Multitargeted Anticancer Strategy through DNA Damage and Mitochondrial Collapse by a Ferrocene-Benzimidazolium Salt

The development of targeted and selective organometallic anticancer drugs is a primary emphasis in contemporary chemotherapeutic research. We provide the synthesis and thorough biological assessment of a new benzimidazolium-derived ferrocenyl compound, N -ferrocenylmethyl- N′ -(2-pyridylmethyl) benzimidazolium iodide ( FBP ). The compound demonstrates significant cytotoxic efficacy against HeLa cervical cancer cells, exhibiting a substantially reduced IC₅₀ value and an elevated selectivity index relative to Doxorubicin and Ferrocene. Biocompatibility evaluations in standard fibroblast cell lines (NIH3/T3 and L929) demonstrated no off-target toxicity within physiologically pertinent dosage ranges. Cellular uptake investigations utilizing Hoechst and MitoTracker Green labeling demonstrated effective internalization and primary mitochondrial location of FBP in HeLa cells. Mechanistic studies, encompassing Live/Dead viability assays, DCFDA-based quantification of reactive oxygen species, singlet oxygen detection through SOSG, and JC-1 analysis of mitochondrial membrane potential, collectively indicate that FBP induces oxidative stress and mitochondrial dysfunction, resulting in apoptotic cell death. Subsequent assessments related to apoptosis revealed distinct nuclear condensation and fragmentation through DAPI staining, along with notable DNA double-strand break production indicated by γ -H2AX expression. Furthermore, 3D multicellular spheroid experiments validated the penetrating ability and prolonged anticancer effectiveness of FBP in a tumor-mimicking environment.