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.
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.