Novel Theranostic Approaches to Identify and Treat Tinnitus Using Functionalized Nanoparticles

描述（由申请人提供）：在美国，约有5000万人患有耳鸣（没有明显声学刺激的声音感知），约有300万人患有耳鸣。 需要治疗的残疾人。 不幸的是，目前耳鸣的治疗方案往往是无效的，并产生不一致的，有时令人失望的结果. 耳鸣的诊断和治疗需要新的创新方法。 在这里，我们引入了一个耳鸣研究和缓解的新概念，该概念基于这样的假设：可以使用基于多功能衣壳的纳米颗粒来识别和靶向与耳鸣相关的神经元活动过度活跃的大脑区域内基因的过度表达。 这里提出的努力利用了一个多功能的基于captain的纳米颗粒平台，代表了我们建立一个基于纳米颗粒的平台，通过衰减过度活跃的神经活动来定位和治疗耳鸣的长期目标的第一步。 我们将）评估纳米颗粒是否可以在体外和体内定位于特定靶点，以及ii）优化设计以在脑内理想定位并穿过血脑屏障（BBB）运输，副作用可忽略不计。 在这里，我们试图评估使用纳米粒子在体内使用的可行性，并建立一个坚实的基础，未来的工作将使更深入的研究，使用动物模型的耳鸣。 具体目标1。 使用定制的纳米颗粒建立下丘（IC）内特定蛋白质的靶向。 纳米颗粒（NPs）将被设计为靶向蛋白质，这些蛋白质被证明在耳鸣相关活动过度的区域中差异表达。 成年Sprague-Dawley大鼠将用于评价NP的靶向特异性。 用结合活性神经元中的受体的抗体修饰并负载荧光团的NP将用于组织学评估期间的可视化。 使用两种方法来验证NP结合的特异性：1. Western印迹将用于确定NP能够结合从IC分离的目的蛋白。 该评估将包括暴露于i）用特异性抗体修饰的NP，ii）没有抗体的NP和iii）单独的抗体。 2. 显微镜检查将用于验证NP与天然IC细胞中的目标蛋白质的结合。 负载有荧光标签的NP 将被定位以量化i）IC的特定分区ii）其他听觉脑区域以及iii）非听觉脑区域内和跨这些分区的NP分布的差异。 具体目标2。 优化MRI可检测定制制造纳米颗粒穿过BBB的剂量、定位和运输。 将给予成年Sprague-Dawley大鼠全身注射定制设计的NP。 它们将用促进BBB转运（通过LPR-1）的短肽进行修饰。 在注射后的多个时间点，具有顺磁性核和缀合的荧光标签的纳米颗粒将被定位：1. T1加权MRI和2. 使用冷冻切片通过基于荧光的显微镜进行交叉验证。

DESCRIPTION (provided by applicant): In the United States, about 50 million individuals suffer from tinnitus (perception of sound without overt acoustic stimulation), with circa 3 million people that are disabled and require treatment. Unfortunately, current treatment options for tinnitus are often ineffective and produce inconsistent and sometimes disappointing results. New and innovative approaches for diagnosis and treatment of tinnitus are urgently needed. Here, we introduce a novel concept for tinnitus research and abatement based on the hypothesis that over-expression of genes within brain regions with hyperactive neuronal activity associated with tinnitus can be identified and targeted using multifunctional capsid-based nanoparticles. The effort proposed here leverage a multifunctional capsid-based nanoparticle platform and represents the initial step toward our long-term goal to establish a nanoparticle-based platform to localize and treat tinnitus by attenuating hyperactive neural activity. We will ) evaluate whether nanoparticles can localize to specific targets in vitro and in vivo and ii) optimize the design for ideal localization within the brain and transport across the blood-brain-barrier (BBB) with negligible side effects. Here we seek to evaluate the feasibility of using nanoparticles for in vivo use and establish a solid foundation where future work will enable more in-depth studies using animal models of tinnitus. Specific Aim 1. To establish targeting of specific proteins within the inferior colliculus (IC) using custom-fabricated nanoparticles. Nanoparticles (NPs) will be designed to target proteins demonstrated to be differentially expressed in areas with tinnitus related hyperactivity. Adult Sprague-Dawley rats will be used to evaluate targeting specificity of the NPs. NPs decorated with antibodies that bind receptors in active neurons and loaded with fluorophore will be used for visualization during histological assessment. Two approaches to validate the specificity of NP binding are used: 1. Western blotting will be used to establish that the NPs are capable of binding the protein-of-interest isolated from the IC. This assessment will include, exposure to i) NPs modified with the specific antibody, ii) NPs without the antibody and iii) antibody alone. 2. Microscopy will be used to verify NP binding to the protein of interest in native IC cells. NPs loaded with a fluorescent tag will be localized to quantify differences in NP distribution within and across i) specific subdivisions of the IC ii) other auditory brain regions, as well as, iii) non-auditory brain region. Specific Aim 2. To optimize dose, localization, and transport of MRI detectable custom fabricated nanoparticles across the BBB. Adult Sprague-Dawley rats will be given a systemic injection of custom- designed NPs. They will be modified with a short peptide that facilitates BBB transport (through LPR-1). At multiple time points following injection, nanoparticles with a paramagnetic core and a conjugated fluorescent tag, will be localized: 1. with T1-weighted MRI and 2. cross-validated by fluorescence-based microscopy using frozen sections.