Peptides are implicated in various physiological responses and hold significant potential as targeting molecules, especially in cancer diagnosis or treatment. Radiolabeled peptides have been investigated for their potential as theranostic agents, holding considerable promise for precisely targeting tumorigenic cells. Previous studies indicate that biologically active peptides exhibit a high affinity for the Epidermal Growth Factor receptor (EGFr), which is overexpressed in various tumor cells, including glioblastoma, the most prevalent and aggressive malignant brain tumor.

To evaluate the in vitro interactions involving two radiolabeled peptide inhibitors targeting the EGFr overexpressed in glioblastoma cells.

Two EGFr-targeting peptide inhibitors, anti-EGFr-LP and anti-EGFr-LG, were radiolabeled with [131I]NaI (11.1–14.8 MBq) using the chloramine T method (room temperature; reaction time = 120 s). The radiochemical yield (RCY) (n = 8) and stability (n = 3) were evaluated using ascending chromatography on TLC-SG strips and acetonitrile/water (95:5) as eluent. C6 and U-87 MG glioblastoma cell lines were cultured in supplemented DMEM medium (5% CO2 atmosphere; 37°C) until reaching ∼85% confluence. Subsequently, aliquots of 2 x 10^6 C6 or U-87 MG cells were incubated with each radiopeptide (37°C) under agitation (500 rpm). In vitro binding and internalization percentages were assessed at 1 and 3 h post-incubation (n = 6). Data were expressed as ‘mean ± standard deviation’ and the statistical analysis was performed using GraphPad Prism software.

The RCY of [131I]I-anti-EGFr-LP and [131I]I-anti-EGFr-LG were 92.92 ± 3.42 and 97.80 ± 1.08, respectively. Both 131I-labeled peptides were radiochemically stable over 24 h. The in vitro interaction between C6 cells and [131I]I-anti-EGFr-LP showed binding percentages of 4.80 ± 0.37% (1 h) and 5.87±1.21% (3 h), with no statistically significant difference (p = 0.1519). The internalization percentages, within the bound fractions, increased from 64.45 ± 4.19% (1 h) to 75.15±1.60% (3 h) (p < 0.0001). For the [131I]I-anti-EGFr-LG, the data were of the same order of magnitude. The binding percentages increased from 3.95±0.33% (1 h) to 6.03 ± 0.66 (3 h) (p < 0.0001) and the internalization percentages, among the bound fractions, were 62.57±5.53% (1 h) and 64.04±3.21% (3 h), with no statistically significant difference (p = 0.5959). The in vitro interaction between U-87 MG cells and [131I]I-anti-EGFr-LP showed an increment of the binding percentages from 6.50 ± 0.93% (1 h) to 8.03 ± 0.29% (3 h) (p < 0.0001), but the internalization percentages, within the bound fractions, showed no statistically significant difference (p = 0.2791), 68.98 ± 2.23% (1 h) and 73.02±6.57% (3 h). For the [131I]I-anti-EGFr-LG, the binding percentages were 10.97±1.48 (1 h) and 11.28 ± 0.84 (3 h), with no statistically significant difference (p > 0.6724). The internalization percentages, among the bound fractions, were also statistically similar (p > 0.3596), 68.21 ± 0.16% (1 h) and 65.36 ± 3.56 (3 h).

The in vitro interaction data revealed high affinity of [131I]I-anti-EGFr-LP and [131I]I-anti-EGFr-LG for the C6 and U-87 MG glioblastoma cell lines, which are known to overexpress EGFr. These preliminary findings support the potential use of these peptide inhibitors as specific peptide-based targeting molecules for EGFr, with potential applications as theranostic agents.