NMR and NIR Detection of Phospholipase Activity In Vivo

描述（由申请人提供）：本申请的总体目标是开发体内磷脂酶表达和活性成像的方法。将采用顺序磁共振成像/光谱（MRI/MRS）和使用近红外（NIR）荧光团的光学成像的多模态方法来获得单独使用两种技术无法获得的信息。要解决的关键假设是，体内磷脂酶的激活可以通过确定核磁共振可见脂质代谢物水平的变化以及测量酶水解自猝灭的近红外磷脂荧光团释放的荧光来测量。我们将比较和验证磁共振光谱与近红外光学成像在肿瘤模型中获得的结果，其中胆碱代谢物水平的变化与肿瘤进展和对治疗的反应是众所周知的。由于磷脂酶与包括癌症、关节炎和阿尔茨海默病在内的多种病理有关，因此本研究将为这些疾病中涉及的特定生化途径提供关键的体内数据。该假设将通过实现以下具体目标来验证：目标1)：设计和合成基于手性脂质的自猝灭荧光团，该荧光团含有稳定的焦酚化物近红外荧光团，与暗猝灭剂耦合。荧光团将连接到甘油主链的不同位置，以询问不同磷脂酶同工型的活性。猝灭键将连接在甘油主链的不同点上，以区分磷脂酶类型。结构-活性研究将通过综合插入不同长度的连接体来确定位阻对探针活性的影响。目的2)：合成探针的体外表征。这将包括试管演示探针对各种磷脂酶类型和异构体的敏感性和特异性，以及催化和抑制动力学的详细测量。我们将进一步表征这些自猝灭荧光团在培养细胞系中的传递和激活。目的3)：在前列腺癌和非霍奇金淋巴瘤的肿瘤异种移植模型中展示磷脂酶的体内递送、生物分布和随后的激活，随后使用1H磁共振光谱和光学成像进行双模态分子成像。这些数据将为涉及疾病发病和随后治疗的特定脂质分解代谢途径提供关键信息。公共卫生相关性：开发近红外荧光信标，通过双光学成像和磁共振光谱检测体内磷脂酶活性。

DESCRIPTION (provided by applicant): The overall aim of this application is to develop methods to image the expression and activity of phospholipases in vivo. A multi-modality approach with sequential magnetic resonance imaging/spectroscopy (MRI/MRS) and optical imaging using near infrared (NIR) fluorophores will be employed to obtain information that is not available from either technique separately. The key hypothesis to be addressed is that in vivo activation of phospholipases can be measured by determining the changes in MR-visible lipid metabolite levels together with measuring fluorescence released by enzymatic hydrolysis of a self- quenching NIR phospholipid fluorophore. We will compare and validate the MR spectroscopy with results obtained using NIR optical imaging in tumor models in which changes in choline metabolite levels associated with tumor progression and response to therapy are well known. Since phospholipases have been implicated in a wide range of pathologies including cancer, arthritis and Alzheimer's disease, this study will supply critical in vivo data on the specific biochemical pathways involved in these diseases. The hypothesis will be tested by the achievement of the following specific aims: Aim 1): Design and synthesis of chiral lipid-based self-quenching fluorophores containing a stable pyropheophorbide NIR fluorophore, coupled to a dark quencher. The fluorophore will be attached to different positions of the glycerol backbone to interrogate the activity of different phospholipase isoforms. The quencher linkage will be attached at different points on the glycerol backbone to distinguish between phospholipase types. Structure-activity studies will be performed by synthetically inserting linkers of various length to determine the effects of steric hindrance on probe activity. Aim 2): In vitro characterization of the synthesized probes. This will include test-tube demonstration of the sensitivity and specificity of the probes to various phospholipase types and isoform and detailed measurement of kinetics of catalysis and inhibition. We will further characterize the delivery and activation of these self- quenching fluorophores in cultured cell lines. Aim 3): Demonstration of in vivo delivery, bio-distribution and subsequent activation of phospholipases in tumor xenograft models of prostate cancer and non-Hodgkin's lymphoma, followed by dual modality molecular imaging using 1H MR spectroscopy and optical imaging. These data will supply critical information on the specific lipid catabolic pathways involved in the onset and subsequent treatment of disease. PUBLIC HEALTH RELEVANCE: To develop near-infrared fluorescent beacons for the in vivo detection of phospholipase activity via dual optical imaging and MR spectroscopy.