Abstract
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Objectives NGR (Asn-Gly-Arg) sequence is known as a CD13 receptor-specific motif binding to tumor neovessels. VEGI (Vascular Endothelial Growth Inhibitor) is being developed for the treatment of tumor angiogenesis. In this study, we labeled a NGR-VEGI fusion protein with Cy5.5 and evaluated tumor-targeting efficacy and pharmacokinetics of the resulting Cy5.5-NGR-VEGI probe in tumor-bearing mice.
Methods The NGR-VEGI fusion protein was conjugated with Cy5.5 NHS ester followed by purification with a PD-10 column. In vitro fluorescence staining was performed to determine the binding of Cy5.5-NGR-VEGI to CD13-overexpressed HT-1080 cells. A Cy5.5 filter set was used to acquire the fluorescence signals in an IVIS-200 system. Each mouse bearing an HT-1080 tumor received 1.5 nmol of Cy5.5-NGR-VEGI intravenously and subjected to optical imaging at various time points with or without 20 mg/kg of unlabeled NGR-VEGI. The tumors, tissues, and organs were dissected and subjected to ex vivo fluorescence imaging at 8 h pi.
Results In vitro fluorescence staining revealed dose-dependent binding of Cy5.5-NGR-VEGI to HT-1080 cells. In vivo optical imaging with Cy5.5-NGR-VEGI exhibited rapid HT-1080 tumor targeting. The highest tumor-to-background contrast was achieved at 8 h pi. Tumor specificity was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR-VEGI. Ex vivo studies further confirmed the in vivo imaging findings, demonstrating that Cy5.5-NGR-VEGI displayed an excellent tumor-to-muscle ratio (18.93±2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92±0.75) for the blocking group.
Conclusions The Cy5.5 fluorophore was successfully conjugated with NGR-VEGI to image tumor vasculature. Cy5.5-NGR-VEGI provided highly sensitive and target-specific imaging of HT-1080 tumors. Cy5.5-NGR-VEGI has the potential as a promising probe for longitudinal monitoring of tumor response to treatment with NGR-VEGI.
Research Support This work was supported by the USC Department of Radiology, the Major Program of National Natural Science Foundation of China (Grant No. 81230033), the National Basic Research and Development Program of China (Grant No. 2011CB707704), and the Major Research Instrumentation Program of National Natural Science Foundation of China (Grant No. 81227901).