Injectable Scaffold for Efficient, Tunable siRNA Delivery to Skin Wounds

描述（由申请人提供）：糖尿病伤口愈合在美国和全球都是一个重要且日益严重的问题，但目前还没有有效的治疗方法能够持续地实现溃疡伤口的愈合。我们假设基于小干扰核糖核酸（siRNA）的治疗方法提供了一种合乎逻辑且潜在有利的方法，因为糖尿病伤口的特征是促炎基因的异常过度表达，从而阻碍伤口愈合。尽管siRNA为基因沉默提供了一个强大的工具，但由于siRNA是大的极性分子，无法通过细胞膜扩散到达mRNA目标，因此递送是困难的。为了被内化，siRNA必须被内吞到膜结合的内体中，在那里它们通常与细胞质隔离，并被循环出细胞或被运送到溶酶体降解。在当前的提案中，提出了一种新型的，环境响应的“智能”聚合物纳米颗粒（SPN） siRNA载体的应用。这种创新的基于聚合物的载体响应内体的酸性pH值，触发这些囊泡的破坏，并使siRNA胞质递送。spn和其他siRNA载体的最初应用主要集中在全身或局部注射生理盐水中，以抵消异常基因表达，通常用于抗癌应用。然而，siRNA的活性本质上是短暂的，基于支架的控制释放系统的开发用于局部递送到病理部位（即伤口）尚未得到彻底的探索。本文提出了一种新型的递送系统，该系统由sirna携带的spn嵌入到可注射聚氨酯（PUR）支架中。我们之前已经证明，PUR支架可以被精细地调整，以实现生长因子的爆发或持续释放。通过将spn配制成PUR支架，我们寻求实现siRNA在皮肤伤口的局部和持续的细胞内递送。我们将首先在体外制造、验证和优化PUR-SPN平台技术（目的1）。在Aim 2中，我们将使用荧光素酶报告小鼠进行PUR-SPN基因沉默的体内验证，并将完成在糖尿病皮肤伤口中敲低肿瘤坏死因子α的治疗性研究。这些目标补充了NIBIB的既定使命，即“通过领导生物医学技术的发展和加速应用来改善健康”。我们的跨学科团队包括工程师、病理学家和生物学家，工作内容涵盖高分子科学、化学、生物学、医学和制药科学。拟议的项目将专注于炎症基因沉默在不愈合的皮肤伤口，但我们最终渴望扩大病理应用这种多功能平台技术。

DESCRIPTION (provided by applicant): Diabetic wound healing is a significant and growing problem in the United States and globally, but there are currently no effective treatments that consistently achieve closure of ulcerated wounds. We hypothesize that small interfering ribonucleic acid (siRNA)-based therapeutics provides a logical and potentially advantageous approach because diabetic wounds are characterized by aberrant overexpression of proinflammatory genes that hinder wound healing. Although siRNA provide a powerful tool for gene silencing, delivery is difficult because siRNA are large, polar molecules that are unable to diffuse through cell membranes to reach mRNA targets. To be internalized, siRNA must be endocytosed into membrane-bound endosomes, where they typically remain sequestered from the cytosol and are recycled out of the cell or trafficked for lysosomal degradation. In the current proposal, application of a novel, environmentally-responsive "smart" polymer nanoparticle (SPN) carrier for siRNA is proposed. This innovative polymer-based carrier responds to the acidic pH in endosomes to trigger disruption of these vesicles and enable siRNA cytosolic delivery. Initial applications of SPNs and other types of siRNA carriers have focused on systemic or local injection in saline to counteract aberrant gene expression, commonly for anticancer applications. However, siRNA activity is inherently transient, and development of scaffold-based controlled release systems for local delivery to pathological sites (i.e. wounds) has not been thoroughly explored. Here, a novel delivery system is proposed that consists of siRNA-carrying SPNs embedded into injectable polyurethane (PUR) scaffolds. We have previously shown that PUR scaffolds can be finely tuned to achieve burst or sustained release of growth factors. By formulation of SPNs into PUR scaffolds, we seek to achieve localized and sustained intracellular delivery of siRNA to skin wounds. We will first fabricate, validate, and optimize the PUR-SPN platform technology in vitro (Aim 1). In Aim 2, we will employ a luciferase reporter mouse for in vivo validation of PUR-SPN gene silencing and will complete a therapeutic study on knockdown of tumor necrosis factor alpha in diabetic skin wounds. These aims complement the stated mission of the NIBIB "to improve health by leading the development and accelerating the application of biomedical technologies". Our interdisciplinary team includes engineers, a pathologist, and a biologist, and the work encompasses elements of polymer science, chemistry, biology, medicine, and pharmaceutical sciences. The proposed project will focus on inflammatory gene silencing in nonhealing skin wounds, but we ultimately aspire to expand the pathological applications for this versatile platform technology. PUBLIC HEALTH RELEVANCE: Patients with diabetes are more prone to severe nonhealing skin wounds that, in the most dire cases, require limb amputation. The best biological drug currently available for diabetic wounds cannot stimulate wound healing in over 50% of patients, so there is a very important need for improved treatments. There is a promising new class of drugs called small interfering ribonucleic acids (siRNA) that have the potential to fill this clinical need, but difficult barriers exist for efficient delivery of siRNA to wounds. The goal of this proposal is to invent and validate a system that can efficiently deliver siRNA in order to develop better drugs to treat skin wounds.