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.
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