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

描述（由申请人提供）：目前需要改进的自旋探针，以帮助对活性氧（ROS）介导的疾病进行诊断和基础研究。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，并且许多年龄相关疾病如帕金森病和阿尔茨海默病的特征在于过量ROS的损伤。自旋探针允许使用各种磁共振技术（例如电子顺磁共振（EPR））来检测和鉴定这些不稳定的氧物质。虽然动物中的某些癌症（Mikuni等人，2004）和组织，如分离的大鼠心脏（Zweier等人，1998），已经使用EPR成像（EPRI）成功地成像，但是使用当前一代的自旋探针，不可能检测年龄相关疾病中ROS的产生。开发新的自旋探针，允许在体内检测由帕金森氏症和其他ROS疾病产生的ROS，将代表诊断这些疾病和指导其治疗的重大进展。为了克服目前可用的自旋探针的局限性，我们建议调查自旋探针的基础上封装在C60富勒烯，N@C60的单顺磁性氮原子。在这个物种中，氮被钉在对称富勒烯笼的中心，在那里它的未成对自旋完全被保护不与外部物种反应。富勒烯笼中与外部环境的隔离赋予N@C60最已知的EPR线宽之一（Morton等人，2006），使其检测效率比当前的自旋探针高100至1000倍。此外，与表面N@C60上发生的ROS的相互作用将在光谱中产生可测量的位移，而不与探针直接反应。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和将受益于此类化合物的应用数量，本项目的总体目标是合成水溶性的、生物可利用的N@C60衍生物N@C3，并表征其使用体内磁共振技术测量分子氧和生物重要的ROS（包括超氧化物）的能力。该项目的具体目标是：1）表明我们的N@C60衍生物具有适合用作血氧测定和ROS检测的自旋探针的EPR信号，2）将N@C3与目前可用的自旋探针进行比较，用于血氧测定和检测水和脂质环境中、细胞中和分离的线粒体中的超氧化物和其他ROS，和3）使用Overhauser增强MRI技术来研究自旋探针a）增强MRI图像的空间分辨率，B）显著改善MRI的氧标测，和c）检测和标测体内ROS的能力。拟议的研究是开发基于内嵌富勒烯的化合物作为新型自旋探针的第一步，并可能为诊断和治疗从癌症到阿尔茨海默氏症的疾病开辟新的途径。有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氏病，但目前还没有技术允许ROS（或自由基）在患者或在人类疾病的完整动物模型中进行测量。该项目旨在开发一种新型的“自旋探针”，即能够与ROS相互作用产生信号的分子试剂，该信号可以使用各种磁共振成像技术（如磁共振成像（MRI））进行检测，以帮助早期诊断和治疗各种人类疾病。

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.