Dendrimer nanoparticles for assessment of microglial activation and therapeutic response to neonatal brain injury

项目摘要 围产期窒息（PA）是出生时发病率和死亡率的主要原因。出生于 许多PA患者会出现中度或重度缺氧缺血性脑病（HIE），20 - 30%的患者会出现 长期影响包括脑瘫、癫痫、智力迟钝或学习障碍。PA很难 预防或预测，目前的治疗策略治疗低温只提供了15%的绝对 降低死亡和残疾的风险。因此，确定治疗窗口或改善治疗效果的策略， 具有显著的临床潜力。炎症与HIE的发展有关， 是一个有用的治疗靶点，因为它广泛地描述了多种途径， 受伤的严重程度。在神经炎症中，小胶质细胞，大脑中的常驻免疫细胞，采用激活的 吞噬行为增加的表型。以前的研究利用这种效应来证明 增加的小分子，特别是树枝状聚合物纳米颗粒，在受损组织中的小胶质细胞内的摄取。 载药树枝状聚合物在小胶质细胞中的积累，HIE损伤的介质，允许靶向递送药物。 治疗有效载荷姜黄素，我们提出的治疗，已被我们的实验室表明，有显着的 由于其抗炎和抗氧化特性，在新生儿HIE大鼠模型中具有神经保护作用。我们 将配制两种树枝状聚合物缀合物，荧光Cy 5-树枝状聚合物颗粒和载药Cy 5-树枝状聚合物。 姜黄素颗粒，使用已建立的化学。我们将使用Cy 5-树枝状聚合物来评估以下时间轴： 在新生大鼠模型中，小胶质细胞活化响应于损伤，寻找小胶质细胞活化峰值的时间点 通过免疫组织化学（基于表型和树枝状聚合物-Cy 5共定位的半定量）和 荧光激活细胞分选（基于抗体表达和Cy 5荧光的定量）。使用 这些相同的技术，我们将评估小胶质细胞对治疗性树状聚合物给药后的反应。 全身注射负载姜黄素的缀合物。该项目将提供关键答案，包括（1）理想的 缺血性损伤后提供治疗干预以抑制炎症的时间，和（2） 抗炎疗法来逆转细胞水平上的损伤。该项目的总体目标是 在新生儿缺氧缺血性脑病的治疗过程中， 确定最佳治疗窗。确定损伤时间轴和纳米颗粒平台以利用 该疾病的病理生理学将导致改善新生儿脑损伤的治疗干预， 这一结论可以转化为成人神经系统疾病，其中炎症也起着关键作用。

Project Summary Perinatal asphyxia (PA) is the leading cause of morbidity and mortality around the time of birth. In patients born with PA, many develop moderate or severe hypoxic-ischemic encephalopathy (HIE) and 20 to 30% will develop long term effects including cerebral palsy, epilepsy, mental retardation, or learning disability. PA is difficult to prevent or predict, and the current treatment strategy of therapeutic hypothermia only offers a 15% absolute reduction in the risk of death and disability. Thus, strategies to identify a therapeutic window or improve therapeutic efficacy have significant clinical potential. Inflammation is implicated in the development of HIE and is a useful therapeutic target since it broadly describes multiple pathways which perpetuate and increase the severity of injury. In neuroinflammation, microglia, the resident immune cells of the brain, adopt an activated phenotype with increased phagocytic behavior. Previous studies have leveraged this effect to demonstrate increased small molecule, specifically dendrimer nanoparticle, uptake within microglia in injured tissue. Accumulation of drug-loaded dendrimers in microglia, mediators of HIE injury, allows for targeted delivery of a therapeutic payload. Curcumin, our proposed therapeutic, has been shown by our lab to have significant neuroprotective effect in a rat model of neonatal HIE due to its anti-inflammatory and antioxidant properties. We will formulate two dendrimer conjugates, a fluorescent Cy5-dendrimer particle and a drug loaded Cy5-dendrimer- curcumin particle, using established chemistries. We will use Cy5-dendrimers to evaluate the timeline of microglia activation in response to injury in the neonatal rat model, finding time points of peak microglial activation by immunohistochemistry (semi-quantification based on phenotype and dendrimer-Cy5 co-localization) and fluorescent activated cell sorting (quantification based on antibody expression and Cy5 fluorescence). Using these same techniques, we will then evaluate microglial response to therapeutic dendrimer administration after systemic injection of the curcumin-loaded conjugate. This project will provide key answers including (1) the ideal time to provide therapeutic intervention after ischemic injury for suppression of inflammation, and (2) the ability of anti-inflammatory therapeutics to reverse injury on a cellular level. The overall goal of this project is to demonstrate the therapeutic potential of engineered dendrimer nanoparticles in neonatal HIE during a determined optimal therapeutic window. Identifying the injury timeline and a nanoparticle platform to leverage the disease pathophysiology will lead to improved therapeutic intervention in neonatal brain injury, with implications that can be translated to adult neurological disorders, where inflammation also plays a critical role.