68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET.
Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine-glycine-arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a (68)Ga-labeled NGR peptide as a new molecular probe that binds to APN.


METHODS
NGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with (68)Ga at 95°C for 10 min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of (68)Ga-DOTA-NGR was determined in normal mice by dissection method. (68)Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry.


RESULTS
The radiochemical purity of (68)Ga-DOTA-NGR was 98.0% ± 1.4% with a specific activity of about 17.49 MBq/nmol. The uptake of (68)Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that (68)Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1 h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58 ± 1.26 at 1.5 h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature.


CONCLUSIONS
(68)Ga-DOTA-NGR was easily synthesized and showed favorable biodistribution and kinetics. (68)Ga-DOTA-NGR could also specifically bind to the APN receptor in vitro and in vivo, and might be a potential molecular probe for the noninvasive detection of APN-positive tumors and neovasculature.
Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China, 210006; Taizhou People's Hospital, Taizhou, Jiangsu, China, 225300.
Jun
J
Zhang
Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China, 210006.
Xiaoli
X
Lu
Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China, 210006.
Nan
N
Wan
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing, Jiangsu, China, 210000.
Zichun
Z
Hua
Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China, 210006. Electronic address: zzwang136@aliyun.com.
Zizheng
Z
Wang
Key Laboratory of Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, China, 210463.
Hongbo
H
Huang
Key Laboratory of Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, China, 210463.
Min
M
Yang
Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China, 210006; State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing, Jiangsu, China, 210000. Electronic address: fengwangcn@hotmail.com.
Feng
F
Wang
D000818:Animals; D001692:Biological Transport; D018826:CD13 Antigens / Q000378:metabolism*; D045744:Cell Line, Tumor; D005240:Feasibility Studies; D005260:Female; D005710:Gallium Radioisotopes; D015972:Gene Expression Regulation, Neoplastic; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D008175:Lung Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D049268:Positron-Emission Tomography / Q000379:methods*; D011854:Radiochemistry; D055752:Small Cell Lung Carcinoma / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology
D005710:Gallium Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; C526428:NGR peptide; D009842:Oligopeptides; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D018826:CD13 Antigens
(68)Ga labeling; APN; CD13; NGR peptide; Tumor angiogenesis