Targeted Imaging of Estrogen Receptor‐Positive Cancer Cells Using Fluorescent Estradiol Probes

Abstract Breast cancer remains the second most common cause of cancer‐related deaths in women worldwide, with ≈70% of cases linked to the overexpression of Estrogen Receptor (ERα). Existing imaging tools often fail to reliably differentiate between ER‐positive and ER‐negative cancer cells. To address this limitation, two novel fluorescent probes, E2N and E2R , are synthesized by conjugating estradiol to styryl and rhodamine‐based fluorophores using click chemistry. These probes are characterized by their photophysical properties, biocompatibility, and selective targeting of ER‐positive cells. Cellular uptake studies demonstrate preferential internalization of E2N and E2R in ER‐positive MCF‐7, ZR‐75‐1, and T‐47D cells, with minimal uptake in ER‐negative MDA‐MB‐231, MDA‐MB‐468, and healthy COS‐7 and NIH‐3T3 cell lines. Kinetic studies reveal efficient and rapid uptake of E2N in ER‐positive MCF‐7 cells, while mechanistic investigations identified clathrin‐mediated endocytosis as the receptor‐mediated pathway for both probes. Localization studies further confirm their mitochondrial specificity in ER‐positive cells, with E2R displaying higher mitochondrial selectivity. These findings underscore the potential of E2N and E2R as powerful tools for distinguishing ER‐positive from ER‐negative breast cancer cells. Their receptor‐mediated targeting and precise imaging capabilities make them promising candidates for advancing breast cancer diagnostics and enabling more targeted therapeutic strategies.