Molecular Insights into Doxorubicin‐Human Serum Albumin Interactions: Binding Sites, Conformational Changes, and Pharmacokinetic Implications

Abstract Doxorubicin, a potent antineoplastic agent, is widely utilized in the treatment of various malignancies, including breast cancer, malignant lymphomas, and soft tissue sarcomas. This study investigates the interaction between doxorubicin and human serum albumin (HSA) under physiological conditions using a combination of biophysical techniques, including circular dichroism (CD), Fourier‐transform infrared spectroscopy (FT‐IR), equilibrium dialysis, molecular docking, and UV–visible (vis) absorption spectroscopy. The results indicate that doxorubicin quenches the intrinsic fluorescence of HSA via a static quenching mechanism. Binding constants ( K b ), Stern–Volmer quenching constants ( K sv ), the number of binding sites ( n ), and thermodynamic parameters were determined at multiple temperatures. Data analysis revealed cooperative binding, wherein the binding of one doxorubicin molecule enhances the affinity for subsequent molecules. Molecular docking studies further demonstrated that doxorubicin interacts primarily with the IIA, IIIA, and IIB subdomains of HSA, inducing subtle conformational changes. Both fluorescence spectroscopy and molecular docking analyses suggest that hydrophobic interactions are a dominant force in the doxorubicin‐HSA binding process.