A Dual-Function Indole-Benzimidazole Hybrid: Lipid Droplet Imaging and Anticancer Potential.

A novel indole-fused benzimidazole fluorescent probe, I-BZ, was designed, synthesized, and characterized for selective imaging of intracellular lipid droplets (LDs) in human breast cancer cells (MDA-MB-231). The probe was prepared via condensation of 3-methyl-1H-indole-2-carbaldehyde with 4-methylbenzene-1,2-diamine and confirmed by 1H/13C NMR and LC-MS/MS. Photophysical studies indicated that I-BZ exhibits ESIPT-based fluorescence, enabling sensitive detection of lipid-rich compartments. Cytotoxicity assays showed that low concentrations (0.5 μM) were biocompatible for live-cell imaging, whereas higher doses exhibited anticancer activity, with IC50 values of 35 μM at 24 h and 15 μM at 48 h. Fluorescence microscopy in fixed and live cells demonstrated selective accumulation of I-BZ in LDs, validated by ethanol disruption and colocalization with Nile Red. Nuclear costaining with DAPI provided spatial context for precise visualization of cytoplasmic LDs. I-BZ shows great potential as a fluorescent probe for lipid droplet imaging, offering a robust tool for investigating lipid-associated processes in cancer biology.