（99m）Tc-表皮生长因子受体（EGFR）-肽核酸的制备及评价用于恶性肿瘤中 EGFR 信使 RNA 表达的可视化。
Preparation and Evaluation of (99m)Tc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors.
机构信息
Department of Nuclear Medicine, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China
Department of Nuclear Medicine, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China.
出版信息
J Nucl Med. 2014 Jun;55(6):1008-16. doi: 10.2967/jnumed.113.136101. Epub 2014 Apr 17.
UNLABELLED
Epidermal growth factor receptor (EGFR) is overexpressed in many carcinomas and remains a prime target for diagnostic and therapeutic applications. There is a need to develop noninvasive methods to identify the subset of patients that is most likely to benefit from EGFR-targeted treatment. Noninvasive imaging of EGFR messenger RNA (mRNA) expression may be a useful approach. The aim of this study was to develop a method for preparation of single-photon-emitting probes, (99m)Tc-labeled EGFR mRNA antisense peptide nucleic acid (PNA) ((99m)Tc-EGFR-PNA), and nontargeting control ((99m)Tc-CTL-PNA) and to evaluate their feasibility for imaging EGFR mRNA overexpression in malignant tumors in vivo.
METHODS
On the 5' terminus of synthesized single-stranded 17-mer antisense EGFR mRNA antisense PNA and mismatched PNA, a 4-amino-acid (Gly-(D)-Ala-Gly-Gly) linker forming an N4 structure was used for coupling (99m)Tc. Probes were labeled with (99m)Tc by ligand exchange. The radiochemical purity of these (99m)Tc-labeled probes was determined by reversed-phase high-performance liquid chromatography. Cellular uptake, retention, binding specificity, and stability of the probes were studied either in vitro or in vivo. Biodistribution and radionuclide imaging were performed in BALB/c nude mice bearing SKOV3 (EGFR-positive) or MDA-MB-435S (EGFR-negative) carcinoma xenografts, respectively.
RESULTS
The average labeling efficiencies of (99m)Tc-EGFR-PNA and (99m)Tc-CTL-PNA were 98.80% ± 1.14% and 98.63% ± 1.36% (mean ± SD, n = 6), respectively, within 6 h at room temperature, and the radiochemical purity of the probes was higher than 95%. (99m)Tc-EGFR-PNA was highly stable in normal saline and fresh human serum at 37°C in vitro and in urine and plasma samples of nude mice after 2-3 h of injection. Cellular uptake and retention ratios of (99m)Tc-EGFR-PNA in SKOV3 cells were higher than those of (99m)Tc-CTL-PNA and the EGFR-negative control. Meanwhile, EGFR mRNA binding (99m)Tc-EGFR-PNA was blocked with an excess of unlabeled EGFR-PNA in SKOV3 cell lines. The biodistribution study demonstrated accumulation of (99m)Tc-EGFR-PNA primarily in the SKOV3 xenografts and in EGFR-expressing organs. Radionuclide imaging demonstrated clear localization of (99m)Tc-EGFR-PNA in the SKOV3 xenografts shortly after injection but not in (99m)Tc-CTL-PNA and the EGFR-negative control.
CONCLUSION
(99m)Tc-EGFR-PNA has the potential for imaging EGFR mRNA overexpression in tumors.
目的
制备单光子发射探针，即（99m）Tc 标记的表皮生长因子受体（EGFR）mRNA 反义肽核酸（PNA）（（99m）Tc-EGFR-PNA）和非靶向对照探针（（99m）Tc-CTL-PNA），并评估其在体内用于成像恶性肿瘤中 EGFR mRNA 过表达的可行性。
方法
在合成的 17 个碱基对的单链反义 EGFR mRNA 反义 PNA 和错配 PNA 的 5'末端，使用 4 个氨基酸（甘氨酸-（D）-丙氨酸-甘氨酸-甘氨酸）连接子形成 N4 结构，用于（99m）Tc 偶联。探针通过配体交换进行（99m）Tc 标记。通过反相高效液相色谱法确定这些（99m）Tc 标记探针的放射化学纯度。在体外或体内研究了探针的细胞摄取、保留、结合特异性和稳定性。BALB/c 裸鼠分别携带 SKOV3（EGFR 阳性）或 MDA-MB-435S（EGFR 阴性）癌异种移植，进行生物分布和放射性核素成像研究。
结果
（99m）Tc-EGFR-PNA 和（99m）Tc-CTL-PNA 的平均标记效率分别为 98.80%±1.14%和 98.63%±1.36%（n=6），在室温下 6 小时内，探针的放射化学纯度高于 95%。（99m）Tc-EGFR-PNA 在 37°C 下在生理盐水和新鲜人血清中以及在注射后 2-3 小时的裸鼠尿液和血浆样本中非常稳定。（99m）Tc-EGFR-PNA 在 SKOV3 细胞中的细胞摄取和保留率高于（99m）Tc-CTL-PNA 和 EGFR 阴性对照。同时，（99m）Tc-EGFR-PNA 在 SKOV3 细胞系中与过量未标记的 EGFR-PNA 结合，阻断了 EGFR mRNA 结合。生物分布研究表明，（99m）Tc-EGFR-PNA 主要在 SKOV3 异种移植瘤和 EGFR 表达器官中积聚。放射性核素成像显示，（99m）Tc-EGFR-PNA 在注射后不久即可在 SKOV3 异种移植瘤中清晰定位，但在（99m）Tc-CTL-PNA 和 EGFR 阴性对照中则没有。
结论
（99m）Tc-EGFR-PNA 具有成像肿瘤中 EGFR mRNA 过表达的潜力。
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