Breast cancer represents a major public health challenge worldwide, with projections indicating more than 35 million new cancer diagnoses by 2050. The search for molecular probes capable of selectively targeting tumor cells has therefore intensified. Among these, biologically active peptides derived from laminin-111 have attracted attention due to their role in regulating cellular behavior, including gene expression, adhesion, migration, and proliferation in breast cancer cells. Notably, the laminin-111–derived sequences YIGSR and IKVAV have been reported to interact with tumor-associated receptors and modulate key pathways involved in cancer progression.

This study aimed to investigate the biological behavior of the laminin-111–derived peptides HYIGSR and HYIKVAV radiolabeled with technetium-99m (99mTc), by assessing their in vitro interaction with breast cancer cells (MDA-MB-231) and their ex vivo biodistribution in mice.

Radiolabeled peptides [99mTc]Tc-HYIGSR and [99mTc]Tc-HYIKVAV were incubated with 2 × 106 breast cancer cells (MDA-MB-231) at 37°C under gentle agitation (500 rpm). In vitro binding and internalization were evaluated after 1h and 2h of incubation. Radioactivity in the cell pellet and supernatant was measured using an automatic gamma counter, and binding percentages were calculated. Subsequently, the pellets were resuspended in 0.5 mL of acid buffer to evaluate internalization. For biodistribution studies, [99mTc]Tc-HYIGSR and [99mTc]Tc-HYIKVAV were intravenously injected into normal female BALB/c nude mice (n = 4 per peptide). Ex vivo biodistribution was performed 2h post-injection. At the selected time point, organs were harvested, weighed, and counted in a Wizard2™ 3”-2480 gamma counter (PerkinElmer). Results were expressed as the mean percentage of injected dose per gram of tissue (%ID/g).

In vitro assays demonstrated that both radiopeptides exhibited affinity for MDA-MB-231 breast cancer cells. For [99mTc]Tc-HYIGSR, binding values were 3.61 ± 0.96% at 1h and 2.63 ± 0.80% at 2h, while internalization reached 67.42 ± 4.27% at 1h and 35.18 ± 7.49% at 2h (n = 6). For [99mTc]Tc-HYIKVAV, binding percentages were 5.38 ± 1.02% at 1h and 5.25 ± 0.50% at 2h, and internalization values were 64.39 ± 7.90% at 1h and 57.77 ± 4.44% at 2h (n = 6). Ex vivo biodistribution in normal female BALB/c nude mice (n = 4) showed rapid blood clearance and low uptake in most evaluated organs, including heart, spleen, lungs, kidneys, and muscle, with values below 5% of the injected dose (%ID/g). In contrast, liver, intestines, stomach, and thyroid exhibited pronounced accumulation of both [99mTc]Tc-HYIGSR and [99mTc]Tc-HYIKVAV, with %ID values exceeding 23%.

In conclusion, [99mTc]Tc-HYIGSR and [99mTc]Tc-HYIKVAV showed relevant interaction with breast cancer cells, characterized by moderate binding and high internalization. Biodistribution results indicate the need for further optimization to improve stability and in vivo targeting, supporting the potential of these peptides as candidates for breast cancer imaging.