Synergistic antitumor activity of amino acid–derived ionic liquid binary mixtures: Nanomolar potency with high selectivity

Synergistic antitumor effects were investigated for binary mixtures composed of two ionic liquids derived from amino acids (IL–IL mixtures), as well as for mixtures combining a single ionic liquid with methotrexate (MTX), a clinically approved chemotherapeutic agent. These combinations were tested against two human cancer cell lines, A-549 (lung adenocarcinoma) and HT-29 (colon adenocarcinoma). In general, the binary mixtures exhibited enhanced antitumoral activity and selectivity compared to their individual components under both physiological and acidic pH conditions. Notably, these mixtures achieved IC₅₀ values in the nanomolar range, with selectivity indexes in some cases exceeding 100, representing improvements of one to two orders of magnitude over the individual compounds. Supramolecular interaction studies revealed cooperative interactions between long- and short-chained imidazolium derivatives, underscoring the critical role of the amino acid moiety and hydrogen bonding involving amide groups in driving the observed synergistic effects. Furthermore, ¹H NMR and ESI-MS analyses confirmed molecular interactions between the long-chained IL (IL 1 ) and methotrexate. Confocal fluorescence microscopy, using a fluorescently labelled IL (IL 2 ), both alone and in combination with IL 1 , demonstrated membrane disruption and mitochondrial accumulation, providing further insight into the mechanism of action. In addition, the affinity of individual ILs and IL–IL binary mixtures for salmon sperm DNA was assessed, supporting potential DNA-related mechanisms. Altogether, these findings highlight the potential of these imidazolium-based compounds as promising candidates for combination chemotherapy strategies, and they pave the way for novel applications of ionic liquids in cancer treatment.