Spin Probes in Semipermeable Nanospheres: EPR spectroscopy & imaging of tumor pH

描述（由申请人提供）：该项目的总体目标是开发新的功能性EPR探针，增强其在体内EPR光谱和pH成像的稳定性，pH是生物体生物化学中最重要的参数之一。对ph敏感的硝基自由基之前已经被P.I.和同事们开发出来，但在活体组织中往往缺乏稳定性。在这个项目中，将采用两种不同的策略来开发具有体内稳定性的顺磁探针，这是基于用结合的氮氧化物或纳米自旋蛋白构建纳米级粒子的原始想法。纳米自旋蛋白的半透膜将区分感应氮氧化物和生物还原剂，同时允许分析氢离子自由渗透。这将填补荧光pH探针和NMR/MRI之间的空白，前者在细胞和亚细胞检测方面取得了进展，后者在活体动物和人类中得到了应用，但这些目前的技术往往缺乏灵敏度（比EPR低1000倍或更低）和特异性。具体目标是：（SA1）为ph敏感纳米自旋蛋白的设计提供有效的方法。将使用两种替代策略将氮氧化物掺入半透性纳米球，即掺入磷脂脂质体和聚酰胺胶囊。（SA2）定义ph敏感纳米自旋蛋白的光谱和物理化学特性。所获得的纳米ospin的定量表征对于制备工艺的优化和其进一步应用的效率至关重要。（SA3）利用开发的纳米旋旋蛋白（nanoospins）在PyMT荷瘤小鼠体内测量pHe的EPR。利用已开发的pH敏感纳米自旋蛋白（nanoospins）测量活小鼠乳腺肿瘤中细胞外pHe，将为相关生化过程提供新的见解，包括更好地理解所观察到的粒细胞巨噬细胞集落刺激因子（GM-CSF）的抗肿瘤活性，这是一种目前备受关注的治疗方法。该结果可能为设计其他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功能性EPR光谱和生物成像在医学上的应用产生重大影响。该项目将开发基于将氮氧化物封装成半渗透纳米球的ph敏感顺磁探针，增强其稳定性。这些探针被称为nanoSPINs，将允许在体内EPR光谱和pH成像。在活体小鼠PyMT乳腺肿瘤中使用pH敏感的nanoSPINs的实验计划有助于理解实体肿瘤中细胞外酸中毒的机制，并利用细胞外pH监测肿瘤进展，从而评估抗肿瘤药物的疗效，并为设计相应的治疗方法提供机会。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop new functional EPR probes of enhanced stability for in vivo EPR spectroscopy and imaging of pH, one of the most important parameters in the biochemistry of living organisms. pH-sensitive nitroxyl radicals have been previously developed by the P.I. and colleagues but often suffer insufficient stability in living tissues. In this project two different strategies will be used to develop paramagnetic probes with stability in vivo based on the original idea of constructing nano-Sized Particles with the Incorporated Nitroxides, or nanoSPINs. The semipermeable membrane of the nanoSPINs will differentiate sensing nitroxides from biological reductants while allowing free penetration of the analyze, H+. This will fill a niche between fluorescent pH probes, which have provided advances in applications for cellular and subcellular detection, and NMR/MRI, which have provided applications in living animals and humans, but these current techniques often suffer from the lack of sensitivity (1000 fold or lower than EPR) and specificity. The specific aims are: (SA1) To develop effective approaches for the design of pH-sensitive nanoSPINs. Two alternative strategies for the incorporation of the nitroxides into semipermeable nanospheres will be used, namely incorporation into phospholipids liposomes and polyamide capsules. (SA2) To define spectroscopic and physicochemical characteristics of pH-sensitive nanoSPINs. Quantitative characterization of the obtained nanoSPIN is absolutely crucial both for the optimization of the preparation procedures and for efficiency of their further applications. (SA3) To apply in vivo EPR measurements of pHe in PyMT tumor-bearing mice using developed nanoSPINs. The measurement of the extracellular pHe in the PyMT mammary tumors in living mice using developed pH- sensitive nanoSPINs will provide new insights into related biochemical processes, including better understanding of the observed anti-tumor activity of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), a therapeutic approach which is currently of much interest. The results may provide an opportunity for the design of other corresponding therapeutic approaches. In summary, the success of this project may have a significant impact on the future of functional in vivo EPR spectroscopy and bioimaging applications to medicine. This project will develop pH-sensitive paramagnetic probes of enhanced stability based on encapsulation of the nitroxides into semipermeable nanospheres. These probes, termed nanoSPINs, will allow in vivo EPR spectroscopy and imaging of pH. The experiments using pH-sensitive nanoSPINs in PyMT mammary tumors in living mice are planned to contribute to the understanding of the mechanisms of extracellular acidosis in solid tumors and to use extracellular pH to monitor tumor progression and thus evaluate the efficacy of anti-tumor drugs, and provide opportunities for designing corresponding therapeutic approaches.