Although to date, no antiangiogenic agents can eradicate tumors alone, effective inhibition of tumor angiogenesis might arrest tumor progression and has been shown to synergize with other cancer treatments [16–17]. As one targeted tumor therapy, only a few patients can actually benefit from antiangiogenic therapy due to the biological heterogeneity of tumors [18–19]. Molecular imaging using radioisotopes labeled with targeted drugs or its analogs makes it possible to select patients, predict efficacy, and dynamically assess response to therapy [20]. Detection of specific molecular targets is an important application of molecular imaging in the management of malignancies.
In nuclear medicine, PET has become a very important imaging technique for the diagnosis and treatment efficacy evaluation of cancer. Currently, 18F-FDG is still the most widely used radiotracer for PET imaging. The acquisition of 18F-FDG depends on the availability of a nearby cyclotron. Due to the high price and operating cost of the cyclotron, only a small number of medical institutions has both a PET and cyclotron. Moreover, 18F-FDG traces tumor cell glucose metabolism and is used for the diagnosis and evaluation of therapeutic effects. It is a non-specific tumor imaging agent, which limits its application to guide targeted therapy, especially selecting the right patients.
68Ga, a generator-based positron radioisotope, is relatively easy to obtain by 68Ge/68Ga radionuclide generators and shows equally high-quality images on PET. 68Ga3+ can form stable complexes with many ligands containing oxygen and nitrogen as donor atoms by aid of chelators, which makes it an appealing alternative to 18F [21].
Radioisotope-labeled RGD peptides have been widely studied as molecular probes for tumor angiogenesis imaging [22–25]. However, RGD targets integrin αvβ3, which works well for tumors with positive expression of integrin αvβ3, but cannot be used for tumors that do not express or have low expression of αvβ3. CD13 is another important target of tumor and angiogenesis, and NGR can be specifically combined with CD13 to form a new probe, which can be an important complement to the RGD probe. To improve the targeting and biological metabolic characteristics of the NGR probe, we refer to the successful experience of RGD modification. The cyclic dimer peptide has a more stable structure and higher affinity than the monomer, and PEG modification can increase the hydrophobicity of peptides and thus improve the pharmacokinetics of the probe [13, 26]. In this study, we designed a cyclic NGR dimer linked by PEG-4 and labeled it with 68Ga to form a new radiolabeled probe targeting CD13.
68Ga-DOTA-c(NGR)2 with high radiochemical purity was obtained after relatively simple labeling steps without further purification, and the yield maintained stability in bovine serum beyond 3 h. To verify the specificity and cellular binding kinetics of the probe, two ovarian cancer cell lines, ES2 cells and SKOV3 cells, expressing CD13 at significantly different levels were utilized to mimic heterogeneity from the same tumor. An in vitro binding assay demonstrated that the binding of 68Ga-DOTA-c(NGR)2 to ES2 cells was significantly higher than that in SKOV3 cells and could be inhibited with unlabeled DOTA-c(NGR)2 in a dose-dependent relationship, which revealed the specificity of 68Ga-DOTA-c(NGR)2 to CD13-positive tumor cells in vitro. The results suggested that 68Ga-DOTA-c(NGR)2 could be used to identify tumors with high expression of CD13 by PET imaging in vivo.
Biodistribution studies demonstrated that a large proportion of 68Ga-DOTA-c(NGR)2 was metabolized through the urinary system and rapidly cleared from blood, whereas its early uptake in other organs such as liver, lungs, and spleen was probably correlative to the blood supply of these organs. The distribution characteristics were also confirmed by subsequent imaging studies of tumor xenografts. Series of microPET images of ES2 tumor model showed that focal uptake in the tumor was distinctly observed from 1 h post-injection of 68Ga-DOTA-c(NGR)2. Meanwhile, 68Ga-DOTA-c(NGR)2 started to accumulate in the bladder from 30 min. Clear and high-contrast tumor visualization occurred at 1 h, which corresponded to the highest T/B ratio of 10.30 ± 0.26. Moreover, accumulation of ES2 tumor apparently declined by pretreatment of unlabeled peptide, which further proved the specificity of 68Ga-DOTA-c(NGR)2. In SKOV3 tumor models, the tumor was not clearly seen under the same imaging conditions.
In our previous study, 68Ga labeling linear NGR conjugated with DOTA, 68Ga-DOTA-NGR, was synthesized, which showed high selectivity and affinity for CD13 and in significant uptake in CD13-positive lung tumors [12]. Compared with this study, a cNGR dimer was used and its higher affinity to CD13 and higher uptake in tumors were observed, consistent with our prediction. However, we noted that the uptake decreased in the tumor at 2 h post-injection. Further optimization of the structure of the probe may improve its pharmacokinetic characteristics and increase affinity to CD13-positive tumors.
It is worth noting that our work is only a preliminary study on the physicochemical and biological characteristics of a newly designed cyclic dimer probe based on the NGR sequence. It was not compared with other probes of NGR derivatives in previous studies under the same experimental conditions.