Radioisotope Labeled With Tumoral Neurovascular Targeted NGR Peptide Study In Vitro And In Vivo

ObjectivePeptides containing Asn-Gly-Arg（NGR）sequence were radiolabeled by¹³¹Iand⁹⁹mTc and evaluated their radiochemical characteristics for screening CD13expression on different tumor cell lines in vitro and investigating biodistributionand SPECT imaging of⁹⁹mTc-NGR in nude mice bearing human HepG2hepatoma in vivo. Furthermore，monomeric NGR peptide [NGR1:（GNGRG）]and dimeric NGR peptide [NGR2:（GNGRG）2KGK] were developed and labeledwith⁹⁹mTc for relative study in vitro and in vivo.MethodsNGR peptides were labeled with¹³¹I by Iodogen method and with⁹⁹mTc bydirect labeling method. Probes’ radiochemical character was evaluated by TLCwith100%acetone and Vethanol:Vwater:Vammonia water=2:1:5as the eluents.Immunofluorescence experiment and Western blot were used to detect CD13 expression on HT-1080, HUVEC, HepG2, U87Mg, PC-3, HT-29and MCF-7tumor cell lines. Binding affinity of¹³¹I-NGR with CD13positive cell lines wasdetected by receptor assay in vitro. The lethal effect of various dosage of¹³¹I-NGR,Na¹³¹I and NGR on positive and negative expression cells after24,48and72hincubation were measured by MTT test. HepG2oxengraft mice were acquired%ID/g and dynamic T/NT ratio（tumor to non tumor） change by ROI method at1,2,4,8,12h after injection of7.4MBq（⁰.2mL）⁹⁹mTc-NGR. The blocking groupwas injected with free NGR100μg（50μL） before⁹⁹mTc-NGR. Both NGRmonomer and dimer were synthesized and labeled with⁹⁹mTc for cell uptakeexperiments to evaluate the CD13-binding affinity of the⁹⁹mTc labeled NGRpeptide-based conjugates in vitro. For biodistribution and SPECT imaging studies,nude mice bearing HepG2human hepatoma xenograft were injected with7.4MBq⁹⁹mTc-NGR1and⁹⁹mTc-NGR2respectively and images were acquired at1,4,12and24h p.i.. In addition, blocking experiment was achieved byco-injection with radiolabeled probe with excess non-radiolabeled NGR2peptide（20mg/kg） for⁹⁹mTc-NGR2to evaluate its specificity in vivo.ResultsNGR peptide was produced and successfully labeled with¹³¹I. The bestcomponent ratio of labeling was18.5MBq¹³¹I,10μg NGR and20μg Iodogen.The label yield of¹³¹I-NGR was （93.7±2.5）%and radioactivity was1.41TBq/mmol. The probe was stable and its radiochemical purity was more than87%at24h. Cell immunofluorescence and western blot experiment showed thatCD13expression of HT-1080, HUVEC, HepG2, U87Mg and PC-3cell lines werepositive, while MCF-7and HT-29were negative. Receptor binding assay onHT-1080cell line showed that Kd was7.3nmol/L and Bmax was0.302nmol/L.MTT test verified that¹³¹I-NGR had stronger growth inhibit effect on HT-1080 cells than HT-29cells （P<0.01）. The inhabiting ratio of¹³¹I-NGR to HT-1080was（67.9±3.4）%at72h and3700MBq/L, while the ratio¹³¹I-NGR to HT-29was（4.0±0.5）%（P<0.0001）.⁹⁹mTc-NGR’s labeling yield was more than90%, withRCP more than95%. Biodistribution results showed that kidney and liver had thehigher uptake, tumor uptake was （2.52±0.62）%ID/g at1h p.i.,（7.26±2.71）%ID/gat the highest at8h and remains （3.93±1.93）%ID/g at12h p.i., while the blockinggroup was （1.29±0.85）%ID/g. The SPECT static imaging showed the decreasingtumor uptake with time and tumor/muscle value was obtained. The highest T/NTvalue was3.25at4h. The oxengrafted tumor became visible at1h and the mostclearly at12h. The in vitro experiment demonstrated that⁹⁹mTc-NGR1and⁹⁹mTc-NGR2are stable in PBS（phosphate buffered saline） with more than92%of remaining intact after12h of incubation. Cellular uptake studies revealed thatuptake of⁹⁹mTc-NGR2（1.73±0.12%） in HepG2cells was higher than that of⁹⁹mTc-NGR1（1.27±0.18%） after4h incubation. For SPECT scan, thesubcutaneous HepG2tumors were all clearly visible with good contrast tocontralateral background at all measured time points after injection of⁹⁹mTc-NGR1or⁹⁹mTc-NGR2. At1h pi., the HepG2tumor uptake of⁹⁹mTc-NGR2was determined to be the highest （6.22±1.22%ID）, whereas⁹⁹mTc-NGR1was3.46±0.47%ID. As compared to⁹⁹mTc-NGR1,⁹⁹mTc-NGR2displayed rapidHepG2tumor uptake and better tumor retention. In addition, blocking experimentof⁹⁹mTc-NGR2suggested⁹⁹mTc-NGR2is a CD13receptor-specific probe.Furthermore, the biodistribution results were consistent with the quantification ofSPECT imaging, demonstrating⁹⁹mTc-NGR2predominantly renal and hepaticexcretion and the highest ratio （6.37±0.54） of tumor/muscle uptake of⁹⁹mTc-NGR2at12h p.i. for non-blocking group and significantly decreased ratio（1.85±0.61） for blocking group. Conclusion¹³¹I-NGR and⁹⁹mTc-NGR can be efficiently prepared and favorably target CD13positive cell lines, which would be fundamental for following researches ontumor targeted imaging and biotherapy with NGR compounds. Our study showedthat dimerization of NGR peptide results in moderate improvement of imagingcharacteristics as compared to monomeric counterpart for better specificity.⁹⁹mTc-NGR2is a promising probe for imaging CD13-positive tumors.