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，许多与年龄相关的疾病，如帕金森氏症和阿尔茨海默病，其特征是ROS过多造成的损害。自旋探针允许使用各种磁共振技术来检测和识别这些不稳定的氧物种，例如电子顺磁共振(EPR)。虽然动物(Mikuni等人，2004年)和组织中的某些癌症，如离体鼠心脏(Zweier等人，1998)，已经使用EPR成像(EPRI)成功地进行了成像，但使用当前一代自旋探针，不可能在年龄相关疾病中检测到ROS的产生。新的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和指导其治疗的重大进步。为了克服目前可用的自旋探针的局限性，我们建议研究基于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，氮被钉在对称的富勒烯笼子的中心，在那里它的不成对的自旋完全被保护，不与外部物种反应。在富勒烯笼子中与外部环境隔离使N@C60具有已知的最窄的EPR谱线宽度之一(Morton等人，2006年)，使其检测效率比目前的自旋探针高100至1000倍。此外，在N@C60表面与ROS发生的相互作用将在不与探针直接反应的情况下在光谱中产生可测量的位移。这些组合特征使N@C60成为一种潜在的理想自旋探测器。鉴于这类化合物作为自旋探针的潜力以及此类化合物的应用数量，该项目的总体目标是合成一种水溶性、生物可用的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包括超氧化物在内的生物重要ROS的能力。该项目的具体目标是：1)显示我们的N@C60衍生物具有EPR信号，适合用作血氧仪和ROS检测的自旋探针；2)比较N@C3与目前可用的自旋探针在水溶液和脂质环境、细胞以及孤立线粒体中检测超氧化物和其他ROS的能力；3)使用Overhauser增强磁共振技术来研究自旋探针的能力，a)提高MRI图像的空间分辨率，b)显著改进MRI的氧图，以及c)在体内检测和绘制ROS。这项拟议的研究是开发基于富勒烯的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氏症的各种疾病的诊断和治疗开辟新的途径。越来越多的证据表明，活性氧物种(ROS)可能导致许多人类疾病的发生，包括癌症、糖尿病、阿尔茨海默氏症和帕金森病，但目前还没有技术可以在患者身上或在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”，即能够与ROS相互作用的分子试剂，以产生可以使用各种磁共振成像技术(如磁共振成像)检测到的信号，以帮助对广泛的人类疾病进行早期诊断和治疗。

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.