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, <i>N</i>-ferrocenylmethyl-<i>N'</i>-(2-pyridylmethyl) benzimidazolium iodide (<b>FBP</b>). 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 <b>FBP</b> 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 <b>FBP</b> 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 <i>γ</i>-H2AX expression. Furthermore, 3D multicellular spheroid experiments validated the penetrating ability and prolonged anticancer effectiveness of <b>FBP</b> in a tumor-mimicking environment.