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

描述（由申请人提供）：本项目的总体目标是开发新的功能性EPR探针，其具有增强的稳定性，用于体内EPR光谱和pH成像，pH是活生物体生物化学中最重要的参数之一。pH敏感的硝酰基自由基先前已由P.I.但是在活组织中经常遭受稳定性不足。在这个项目中，将使用两种不同的策略来开发具有体内稳定性的顺磁性探针，其基于构建具有并入的氮氧化物的纳米尺寸颗粒或nanoSPIN的原始想法。nanoSPIN的半透膜将区分感测氮氧化物与生物还原剂，同时允许分析物H+自由渗透。这将填补荧光pH探针和NMR/MRI之间的空白，荧光pH探针已经在细胞和亚细胞检测的应用中取得了进展，NMR/MRI已经在活体动物和人类中提供了应用，但这些现有技术通常缺乏灵敏度（EPR的1000倍或更低）和特异性。具体目标是：（SA 1）开发用于设计pH敏感的nanoSPIN的有效方法。将使用将氮氧自由基掺入半透性纳米球的两种替代策略，即掺入磷脂脂质体和聚酰胺胶囊。(SA2)确定pH敏感性nanoSPIN的光谱和理化特性。所获得的nanoSPIN的定量表征对于制备过程的优化和其进一步应用的效率都是绝对关键的。(SA3)使用开发的nanoSPIN在PyMT荷瘤小鼠中应用pHe的体内EPR测量。使用开发的pH敏感性nanoSPIN测量活小鼠中PyMT乳腺肿瘤中的细胞外pHe将提供对相关生化过程的新见解，包括更好地理解粒细胞巨噬细胞集落刺激因子（GM-CSF）的观察到的抗肿瘤活性，这是一种目前非常感兴趣的治疗方法。这些结果可能为设计其他相应的治疗方法提供了机会。总之，该项目的成功可能会对未来的功能性体内EPR光谱和医学生物成像应用产生重大影响。该项目将开发基于将氮氧化物封装到半渗透性纳米球中的增强稳定性的pH敏感的顺磁探针。这些探针，称为nanoSPIN，将允许体内EPR光谱和pH成像。计划在活体小鼠的PyMT乳腺肿瘤中使用pH敏感的nanoSPIN的实验有助于理解实体瘤中细胞外酸中毒的机制，并使用细胞外pH来监测肿瘤进展，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.