RADIOLABELING OF THE DOTA-C6-ANTI-EGFR PEPTIDE WITH GALLIUM-68: A PRELIMINARY STUDY OF SYNTHESIS, COMPLEXATION, AND RADIOCHEMICAL STABILITY

Introduction/Justification Peptides with high affinity for molecular targets overexpressed in tumor tissues have been widely explored for the development of radiopharmaceuticals with theranostic potential. Previous studies from our group demonstrated that the EEEEYFELV peptide fragment exhibits high affinity for the epidermal growth factor receptor (EGFr), with promising cellular internalization and tumor specificity. Structural modification using spacers and chelators is essential to enable stable complexation with metallic radionuclides while preserving receptor interaction, supporting the development of peptide-based radiotracers. Objectives To evaluate the radiolabeling of the DOTA-C6-anti-EGFr peptide with gallium-68 and to investigate labeling yield, radiochemical purity, and preliminary stability. Materials and Methods The anti-EGFr peptide was synthesized by solid-phase synthesis (SPFS) and modified with the e-aminocaproic acid (C6) spacer. The DOTA chelating agent was conjugated to the peptide using DIPEA/TBTU in DMF/DCM (1:1). Radiolabeling of DOTA-C6-anti-EGFr was performed manually with [68Ga]GaCl3 eluted from a 68Ge–68Ga with 6 mL of 0.1 M HCl (∼12.21 mCi/mL) and purified in a cationic filter with acetone. Approximately 500 µL of eluate was added to 40 µL of peptide solution (1 mg/mL) and 1 mL of 0.2 M sodium acetate buffer (pH 4.0), followed by heating at 95°C for 15 minutes. The radiolabeled product was purified using a Sep-Pak C18 cartridge with 50% ethanol. Radiochemical purity of the final product was determined using ascending thin-layer chromatography (iTLC) with 0.1 M ammonium acetate in methanol (1:1) as the mobile phase. Results The DOTA-C6-anti-EGFr peptide was successfully synthesized with a yield of 18.3%. After purification step, preliminary radiolabeling with 68Ga resulted in a radiochemical yield of approximately 78%. iTLC analysis showed a retention factor (Rf) of 0.9–1.0 for the radiolabeled peptide, while free [68Ga]GaCl3 presented an Rf of 0.0–0.1, indicating effective separation between the product and radiochemical impurities. A preliminary stability study demonstrated maintenance of radiochemical purity over time, with values of 93.84 ± 0.00% at time zero, 92.84 ± 7.16% after 30 minutes, and 87.62 ± 3.97% after 90 minutes. These results suggest efficient formation and satisfactory short-term stability of the DOTA–68Ga complex under the evaluated conditions. Conclusion Preliminary data indicate that the DOTA-C6-anti-EGFr conjugate exhibits favorable behavior for radiolabeling with gallium-68, reinforcing the potential of this conjugation strategy for applications in molecular radiopharmacy. Further studies optimizing labeling parameters, purification, and stability, as well as cell binding and internalization assays in glioblastoma models, are planned to confirm and further develop the results obtained.