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、面向功能的探测器，以满足电子顺磁共振(EPR)光谱和成像的需要，特别是用于活体应用。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可用。广泛种类的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沉默的产物所抹去。在这个项目中，将使用几种新的方法来开发具有更高体内稳定性的顺磁探针，该探针基于最初的想法，即用含氮氧化物或纳米SPIN构建纳米颗粒。这些纳米SPIN对小分析物具有渗透性，将敏感氮氧化物从生物还原剂中分离出来。NanoSpin传感器将用于检测生理上重要的物种，即H+(PH)和一氧化氮(NO)。这将填补荧光探针和核磁共振/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的检测能力，后者在完整的活体动物和人类中提供了光谱和成像能力。然而，核磁共振/磁共振成像缺乏敏感性(1000倍或低于EPR)和特异性。具体目标是：(SA1)开发有效的纳米自旋设计方法。所提出的策略是基于氮氧化物的两种基质，即溶胶-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加入“造孔剂”，如革兰菌素A(SA2)和对pH和不敏感的纳米SPIN的物理化学表征来确保。对纳米SPIN的定量表征，特别是生物组织中的功能敏感性和稳定性，对于优化制备过程和应用效率都是绝对关键的。(SA3)研究心肌酸中毒和NO生成在缺血心脏中的作用以及在使用已开发的纳米SPIN进行缺血预适应模型中的作用。我们推测，pH动态平衡的改变和NO的产生在缺血预适应(IPC)中起着至关重要的作用。为了验证这一假设，EPR将在缺血对照和预适应心脏中无创地监测心肌酸中毒和一氧化氮的产生。(SA4)应用EPR测定缺血预适应小鼠心脏局部缺血再灌流模型的pH和NO生成。为了验证我们在IPC中的pH和NO假说，我们将利用这个在体小鼠心脏模型，使用开发的纳米SPIN来无创性地监测心肌pH和NO产生的变化及其与IPC保护机制的相关性。这一结果可能为设计相应的治疗方法提供机会。综上所述，该项目的成功可能会对活体EPR波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的探针，称为纳米SPIN，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。应用实验将在大鼠离体心和活体小鼠心脏局部缺血再灌流模型中使用pH和非敏感纳米SPIN，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.