INVESTIGATION OF THE IN VITRO ASSESSMENT OF 99MTC-LABELED LAMININ-111 PEPTIDES AS PROSPECTIVE BIOMARKERS FOR BREAST CANCER

Abstract

Introduction/Justification: Breast cancer constitutes a significant public health issue as the second most prevalent type of tumor among women. In the past decade, radiolabeled peptides have been employed in both therapeutic interventions and tumor imaging, representing a substantial promise in the specific targeting of tumorigenic cells. Several studies demonstrate that biologically active peptides derived from laminin-111 regulate gene expression in breast cancer-derived cells, including the YIGSR and IKVAV peptides. Objectives: To synthesize the HYIGSR and HIKVAV fragments, derived from laminin-111, standardize and optimize their radiolabeling process with technetium-99m (99mTc), as well as, to assess the in vitro biological characteristics of these radiolabeled peptides as potential biomarkers for breast cancer. Materials and Methods: The HYIGSR and HIKVAV peptides were synthesized employing the solid-phase peptide synthesis through the Fmoc/tBut strategy, characterized, and purified utilizing high-performance liquid chromatography (HPLC). By using the tricarbonyl method, it was possible to label the histidine residue of the peptide fragments with the organometallic aqua-ion [99mTc(H2O)3(CO)3]+, abbreviated as [99mTc]TcCO3, directly from reaction of [99mTc]TcO4- under 1 atm of CO for 30 min at 70°C. Subsequently, both peptides were labeled with approximately 148 MBq and incubated for 30 min at 85°C. The stability of the radiolabeled peptides in saline and serum was assessed at 1, 2, 3, and 4 h and evaluated using HPLC. The partition coefficient was determined for both radiopeptides. Studies to assess the percentage of binding to serum proteins were conducted at 60 min. The binding and internalization of radiolabeled peptides with tumorigenic cells derived from breast cancer (MCF-7) were assessed at 1, 2, and 4 h. Results: The HYIGSR and HIKVAV peptides were efficiently synthesized and characterized. The radiolabeling process with [99mTc]TcCO3 was optimized and the [99mTc]TcCO3-HYIGSR and [99mTc]TcCO3- HYIKVAV were successful obtained with radiochemical yields of 95.53% ± 1.19 and 95.13% ± 1.96 (n = 6), respectively. Notably, stability studies revealed that both radiopeptides exhibited stability within a four-hour timeframe when stored in either saline or serum. The [99mTc]TcCO3-peptides demonstrated hydrophilic properties, as indicated by Log P values of -2.12 ± 0.16 and -1.39 ± 0.19 (n = 3) for [99mTc]TcCO3-HYIGSR and [99mTc]TcCO3-HYIKVAV, respectively. Additionally, the binding percentage to serum proteins for [99mTc]TcCO3-HYIGSR and [99mTc]TcCO3-HYIKVAV was found to be 46.08% ± 3.75 and 24.87% ± 6.24 (n = 3) within 60 min, respectively. Furthermore, binding and internalization studies conducted with MCF-7 cells (n = 4) demonstrated a higher percentage of binding and internalization for [99mTc]TcCO3-HYIKVAV, with values of 9.20% ± 2.87 and 51.74% ± 8.00, respectively. In contrast, [99mTc]TcCO3-HYIGSR exhibited percentages of 2.96% ± 0.60 and 25.85% ± 3.33 for binding and internalization within a 1-hour period. Conclusion: Both peptides exhibited good radiochemical yields and demonstrated sustained stability over the course of the study. Both peptides showed hydrophilic characteristics and our findings specifically underscored the higher affinity of [99mTc]TcCO3-HYIKVAV towards human breast cancer cells. Nevertheless, it is imperative to note that further in vivo investigations are necessary.

Keywords

• Breast cancer
• Laminin-111
• MCF-7 cells.
• Tricarbonyl-labeled peptides