NanoSPINs for In Vivo EPR-Based Spectroscopy and Imaging

描述（由申请人提供）：该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，特别是用于体内应用。基于EPR的技术远未达到其最大潜力，主要是因为缺乏稳定的体内外源自旋探针。各种氮氧化物探针应用于生物医学的所有优势，在很大程度上被它们在组织中迅速降解为epr沉默产物所消灭。在这个项目中，几种新的方法将被用于开发顺磁探针，增加体内稳定性，这是基于构建含有氮氧化物的纳米级粒子的原始想法。这些纳米自旋蛋白可以渗透到小的分析物中，将传感氮氧化物从生物还原剂中分离出来。纳米螺旋传感器将用于检测生理上重要的物质，即氢离子（pH）和一氧化氮（NO）。这将填补荧光探针和核磁共振之间的空白，前者提高了我们在细胞和亚细胞水平上的检测能力，后者提供了对完整的活体动物和人类的光谱和成像能力。然而，NMR/MRI缺乏灵敏度（比EPR低1000倍或更低）和特异性。具体目标是：（SA1）开发有效的纳米自旋线设计方法。提出的策略是基于两种基质的氮氧化物包封，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“孔隙形成物”如gramicidin a （SA2）来保证。纳米自旋蛋白的定量表征，特别是在生物组织中的功能敏感性和稳定性，对于制备工艺的优化和应用效率的提高都是至关重要的。（SA3）利用纳米螺旋蛋白研究心肌酸中毒和一氧化氮生成在缺血心脏及缺血预处理模型中的作用。我们假设pH稳态的改变和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，在缺血控制和预处理心脏中，心肌酸中毒和NO的产生将通过EPR无创监测。（SA4）应用缺血预处理小鼠心脏局部缺血再灌注模型的体内EPR测量pH和NO生成。为了验证我们在IPC中的pH和NO假设，我们将使用这种小鼠体内心脏模型来无创监测心肌pH和NO生成的变化及其与IPC保护机制的相关性。该结果可能为设计相应的治疗方法提供机会。综上所述，该项目的成功可能对体内EPR光谱和生物成像在医学上的应用产生重大影响。公共卫生相关性：该项目将开发新一代顺磁功能导向探针，称为纳米旋蛋白，以满足电子顺磁共振（EPR）光谱和生物成像的需要，特别是用于体内医学应用。应用实验将在离体大鼠心脏和小鼠心脏局部缺血再灌注缺血预处理模型中使用pH和no敏感的nanoSPINs，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.