siRNA nanoparticles and buffer treated mice was not. This confirms specific TNF-α silencing effects of the siRNA.Figure 3Therapeutic 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The survival curves of the animals showed a clear distinction between groups treated either with TNF-α DsiRNA nanoparticles (modified and unmodified) and dexamethasone (100% survival) or the control siRNA nanoparticles and buffer (60% and 40% survival, respectively) (Figure 3b). Histological analysis was performed on paws taken from treatment groups (5 days after treatment) and evaluated for joint destruction defined as cartilage and bone erosion, and joint inflammation that measures hyperplasia of the synovial lining layer and cellular infiltration characteristic of this arthritic model. Joint and cartilage integrity was maintained in animals treated with nanoparticles containing unmodified anti-TNF-α DsiRNA (Figure 4c) and exhibited similar effects to the dexamethasone control (Figure 4a). No panus was evident in this group suggesting noninfiltration by inflammatory cells. In comparison, groups treated with chitosan/control siRNA (Figure 4e) and buffer (Figure 4b) exhibited extreme cartilage and bone destruction occupied predominately by mono- and polymorphonuclear leukocytes. Interestingly, mice treated with 2′-O-Me anti-TNF-α DsiRNA showed some degree of cartilage damage and evidence of cellular infiltration (Figure 4d). This trend is confirmed by histological scoring (scale: 0 = normal, 1 = mild, 2 = moderate, 3 = severe, 4 = maximal) for cartilage damage and inflammation shown in Figure 4b; however, control siRNA and buffer showed higher scores than the modified-treated group. It is possible that these abnormalities would improve if higher dosing were used with the modified duplexes.Figure 4Histopathological 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The prophylactic application of TNF-α silencing was investigated in the CIA model (Figure 5). The rational for this experiment was to silence TNF-α production during the inductive phase of arthritis (day 0 to 28 after collagen immunization) to delay the onset of inflammation. Animals were dosed i.p. with either 2′-O-Me TNF-α-DsiRNA or modified control siRNA nanoparticles (5 μg in 200 μl) 2 days before collagen immunization and after immunization on days 1, 5, 9, 13, 17, and 21. From day 26, control siRNA nanoparticles and buffer-treated mice indicated signs of joint inflammation with scores of 1.3 and 1.5 compared to the modified formulation (0.3). Interestingly, 2 days after removal of the mice from 2′-O-Me TNF-α DsiRNA nanoparticle treatment, accelerated progression of joint inflammation resulted (the arthritic score rising from ~0.3 on day 26 to ~5.3 by day 31) compared to the control siRNA nanoparticles (1.5–2.5) and untreated group (1.5–3.2) during the same period.Figure 5Prophylactic 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThis work describes a novel approach for inflammatory reduction in rheumatoid arthritis by chitosan/siRNA nanoparticle–mediated TNF-α silencing in peritoneal macrophages. Rheumatoid arthritis is a systemic inflammatory disease, manifested locally as erosion of the joints. Pathogenesis from early to chronic rheumatoid synovitis involves systemic-derived cellular infiltration and cytokine production that modulate cellular differentiation, recruitment, and activation responsible for cartilage and bone destruction.1Arend WP Physiology of cytokine pathways in rheumatoid arthritis.Arthritis Rheum. 2001; 45: 101-106Crossref PubMed Google Scholar Recruited peripheral macrophages23Shahrara S Proudfoot AE Woods JM Ruth JH Amin MA Park CC et al.Amelioration of rat adjuvant-induced arthritis by Met-RANTES.Arthritis Rheum. 2005; 52: 1907-1919Crossref PubMed Scopus (98) Google Scholar,24Kirino Y Takeno M Murakami S Kobayashi M Kobayashi H Miura K et al.Tumor necrosis factor α acceleration of inflammatory responses by down-regulating heme oxygenase 1 in human peripheral monocytes.Arthritis Rheum. 2007; 56: 464-475Crossref PubMed Scopus (46) Google Scholar play a predominant role throughout this process by (i) expression in the joints of cytokines, chemokines (including IL-1, TNF-α, IL-8), and matrix-degrading enzymes and (ii) systemically secreted IL-1 and TNF-α-mediated control of IL-6 expression involved in the acute phase response.11Kinne RW Brauer R Stuhlmuller B Palombo-Kinne E Burmester GR Macrophages in rheumatoid arthritis.Arthritis Res. 2000; 2: 189-202Crossref PubMed Scopus (590) Google Scholar Reduced inflammation shown in an arthritic rat model by depleting systemic and joint macrophages with chlodronate-loaded liposomes further supports these cells as therapeutic targets.25Richards PJ Williams AS Goodfellow RM Williams BD Liposomal clodronate eliminates synovial macrophages, reduces inflammation and ameliorates joint destruction in antigen-induced arthritis.Rheumatology (Oxford). 1999; 38: 818-825Crossref PubMed Scopus (83) Google Scholar Furthermore, the improvement in clinical effects with competitive receptor-based immunotherapies against TNF-α5Taylor PC Williams RO Maini RN Immunotherapy for rheumatoid arthritis.Curr Opin Immunol. 2001; 13: 611-616Crossref PubMed Scopus (61) Google Scholar provides the rational for the TNF-α target in this study.A significant TNF-α knockdown was observed in vitro in stimulated primary murine peritoneal macrophages 24 and 48 hours after transfection with chitosan/TNF-α-specific nanoparticles compared to untreated and control siRNA nanoparticles. A slight increase in TNF-α level seen with the control formulation (but not in non-LPS stimulated macrophages) is likely due to unspecific effects or macrophage activation shown to occur in isolated peritoneal macrophages26Mori T Murakami M Okumura M Kadosawa T Uede T Fujinaga T Mechanism of macrophage activation by chitin derivatives.J Vet Med Sci. 2005; 67: 51-56Crossref PubMed Scopus (76) Google Scholar and TNF-α stimulation demonstrated in macrophage cell lines with certain soluble chitosan derivatives.27Feng J Zhao L Yu Q Receptor-mediated stimulatory effect of oligochitosan in macrophages.Biochem Biophys Res Commun. 2004; 317: 414-420Crossref PubMed Scopus (123) Google Scholar The effect of TNF-α-specific knockdown, however, seemingly negates this problem.The ability to target peripheral macrophages after intravenous administration is compromised by the serum instability of polyplexes.13Dash PR Read ML Fisher KD Howard KA Wolfert M Oupicky D et al.Decreased binding to proteins and cells of polymeric gene delivery vectors surface m