Nanoparticle-Based Drug Delivery Targeting the Respiratory Neural Network

摘要 迫切需要新的治疗策略来改善呼吸和气道保护功能， 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、ALS、脑炎和神经毒性等。呼吸神经靶向给药 网络是有效治疗这些神经肌肉疾病的关键目标。不幸的是，药物输送到 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 为了维持/恢复呼吸能力的治疗策略，目前可用的选择很少。根本 该提案的目标是测试一种有前途的、高度新颖的基于纳米颗粒的药物递送策略 呼吸运动神经元和相关的神经回路。 运动神经元是独特的，因为它们的轴突投射到达外周，使其有可能绕过 通过逆行轴突药物转运的BBB。然而，目前尚不清楚运动神经元轴突运输是否 可以利用这种机制将治疗药物运送到膈运动神经元及其相关的前 突触神经网络运动神经元将某些物质逆行运输到其细胞体，包括 霍乱毒素β亚单位（CtB），一种广泛用于标记呼吸运动神经元的无毒蛋白质。CtB- 共轭荧光团也被逆行转运到膈运动神经元胞体。然而，CtB- 缀合物不直接影响与靶向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药物递送， 跨运动神经元细胞膜的转运，有效地将治疗剂递送至相关细胞 网络碳量子点（CDots）是具有药物所需特性的纳米颗粒（<10 nm）， 递送，并且可以用作用各种分子修饰的纳米平台。惊人的初步数据显示， 与直接CtB荧光团缀合物不同，通过CDot与荧光团缀合的CtB标记了 脊髓中间神经元超出膈运动神经元本身;这一发现表明， 转运的CtB-CDot-缀合物并不局限于运动神经元内，而是更广泛地分布于 神经网络中的突触前神经元。我们将检验胸膜内注射CtB-CDot- 生物缀合物：1）在整个脊髓和脑干呼吸系统中分布于膈运动神经元之外 神经回路（Aim 1）;和2）将功能性货物运输到膈运动回路（Aim 2）。 该提案符合高风险，高影响的定义，适合R21赠款申请，因为它将： 1)建立选择性药物通过血脑屏障到达呼吸神经系统的新方法 2）产生关于CDot-缀合物的神经元间转运机制的基础知识; 以及3）指导新策略的开发以治疗危害呼吸的破坏性临床病症， 包括创伤性、遗传性、感染性和毒性神经肌肉疾病，这些疾病会影响呼吸。

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.