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.
J Nucl Med. 2014 Jun;55(6):1008-16. doi: 10.2967/jnumed.113.136101. Epub 2014 Apr 17.
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.
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.
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.
(99m)Tc-EGFR-PNA has the potential for imaging EGFR mRNA overexpression in tumors.
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。
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。
（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。
（99m）Tc-EGFR-PNA 具有成像肿瘤中 EGFR mRNA 过表达的潜力。
