Preparation and Evaluation of ^99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

Abstract

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 singlestranded 17-mer antisense EGFR mRNA antisense PNA and mismatched PNA, a 4-amino-acid (Gly-(D)-Ala-Gly-Gly) linker forming an N 4 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 5 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.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma

“…According to the biological distribution study, Lip- 99m Tc-HYNIC- ASON has clearly accumulated in the tumor, meanwhile, the most radioactivity is accumulated in the kidney and bladder, indicating that the probe is mainly excreted through the urinary and digestive system. However, there is not much radioactivity accumulation in the liver, which is different from other reports that the liver has the highest radioactivity uptake in all organs [ 12 , 13 , 20 , 23 ]. The reason might be that the molecular of Lip- 99m Tc-HYNIC-ASON probe is small and does not need to be digested by macrophages when passing through the liver, hence, the radiolabeled antisense probe we developed caused a short retention time and mild potential toxicity to the liver.…”

“…Antisense imaging is an attractive and non-invasive method to detect the expression of HOTAIR in gliomas. Because the antisense oligonucleotide sequence can combine with the target gene through base complementary pairing, we attach radionuclides to antisense oligonucleotides so that radiolabeled oligonucleotides can noninvasively detect the expression of HOTAIR with SPECT and directly express and quantify biological processes at the cellular or subcellular level [ 12 , 13 ].…”

Application and Evaluation of [99mTc]-Labeled Peptide Nucleic Acid Targeting MicroRNA-155 in Breast Cancer Imaging

“…For the purpose of decreasing steric hindrance, a 4-aminobutyric acid was introduced. 22 Both antisense and mismatched PNA precursors were commissioned by Shanghai Science Peptide Biological Technology Co, Ltd (Shanghai, China) for synthesis and purification.…”

“…Radiolabeling was performed referring to the method of Zhao et al 22 In brief, 10 mL NaOH (0.15 M) was added to the PNA solution (1.7 nmol, 20 mL) and mixed. Subsequently, 200 mL (148-185 MBq) fresh [ 99m Tc]-pertechnetate and 10 mL SnCl 2 Á2H 2 O (1 mg/mL, 0.01 M HCl) were added immediately in sequence.…”

Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model

“…Nucleic acid mimics such as peptide nucleic acid (PNA), locked nucleic acid (LNA) and 2’-O-methyl-RNA (2OMe) have been studied as diagnostic probes of infectious diseases and are capable of replacing conventional DNA or RNA probes [ 13 ]. The key advantages of these mimics are the higher stability in vivo [ 14 , 15 , 16 ] and the more favorable diffusion and hybridization properties than the corresponding unmodified DNA or RNA probes [ 17 , 18 ]. However, the unsolved question of how these types of probes can be used to detect clinically relevant bacteria remains.…”