Neuron-specific nanotherapeutics for spinal cord injury repair

由于有限的内在愈合能力和缺乏有效的治疗方法，脊髓损伤 (SCI)通常导致病变水平以下的运动和感觉功能的永久性丧失。SCI展品 这是一种复杂的病理生理学，对功能恢复造成多种障碍。这些包括缺血和 引起进行性神经元和神经胶质细胞死亡以及细胞外生长的炎症 抑制剂和内在缺陷的神经元生物化学，限制了成年轴突的能力， 可塑性/再生使用靶向两种或多种这些屏障的治疗方式的联合疗法 已经取得了有希望的临床前结果，但它们的应用是复杂的，往往需要多个 干预措施。因此，需要开发能够同时 递送多种生物活性分子。这项提案的目的是开发神经元特异性 用于SCI修复的纳米治疗剂。假设是调节cAMP的药物的组合递送 RhoA信号通路通过协同作用提供神经保护，促进功能恢复 在挫伤性脊髓损伤模型中。之所以选择这些治疗靶点，是因为它们被认为是 信号分子参与SCI病理生理学的多个方面，包括炎症、神经细胞 死亡，以及轴突再生的外在和内在障碍。我们的方法是基于一本小说 本实验室研制的阳离子两亲性共聚物聚（丙交酯-乙交酯）-接枝-聚乙烯亚胺（PgP）。 PgP形成聚合物胶束，为组合药物递送提供三个关键特征： 用于装载咯利普兰（Rm）的核，咯利普兰是一种能够恢复cAMP水平的磷酸二酯酶4抑制剂，2）一种阳离子， 用于静电结合靶向RhoA的siRNA（siRhoA）的亲水性壳，和3）表面官能团 其将用于L1神经细胞粘附分子的缀合以增加神经元靶向。在 我们的初步研究表明，PgP/siRhoA和PgP/Rm各自能够在 动物损伤模型。在目标1中，我们将制定神经元特异性纳米治疗药物，将L1作为靶向 配体、新型设计的纳米结构中的siRhoA和Rm（L1-PgP/siRhoA/Rm），并测试它们在一种新型纳米结构中的功效。 体外缺氧脊髓损伤模型。在目标2中，我们将在大鼠中度挫伤中测试最有希望的候选物 模型，结合了生物分布以及重复和延迟给药的研究。治疗功效 结果将包括cAMP/RhoA的生化分析，组织学分析，运动恢复， 神经性疼痛/烯丙缺乏症。这项工作是创新的，因为它使用了一种新型材料， 在单一制剂中同时递送两种化学上不同的药物，cAMP/RhoA作为一种新的 组合疗法、L1靶向和新型siRNA纳米结构。拟议的工作意义重大，因为 它解决了有效治疗递送的关键和研究不足的挑战， 本发明还可应用于其它CNS损伤和病症，包括创伤性脑损伤、阿尔茨海默病和中风。

Due to limited intrinsic healing capacity and the absence of effective therapeutic treatment, spinal cord injury (SCI) usually results in permanent loss of motor and sensory function below the level of the lesion. SCI exhibits a complex pathophysiology that presents multiple barriers to functional recovery. These include ischemia and inflammation that cause progressive neuronal and glial cell death, as well as the presence of extracellular growth inhibitors and intrinsic deficiencies in neuronal biochemistry that limit the capacity of adult axons for plasticity/regeneration. Combination therapies using treatment modalities targeting two or more of these barriers have achieved promising preclinical results, but their application is complex and often requires multiple interventions. Thus, there is a need for the development of new technologies capable of simultaneously delivering multiple bioactive molecules. The objective of this proposal is to develop neuron-specific nanotherapeutics for SCI repair. The hypothesis is that combinatorial delivery of drugs modulating the cAMP and RhoA signaling pathways will provide neuroprotection and promote functional recovery by synergistic effect in contusion SCI model. These therapeutic targets have been chosen because of their recognition as key signaling molecules implicated in multiple aspects of SCI pathophysiology including inflammation, neuronal cell death, and both extrinsic and intrinsic barriers to axonal regeneration. Our approach is based upon a novel cationic amphiphilic co-polymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) developed in our lab. PgP forms polymeric micelles offering three critical features for combinatorial drug delivery: 1) a hydrophobic core for loading of rolipram (Rm), a phosphodiesterase 4 inhibitor capable of restoring cAMP levels, 2) a cationic, hydrophilic shell for electrostatic binding of siRNA targeting RhoA (siRhoA), and 3) surface functional groups that will be used for conjugation of the L1 neural cell adhesion molecule to increase neuronal targeting. In preliminary studies we show that PgP/siRhoA and PgP/Rm are each capable of achieving therapeutic effects in animal injury models. In Aim 1, we will formulate neuron-specific nanotherapeutics incorporating L1 as a targeting ligand, siRhoA in novel, designed nanostructures, and Rm (L1-PgP/siRhoA/Rm) and test their efficacy in an in vitro hypoxia SCI model. In Aim 2, we will test the most promising candidate in a rat moderate contusion injury model, incorporating studies of biodistribution and repeat and delayed administration. Therapeutic efficacy outcomes will include biochemical analysis of cAMP/RhoA, histological analysis, locomotor recovery, and neuropathic pain/allydonia. The proposed work is innovative because it uses a novel material capable of simultaneously delivering two chemically different drugs in a single formulation, cAMP/RhoA as a new combination therapy, L1 targeting and novel siRNA nanostructures. The proposed work is significant, because it addresses the critical and under-studied challenge of effective therapeutic delivery and may have widespread application to other CNS injury and disorders including traumatic brain injury, Alzheimer's disease, and stroke.