Chemotherapy-induced immunosuppression frequently necessitates the co-administration of antibiotics, raising concerns about potential drug-drug interactions at the level of plasma protein binding. While binary interactions of doxorubicin (DOX) or ciprofloxacin (CIP) with human serum albumin (HSA) have been individually explored, their simultaneous and order-dependent binding behavior remains poorly understood. This study presents a comprehensive comparative investigation of the ternary (HSA-DOX-CIP) systems using multi-spectroscopic techniques and molecular docking. In binary systems, DOX exhibited higher binding affinity than CIP, with both interactions being predominantly entropy-driven. In contrast, ternary complexes displayed pronounced order-dependent behavior. Critically, the order of binding dictated the mechanistic outcome: pre-binding of CIP converted the HSA-DOX interaction into an enthalpy-driven process characterized by a decrease in the Stern-Volmer constant (Ksv) with increasing temperature, whereas DOX pre-association markedly enhanced CIP binding at elevated temperatures, indicating an entropy-driven cooperative effect. These shifts were accompanied by more pronounced microenvironmental perturbations around tyrosine/tryptophan residues and greater α-helix loss, as confirmed by synchronous fluorescence and circular dichroism. Molecular docking suggested partial binding site overlap in domain I upon co-binding. Collectively, these results demonstrate that the HSA-DOX-CIP interaction is not a simple sum of its binary parts but a complex, order-dependent process that can fundamentally reprogram binding affinity, mechanism, and associated structural changes. This insight underscores the necessity of evaluating ternary interactions to accurately predict drug disposition and potential interactions in polypharmacy scenarios.

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