NanoSPINs for In Vivo EPR-Based Spectroscopy and Imaging

描述（由申请人提供）：本项目将开发新一代顺磁性、功能导向探针，以满足电子顺磁共振（EPR）光谱和成像的需求，特别是体内应用。基于EPR的技术远未达到其最大潜力，主要是因为缺乏可用的稳定的体内外源性自旋探针。氮氧化合物探针在生物医学中应用的所有优势在很大程度上都被它们在组织中快速降解为EPR沉默产物所抵消。在这个项目中，几种新的方法将被用来开发顺磁性探针，增加体内稳定性的基础上的原始想法的纳米尺寸的粒子与合并的氮氧化物，或nanoSPIN的建设。这些nanoSPIN可渗透小的分析物，将从生物还原剂中分离感测氮氧化物。nanoSPIN传感器将用于检测生理上重要的物质，即H+（pH）和一氧化氮（NO）。这将填补荧光探针和NMR/MRI之间的空白，荧光探针在细胞和亚细胞水平上提高了我们的检测能力，NMR/MRI在完整的活体动物和人类中提供了光谱和成像能力。然而，NMR/MRI缺乏灵敏度（EPR的1000倍或更低）和特异性。具体目标是：（SA 1）开发nanoSPIN设计的有效方法。所提出的策略是基于两个矩阵的氮氧化物封装，溶胶凝胶“玻璃”和磷脂双层囊泡，包括使用聚合脂质体。脂质体的小离子渗透性将通过掺入“成孔剂”如短杆菌肽A来确保。(SA2)pH和NO敏感性纳米SPIN的物理化学表征。nanoSPIN的定量表征，特别是在生物组织中的功能灵敏度和稳定性，对于制备过程的优化和应用效率都是至关重要的。(SA3)研究心肌酸中毒和NO生成在缺血心脏中的作用，以及使用开发的nanoSPIN的缺血预处理模型。我们推测，pH值的动态平衡和NO的产生在缺血预处理（IPC）中起着至关重要的作用。为了验证这一假设，将在缺血对照和预处理心脏中通过EPR无创监测心肌酸中毒和NO产生。(SA4)应用EPR技术在体检测缺血预处理对小鼠心脏局部缺血再灌注模型的pH值和NO生成的影响。为了测试我们的pH和NO假设IPC，我们将使用这种在体内小鼠心脏模型，以非侵入性监测心肌pH和NO生成的变化及其相关性IPC使用开发的nanoSPINs的保护机制。这一结果可能为设计相应的治疗方法提供了机会。总之，该项目的成功可能对体内EPR光谱和医学生物成像应用的未来产生重大影响。 公共卫生相关性：该项目将开发新一代顺磁功能导向探针，称为nanoSPIN，用于电子顺磁共振（EPR）光谱和生物成像的需要，特别是用于医学的体内应用。应用实验将在离体大鼠心脏中和在体内使用pH-和NO-敏感的nanoSPIN {具有缺血预处理的小鼠心脏局部缺血再灌注模型}，并将为设计相应的治疗方法提供新的机会。

DESCRIPTION (provided by applicant): This project will develop a new generation of paramagnetic, functionally oriented probes for the needs of electron paramagnetic resonance (EPR) spectroscopy and imaging, particularly for in vivo applications. EPR- based techniques are far from attaining their maximum potential, predominantly because of a lack of stable in vivo exogenous spin probes available. All the advantages of application of wide classes of nitroxide probes to biomedicine are largely wiped out by their rapid degradation in tissues to EPR-silent products. In this project several new approaches will be used to develop paramagnetic probes with increased in vivo stability based on the original idea of the construction of the nano-Sized Particles with the Incorporated Nitroxides, or nanoSPINs. These nanoSPINs, being permeable to small analytes will separate sensing nitroxides from biological reductants. The nanoSPIN sensors will be used to detect physiologically important species, namely H+ (pH) and nitric oxide (NO). This will fill a niche between fluorescent probes, which have advanced our detection capabilities at cellular and subcellular levels, and NMR/MRI, which have provided spectroscopic and imaging capabilities in intact living animals and humans. However, NMR/MRI suffers from the lack of sensitivity (1000 fold or lower than EPR) and specificity. The specific aims are: (SA1) Development of effective methods for the nanoSPIN design. The proposed strategies are based on two matrixes for nitroxide encapsulation, sol gel "glasses" and phospholipid bilayer vesicles, including use of polymerized liposomes. The small ion permeability of the liposomes will be ensured by incorporation of "pore formers" such as gramicidin A. (SA2) Physicochemical characterization of pH- and NO-sensitive nanoSPINs. Quantitative characterization of the nanoSPINs, particularly functional sensitivity and stability in biological tissues, is absolutely crucial, both for the optimization of the preparation procedures and for efficiency of their applications. (SA3) To study the role of myocardial acidosis and NO generation in ischemic hearts and in the model of ischemic preconditioning using developed nanoSPINs. We hypothesize that alterations in pH homeostasis and NO production play critically important roles in ischemic preconditioning (IPC). To test the hypothesis, myocardium acidosis and NO production will be monitored noninvasively by EPR in ischemic control and preconditioned hearts. (SA4) To apply in vivo EPR measurements of pH and NO generation in models of mouse heart regional ischemia reperfusion with ischemic preconditioning. In order to test our pH and NO hypothesis in IPC, we will use this in vivo mouse heart model to noninvasively monitor the variations of myocardial pH and NO generation and their correlations to the protective mechanisms of IPC using developed nanoSPINs. The results may provide an opportunity for the design of corresponding therapeutic approaches. In summary, the success of this project may have a significant impact on the future of in vivo EPR spectroscopy and bioimaging applications to medicine. PUBLIC HEALTH RELEVANCE: This project will develop a new generation of paramagnetic functionally oriented probes, termed nanoSPINs, for the needs of electron paramagnetic resonance (EPR) spectroscopy and bioimaging, particularly for in vivo applications to medicine. Application experiments will use pH- and NO-sensitive nanoSPINs in isolated rat hearts and in vivo in a {models of mouse heart regional ischemia reperfusion with ischemic preconditioning} and will provide new opportunities for designing corresponding therapeutic approaches.