J Drug Target. 2017 Feb;25(2):172-178. 

Development of a double-stranded siRNA labelling method by using 99mTc and single photon emission computed tomography imaging.

Kano D1, Nakagami Y2,3, Kurihara H4, Hosokawa S5, Zenda S5, Kusumoto M2, Fujii H6, Kaneta T3, Saito S1, Uesawa Y7, Kagaya H7.

1Department of Pharmacy, National Cancer Center Hospital East, Japan

2Radiology Division, National Cancer Center Hospital East, Japan

3Department of Radiology, Yokohama City University, School of Medicine, Yokohama City University Graduate School of Medicine, Japan

4Radiology Division, National Cancer Center Hospital, Japan

5Department of Radiation Oncology, National Cancer Center Hospital East, Japan

6Division of Functional Imaging, Research Center for Innovative Oncology, National Cancer Center Hospital East, Japan

7Department of Clinical Pharmaceutics, Meiji Pharmaceutical University, Japan



In vivo biodistribution of small interfering RNAs (siRNAs) is important to develop them for medical use. Therefore, novel single photon emitter-labelled siRNA was prepared by using diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and poly(A) polymerase, and subsequently, real-time analysis of siRNA trafficking was performed by using single photon emission computed tomography (SPECT). This study aimed at assessing the use of 99mTc-radiolabelled siRNA targeting lacZ to detect lacZ expression in vivo. siRNA targeting lacZ was radiolabelled with 99mTc by using the bifunctional chelator DTPA, and the labelling efficiency and specific activity were determined. The probe stability in RNaseA was assessed. SPECT imaging was performed in mice overexpressing the lacZ gene in the liver. Radiolabelled siRNA remained highly stable in RNaseA solution at 37°C. In SPECT imaging, significant 99mTc accumulation in the liver was observed in mice overexpressing the lacZ gene. 99mTc-labelled lacZ siRNA shows β-galactosidase-specific accumulation and appears promising for the visualisation of lacZ expression in vivo. Our labelled siRNA should be deliverable to specific regions overexpressing the target gene.


gene therapy; genomics; in vivo imaging; lacZ; nanotechnology; systemic delivery; vectors; β-galactosidase

PMID: 27588821



Among several tracing agents developed in recent years, siRNA radiolabelling has attracted attention for its ability to localise and quantify siRNAs by using noninvasive imaging methods.1, 2

We developed a novel technique for labelling siRNA with 99mTc, in which double-stranded siRNA was labelled to obtain conformational accuracy for examining the pharmacokinetics of siRNA by using diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and labelling reagent.

The lacZ gene was overexpressed by introducing pSV-β-galactosidase control vector into 293T cells and by introducing this vector into the liver of mice by using the TransIT-QR Hydrodynamic Delivery Starter Kit.3 We used radiolabelled siRNA to measure lacZ mRNA expression directly by noninvasive methods.

Using poly(A) polymerase, several adenines were added to the 3′ end of siRNA and we joined the -NH2 group of the adenine and the -COOH group of DTPA. Furthermore, we succeeded in coupling 99mTc with siRNA by using the chelating action of DTPA (Fig. 1).

The molecular probe was synthesised in two steps as follows: siRNA was chelated with DTPA-conjugated ATP, which provides coordinate groups for labelling, and then siRNA was labelled with 99mTc. We used the Poly(A) Tailing Kit in order to add several adenines to the 3′-end of siRNA. This kit contains a set of reagents designed to add a ≥150-base poly(A) tail to RNA transcripts. This is accomplished by using Escherichia coli poly(A) polymerase (E-PAP) and ATP. The resulting capped and tailed RNA can then be used in transfection or micro-injection experiments, in which enhanced translation compared with that of tailless mRNAs may be seen due to increased mRNA stability and translation efficiency.4-6

We forcibly expressed the lacZ gene in mouse liver by using TransIT-QR Hydrodynamic Delivery Starter Kit and pSV-β-galactosidase control vector. We mixed 99mTc-labelled siRNA solution (0-200 bp) with 2 ml of transfection reagent TransIT-QR, and administered the solution to the mouse intravenously via the tail vein. At 30 min after injection, we performed imaging by using a SPECT camera. Significant 99mTc accumulation was observed in the liver (Fig. 2-a). On the other hand, 99mTc accumulation in mouse liver without transfection was low and nonspecific (Fig. 2-b). At 30 min after the injection of 99mTc-DTPA-poly(A)-siRNA targeting lacZ mRNA into the mouse that overexpressed lacZ in the liver, SPECT imaging by using the radiolabelled probes showed that the probe was cleared rapidly from the major organs, but not from the liver. At 30-min after injection, probe uptake in the liver of the mouse overexpressing the lacZ gene was higher than probe uptake in the liver of the normal control mouse.

This supports the specific hybridisation of the 99mTc-DTPA-poly(A)-siRNA probe to lacZ mRNA and the specific accumulation of the radiolabelled siRNA in cells overexpressing the lacZ gene.

Our results show that 99mTc-DTPA-poly(A)-siRNA targeting lacZ mRNA can detect β-galactosidase in vivo. However, β-galactosidase is not an important protein clinically, so we plan to investigate the use of 99mTc-DTPA-poly(A)-siRNA targeting oncogenes, potentially enabling the novel imaging of tumours.

We developed a novel single photon emitter-labelling methodology for siRNA and evaluated the in vivo trafficking of 99mTc-labelled siRNA by using a SPECT camera.



 Figure 1.Synthesis: DTPA anhydride was added to an ATP solution and DTPA-conjugated ATP was produced. Several adenines were added to the 3′-end of siRNA by an enzymatic reaction of poly(A) polymerase (E-PAP), and DTPA-poly(A)-siRNA was generated. 99mTcO4- and a little stannous chloride(II) were added to the solution and incubated. 99mTc-DTPA-poly(A)-siRNA was generated.



Figure 2.SPECT imaging: (a) Significant 99mTc accumulation in the liver was observed. (b)The 99mTc accumulation in the liver of a mouse without transfection of the vector into this organ was low and nonspecific.



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