Silencing TNFα with lipidoid nanoparticles downregulates both TNFα and MCP-1 in an in vitro co-culture model of diabetic foot ulcers

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 alpha (TNF alpha), 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 TNF alpha in the wound bed. Although TNF alpha is a challenging gene silencing target, our lipidoid nanoparticles potently silence TNFa mRNA and protein expression at siRNA doses of 5-100 nM 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 TNF alpha and monocyte chemotactant protein-1 (MCP-1/CCL2) cross-talk that exists in diabetic wounds. Because TNF alpha induces fibroblasts to produce MCP-1, we show that silencing TNF alpha 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 100 nM siTNF alpha downregulated TNF alpha 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. Statement of significance 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 alpha (TNF alpha) in inflammatory macrophages which leads to a subsequent downregulation of fibroblast produced macrophage chemotactant protein-1 (MCP-1). Both TNF alpha 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 TNFa in inhibiting two key inflammatory targets in chronic wounds. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.