| Objective: The epidermal growth factor receptor (EGFR/HER1),amplified or overexpressed in a number of malignant tumours, has beensuggested to be a novel target for cancer therapy. Consequently, EGFR is oneof the most attractive molecular markers available. Radionuclide molecularimaging of EGFR expression in cancer could not only provide importantdiagnostic information but influence patient management. so imaging ofEGFR expression also has prognostic value. Watson-Crick mechanism ofantisense oligonucleotides has been used as the basis to deliver radiolabeledoligonucleotides for imaging of gene expression in the target sites, calledantisense imaging, using non-invasive imaging modalities such as singlephoton emission computed tomography (SPECT) or positron emissiontomography (PET). The development of antisense imaging has gained greatattention, which have led to a new area of molecular imaging and targetingutilizing radiolabeled antisense oligonucleotides.The overall aim of this studywas to evaluate if99Tcm-labeled antisense peptide nucleic acid (PNA)targeting EGFR messenger RNA (mRNA) might image EGFR expression intumours.Methods: A17mer single-strand antisense PNA (5′-AGGGTCGCATCGCTGCT-3′) targeting EGFR mRNA and mismatch PNA(5′-AGTAGAGATGTGACGAT-3′) were synthesized. Of that, four amino acids(Gly-(D)-Ala-Gly-Gly-) and γ-Aba(4-aminobutyric acid), linked to the5′terminal of PNA, served as a powerful chelator of99Tcmand a spacer tominimize steric hindrance respectively. Antisense PNA or mismatch PNA wasradiolabeled with99Tcmby the way of ligands exchange. Then the radiolabeledprobes were characterized in vitro, High Performance Liquid Chromatography (HPLC) and Instant Thin-Layer Chromatography (ITLC) measure labelingefficiencies and stability of probes at room temperature and in fresh37℃human serum. Reverse-transcriptase polymerase chain reaction (RT-PCR) wasperformed to assay the mRNA level of EGFR expression in the Lewis lungCancer tissues of the tumor.99Tcm-labeled antisense PNA targeting EGFRmRNA or mismatch PNA was injected intravenously via tail vein of lungtumor-bearing C57BL/6mices. In vivo tumor scintigraphic images wereacquired periodically. The ratios of target to nontarget (T/NT) at2h,6h,12h,18h were measured by the way of region of interest (ROI). Allvariables were expressed as average±SD(x±SD) and all data was analyzed bystatistical sofrware, P＜0.05was considered significant.Results: The labeling efficiency of99Tcm-labeled antisense PNAtargeting EGFR mRNA reached(95.64±2.48)%(n=5),it showed completestability at room temperature and in fresh37℃human serumthe same asmismatch PNA. RT-PCR confirmed that Lewis lung Cancer tissues of tumoroverexpressed the messenger RNA of EGFR. After administration of99Tcm-labeled antisense PNA targeting EGFR mRNA in lung Cancer tumor-bearingmices, the xenografts were visualized clearly and had significantly highaccumulation in xenografts and high T/NT ratios. At12h, the T/NT ratio was6.05±0.18, which was much higher than that of mismatch group(1.29±0.12)(t=53.392, p<0.05).As for that of mismatch probes, the uptake ofxenografts,which showed decrease at later time phases (T/NT ratios:18h1.02±0.05, P＜0.05), was light.Conclusions:99Tcm-labeled antisense PNA targeting EGFR mRNA,anovel molecular probe, was of both high labeling efficiency and outstandingbiological characteristic as well as easy radiolabeling strategies, perserving thepowerful capacity to bind target gene,compared with mismatch probes. Theantisense imaging with99Tcm-labeled antisense PNA targeting EGFR mRNAmay be a promising method for visualization of EGFR expression in Lewislung carcinoma. |
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