Noninvasive visualization of RNA delivery with 99mTc-radiolabeled small-interference RNA in tumor xenografts.
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Kang
Lei
L
Department of Nuclear Medicine, Peking University First Hospital, Beijing, China.
Wang
Rong Fu
RF
Yan
Ping
P
Liu
Meng
M
Zhang
Chun Li
CL
Yu
Ming Ming
MM
Cui
Yong Gang
YG
Xu
Xiao Jie
XJ
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D005260:Female; D015902:Gamma Cameras; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D057054:Molecular Imaging / Q000379:methods*; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000097:blood / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D034741:RNA, Small Interfering / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D011877:Radionuclide Imaging; D013667:Technetium; D019098:Telomerase / Q000172:deficiency / Q000235:genetics
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D013667:Technetium; D019098:Telomerase