NMR and NIR Detection of Phospholipase Activity In Vivo

描述(申请人提供)：本申请的总体目标是开发成像磷脂酶在体内的表达和活性的方法。将使用顺序磁共振成像/波谱(MRI/MRS)和使用近红外(NIR)荧光团的光学成像的多模式方法来获得单独从这两种技术中无法获得的信息。需要解决的关键假设是，在体内磷脂酶的激活可以通过测定MR可见的脂类代谢物水平的变化以及测量自猝灭的近红外磷脂荧光团的酶解释放的荧光来测量。我们将在肿瘤模型中将磁共振波谱与近红外光学成像的结果进行比较和验证，在肿瘤模型中，与肿瘤进展和治疗反应相关的胆碱代谢产物水平的变化是众所周知的。由于磷脂酶与包括癌症、关节炎和阿尔茨海默病在内的广泛的病理过程有关，这项研究将提供关键的体内数据，了解这些疾病涉及的特定生化途径。这一假设将通过以下具体目标的实现来验证：目标1)：设计和合成含有稳定的焦脱镁叶绿体的手性脂基自猝灭荧光团，并与暗猝灭剂偶联。荧光团将附着在甘油骨架的不同位置，以询问不同磷脂酶亚型的活性。猝灭连接将连接在甘油骨架上的不同位置，以区分磷脂酶类型。结构-活性研究将通过合成插入不同长度的连接基来确定空间位阻对探针活性的影响。目的2)：对合成的探针进行体外表征。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：在前列腺癌和非霍奇金淋巴瘤异种移植瘤模型中展示磷脂酶的体内传递、生物分布和随后的激活，然后用1H磁共振波谱和光学成像进行双模式分子成像。这些数据将提供有关疾病发生和随后治疗所涉及的特定脂类分解代谢途径的关键信息。与公众健康相关：开发近红外荧光信标，通过双重光学成像和磁共振光谱学在体内检测磷脂酶活性。

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.