The development of novel peptide-associated chelators that exhibit greater radiochemical stability compared to classical systems is of great interest in radiopharmaceutical chemistry, especially due to the challenges observed in in vitro and in vivo biological stability studies. Although the molecular development step is complex and laborious, evaluating the feasibility of complexation with radionuclides is fundamental to determining the potential use of these molecules as radiotracers. In this context, the NAQ chelator was developed to promote a stable bond with technetium-99m in the tricarbonyl form, exploiting an amide binding site, in a manner analogous to peptide-histidine interactions, but as a promising alternative. To evaluate the radiolabeling efficiency of an anti-EGFR peptide modified with the chelating agent NAQ, as well as to investigate the radiochemical purity obtained under different peptide concentrations and pH conditions. The tricarbonyl-Tc-99m precursor was prepared from a solid mixture of sodium borohydride, potassium carbonate and sodium potassium tartrate under a CO atmosphere, followed by the addition of sodium pertechnetate (∼14.9 mCi) and heating at 80°C for 30 minutes. The anti-EGFR peptide conjugated to the C6 spacer and to NAQ chelator was prepared in 1 mM 20% acetonitrile and aliquoted at 158, 474, and 948 µg/mL. Radiolabeling was performed by the addition of tricarbonyl-Tc-99m (∼9 mCi), followed by heating (70°C, 30 minutes). Quality control of the tricarbonyl precursor (pH 8) and radiolabeled samples were performed by radio-HPLC. A second experiment was conducted with pH adjustment to 6.5, followed by purification in a Sep-Pak C18 cartridge. Autoradiography was performed on 20 µm histological brain sections containing C6 glioblastoma at 15 and 60 minutes of incubation with the purified radiolabeled peptide (10 µL, 105 µCi/mL). After successive washes in water, the slides were dried at room temperature and exposed to a phosphor imaging plate for 10 minutes, followed by reading on a photoluminescence scanner. The tricarbonyl-Tc-99m complex exhibited a radiochemical purity of 97 ± 0.4%. The radiolabeled peptides showed radiochemical purity of approximately 68% for concentrations of 158 and 474 µg/mL and 78% for 948 µg/mL. Additional chromatographic peaks were visually observed, mainly at the highest concentration, although not identified. After pH adjustment and purification of the radiolabeled peptide at 158 µg/mL, a radiochemical yield of 76% was obtained, with 7% of the activity retained in the cartridge. Additionally, the preliminary autoradiography study demonstrated accumulation of the radiolabeled peptide in the tumor region, indicating that the peptide's affinity for the target was maintained after conjugation with the chelating agent NAQ; however, further assays are needed to confirm and validate this finding. The results indicate that the NAQ chelator shows potential for the radiolabeling of peptides with technetium-99m, although the methodology still needs optimization. Further evaluations involving pH and temperature adjustments and purification strategies are necessary to improve the efficiency and radiochemical purity of the conjugate, enabling its application in future biological studies.
Souza et al. (Sun,) studied this question.