Amino Acids

, Volume 46, Issue 6, pp 1547–1556 | Cite as

Near-infrared fluorescence imaging of CD13 receptor expression using a novel Cy5.5-labeled dimeric NGR peptide

  • Guoquan Li
  • Yan Xing
  • Jing WangEmail author
  • Peter S. Conti
  • Kai ChenEmail author
Original Article


In this study, we synthesized a novel Cy5.5-labeled dimeric NGR peptide (Cy5.5-NGR2) via bioorthogonal click chemistry, and evaluated the utility of Cy5.5-NGR2 for near-infrared fluorescence imaging of CD13 receptor expression in vivo. The dimeric NGR peptide (NGR2) was conjugated with an alkyne-containing PEG unit followed by mixing with an azide-terminated Cy5.5 fluorophore (Cy5.5-N3) to afford Cy5.5-NGR2. The probe was subject to in vitro and in vivo evaluations. The bioorthogonal click chemistry provided a rapid conjugation of the alkyne-containing NGR2 with Cy5.5-N3 in a quantitative yield within 15 min. The laser confocal microscopy revealed that binding of Cy5.5-NGR2 to CD13 receptor is target-specific as demonstrated in CD13-positive HT-1080 cells, CD13-negative MCF-7 cells, and a blocking study in HT-1080 cells. For in vivo optical imaging, Cy5.5-NGR2 exhibited rapid HT-1080 tumor targeting at 0.5 h postinjection (pi), and highest tumor-to-background contrast at 2 h pi. The CD13-specific tumor accumulation of Cy5.5-NGR2 was accomplished by a blocking study with unlabeled NGR peptide in HT-1080 tumor bearing mice. The tumor-to-muscle ratio of Cy5.5-NGR2 at 2 h pi reached 2.65 ± 0.13 in the non-blocking group vs. 1.05 ± 0.06 in the blocking group. The results from ex vivo imaging were consistent with the in vivo findings. We concluded that Cy5.5-NGR2 constructed by bioorthogonal click chemistry is a promising molecular probe, not only allowing the NIR optical imaging of CD13 overexpressed tumors, but also having the potential to facilitate noninvasive monitoring of CD13-targeted tumor therapy.


Molecular imaging probe NGR peptide Fluorescence imaging CD13 receptor Tumor vasculature 



Near-infrared fluorescence


Positron emission tomography


Single photon emission computed tomography


Computed tomography


Magnetic resonance imaging


Aminopeptidase N




High-performance liquid chromatography


Phosphate-buffered saline






Trifluoroacetic acid


Dimethyl sulfoxide















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).

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  1. 1.Department of Nuclear MedicineXijing Hospital, The Fourth Military Medical UniversityXi’anChina
  2. 2.Molecular Imaging Center, Department of Radiology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA

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