Nanoparticle-Based Drug Delivery Targeting the Respiratory Neural Network

摘要 迫切需要新的治疗策略来改善患者的呼吸和呼吸道保护功能 导致呼吸障碍、呼吸机依赖和死亡的神经肌肉疾病，如颈椎病 脊髓损伤、肌萎缩侧索硬化症、脑炎和神经毒性等。呼吸神经的靶向给药 网络是有效治疗这些神经肌肉疾病的关键目标。不幸的是，将药物运送到 中枢神经系统受到血脑屏障(BBB)的限制。尽管迫切需要有效的 治疗策略，以保持/恢复呼吸能力，目前几乎没有可用的选择。最基本的 这项提议的目标是测试一种前景看好的高度新颖的纳米药物输送策略。 呼吸运动神经元和相关的神经回路。 运动神经元是独一无二的，因为它们的轴突投射延伸到外周，使其有可能绕过 血脑屏障通过逆行轴突药物转运。然而，目前尚不清楚运动神经元轴突是否运输 可以利用机制将治疗药物运送到膈运动神经元及其相关的前运动神经元。 突触神经网络。运动神经元将某些物质逆行运输到细胞体，包括 霍乱毒素β亚单位(CTB)，一种无毒的蛋白质，广泛用于标记呼吸运动神经元。CTB- 共轭荧光团也被逆行运输到膈运动神经元胞体。然而，CTB- 共轭化合物不会直接影响与目标运动神经元相关的神经/神经胶质网络。 我们建议通过CTB和具有独特性质的纳米颗粒实现逆行给药，从而使 跨运动神经元细胞膜运输，有效地将治疗药物输送到相关的细胞 网络。碳量子点(CDots)是具有药物必备特性的纳米颗粒(&lt；10 nm) 传递，并可用作装饰各种分子的纳米平台。惊人的初步数据显示 与直接的CTB荧光团结合物不同，CTB通过CDot标记为 脊髓中间神经元超越了膈运动神经元本身；这一发现表明 转运的CTB-CDOT结合物并不局限于运动神经元，而是更广泛地分布到 神经网络中的突触前神经元。我们将检验胸腔内注射CTB-CDOT的假设- 生物结合物：1)横隔运动神经元以外分布于脊髓和脑干呼吸 神经回路(目标1)；和2)将功能货物输送到膈运动神经回路(目标2)。 该提案符合适用于R21赠款申请的高风险、高影响的定义，因为它将： 1)建立跨血脑屏障选择性给药至呼吸神经的新方法 网络；2)产生有关CDOT-偶联物的神经元间转运机制的基础知识； 以及3)指导开发新的策略来治疗危及呼吸的破坏性临床疾病， 包括影响呼吸的创伤性、遗传性、传染性和毒性神经肌肉疾病。

ABSTRACT New treatment strategies are desperately needed to improve respiratory and airway protective functions in neuromuscular disorders that cause breathing impairment, ventilator-dependence and death, such as cervical spinal injury, ALS, encephalitis and neurotoxicity, among others. Targeted drug delivery to the respiratory neural network is a critical goal to effectively treat these neuromuscular disorders. Unfortunately, drug delivery to the central nervous system is restricted by the blood-brain barrier (BBB). Despite the critical need for effective treatment strategies to preserve/restore breathing ability, few options are currently available. The fundamental goal of this proposal is to test a promising and highly novel strategy of nanoparticle-based drug delivery to respiratory motor neurons and associated neural circuits. Motor neurons are unique since their axonal projections reach into the periphery, making it possible to bypass the BBB via retrograde axonal drug transport. However, it is not yet known if motor neuron axon transport mechanisms can be harnessed to carry therapeutic drugs to phrenic motor neurons and their associated pre- synaptic neural network. Motor neurons retrogradely transport certain substances to their cell bodies, including cholera toxin subunit beta (CtB), a non-toxic protein extensively used to label respiratory motor neurons. CtB- conjugated fluorophores are also retrogradely transported to phrenic motor neuron somata. However, CtB- conjugates do not directly affect neural/glial networks associated with targeted motor neurons. We propose to enable retrograde drug delivery via CtB and nanoparticles with unique properties that enable transport across motor neuron cell membranes, effectively delivering therapeutics to the relevant cellular network. Carbon quantum dots (CDots) are nanoparticles (<10 nm) with requisite characteristics for drug delivery, and can be utilized as nanoplatforms decorated with various molecules. Striking preliminary data show that, unlike direct CtB fluorophore conjugates, CtB conjugated to fluorophores via CDots label a subset of spinal interneurons beyond the phrenic motor neurons per se; this finding suggests that retrogradely transported CtB-CDot-conjugates do not remain confined within motor neurons, but distribute more broadly to pre-synaptic neurons in the neural network. We will test the hypotheses that intrapleurally injected CtB-CDot- bioconjugates: 1) distribute beyond phrenic motor neurons throughout spinal and brainstem respiratory neural circuitry (Aim 1); and 2) transport functional cargo to the phrenic motor circuit (Aim 2). This proposal fits the definition of high risk, high impact, appropriate for an R21 grant application since it will: 1) establish new methods of selective drug delivery across the blood brain barrier to the respiratory neural network; 2) yield fundamental knowledge concerning inter-neuronal transport mechanisms of CDot-conjugates; and 3) guide development of new strategies to treat devastating clinical disorders that compromise breathing, including traumatic, genetic, infectious and toxic neuromuscular disorders that compromise breathing.