To investigate the feasibility of in vivo imaging study of atherosclerotic plaque and restenosis using antisense probe, we evaluated the uptake kinetics of radiolabeled oligonucleotides to the messenger RNA (mRNA) of proliferating cell nucleus antigen (PCNA) in vascular smooth muscle cells (VSMCs) and the effect on gene expression.
Methods: The antisense oligonucleotide to PCNA was radiolabeled with (99m)Tc through bifunctional chelator mercaptoacetyltriglycine (MAG(3)). The labeling efficiency was assessed by Sephadex G25 chromatography. The radiochemical purity, in vitro stability, and ability of the labeled antisense oligonucleotide to hybridize to its complement were analyzed by Sep-Pak C18 column chromatography. The uptake kinetics of (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide were studied in VSMCs in log and plateau phases. To study whether the antisense probe can hybridize to a respective sequence on the whole PCNA mRNA strand after radiolabeling, we performed reverse-transcriptase polymerase chain reaction to assay the PCNA mRNA level after the VSMCs had been incubated with the (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide.
Results: The labeling efficiency of (99m)Tc-MAG(3)-antisense oligonucleotide was 60.1% (n = 5), the specific activity was 1,960 kBq/microg, and the radiochemical purity was more than 95% after purification. (99m)Tc-MAG(3)-antisense oligonucleotide was stable in vitro and retained the ability to hybridize with its complementary chain. Antisense oligonucleotide showed significantly higher accumulation than sense oligonucleotide in log phase, with peak values of 15.2% +/- 0.58% and 5.6% +/- 0.42%, respectively (P < 0.05). No significant difference was found between uptake of antisense oligonucleotide and uptake of sense oligonucleotide in plateau phase (P > 0.05), but higher accumulation of antisense oligonucleotide was found in log phase than in plateau phase (P < 0.05). The retention rate of antisense oligonucleotide in log phase was much higher than that of sense oligonucleotide (79.6% +/- 0.96% vs. 59.8% +/- 0.75%, P < 0.05) at 240 min. The 2 probes did not significantly differ in plateau phase (P > 0.05). The efflux of antisense oligonucleotide was obviously slower in log phase than in plateau phase (P < 0.05). Compared with (99m)Tc-MAG(3)-sense oligonucleotide, (99m)Tc-MAG(3)-antisense oligonucleotide could inhibit the expression of PCNA mRNA significantly.
Conclusion: This in vitro study in VSMCs provided evidence that the (99m)Tc-labeled antisense oligonucleotide to PCNA can be used for in vivo imaging of atherosclerotic plaque and restenosis in further study.