Folate-targeted, ethylenediaminetetraacetic acid-embedded, methotrexate-loaded albumin nanoparticles: Molecular modeling, design optimization, and in vitro anticancer evaluation in breast cancer cells.

BACKGROUND: The prevalence of breast cancer has catalyzed a surge in the design of sophisticated nanomedicine-based strategies aimed at overcoming the limitations of conventional treatments. Methotrexate (MTX) as standard breast cancer treatments often have limited efficacy. Integrating innovative combinatorial approaches may yield synergistic effects that overcome the therapeutic limitations of traditional single drug protocols. Cellular proliferative capacity is heavily reliant on the presence of essential metal ions, which metal chelating agents can sequester. Consequently, co-treatment involving the chelating agent ethylenediaminetetraacetic acid (EDTA) and the therapeutic agent MTX has shown marked antiproliferative activity against breast cancer.

OBJECTIVE: This study aimed to elucidate the antitumor potential of the chelating agent EDTA as a coadjuvant with MTX, a standard chemotherapeutic agent for breast cancer, using in vitro cell line data analysis.

METHODS: Based on the review of the available literature, the present study was designed to investigate binding efficacy of selected ligands MTX, and folic acid (FA) using molecular docking approach against 11 breast cancer related target proteins [PDB IDS:4LRH, 5IZQ, 5TK6, 1EW8, 1HV5, 1XBT, 1CXV, 4KNO, 1U72, 5HEX, 5H2U] and EDTA against 12 breast cancer related target proteins [PDB IDs: 5ZJB, 7DBL, 5IZQ, 5TK6, 1EW8, 1HV5, 1XBT, 1CXV, 4KNO, 1U72, 5HEX, 5H2U]. MTX-BSA NPs were prepared by desolvation method and optimized using box-behnken design (BBD). Characterization included particle size, zeta potential, % entrapment efficiency, % drug loading, % NPs yield, morphology, in vitro drug release study, FTIR, and in vitro cell line study. Optimized MTX-BSA NPs were further embedded with chelating agent EDTA, followed by surface conjugation with folic acid to yield targeted FA-MTX-EDTA-BSA NPs. The cytotoxic potential of the prepared formulations, coded as MTX-BSA NPs, MTX-EDTA-BSA NPs, and FA-MTX-EDTA-BSA NPs, was investigated and compared for antitumor efficacy against MCF-7 human breast cancer cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cellular uptake, ROS generation, apoptosis, and cell-cycle analysis.

RESULTS: The highest binding docking score of folic acid and MTX was found to be -15.03 kcal/mol, -14.86 kcal/mol, against (PDB ID: 4LRH), and the highest docking score of EDTA was revealed to be -9.296 kcal/mol, against (PDB ID: 7DBL). Optimized BSA NPs (MTX-BSA NPs) showed particle size 69.90 ± 8.37 nm, zeta potential -14.9 ± 0.67 mV, and % entrapment efficiency 91.45 ± 0.56%. EDTA-embedded variants (MTX-EDTA-BSA NPs) measured particle size 123.9 ± 0.43 nm, zeta potential -18.7 ± 0.57 mV, and % entrapment efficiency 93.45 ± 0.74%. The loading efficiency (%) for EDTA loaded NPs (EDTA-BSA NPs) was found to be 6.2 ± 0.11% and for MTX loaded BSA NPs it was found to be 10 ± 0.15%. The final formulation also demonstrate good NPs yield (85 ± 0.21%). Based on qualitative and quantitative data analysis, it was revealed that FA-MTX-EDTA-BSA NPs displayed greater cytotoxicity against MCF-7 cell line with IC50 value of 20 μg/ml after 48 h of treatment, reduced viability, enhanced uptake, increased ROS levels, increased apoptosis, and cell-cycle arrest as compared to other formulations, such as MTX-BSA NPs and MTX-EDTA-BSA NPs. The hemolysis assay results indicated that the developed BSA nanoparticles (NPs) were biocompatible.

CONCLUSION: In the current study, in vitro cell line (MCF-7) results revealed that FA-MTX-EDTA-BSA NPs demonstrated significant anticancer activity and greater efficacy as compared to plain MTX and other formulations (MTX-BSA NPs, MTX-EDTA-BSA NPs) because of the potential action of the chelating agent EDTA as a coadjuvant with the MTX in weakening the cancer cell membrane and targeting of FA-MTX-EDTA-BSA NPs precisely to the diseased cells while minimizing off-target effect. The findings suggest that targeting MTX-EDTA-BSA NPs could significantly improve breast cancer treatment outcomes.