Synthesis and Evaluation of 64Cu-DOTA-NT-Cy5.5 as a Dual-Modality PET/Fluorescence Probe to Image Neurotensin Receptor-Positive Tumor

Huaifu Deng, Hui Wang, Mengzhe Wang, Zibo Li, Zhanhong Wu, Huaifu Deng, Hui Wang, Mengzhe Wang, Zibo Li, Zhanhong Wu

Abstract

Overexpression of neurotensin receptors (NTRs) has been suggested to play important roles in the growth and survival of a variety of tumor types. The aim of this study is to develop a dual-modality probe (64Cu -DOTA-NT-Cy5.5) for imaging NTR1 expression in vivo with both positron emission tomography (PET) and fluorescence. In this approach, the thiol group and N terminal amino group of neurotensin analogue (Cys-NT) were chemically modified with Cy5.5 dye and DOTA chelator, respectively. After radiolabeling with 64Cu, the resulting probe (64Cu-DOTA-NT-Cy5.5) was evaluated in NTR1 positive HT-29 tumor model. Small animal PET quantification analysis demonstrated that the tumor uptake was 1.91±0.22 and 1.79±0.16%ID/g at 1 and 4 h postinjection (p.i.), respectively. The tumor-to-muscle ratio was 17.44±3.25 at 4 h p.i. based on biodistribution. Receptor specificity was confirmed by the successful blocking experiment at 4 h p.i. (0.42±0.05%ID/g). In parallel with PET experiment, fluorescence imaging was also performed, which demonstrated prominent tumor uptake in HT-29 model. As a proof of concept, an imaging guided surgery was performed to the fluorescent moiety of this probe and could provide potential surgery guidance for NTR positive patients. In summary, our results clearly indicated that the dual-modality probe, 64Cu-DOTA-NT-Cy5.5, could serve as a promising agent to image NTR positive tumors in vivo.

Keywords: 64Cu; dual-modality; fluorescence imaging; neurotensin; positron emission tomography.

Figures

Figure 1
Figure 1
HPLC profiles of 64Cu-DOTA-NT-Cy5.5 at 30 min, 2 h, and 6 h after purification with radiochemical purity more than 98.9%, 96.9%, and 90.8%, respectively.
Figure 2
Figure 2
Competitive cell binding assays of 125I-NT(8–13) and DOTA-NT-Cy5.5 or Cys-NT in HT-29 cells. X-axis stands for the concentration of nonradiolabeled competitor. IC50 values for DOTA-NT-Cy5.5 and Cys-NT were 0.65 ± 0.35 and 0.43 ± 0.15 nmol/L, respectively.
Figure 3
Figure 3
PET/CT images of HT-29 tumor-bearing mice at 1 and 4 h postinjection of 64Cu-DOTA-NT-Cy5.5 (A) without and (B) with a blocking dose of NT peptide. (C) The quantitative tumor and muscle uptakes derived from PET images. (D) In vivo fluorescent images of HT-29 tumor-bearing mice.
Figure 4
Figure 4
Biodistribution of 64Cu-DOTA-NT-Cy5.5 in mice bearing HT-29 xenograft at 4 h p.i.
Figure 5
Figure 5
Ex vivo fluorescent images of major organs harvested at 4 h p.i. of 64Cu-DOTA-NT-Cy5.5 (A) without and (B) with coinjection of Cys-NT.
Figure 6
Figure 6
Athymic female nude mouse bearing HT-29 tumor at 4 h postinjection of 64Cu-DOTA-NT-Cy5.5. The tumor lesion was subsequently removed by fluorescent image-guided surgery. (A–D) Digital pictures of the mouse and the tumors during surgery. (E–H) Fluorescent images as a visual guide during surgery.
Figure 7
Figure 7
In vivo functional targeting of the novel DOTA-NT-Cy5.5. Representative serial sections of HT-29 xenograft tumors stained with DAPI (blue), CD31 antibody (green), and DOTA-NT-Cy5.5 (red).
Scheme 1. Synthesis of 64 Cu-DOTA-NT-Cy5.5 a
Scheme 1. Synthesis of 64Cu-DOTA-NT-Cy5.5a
aReagents and conditions: a. pH 7.5, 30 min RT. b. EDC/SNHS, pH = 5.5. c. borate buffer (pH 8.5), 30 min RT. d. NH4OAc (pH 5.5), 37 °C, 60 min.

Source: PubMed

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