Department of Nuclear Medicine, Peking University First Hospital, Beijing, China.
J Nucl Med. 2010 Jun;51(6):978-86. doi: 10.2967/jnumed.109.069906. Epub 2010 May 19.
Small-interference RNAs (siRNAs) are short, double-strand RNA molecules that target specific messenger RNAs for degradation via the process termed RNA interference. The efficacy of RNA interference depends greatly on effective delivery of siRNA, which calls for noninvasive methods for tracing siRNA in vivo. The purpose of this work was to develop a novel (99m)Tc-radiolabeled method to visualize siRNA targeting of a tumor biomarker of human telomerase reverse transcriptase (hTERT) in HepG2 tumor xenografts.
After conjugation with S-acetyl N-hydroxysuccinimide-mercaptoacetyltriglycine (NHS-MAG3), antisense RNA with 2'-O-methyl modification was annealed with sense strand to form a duplex and then radiolabeled with (99m)Tc. (99m)Tc-siRNAs were tested for stability in serum by measurement of radiochemical purity and for inhibitory activity by reverse-transcriptase polymerase chain reaction and Western blotting. In vitro cellular uptake was evaluated in HepG2 cells. Biodistribution studies and static imaging were performed in HepG2 tumor-bearing mice.
Radiochemical purity remained highly stable in saline and fresh human serum at room temperature and 37 degrees C. Radiolabeled siRNA demonstrated strong inhibitory effects similar to those of unlabeled siRNA on both hTERT messenger RNA and protein in vitro. (99m)Tc-hTERT siRNA showed more uptake than did control siRNA in HepG2 cells after 1 h of incubation. After administration in HepG2 tumor-bearing mice, (99m)Tc-hTERT siRNA had significantly higher accumulation in tumors and a higher tumor-to-blood ratio than did control siRNA (P < 0.05). Scintigraphy of (99m)Tc-hTERT siRNA showed clear tumor images at 0.5, 1, 3, and 6 h after injection. In contrast, (99m)Tc-control siRNA failed to visualize the tumor. Ratios of uptake in tumor to uptake in contralateral region of hTERT-targeted siRNA were significantly higher than those of control siRNA (P < 0.05) at each time point.
The (99m)Tc radiolabeling method with NHS-MAG3 chelator can be used successfully in siRNA radiolabeling, allowing for the noninvasive visualization of siRNA delivery in vivo.
开发一种新型的（99m）Tc 放射性标记方法，以可视化 HepG2 肿瘤异种移植中人类端粒酶逆转录酶（hTERT）肿瘤生物标志物的 siRNA 靶向。
经 S-乙酰 N-羟基琥珀酰亚胺巯基乙酰三甘氨酸（NHS-MAG3）缀合后，用 2'-O-甲基修饰的反义 RNA 与有义链退火形成双链体，然后用（99m）Tc 标记。通过放射性化学纯度测量和逆转录酶聚合酶链反应和 Western blot 评估（99m）Tc-siRNAs 在血清中的稳定性和抑制活性。在 HepG2 细胞中评估细胞摄取。在 HepG2 荷瘤小鼠中进行生物分布研究和静态成像。
放射性化学纯度在室温下和 37°C 的生理盐水和新鲜人血清中保持高度稳定。放射性标记的 siRNA 在体外对 hTERT 信使 RNA 和蛋白质均表现出与未标记 siRNA 相似的强烈抑制作用。与对照 siRNA 相比，（99m）Tc-hTERT siRNA 在 HepG2 细胞孵育 1 小时后摄取更多。在 HepG2 荷瘤小鼠中给药后，（99m）Tc-hTERT siRNA 在肿瘤中的积累明显高于对照 siRNA，肿瘤与血液的比值也高于对照 siRNA（P<0.05）。（99m）Tc-hTERT siRNA 的闪烁显像术在注射后 0.5、1、3 和 6 小时显示出清晰的肿瘤图像。相比之下，（99m）Tc-对照 siRNA 未能可视化肿瘤。靶向 hTERT 的 siRNA 在肿瘤中的摄取与对侧区域的摄取之比在每个时间点均明显高于对照 siRNA（P<0.05）。
NHS-MAG3 螯合剂的（99m）Tc 放射性标记方法可成功用于 siRNA 放射性标记，从而允许在体内无创可视化 siRNA 递呈。
