Nanomed J. 12(4): 673-689, Autumn 2025 Evaluation of chemo-photodynamic therapy of multi-drug (metformin- methylene blue) conjugated to ferrofluidic nanocarrier on triple-negative breast cancer cells in hyperglycemic conditions Samaneh B. Chalaki1, Mostafa Gholizadeh*1, Batool Akhlaghinia1, Armin Imanparast2,3, Ameneh Sazgarnia2,3 1 Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran 2 Medical Physics Research Center, Basic Sciences Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran 3 Department of Medical Physics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran ABSTRACT Objective(s): Previous studies have established a link between hyperglycemia (high blood sugar) and hyperinsulinemia (elevated insulin levels) in diabetes, and the subsequent promotion of cancer cell growth. Among various cancers, triple-negative breast cancer (TNBC) is one of the most aggressive types, which can exhibit heightened malignancy in the presence of diabetes. The objective of this study is to introduce a novel approach to combined chemo-photodynamic therapy (Ch-PDT) for TNBC, utilizing metformin (MET) and methylene blue (MB) conjugated to a ferrofluidic nanocarrier, targeting MDA-MB-231 breast cancer cells under conditions that simulate high blood sugar. Material and methods: To this end, Fe₃O₄f nanoparticles were synthesized via the hydrothermal method and subsequently modified with polyethylene glycol and 3-aminopropyltriethoxysilane (APTES) to enhance drug loading efficiency. The physicochemical properties of the nanoparticles were characterized using FT-IR, XRD, FE-SEM, TEM, EDX, ¹H-NMR, VSM, and UV-VIS spectroscopy. For the Ch-PDT application, the cells were incubated with the different formulations and subjected to various irradiation conditions. Cell viability was then assessed for all treatment groups using the MTT assay. Results: Increased exposure in the MB-containing group resulted in reduced cell survival, likely due to the photosensitizing properties of methylene blue. However, in the Fe₃O₄f-PEG-MET-MB group, cell death increased significantly as the irradiation time was extended. This synergistic effect is attributed to the enhanced efficacy of combined chemo-photodynamic therapy (Ch-PDT), which optimizes the therapeutic potential of metformin (P < 0.01). Conclusion: Based on the results, Ch-PDT can be an effective method in increasing the efficiency of metformin in inhibiting metastatic breast cancer tumor cells in hyperglycemic conditions

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