siRNA-gold nanoparticle mediated ganglioside depletion for diabetic wound healing

描述(由申请人提供)：改善伤口愈合的管理在美国是一个重要的未得到满足的需求，特别是在肥胖症和2型糖尿病患者中。此外，导致胰岛素抵抗的分子事件仍然知之甚少。最近的研究表明，神经节苷脂GM3是一种唾液酸化的膜糖鞘糖脂，是胰岛素抵抗的关键介质，在培养的脂肪细胞和糖尿病小鼠模型中，神经节苷脂耗尽后胰岛素抵抗的逆转就是明证。我们发现GM3积聚在糖尿病小鼠的角质形成细胞(KC)膜上，而GM3的缺失可以逆转它们的伤口愈合缺陷。我们认为，通过使用一种新的纳米技术方法对神经节苷脂合成的遗传抑制将逆转高糖条件下KCs和糖尿病小鼠受损的伤口愈合。该项目的长期目标是应用局部应用的阻断神经节苷脂生物合成的基因抑制纳米颗粒作为一种新的手段来解决糖尿病患者伤口愈合受损的问题，并更好地了解神经节苷脂如何影响KC的增殖和运动。我们将使用我们独特的寡核苷酸连接金纳米颗粒(Au NPs)，这是一种在细胞中表现出普遍摄取和高效基因敲除的单一试剂。我们将首先评估局部应用的GM3合成酶siRNA-Au纳米粒在促进糖尿病小鼠模型愈合方面的有效性和安全性。接下来，我们将确定神经节苷脂是如何影响KC运动的。用DNA-和siRNA-Au纳米粒分别增加和减少神经节苷脂GM3，我们将在体外评估KC的增殖和伤口闭合。然后，我们将研究神经节苷脂对胰岛素受体(IR)、胰岛素样生长因子-1受体-整合素的影响 IGF-1R和表皮生长因子受体(EGFR)的激活，所有这些都影响KC伤口的愈合。最后，我们将评估GM3缺失对葡萄糖诱导的胰岛素抵抗的影响。这些研究将增加我们对神经鞘糖脂在伤口愈合中的作用的了解。此外，局部应用我们的纳米颗粒结合的神经节苷脂合成核酸抑制剂来逆转肥胖糖尿病小鼠的伤口愈合缺陷，将是促进慢性伤口愈合的一种创新方法。这些研究有望对人类伤口的治疗产生重大影响，特别是对患有胰岛素抵抗的糖尿病患者。

DESCRIPTION (provided by applicant): Improved management of wound healing represents a significant unmet need in the United States, particularly in individuals with obesity and type 2 diabetes. In addition, the molecular events that lead to insulin resistance remain poorly understood. Recent studies suggest that ganglioside GM3, a sialylated membrane glycosphingolipid, is a critical mediator of insulin resistance, as evidenced by the reversal of insulin resistance following ganglioside depletion in cultured adipocytes and diabetic mouse models. We have discovered that GM3 accumulates in keratinocyte (KC) membranes in diabetic mice, and that depletion of GM3 reverses their wound healing defect. We propose that genetic inhibition of ganglioside synthesis through the use of a novel nanotechnology approach will reverse impaired wound healing in KCs under high glucose conditions and in diabetic mice. The long-term goals of this project are to apply gene-suppressing topically-applied nanoparticles that block ganglioside biosynthesis as a new means to address the impaired wound healing in diabetics and to better understand how gangliosides impact KC proliferation and motility. We will use our unique oligonucleotide-conjugated gold nanoparticles (Au NPs), single agents that show universal uptake in cells and highly efficient gene knockdown. We will first evaluate the efficacy and safety of topically-applied GM3 synthase siRNA-Au NPs, which deplete gangliosides, in accelerating healing in diabetic mouse models. Next, we will determine how gangliosides impact KC motility. Using DNA- and siRNA-Au NPs to increase and deplete ganglioside GM3, respectively, we will assess KC proliferation and wound closure in vitro. We will then examine the effect of gangliosides on insulin receptor (IR), insulin-like growth factor-1 receptor-integrin (IGF- 1R), and epidermal growth factor receptor (EGFR) activation, all of which impact KC wound healing. Finally, we will evaluate the impact of GM3 depletion on glucose-induced insulin resistance. These studies will increase our understanding of the role of glycosphingolipids in wound healing. In addition, reversal of the wound healing defect in obese diabetic mice by topical administration of our nanoparticle-conjugated nucleic acid inhibitors of ganglioside synthesis will be an innovative means to promote wound healing in chronic wounds. These studies promise to have great impact in the treatment of wounds in humans, particularly in individuals with insulin-resistant diabetes.