| Background:Cancer-specific molecular imaging, which visualizes distinctive molecular markers in various cancers, is a promising and essential tool for the specific detection, therapeutic target screening, and therapeutic response monitoring of cancer. Molecular markers of angiogenesis, which are overexpressed early in cancer progression, have already proven to be an effective method of cancer-specific molecular imaging.CD13 is a membrane-bound exopeptidase which has been shown to be overexpressed on the neovascular endothelial cells and in a variety of tumor cells, and is a widely acknowledged target for cancer diagnosis and therapy. The polypeptide containing NGR motif, which has been identified as a specific ligand of CD13, is a potential targeting agent for CD13 imaging. Labeled by 64 Cu, 99 m Tc and 68 Ga, the efficiency and specificity of radiolabeled NGR probes have been well established. However, compared with traditional imaging agents such as 18F-FDG, the NGR probe demonstrates relatively low uptake and short retention duration in the cancer tissue, which are some of its major limitations. Such shortcomings are not unique to the NGR probe, as other small molecule peptide probes face similar challenges. The problem is mainly due to the following two reasons. Firstly, because of their small molecular weight and high polarity, the peptide probes are quickly excreted by the kidneys in a physiological system, thus the half-life for the circulation and distribution of the peptides is quite limited. Secondly, because of the poor penetration of the polypeptide tracers into the tumor, the NGR can only temporarily bind to the limited CD13 on the surface of neovascular endothelial cells, resulting in a low uptake rate and short retention duration within the cancer tissue.The Cend R motif(R/KXXR/K) is a C-terminal sequence containing a C-terminal arginine or lysine with a free carboxyl group that can guide the peptide to pass through biological barriers via interaction with NRP-1(neuropilin-1). By combining the Cend R motif with another cancer-specific angiogenesis targeting polypeptide RGD [namely internalizing RGD(i RGD)], Sugahara et al. demonstrated that the i RGD-coated nanoparticles could specifically penetrate from tumor neovasculature into cancer tissue and cells, thus, significantly improving the sensitivity of tumor-imaging agents and providing a mechanism to potentially enhance the efficiency of antitumor drug delivery. Subsequently, the same group conjugated i NGR(internalizing NGR, containing the Cend R motif and polypeptide NGR) to therapeautic nanoparticles and achieved a similar effect.In our research, we intend to synthesize i NGR inserted with Cend R motif, which can specifically bind to NRP-1 and mediate NGR peptide penetration into tumor tissue. By using this tumor-homing strategy, the uptake and retention of the NGR probe in tumors can be significantly enhanced, thus providing a new method for improving the performance of the radiolabeled peptides for tumor targeting.Objective:To evaluate the efficacy of 68Ga-labeled i NGR, containing Asn-Gly-Arg(NGR) homing sequence and Cend R(R/KXXR/K) penetrating motif, as a new molecular probe for micro PET imaging of CD13-positive xenografts, and elucidate the mechanism role of NRP-1 by blocking imaging experiment.Methods:1. DOTA-i NGR(DOTA-GGGCRNGRGPDC, Cys4 and Cys12 conjugated by a disulfide bridge), with the Cend R motif(RNGR) inserted, was designed based on the researches by Sugahara et al.[1, 2], and then synthesized by GL Biochem Ltd.(Shanghai, China). The purity was analyzed by HPLC.2. 68 Ga fresh eluent 200 μL(92.5-129.5 MBq) obtaining with 68Ge/68 Ga radionuclide generator was used to label DOTA-i NGR. The optimal conditions for labeling including p H, temperature, reaction time and concentration of DOTA-i NGR were determined. Then, the in vitro/in vivo stability and octanol/water partition coefficient of the product, 68Ga-DOTA-i NGR were further analyzed. Its biodistribution in normal Kunming mice was examined at time intervals at 10, 20, 40, 60 and 120 min after its intravenous injection.3. The protein levels of CD13 [Aminopeptidase N(APN)] and NRP-1 in HT-1080 and HT-29 cell lines were determined by western blot and immunofluorescence.Compared In vitro affinity to CD13-positive HT-1080 cells between DOTA-i NGR and DOTA-c NGR by competitive cell binding affinity study. The HT-1080 or HT-29 cell uptakes of 68Ga-DOTA-i NGR and 68Ga-DOTA-c NGR were compared by the cell binding assay. In cell blocking studies, the HT-1080 cells were incubated with 1 μg/m L of neutralizing NRP-1 antibody for 15 minutes before adding 68Ga-DOTA-i NGR.4. Xenograft models were generated by subcutaneous injection of 2×106 tumor cells into the upper flanks of nude mice(left: HT-1080; right: HT-29).In vivo imaging of 68Ga-DOTA-c NGR and 68Ga-DOTA-i NGR was compared by mrico PET imaging of the same nude mouse bearing HT1080 and HT29 tumors. The in vivo CD13 affinity was verified by competitive micro PET imaging of the same mouse with or without unlabeled NGR blocking. The mechanism of the theoretically improved performance of i NGR via NRP1 was verified by comparison micro PET imaging by using neutralizing NRP-1 antibody. Tumor uptake was measured by ROI method and quantified by %ID/g.Results:1. DOTA-i NGR was synthesized by GL Biochem Ltd., with a purity of > 95% conformed by HPLC.2. The optimal conditions for labeling was mixing 2 μg DOTA-i NGR peptide with 200 μL 68Ga(92.5-129.5 MBq) at p H 4.0, temperature 90-100 ℃ for 5-10 min. With this condition, our labeling rate reached 97.5 ± 1.3 % and specificity 54-75 MBq/nmol. The radiochemical purity of 68Ga-DOTA-i NGR in both saline and mouse serum were both above 95 % after 4 hours incubation, and the radiochemical purity in urine were greater than 85 % after 1 h metabolism in vivo. The partition coefficient was-2.71 ± 0.18. In normal mice, majority of 68Ga-DOTA-i NGR was excreted from kidneys, with a rapid clearance from blood steam.3. The results revealed that CD13 and NRP-1 were both expressed in HT-1080 cells but not in HT-29 cells.By using a competitive cell binding affinity study, the IC50 values of DOTA-i NGR and DOTA-c NGR were determined as(4.98 ± 1.91) × 10-8 M and(5.04 ± 1.17) × 10-8 M respectively, without significant difference(P > 0.05) between the two values, indicating that the CD13 binding affinity of DOTA-i NGR was comparable to that of DOTA-c NGR.The results of the cell uptake study revealed that both i NGR and NGR could bind to HT-1080 cells, and that their uptake rates were both positively related to the incubation time. Furthermore, the maximum uptake rate of 68Ga-DOTA-i NGR in HT-1080 cells was about 2-fold of that of 68Ga-DOTA-c NGR(1.78 ± 0.14% v.s. 0.90± 0.12%, P < 0.05) after 2 h incubation. While in the CD13-negative HT-29 cells, uptake rates of both 68Ga-DOTA-i NGR and 68Ga-DOTA-c NGR were persistently as low as 0.38 ± 0.07 % and 0.26 ± 0.05 %(P > 0.05), which were significantly lower than those in the HT-1080 cells (P < 0.01). In cell blocking experiments, the uptake rates of 68Ga-DOTA-i NGR in HT-1080 cells were reduced at all time points by preincubation with neutralizing NRP-1 antibody, with the values decreasing from 0.50 ± 0.08, 1.08 ± 0.07, 1.54 ± 0.10, 1.78 ± 0.14 % to 0.26 ± 0.09, 0.58 ± 0.10, 0.86 ± 0.11, 0.96 + 0.12 % respectively(P < 0.05). And after being blocked by NRP-1 antibody, the uptake rate of 68Ga-DOTA-i NGR in HT-1080 cells was similar to that of 68Ga-DOTA-c NGR(P > 0.05).4. The tumors were allowed to grow for 2-3 weeks until they reached 0.5-1 cm3 in volume for micro PET imaging and biodistribution experiments.The efficacy of 68Ga-DOTA-i NGR and 68Ga-DOTA-c NGR were evaluated and compared in nude mice bearing both HT-1080 and HT-29 xenografts(n = 5) at multiple time points(0.5, 1 and 1.5 h) with static micro PET/CT scans. Except for the kidneys, the CD13-positive HT-1080 tumors were clearly visible with much high contrast to HT-29 tumors at all time points after injection of 68Ga-DOTA-i NGR or 68Ga-DOTA-c NGR. Of interest, the uptakes of 68Ga-DOTA-i NGR in HT-1080 tumors were much higher than those of 68Ga-DOTA-c NGR at all time points revealed by self-control study in the following day, with the tumor uptake calculated to be 3.41 ± 0.28, 2.97 ± 0.30, 2.64 ± 0.31 %ID/g at 0.5, 1, 1.5 h p.i. for 68Ga-DOTA-i NGR and 2.68 ± 0.35, 1.91 ± 0.32, 1.45 ± 0.30 %ID/g for 68Ga-DOTA-c NGR respectively(P < 0.05). Comparing the uptake dynamic tendency in the HT-1080 tumors, the decreasing slope from 0.5 h to 1.5 h p.i. was-0.74 ± 0.22 for 68Ga-DOTA-i NGR and-1.23 ± 0.25 for 68Ga-DOTA-c NGR, indicated that the tumor retention time of 68Ga-DOTA-i NGR was 1.7-fold of that of 68Ga-DOTA-c NGR(P < 0.05).Furthermore, 68Ga-DOTA-i NGR uptake in HT-1080 tumors could be almost completely blocked by co-injecting unlabeled NGR peptide at a dosage of 20 mg/kg, with the uptake of tumors decreased from 2.92 ± 0.40 to 0.57 ± 0.26 %ID/g(P < 0.01). However only partial accumulation of 68Ga-DOTA-i NGR in HT-1080 tumors could be blocked by concomitant administration of neutralizing NRP-1 antibody, with the uptake decreasing from 2.96 ± 0.43 to 1.82 ± 0.34 %ID/g(P < 0.05). Interestingly, after being blocked by NRP-1 antibody, the uptake of 68Ga-DOTA-i NGR in HT-1080 tumors was similar to the HT-1080 tumors injected by 68Ga-DOTA-c NGR(P > 0.05) at 1 h p.i.. Conclusion:68Ga-DOTA-i NGR has a higher tumor uptake and better tumor retention than 68Ga-DOTA-c NGR through NRP-1, indicating that Cend R motif modification is a promising method for improving the efficiency of tumor targeting tracers. |
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