Technetium-99m radiolabeled nucleolin-targeted aptamer for glioma tumor imaging in murine models

Aptamers are small molecule DNA or RNA fragments that fold into well-defined 3D structures with high spec-ificity and affinity towards target molecules, therefore emerging as a new class of probes for radiopharmaceutical applications. In this study, a technetium-99m (99mTc) radiolabeled nucleolin-targeted aptamer AS1411 was developed for identifying glioma in murine models.Methods: A six-carbon amine group was linked at the 5 ' end of AS1411 by DNA synthesis. Following the conjugation to NHS-MAG3, AS1411 was radiolabeled with 99mTc. Radiochemical purity and stability were evaluated by radio-TLC and gel electrophoresis. Cellular uptake and MTT assays were carried out in the nucleon-overexpressed U87 cell line to assess the biological ability of AS1411 after conjugating with FITC or radiolabeling with 99mTc. In vivo imaging of 99mTc-AS1411 was performed in glioma tumor xenografts and 99mTc and a 99mTc-labeled non-specific negative control (NC) nucleotide were used as control groups. Biodistribution studies were performed at the terminal time point of SPECT imaging.Results: AS1411 was successfully labeled with 99mTc with the labeling yields of 81.5% +/- 3.6% (n = 5) and was verified by gel electrophoresis and gel imaging. 99mTc-AS1411 was proved to be stable in fresh human serum at room temperature for 12 h. In U87 cells, 99mTc-AS1411 showed significantly higher cellular uptake than 99mTc-NC and 99mTc (9.4% +/- 2.1% vs 1.9% +/- 0.4% and 0.9% +/- 0.1%, P < 0.01, n = 3). FITC-conjugated AS1411 demonstrated more fluorescent signals in U87 cells than NC. In addition, 99mTc-AS1411 displayed similar cellular inhibitory ability with AS1411, which was measured by MTT assay. In U87 tumor-bearing mice, 99mTc-AS1411 demonstrated high radioactive accumulation, whereas 99mTc-NC and 99mTc only showed weak tumor uptake, suggesting the specific uptake of 99mTc-AS1411 in nucleolin-positive U87 tumors in vivo. Biodistribution results further verified the imaging results. The tumor uptake of 99mTc-AS1411 was significantly higher than that of 99mTc-NC (5.81 +/- 1.55%ID/g vs 0.51 +/- 0.16%ID/g, n = 4, P < 0.01).Conclusion: 99mTc-AS1411 can be successfully prepared via the conjugation of NHS-MAG3 and be considered a promising imaging agent capable of identifying nucleolin-positive glioma tumors.