Chang-bin Liu1, Jun-qing Xu2, Bai-xuan Xu1, Jin-ming Zhang1, Ying-mao Chen1, Rui-min Wang1, Jia-he Tian3. 1. Department of Nuclear Medicine, The Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, People's Republic of China. 2. Department Of Radiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, Shaanxi, People's Republic of China. 3. Department of Nuclear Medicine, The Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, People's Republic of China. jiahetiantian@163.com.
Abstract
PURPOSE: We aimed to explore the feasibility of transfection methods for antisense imaging. PROCEDURES: Antisense oligonucleotides (ASON) targeted to the mRNA of hTERT gene were synthesized and labeled with Technetium-99m and fluorescein isothiocyanate (FITC), respectively. Then, ASON was combined with transfection reagent Lipofectamine 2000 and Xfect(TM), named Lipo-ASON and Xfect-ASON, respectively. After transfection, the labeled ASON was characterized in hNPCs-G3 and hRPE cells. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to assay the hTERT mRNA and protein levels after hNPCs-G3 cells were incubated with Lipo-ASON, Xfect-ASON, and naked ASON. In addition, Lipo-ASON, Xfect-ASON, and naked ASON were injected into tumor-bearing mice, and the biodistribution in vivo was performed. RESULTS: The presence of two transfection reagents significantly increased intracellular uptake of radiolabeled ASON in both cell lines compared with naked ASON (p < 0.05). However, there was no significant difference in cellular uptake rates of Lipo-ASON and Xfect-ASON between hNPCs-G3 and hRPE cells. In comparison with naked ASON, the fluorescence intensity was strongly enhanced after binding to transfection reagents. Furthermore, the levels of hTERT mRNA and protein were significantly reduced in cells treated with Lipo-ASON and Xfect-ASON (p < 0.05), but naked ASON had no significant effect on hTERT expression level. The biodistribution study indicated that tumor radioactivity uptake of radiolabeled ASON for naked ASON, Lipo-ASON, and Xfect-ASON group was low and shown no significant difference in vivo. CONCLUSIONS: Lipofectamine transfection and Xfect(TM) transfection were not effective delivery methods of ASON for antisense imaging.
PURPOSE: We aimed to explore the feasibility of transfection methods for antisense imaging. PROCEDURES: Antisense oligonucleotides (ASON) targeted to the mRNA of hTERT gene were synthesized and labeled with Technetium-99m and fluorescein isothiocyanate (FITC), respectively. Then, ASON was combined with transfection reagent Lipofectamine 2000 and Xfect(TM), named Lipo-ASON and Xfect-ASON, respectively. After transfection, the labeled ASON was characterized in hNPCs-G3 and hRPE cells. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to assay the hTERT mRNA and protein levels after hNPCs-G3 cells were incubated with Lipo-ASON, Xfect-ASON, and naked ASON. In addition, Lipo-ASON, Xfect-ASON, and naked ASON were injected into tumor-bearing mice, and the biodistribution in vivo was performed. RESULTS: The presence of two transfection reagents significantly increased intracellular uptake of radiolabeled ASON in both cell lines compared with naked ASON (p < 0.05). However, there was no significant difference in cellular uptake rates of Lipo-ASON and Xfect-ASON between hNPCs-G3 and hRPE cells. In comparison with naked ASON, the fluorescence intensity was strongly enhanced after binding to transfection reagents. Furthermore, the levels of hTERT mRNA and protein were significantly reduced in cells treated with Lipo-ASON and Xfect-ASON (p < 0.05), but naked ASON had no significant effect on hTERT expression level. The biodistribution study indicated that tumor radioactivity uptake of radiolabeled ASON for naked ASON, Lipo-ASON, and Xfect-ASON group was low and shown no significant difference in vivo. CONCLUSIONS:Lipofectamine transfection and Xfect(TM) transfection were not effective delivery methods of ASON for antisense imaging.
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