Improved Spin Probes for Magnetic Resonance Imaging of Oxygen and Reactive Oxygen

描述（由申请人提供）：目前需要改进自旋探针，以帮助诊断和基础研究由活性氧（ROS）介导的疾病。对于年龄相关疾病的研究尤其如此，因为氧化损伤在衰老过程中积累，许多年龄相关疾病，如帕金森病和阿尔茨海默病，其特征是过量的ROS损伤。自旋探针允许使用各种磁共振技术（如电子顺磁共振（EPR））检测和识别这些不稳定的氧。虽然动物（Mikuni et al., 2004）和组织(如分离的大鼠心脏（Zweier et al., 1998）中的某些癌症已经成功地使用EPR成像成像（EPRI），但使用当前一代的自旋探针，不可能检测到年龄相关疾病中ROS的产生。新型自旋探针的开发可以在体内检测帕金森氏症和其他ROS疾病产生的ROS，这将是诊断这些疾病并指导治疗的重大进步。为了克服现有自旋探针的局限性，我们提出研究基于包裹在C60富勒烯中的单个顺磁性氮原子的自旋探针，N@C60。在这个物种中，氮被固定在对称富勒烯笼的中心，在那里它的不成对的自旋完全被保护，不与外部物种发生反应。在富勒烯笼中与外界环境隔离，使N@C60具有已知最窄的EPR线宽之一（Morton et al., 2006），使其检测效率比目前的自旋探针高100至1000倍。此外，与表面上发生的ROS的相互作用N@C60将在没有与探针直接反应的情况下产生可测量的光谱位移。这些综合特性使N@C60成为潜在的理想自旋探针。考虑到这类化合物作为自旋探针的潜力以及从这些化合物中受益的应用数量，该项目的总体目标是合成一种水溶性、生物可利用的N@C60衍生物N@C3，并表征其在体内使用磁共振技术测量分子氧和生物重要ROS（包括超氧化物）的能力。该项目的具体目标是：1)表明我们N@C60导数EPR信号,适合作为自旋探针对血氧定量法和活性氧检测,2)目前比较N@C3和自旋探针对血氧定量法检测超氧化物和其他活性氧在水和脂质环境中,在细胞,分离线粒体,和3)使用Overhauser-enhanced MRI技术研究能力的自旋探针)增强MRI图像的空间分辨率,b)大幅度提高MRI氧定位，c)检测和绘制体内ROS。这些研究是开发基于富勒烯的内源性化合物作为新型自旋探针的第一步，并可能为从癌症到阿尔茨海默病的诊断和治疗开辟新的途径。越来越多的证据表明活性氧（ROS）可能有助于许多人类疾病的发展，包括癌症，糖尿病，阿尔茨海默氏痴呆症和帕金森病，但目前还没有技术允许在患者或完整的人类疾病动物模型中测量ROS（或自由基）。该项目旨在开发一种新型的“自旋探针”，这种分子制剂能够与活性氧相互作用，产生一种信号，这种信号可以通过磁共振成像（MRI）等各种磁共振成像技术检测到，以协助早期诊断和治疗广泛的人类疾病。

DESCRIPTION (provided by applicant): There is currently a need for improved spin probes to help with the diagnosis of and fundamental research into diseases mediated by reactive oxygen species (ROS). This is especially true for the study of age related diseases, since oxidative damage accumulates during the aging process, and many age related disorders such as Parkinson's and Alzheimer's disease are characterized by damage from excess ROS. Spin probes allow these unstable oxygen species to be detected and identified using various magnetic resonance techniques, such as electron paramagnetic resonance (EPR). While certain cancers in animals (Mikuni et al., 2004) and tissues, such as an isolated rat heart (Zweier et al., 1998), have been successfully imaged using EPR imaging, (EPRI), with the current generation of spin probes it is not possible to detect the generation of ROS in age related disorders. The development of new spin probes that allow in vivo detection of ROS produced by Parkinson's and other ROS diseases would represent a significant advance for diagnosing these conditions and for guiding their treatment. To overcome limitations of currently available spin probes, we propose to investigate spin probes based upon single paramagnetic nitrogen atoms encapsulated in C60 fullerenes, N@C60. In this species, the nitrogen is pinned at the center of the symmetric fullerene cage where its unpaired spins are completely protected from reaction with external species. Isolation from the outside environment in the fullerene cage endows N@C60 with one of the narrowest known EPR line widths, (Morton et al., 2006), giving it detection efficiency 100 to 1000 times better than the current spin probes. In addition, interactions with ROS occurring on the surface N@C60 will produce measurable shifts in the spectrum without direct reaction with the probe. These combined features make N@C60 a potentially ideal spin probe. Given the potential of this class of compounds as spin probes and the number of applications that would benefit from such compounds, the overall goal of this project is to synthesize a water-soluble, bioavailable N@C60 derivative, N@C3, and characterize its ability to measure molecular oxygen and biologically important ROS including superoxide using magnetic resonance techniques in vivo. The specific aims of this project are: 1) show that our N@C60 derivative has an EPR signal that is suitable for use as a spin probe for both oximetry and ROS detection, 2) compare N@C3 with currently available spin probes for both oximetry and detection of superoxide and other ROS in aqueous and lipid environments, in cells, and in isolated mitochondria, and 3) Use the technique of Overhauser-enhanced MRI to study the ability of the spin probe to a) enhance spatial resolution of the MRI image, b) substantially improve oxygen mapping by MRI, and c) detect and map ROS in vivo. The proposed studies are the first steps in developing endohedral fullerene-based compounds as novel spin probes, and may open up new avenues for the diagnosis and treatment of diseases ranging from cancer to Alzheimer's. There is growing evidence that reactive oxygen species (ROS) may contribute to the development of many human diseases, including cancer, diabetes, Alzheimer's dementia and Parkinson's disease, but there are currently no techniques which allow ROS (or free radicals) to be measured in patients or in intact animal models of human disease. This project is designed to develop a novel class of "spin probes", molecular agents which are able to interact with ROS to produce a signal which can be detected using various magnetic resonance imaging techniques, such as magnetic resonance imaging (MRI), to assist in early diagnosis and treatment of a broad range of human diseases.