Biodistribution and SPECT Imaging Study of 99mTc Labeling NGR Peptide in Nude Mice Bearing Human HepG2 Hepatoma

A peptide containing Asn-Gly-Arg(NGR) sequence was synthesized and directly labeled with 99mTc. Its radiochemical characteristics, biodistribution, and SPECT imaging were evaluated in nude mice bearing human HepG2 hepatoma. Nude mice bearing HepG2 were randomly divided into 5 groups with 5 mice in each group and injected with ~7.4 MBq 99mTc-NGR. The SPECT images were acquired in 1, 4, 8, and 12 h postinjection via caudal vein. The metabolism of tracers was determined in major organs at different time points, which demonstrated rapid, significant tumor uptake and slow tumor washout. The control group mice were blocked by coinjecting unlabelled NGR (20 mg/kg). Tumor uptake was (2.52 ± 0.83%) ID/g at 1 h, with the highest uptake of (3.26 ± 0.63%) ID/g at 8 h. In comparison, the uptake of the blocked control group was (1.65 ± 0.61%) ID/g at 1 h after injection. The SPECT static images and the tumor/muscle (T/NT) value were obtained. The highest T/NT value was 7.58 ± 1.92 at 8 h. The xenografted tumor became visible at 1 h and the clearest image of the tumor was observed at 8 h. In conclusion, 99mTc-NGR can be efficiently prepared and it exhibited good properties for the potential SPECT imaging agent of tumor.


Introduction
Angiogenic tumor vessels are important element for tumor growth and metastasis and the metalloexopeptidase CD13/ aminopeptidase N (APN) plays a critical role in cancer angiogenesis. Peptides containing NGR have shown high efficiency in targeted cells, tissues, and new vessels with CD13 receptor overexpression [1,2]. Moreover, in tissues that undergo angiogenesis, blood vessels also overexpress APN and proliferation of endothelial cells is well known to be an important factor in tumor angiogenesis [3,4]. It is proved that NGR can bind with new vasculature by aminopeptidase N (CD13) and integrin v 3, although the binding mechanisms are different. Meanwhile, CD13 receptor mediated binding to tumor vasculature is specific but not to the other CD13 rich tissues, which was proved by in vivo studies [5]. Good blood clearance was thought to favor the utilization of imaging techniques. Therefore, NGR peptide was characterized as a promising molecular imaging candidate for early diagnosis, particularly for tumor.
In this study, a new NGR peptide was synthesized and labeled with 99m Tc, then subjected to SPECT imaging of CD13 expression in a subcutaneous mouse HepG2 hepatoma xenograft model, which was proved to show positive CD13 receptor and easy formation of tumor.

Materials and Methods
2.1. General. All chemicals (reagent grade) were obtained from commercial suppliers and used without further purification. NGR (YGGCNGRC) was prepared by SPPS using the Fmoc method on a chlorotrityl chloride resin. 99m TcO 4 − was Tech, China). Water was purified using a Milli-Q ultrapure water system from Millipore (Milford, USA), followed by passing through a Chelex 100 resin before bioconjugation and radiolabeling. Radio-TLC was performed on silica gelcoated plastic sheets (Polygram SIL G, Macherey-Nagel) with acetone and Vethanol : Vammonia water : Vwater = 2 : 1 : 5 as the eluents. The plates were read with Bioscan Mini-scan (USA) and Allchhrom Plus software. The semipreparative high-performance liquid chromatography (HPLC, Aglint, Canada) was employed for peptide analysis. NGR-containing peptide was prepared by solid phase peptide synthesis (SPPS) using the Fmoc strategy on chlorotrityl chloride resins as previously reported [12]. Mass spectra were used to confirm the identity of the products. Mass spectra were obtained on a Q-Tof premier-UPLC system equipped with an electrospray interface (ESI; Waters, USA) or a Thermo Electron Finnigan LTQ mass spectrometer equipped with an electrospray ionization source (Thermo Scientific, USA).

Biodistribution Studies.
Nude mice bearing human HepG2 hepatoma were randomly divided into 5 groups and injected with ∼7.4 MBq of 99m Tc-NGR with or without excess unlabelled NGR peptide (20 mg/kg). After injection of the tracer, mice were sacrificed and dissected. The radioactivity in the HepG2 tumor, major organs, and muscle were collected and weighed wet with tubes (%ID/g). Mean uptake (%ID/g) for a group of animals was calculated with standard deviations. Values were expressed as mean ± SD ( = 5/group).

Chemistry and Radiochemistry.
NGR peptide was well prepared (Figure 1). The analytical HPLC and mass spectroscopy were used to confirm the identity of the products. The mass spectroscopy data and chemical structures for NGR were represented below (Figure 1).  3.08∼6.17 MBq/nmol. The labeling yield of the product was 95 ± 0.35% and the radiochemical purity was greater than 98%. The in vitro stability of 99m Tc-NGR in PBS (pH 7.4) at 37 ∘ C was shown in Figure 2. After 12 h of incubation, more than 92% of 99m Tc-NGR peptide remained intact in mice serum.

Cell Uptake.
Cell uptake study revealed that 99m Tc-NGR bound to HepG2 tumor cells directly. During the first 15 min, about 0.49 ± 0.05% of 99m Tc-NGR uptake in HepG2 cells were determined. After 2 h incubation, the peptide uptake in HepG2 cells reached the maximum of 1.52 ± 0.13% ( Figure  3(a)). About 1.35 ± 0.27% of 99m Tc-NGR were still associated with HepG2 cells after 4 h incubation.

Cell Binding Assay.
Ligand-receptor binding affinities of 99m Tc-NGR to CD13 were determined by a competitive cellbinding assay. 99m Tc-NGR inhibited the binding of NGR peptide to HepG2 cells in a concentration-dependent manner (Figure 3(b)). The IC 50 values for 99m Tc-NGR were calculated to be 287 ± 34 nmol/L.

SPECT Imaging.
The tumor-targeting efficacy of 99m Tc-NGR probe in HepG2 tumor-bearing nude mice was evaluated by static SPECT scans at different time points after injection. Representative decay-corrected images are shown in Figure 4. The HepG2 tumors were clearly visualized with good tumor-to-background contrast for the tracer. Overall, 99m Tc-NGR provided better image quality with the same amount of injected activity.

Biodistribution Studies.
Tissue distribution data for 99m Tc-NGR in mice bearing HepG2 hepatoma tumors are given as percentage administered activity per gram of tissue (%ID/g) in Table 2 and Figure 5. The in vivo biodistribution of with and without coinjection of nonradiolabeled NGR peptide (20 mg/kg of mouse body weight) was examined in HepG2 tumor-bearing mice. For 99m Tc-NGR, the tumor uptake was determined to be 2.52 ± 0.83, 3.03 ± 0.71, 3.26 ± 0.63, and 2.81 ± 0.25% ID/g at 1, 4, 8, and 12 h, respectively. 99m Tc-NGR exhibited 7.93 ± 2.13% ID/g kidney uptake and 4.07 ± 0.76% ID/g liver uptake at 1 h pi. The nonspecific uptake in the muscle was at a very low level. 99m Tc-NGR exhibited high tumor uptake at the early time point (Figure 5), indicating the specific binding and relatively longer circulation time. A decrease of radioactivity was observed in all dissected tissues and organs similar to SPECT imaging results in blocking group (Table 2), with the change of tumor uptake being the most significant reducing markedly from 2.52 ± 0.83% ID/g whereas the presence of nonlabeled NGR peptide significantly reduced to 1.65 ± 0.61% ID/g at 1 h after    injection. For 99m Tc-NGR nonblocking group, 4.07 ± 0.76% ID/g in liver and 7.93 ± 2.13% ID/g in kidney were decreased to 3.27 ± 0.16% ID/g and 5.03 ± 0.97% ID/g by blocking, respectively.

Discussion
In this study, we synthesized a novel NGR peptide and investigated its biological targeting specificity, which turned out to be a promising tumor molecular imaging probe for clinical practice. 99m Tc has favorable chemical and physical properties and can be produced from the generator directly [11]. CD13 receptor is an attractive biological target, which has been found to be overexpressed on newly formed neovasculature and on a wide range of tumor cells types. The high radiochemical purity of the radiotracer (>98%) stimulated further analysis encompassing in vitro and in vivo evaluation, without the time consuming steps of purification and drying of the compound. The 99m Tc-labeled tracer also showed good stability ( Figure 2) and affinity (Figure 3). The results showed that imaging acquisition after injection within 12 h is enough for detecting tumor clearly. The labeling process in this study is so convenient that the probe is practical in future clinical imaging.
The development of radiolabeled peptides for diagnostic and therapeutic applications has expanded exponentially in the last decades [8,13,14]. Peptide-based radiopharmaceuticals can be produced easily and inexpensively and have many favorable properties, including fast clearance, rapid tissue penetration, and low antigenicity [6,9,[15][16][17]. In this study, the cysteine beside NGR motif formed a cyclic via a disulfide linkage and the direct labeling method resulted in a very stable product. At the meantime, breaking the disulfide linkage in the NGR-containing peptide during the directly labeling process may explain the slightly lower binding affinity compared with our previous results [18]. Otherwise, the extra three glycines were added to protect the core motif NGR and may increase peptide half-life and stability [19]. Meanwhile, the liver and kidney uptake was obviously reduced compared with previous study, which may also be caused by adding glycine [18].
SPECT scans of 99m Tc-NGR in nude mice bearing HepG2 hepatoma showed notable uptake in tumor and dominant renal and hepatic clearance. But the unspecific binding on the other tissues was decreasing and the tumor to nontumor ratio was consequently increasing. The receptor specificity of 99m Tc-NGR was further confirmed by effective inhibition of tumor uptake in the presence of excess nonlabeled NGR peptide in biodistribution study (Table 1). Although the 99m Tc-NGR uptake in liver and gastrointestinal tract was lower than previous results, the practice in detecting tumor and metastases in the abdominal area is inapplicable. Since the excretion of the probe was mainly renal, fast blood depuration should be another favorable feature.
In brief, our data demonstrated that synthesizing novel 99m Tc-NGR was a promising synthetic strategy for SPECT imaging in terms of in vitro and in vivo properties. Our future work will continually focus on more optimal approach to reduce liver and gastrointestinal tract uptake by modifying the peptide structure and keep the specificity binding. Additionally, a thorough comparison between various NGR peptides is warranted to screen the better radiotracers.

Conclusion
NGR peptide was successfully labeled with the generatorproduced 99m Tc for SPECT imaging of tumor CD13 receptor. 99m Tc-NGR exhibited good properties in terms of binding affinity, cellular uptake, tumor uptake and retention, and pharmacokinetics. 99m Tc-NGR peptide is a potential SPECT agent for imaging and early diagnosis of tumor.