DEVELOPMENT OF RATIOMETRIC IMAGING PROBE SYSTEM FOR MONITORING INFLAMMATION

描述(申请人提供)：炎症是一种主要的统一综合征，与各种损伤和疾病有关。严重时，炎症性疾病会危及甚至杀死患者。炎症反应程度的直接诊断通常是通过侵入性活检进行组织学检查。此外，经常需要多次活检来评估大伤口的炎症反应程度。这项拟议的工作旨在开发一种新的非侵入性方法，以实时评估体内炎症反应的程度。中性粒细胞释放的活性氧(ROS)已被证明是炎症反应程度的良好指标。近年来，几种ROS反应性生物发光探针被研究用于检测体内的ROS活性。不幸的是，这些探针本身不能用来量化炎症反应的程度，因为生物发光信号的程度也依赖于探针浓度。为了应对这一挑战，我们采用比率探针技术，将ROS敏感剂和ROS不敏感的参考染料连接到生物相容的颗粒载体上。可以计算生物发光/参考荧光强度比，以反映局部ROS活性的程度，同时绕过与ROS探针浓度相关的生物发光强度的变化。我们的初步研究导致了ROS比率探针的开发，该探针可以可靠地检测体内局部的ROS活性和伤口部位的炎症组织。基于这些令人兴奋的观察，拟议的工作将创建一系列ROS比率探针来检测体外ROS活性(目标1)，并评估不同损伤或炎症刺激介导的体内炎症反应的程度(目标2)。最后，由于所有商业成像仪的设计都带有一个封闭的腔室，仅适用于细胞和小动物的成像，因此作为这项研究的一部分，将开发一种用于大型动物和人类的ROS传感的手持式成像仪。这项拟议工作的成功完成将有助于开发一种新的诊断工具，该工具可用于非侵入性地实时监测炎症过程，并辅助开发新的治疗方法，以有效地减轻炎症和改善伤口愈合反应。 公共卫生意义：将制造一系列新型的活性氧物种(ROS)比率成像探针和手持光学设备，以监测和量化体内原位ROS活动和相关的炎症反应。拟议工作的成功完成将有助于开发一种新的诊断工具，该工具可以有效地非侵入性地实时评估炎症过程的程度，并大大提高患者护理的质量。

DESCRIPTION (provided by applicant): Inflammation is a major unifying syndrome associated with a variety of injury and diseases. When severe, inflammatory diseases can endanger and even kill patients. Direct diagnosis of the extent of inflammatory responses is often made histological with an invasive biopsy. Furthermore, multiple biopsies are often required to assess the extent of inflammatory reactions in a large wound. The proposed work is aimed at developing a new non-invasive method to assess the extent of inflammatory responses in real-time in vivo. Reactive oxygen species (ROS) released by polymorphonuclear neutrophils have been shown to be a good indicator of the degree of inflammatory reactions. Several ROS-reactive bioluminescent probes have been investigated in recent years to detect ROS activities in vivo. Unfortunately, these probes alone cannot be used to quantify the degree of inflammatory responses due to the fact that the extent of the bioluminescent signals is also probe-concentration dependent. To address this challenge, we adopt ratiometric probe technique in which both ROS-sensitive agents and ROS-insensitive reference dye are conjugated to biocompatible particle carriers. The bioluminescent/reference fluorescent intensity ratios can be calculated to reflect the extent of localized ROS activities while circumventing the variations of bioluminescent intensities associated with the ROS probe concentrations. Our preliminary studies have led to the development of ROS ratiometric probes which can reliably detect localized ROS activities in vivo and inflamed tissue in the wound site. Based on these exciting observations, the proposed work will create a series of ROS ratiometric probes to detect ROS activities in vitro (Aim 1) and to assess the extent of inflammatory responses mediated by different injuries or inflammatory stimuli in vivo (Aim 2). Finally, since all commercial imagers are designed with an enclosed chamber only suitable for cell and small animal imaging, a handheld imager for ROS sensing in large animals and human will be developed as part of this investigation. The successful completion of this proposed work will help the development of a new diagnosis tool which can be applied to non-invasively monitor the inflammatory processes in real-time and assist the development of new treatments to effectively reduce inflammation and improve wound healing reactions. PUBLIC HEALTH RELEVANCE: A series of novel reactive oxygen species (ROS) ratiometric imaging probes and a hand-held optical device will be fabricated to monitor and quantify in situ ROS activities and associated inflammatory responses in vivo. The successful completion of the proposed work will help the development of a new diagnosis tool which can be effectively used to non-invasively assess the extent of inflammatory processes in real-time and substantially improve the quality of patient care.