title: Silencing TNFα with lipidoid nanoparticles downregulates both TNFα and MCP-1 in an in vitro co-culture model of diabetic foot ulcers.
abstract: Diabetes is one of the most formidable diseases facing the world today, with the number of patients growing every year. Poor glycemic control yields a host of complications, such as impaired wound healing. This often results in the formation of diabetic foot ulcers, which carry a poor prognosis because they are notoriously difficult to treat. Current therapies do not address the increased number of infiltrating macrophages to the wound bed that overproduce tumor necrosis factor α (TNFα), which increases fibroblast apoptosis and collagen dismantling and decreases angiogenesis. In this study, we investigated the potential of RNA interference therapy to reduce the inappropriately high levels of TNFα in the wound bed. Although TNFα is a challenging gene silencing target, our lipidoid nanoparticles potently silence TNFα mRNA and protein expression at siRNA doses of 5-100nM without inducing vehicle-related gene silencing or cell death. We also describe the creation of an in vitro macrophage-fibroblast co-culture model, which reflects the TNFα and monocyte chemotactant protein-1 (MCP-1/CCL2) cross-talk that exists in diabetic wounds. Because TNFα induces fibroblasts to produce MCP-1, we show that silencing TNFα results in a downregulation of MCP-1, which should inhibit the recruitment of additional macrophages to the wound. In co-culture experiments, a single lipidoid nanoparticle dose of 100nM siTNFα downregulated TNFα and MCP-1 by 64% and 32%, respectively. These data underscore the potential of lipidoid nanoparticle RNAi treatment to inhibit a positive feedback cycle that fuels the pathogenesis of diabetic foot ulcers.
  
  
  CONCLUSIONS
  Diabetic foot ulcers are a rapidly growing issue worldwide, with current ulcer treatments not as effective as desired. RNA interference therapy represents a largely untapped possible solution to impaired wound healing. We show that siRNA-loaded lipidoid nanoparticles silence the overexpression of tumor necrosis factor α (TNFα) in inflammatory macrophages which leads to a subsequent downregulation of fibroblast-produced macrophage chemotactant protein-1 (MCP-1). Both TNFα and MCP-1 are critical components of the inflammatory feedback loop that exists in chronic wounds. In contrast to the majority of wound drug delivery studies, our study utilizes macrophage/fibroblast co-culture experiments to recapitulate a multicellular wound environment in which cytokine signaling influences inflammation. Results underscore the therapeutic potential of siRNA nanoparticles directed against TNFα in inhibiting two key inflammatory targets in chronic wounds.
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA.
authors.forename: Lisa N
authors.initials: LN
authors.lastname: Kasiewicz
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA; Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Ave, Pittsburgh, PA 15213, USA. Electronic address: kawhite@cmu.edu.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Whitehead
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018932:Chemokine CCL2 / Q000378:metabolism*; D018925:Chemokines / Q000378:metabolism; D018920:Coculture Techniques; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism* / Q000473:pathology; D017719:Diabetic Foot / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D008055:Lipids / Q000737:chemistry*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D019899:Paracrine Communication; D000067996:RAW 264.7 Cells; D034741:RNA, Small Interfering / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D018925:Chemokines; D008055:Lipids; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha
keywords: Chronic wound; Diabetic foot ulcer; Lipid nanoparticle; Lipidoid nanoparticle; Monocyte chemotactant protein (MCP-1/CCL2); Tumor necrosis factor α (TNFα); Wound co-culture; siRNA delivery