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

描述(申请人提供)：该项目的总体目标是开发新的功能性EPR探针，增强体内EPR波谱和pH成像的稳定性，pH是活体生物体生物化学中最重要的参数之一。PH值敏感的硝基自由基以前是由P.I.和他的同事开发出来的，但在活组织中经常遇到稳定性不足的问题。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。纳米SPIN的半透膜将区分敏感的氮氧化物和生物还原剂，同时允许分析物H+的自由穿透。这将填补荧光pH探针和核磁共振/核磁共振之间的空白，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提供了应用，但这些目前的技术往往存在灵敏度(1000倍或低于EPR)和特异性不足的问题。具体目标是：(SA1)开发设计对pH敏感的纳米SPIN的有效方法。将使用两种将氮氧化物并入半透纳米球的替代策略，即并入磷脂脂质体和聚酰胺胶囊。(SA2)确定对pH敏感的纳米SPIN的光谱和物理化学特性。对所获得的纳米Spin进行定量表征对于优化制备工艺和提高其进一步应用效率都是至关重要的。(SA3)使用开发的纳米SPIN在体内应用Phe在携带PYMT肿瘤的小鼠中的EPR测量。利用开发的pH敏感型纳米SPIN对活体小鼠的PYMT乳腺肿瘤细胞外Phe的测量将为相关的生化过程提供新的见解，包括更好地了解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，这是目前非常感兴趣的一种治疗方法。这一结果可能为设计其他相应的治疗方法提供机会。综上所述，该项目的成功可能会对未来功能性在体EPR波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包裹到半透纳米球中来增强稳定性的pH敏感顺磁探针。这些被称为纳米SPIN的探针将允许在体内进行EPR光谱和pH成像。利用pH敏感的纳米SPIN在活体小鼠乳腺肿瘤中进行实验，旨在有助于了解实体瘤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.