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 specificity 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. 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. 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). 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.