siRNA-gold nanoparticle mediated ganglioside depletion for diabetic wound healing

描述（由申请人提供）：在美国，改善伤口愈合的管理是一个显著未满足的需求，特别是在肥胖和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GM 3，唾液酸化膜鞘糖脂，是胰岛素抵抗的关键介导剂，如通过在培养的脂肪细胞和糖尿病小鼠模型中神经节苷脂消耗后胰岛素抵抗的逆转所证明的。我们已经发现，GM 3在糖尿病小鼠的角质形成细胞（KC）膜中积累，并且GM 3的消耗逆转了它们的伤口愈合缺陷。我们建议，通过使用一种新的纳米技术方法的神经节苷脂合成的遗传抑制将逆转受损的伤口愈合在高葡萄糖条件下的KC和糖尿病小鼠。该项目的长期目标是应用基因抑制局部应用的纳米颗粒，阻断神经节苷脂的生物合成，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增殖和运动。我们将使用我们独特的三聚氰胺结合的金纳米粒子（Au NPs），单一的代理人，显示在细胞中的普遍吸收和高效的基因敲除。我们将首先评估局部应用的GM 3合酶siRNA-Au纳米颗粒（其消耗神经节苷脂）在糖尿病小鼠模型中加速愈合的有效性和安全性。接下来，我们将确定神经节苷脂如何影响KC运动。使用DNA和siRNA-Au纳米颗粒分别增加和消耗神经节苷脂GM 3，我们将评估KC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-1 R）和表皮生长因子受体（EGFR）活化，所有这些都影响KC伤口愈合。最后，我们将评估GM 3耗竭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鞘糖脂在伤口愈合中的作用的理解。此外，通过局部施用我们的神经节苷脂合成的纳米颗粒缀合的核酸抑制剂来逆转肥胖糖尿病小鼠中的伤口愈合缺陷将是促进慢性伤口中的伤口愈合的创新手段。这些研究有望对人类伤口的治疗产生重大影响，特别是对胰岛素抵抗型糖尿病患者。

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.