A Cy5.5-labeled dimeric NGR peptide for near-infrared fluorescence imaging of CD13 receptor expression

Objectives 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 (NIRF) imaging of CD13 receptor expression in vivo.

Methods 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. Each mouse bearing an HT-1080 tumor was intravenously injected with 1.5 nmol of Cy5.5-NGR2 and imaged at various time points with or without 20 mg/kg of unlabeled NGR peptide. The animals from non-blocking and blocking groups were euthanized at 4 h pi. The tumors, tissues, and organs were dissected for ex vivo fluorescence imaging.

Results 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 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 non-blocking group vs. 1.05±0.06 in blocking group. The results from ex vivo imaging were consistent with the in vivo findings.

Conclusions We have successfully constructed a Cy5.5-labeled dimeric NGR peptide, which is a promising molecular probe, not only allowing the NIR optical imaging of CD13 overexpressed tumors, but also facilitating noninvasive monitoring of CD13-targeted tumor therapy.

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).