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范围内是一个重要且日益严重的问题，但目前没有持续实现溃疡伤口闭合的有效治疗方法。我们假设，小干扰核糖核酸（siRNA）为基础的治疗提供了一个合乎逻辑的和潜在的有利的方法，因为糖尿病伤口的特点是异常过度表达的促炎基因，阻碍伤口愈合。尽管siRNA提供了基因沉默的有力工具，但递送是困难的，因为siRNA是大的极性分子，其不能通过细胞膜扩散以到达mRNA靶标。为了被内化，siRNA必须被内吞到膜结合的内体中，在那里它们通常保持与胞质溶胶隔离，并从细胞中回收或运输用于溶酶体降解。在目前的建议中，提出了一种新的，环境响应性的“智能”聚合物纳米颗粒（SPN）载体的siRNA的应用。这种创新的基于聚合物的载体响应于内体中的酸性pH，以触发这些囊泡的破坏，并使siRNA胞质递送成为可能。SPN和其他类型的siRNA载体的最初应用集中在盐水中的全身或局部注射以抵消异常基因表达，通常用于抗癌应用。然而，siRNA活性本质上是瞬时的，并且用于局部递送至病理部位（即伤口）的基于支架的控释系统的开发尚未被彻底探索。在这里，提出了一种新的递送系统，该系统由嵌入可注射聚氨酯（PUR）支架中的携带siRNA的SPN组成。我们以前已经表明，PUR支架可以微调，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向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（目标1）。在目标2中，我们将采用荧光素酶报告小鼠进行PUR-SPN基因沉默的体内验证，并将完成糖尿病皮肤伤口中肿瘤坏死因子α敲低的治疗研究。这些目标补充了国家生物医学和生物医学研究所的既定使命，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，工作包括聚合物科学，化学，生物学，医学和制药科学的元素。拟议的项目将专注于不愈合皮肤伤口中的炎症基因沉默，但我们最终希望扩大这种多功能平台技术的病理应用。 公共卫生相关性：糖尿病患者更容易出现严重的不愈合皮肤伤口，在最严重的情况下，需要截肢。目前可用于糖尿病伤口的最佳生物药物不能刺激超过50%的患者的伤口愈合，因此非常需要改进治疗方法。有一类很有前途的新型药物称为小干扰核糖核酸（siRNA），有可能满足这种临床需求，但siRNA有效递送到伤口存在困难的障碍。该提案的目标是发明和验证一种可以有效递送siRNA的系统，以开发更好的药物来治疗皮肤伤口。

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.