Breast cancer remains the most prevalent malignancy among Brazilian women and is frequently characterized by overexpression of the epidermal growth factor receptor (EGFR), a protein closely associated with tumor progression and therapeutic resistance. The MDA-MB-231 (EGFR-overexpressing) cell line is widely used model for investigating receptor-ligand interactions. Due to their high affinity for EGFR, peptides such as GE11 and EEEEYFELV have emerged as promising candidates for targeted diagnostic and therapeutic approaches in tumors expressing this molecular marker.

This study aims to evaluate the interaction of the GE11 and EEEEYFELV peptides toward EGFR in the MDA-MB-231 breast cancer cell line.

Peptides were synthesized by solid-phase peptide synthesis (SPPS) using the Fmoc/tBu strategy and subsequently characterized and purified by HPLC. The GE11 and EEEEYFELV peptides were radiolabeled with Iodine-131 (¹³¹I) using the Chloramine-T method. Radiochemical yields of [¹³¹I]I-GE11 and [¹³¹I]I-EEEEYFELV were evaluated by ascending thin-layer chromatography (TLC). For [¹³¹I]I-GE11, TLC-SG with 95% acetonitrile was used as the stationary and mobile phase, respectively, whereas [¹³¹I]I-EEEEYFELV was analyzed using Whatman 3MM with 95% methanol as the mobile phase. Radiochemical stability was assessed at room temperature for up to 4h. The radiolabeled peptides, [¹³¹I]I-GE11 and [¹³¹I]I-EEEEYFELV were incubated with 2 × 106 MDA-MB-231 and MCF-7 cells at 37°C under gentle agitation (500 rpm) to evaluate binding and internalization at 1 and 2h post-incubation.

To date, the peptides [¹³¹I]I-GE11 and [¹³¹I]I-EEEEYFELV have been successfully synthesized, characterized, and purified, achieving yields of approximately 80% for both peptides. After radiolabeling, radiochemical purities of about 94% were obtained for compounds. Stability assays for [¹³¹I]I-GE11 demonstrated radiolabeling maintenance of 93.09% (1h), 85.87% (2h), and 89% (4h). Similarly, [¹³¹I]I-EEEEYFELV showed stability percentages of 88.78% (1h), 88.54% (2h), and 87.69% (4h). In vitro assays indicated affinity of [¹³¹I]I-GE11 for MDA-MB-231 cells, with binding percentages of 1.77 ± 0.52% (1h) and 2.21 ± 0.26% (2h), and internalization rates of 67.80 ± 5.38% (1h) and 31.77 ± 5.39% (2h). For [¹³¹I]I-EEEEYFELV, binding percentages were 1.22 ± 0.25% (1h) and 0.91 ± 0.22% (2h), while internalization rates reached 43.77 ± 10.86% (1h) and 10.21 ± 5.59% (2h).

Preliminary results of this study demonstrate that GE11 and EEEEYFELV peptides were successfully synthesized and radiolabeled, exhibiting high radiochemical yields and satisfactory stability under the evaluated conditions. In the in vitro assays performed to date, [¹³¹I]I-GE11 showed binding and internalization affinity in MDA-MB-231 cells, consistent with the known EGFR overexpression in this cell line, whereas [¹³¹I]I-EEEEYFELV displayed more limited performance. These preliminary findings highlight the potential of GE11 as a promising targeting vector for EGFR-directed radionuclide therapy. Ongoing studies, including experiments using the MCF-7 cell line and additional comparative analyses, will be essential to confirm selectivity, receptor–ligand interaction profiles, and the broader applicability of these peptides in different breast cancer models.