Novel Theranostic Approaches to Identify and Treat Tinnitus Using Functionalized Nanoparticles

描述(申请人提供)：在美国，约有5000万人患有耳鸣(在没有明显声音刺激的情况下感知声音)，约300万人 残疾人和需要治疗的人。不幸的是，目前耳鸣的治疗选择往往无效，产生不一致的结果，有时甚至令人失望。迫切需要诊断和治疗耳鸣的新的和创新的方法。在这里，我们介绍了一个新的概念，用于耳鸣的研究和减轻，基于这样的假设，即与耳鸣相关的大脑区域中神经元活动过度活跃的基因的过度表达可以通过多功能衣壳纳米颗粒来识别和靶向。这里提出的努力利用了一个基于衣壳的多功能纳米颗粒平台，并代表着朝着我们的长期目标迈出的第一步，即建立一个基于纳米颗粒的平台，通过减弱过度活跃的神经活动来定位和治疗耳鸣。我们将评估纳米颗粒能否在体外和体内定位于特定的靶点，以及ii)优化设计，以实现理想的脑内定位和跨血脑屏障(BBB)运输，且副作用可忽略不计。在这里，我们试图评估使用纳米颗粒在体内使用的可行性，并为未来的工作奠定坚实的基础，以便能够使用耳鸣的动物模型进行更深入的研究。具体目的1.使用定制的纳米颗粒建立下丘(IC)内特定蛋白的靶向。纳米颗粒(NPs)将被设计成靶向在耳鸣相关多动区域差异表达的蛋白质。成年SD大鼠将被用来评估NPs的靶向性。在组织学评估过程中，用结合活跃神经元中受体的抗体装饰的NPS将用于组织学评估。使用两种方法来验证NP结合的特异性：1.Western blotting将用于确定NP能够与从IC分离的目的蛋白结合。这项评估将包括：接触i)用特定抗体修饰的NPs，ii)没有抗体的NPs和iii)单独的抗体。2.显微镜将用于验证NP与天然IC细胞中感兴趣的蛋白质的结合。装有荧光标签的NPS 将被局部化，以量化NP在i)IC的特定分区内和之间的分布差异，ii)其他听觉脑区，以及iii)非听觉脑区。具体目标2.优化MRI可检测的定制纳米颗粒的剂量、定位和跨血脑屏障的传输。成年Spraogue-Dawley大鼠将全身注射定制设计的NP。它们将被一种促进血脑屏障转运的短肽修饰(通过LPR-1)。在注射后的多个时间点，具有顺磁核心和共轭荧光标记的纳米颗粒将被定位：1.使用T1加权MRI和2.通过使用冰冻切片的基于荧光的显微镜交叉验证。

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.