Systemic Application for Injury Site Specific Delivery via Neutrophils to Treat B

描述（由申请人提供）：腰痛是一种地方病，是美国人去看医生的第二大常见原因，仅次于最常见的感冒和流感。腰痛也是第二个最常见的疾病相关的原因，因为错过了工作日和残疾的最常见原因。近年来，大量研究表明，损伤诱导的炎症在椎间盘损伤和退化引起的腰痛中可能发挥作用。因此，自20世纪50年代作为一种治疗方法开始以来，使用荧光镜引导的腰椎硬膜外注射类固醇以减少局部化学炎症反应的情况急剧增加。然而，硬膜外注射的风险包括但不限于硬膜外血肿、硬膜外感染、神经根损伤、硬膜穿刺、空气栓塞和血管迷走性晕厥。此外，硬膜外注射技术要求高。它通常由受过特殊训练的医生在X光下进行。当病人得到预约时，疼痛可能处于亚急性或慢性状态。因此，对急性/慢性背痛进行具有成本效益的治疗非常重要。我们的申请提出了一种新的治疗方法，通过将纳米富勒烯连接到cFLFLF，以特异性靶向迁移到椎间盘突出部位的活化中性粒细胞，并通过外周静脉注射治疗急性背痛，这在技术上要求不高，可以由护士进行。纳米富勒烯具有抗氧化和抗炎的特性，不具有特定的生物靶点，不仅可以减少椎间盘突出引起的疼痛过敏，而且可以消除标准生物靶向药物分子的副作用。然而，需要一种有效的递送系统来克服其高度疏水性，以特异性靶向损伤部位。本研究的总体假设是，表达甲酰肽受体（FPR）的活化中性粒细胞将与蛋白水解抗性合成肽cFLFLF结合，并且它们将一起浸润并积聚到疝状DIC的炎症部位。将纳米富勒烯连接到cFLFLF（cFLFLF-PEG-富勒烯）将确保抗炎剂通过全身注射到损伤部位的事件特异性时间和空间递送， 治疗因椎间盘突出及周围过度炎症引起的疼痛。在本研究的目标1中，我们将使用嗜中性粒细胞特异性小分子显像剂和免疫组织学来描述活化的中性粒细胞浸润到急性椎间盘突出中的特征。将使用FPR敲除小鼠验证成像信号。将分析中性粒细胞和巨噬细胞的FPR表达和定量以及损伤定量。在目标2中，我们将表征纳米富勒烯的水性悬浮液对由椎间盘突出在体外和体内诱导的局部炎症的抑制作用。在目的3中，我们将合成cFLFLF-PEG-富勒烯并在小鼠神经根病模型中评价其向炎症部位的全身递送与局部递送;我们还将在针刺诱导的椎间盘变性模型中使用野生型和FPR敲除小鼠评估损伤部位靶向全身递送与非特异性全身递送。这项研究将提供一个新的具有成本效益的治疗策略，急性和慢性背部/颈部/腿部疼痛涉及全身注射的非蛋白质，纳米结构，生物相容性富勒烯共轭cFLFLF，一个嗜中性粒细胞特异性结合肽的有用的见解。这种方法比硬膜外类固醇注射更具成本效益。

DESCRIPTION (provided by applicant): Low back pain is an endemic problem and is the second most common reason that Americans go to see their doctor, second only to the most common cold and the flu. Low back pain is also the second most common illness-related reason given for a missed workday and the most common cause of disability. In recent years, a body of research has suggested a possible role of injury-induced inflammation in low back pain caused by damaged and degenerating intervertebral discs. As such, the use of fluoroscopy-guided lumbar epidural injections of steroids to reduce the local chemo-inflammatory response has increased dramatically since their inception as a treatment in the 1950s. However, the risks of epidural injections include, but are not limited to, epidural hematoma, epidural infection, nerve root injury, dural puncture, air embolism and vasovagal syncope. Moreover, the epidural injection is technically demanding. It is usually performed under x-ray by doctors with special training. When a patient gets an appointment, the pain is likely in a more subacute or chronic state. Therefore, a cost-effective treatment for acute/chronic back pain is of great importance. Our application proposes a novel treatment by linking nano-fullerene to cFLFLF in order to specifically target activated neutrophils that migrate into the disc herniation site and treat acut back pain via peripheral vein injection, which is not technically demanding and could be performed by a nurse. Nano-fullerene, which has potent anti-oxidative and anti-inflammatory properties, without specific biological target could not only reduce hyperalgesia induced by the extrusion of nucleus pulposus tissue from herniated discs, but also eliminate the side effects associated with standard biological target-oriented drug molecules. However, an effective delivery system is needed to overcome its highly hydrophobic properties to specifically target the site of injury. The overall hypothesis of this study is that the activated neutrophils expressig formyl peptide receptor (FPR) will bind to proteolytically resistant synthetic peptide cFLFLF and that together they will infiltrate and accumulate into the site of inflammation in the herniated dic. Linking nano-fullerene to a cFLFLF (cFLFLF-PEG-fullerene) will ensure event-specific temporal and spatial delivery of the anti-inflammatory agent to the site of injury via systemic injection to treat the pain caused by excessive inflammation in and around herniated discs. In Aim 1 of the proposed study, we will characterize the infiltration of activated neutrophils into an acute disc herniation using neutrophil-specific small molecule imaging agents and immunohistology. The imaging signal will be validated using FPR knockout mice. The neutrophils and macrophages will be analyzed for expression and quantification of FPR and for the quantification of injury. In Aim 2, we will characterize the inhibitory effect of aqueous suspensions of nano-fullerene on local inflammation induced by disc herniation in vitro and in vivo. In Aim 3, we will synthesize cFLFLF-PEG-fullerene and evaluate its systemic vs. local delivery to a site of inflammation in a mouse radiculopathy model; we will also assess injury site target systemic delivery vs. non- specific systemic delivery using wild type and FPR knockout mice in a disc degeneration model induced by needle puncture. This study will provide useful insights into a novel cost-effective therapeutic strategy for acute and chronic back/neck/leg pain involving systemic injection of non-protein, nano-structured, biocompatible fullerene conjugated with cFLFLF, a neutrophil-specific binding peptide. This method is much more cost- effectiveness compared to epidural steroid injection.