Assessing Angiogenesis by Near-Infrared Imaging

本研究的长期目标是开发针对血管生成血管的近红外（NIR）可激发荧光造影剂，并使用无害光对抗血管生成治疗进行无创评估。然而，为了成功，造影剂必须提供显著的对比度增强。我们假设体内噬菌体展示技术衍生的RGD4C和CNGRC肽可用于在血管生成的肿瘤血管中分别将近红外荧光染料定位于α v β 3整合素和CD13受体。我们进一步提出，使用水溶性树突状聚合物，在聚合物链的尖端附着多个归甲肽，将显著增加配体的结合亲和力，并增强肿瘤组织与正常组织的对比。为了验证这些假设，将合成携带荧光吲哚菁绿的星形聚l -谷氨酸。RGD-4C和CNGRC肽将偶联到聚合物位点的末端。缀合方案、聚合物载体和配体将会改变，以优化荧光特性、受体结合和近红外检测。为了建立新合成的近红外造影剂在血管生成血管近红外成像中的应用，我们将使用频域光子迁移ICCD相机对携带人类肿瘤的裸鼠进行成像，获取每种造影剂静脉注射后荧光强度随时间的函数，并获得肿瘤和正常组织中的药代动力学参数。此外，聚合物造影剂将用In- i11标记，用于同时光学和核成像和比较。评估造影剂在肿瘤内的空间分布，以确定对血管床的特异性靶向。最后，选择靶向alphavbeta3和CD13受体的近红外造影剂来评估抗alphavbeta3和抗CD13治疗的治疗反应。将尝试将对比增强和光学成像得出的药代动力学参数与治疗结果、微血管密度计数以及免疫组织化学研究中测量的肿瘤血管中mvg3和CD13受体的表达水平联系起来。拟议的研究将有助于确定近红外造影剂的关键设计特征，以成功地利用近红外成像肿瘤血管系统。

DESCRIPTION (provided by applicant) The long-term goal of this research is to develop near-infrared (NIR) excitable fluorescent contrast agents that target angiogenic blood vessels and enable noninvasive assessment of antiangiogenic therapy using harmless light. To be successful, however, contrast agents must provide significant contrast enhancement. We hypothesize that the RGD4C and CNGRC peptides derived from in vivo phage-displaying technology can be used to home NIR fluorochromes to alpha v beta 3 integrin and CD13 receptors, respectively, in angiogenic tumor blood vessels. We further propose that use of water-soluble dendritic polymers with multiple homing peptides attached to the tips of polymer chains will significantly increase the ligand's binding affinity and enhance the contrast between tumor and normal tissues. To test these hypotheses, star-shaped poly(L-glutamic acid) carrying fluorochrome indocyanine green will be synthesized. RGD-4C and CNGRC peptides will be coupled to the end of the polymers site-specifically. The conjugation scheme, polymeric carriers, and ligands will be varied to enable optimization of fluorescent properties, receptor binding, and NIR detection. To establish the utility of newly synthesized NIR contrast agents in the NIR imaging of angiogenic blood vessels, nude mice bearing human tumors will be imaged using a frequency-domain photon migration ICCD camera to acquire fluorescent intensity as a function of time and obtain pharmacokinetic parameters in tumor and normal tissues following intravenous injection of each contrast agent. In addition, polymeric contrast agent will be labeled with In-I 11 for simultaneous optical and nuclear imaging and comparison. The spacial distribution of the contrast agents in the tumors will be evaluated to confirm the specific targeting to vascular beds. Finally, selected NIR contrast agents targeted to alphavbeta3 and CD13 receptors will be used to evaluate treatment responses to anti-alphavbeta3 and anti-CD13 therapy. Attempts will be made to correlate contrast enhancement and pharmacokinetic parameters derived from optical imaging with treatment outcome, microvessel density counts, and levels of expression ofmvg3 and CD13 receptors in tumor vasculature measured in immunohistochemical studies. The proposed studies will help define critical design features of NIR contrast agents for successfullv imaging tumor vasculature using NIR.