Synergistic Efficacy of Dual-Target HSA-Based Delivery for HER and VEGFR Co-Blockade in Triple-Negative Breast Cancer

Resistance to monotherapies targeting receptor tyrosine kinases remains a major challenge in triplenegative breast cancer (TNBC). Here, we developed a dualtarget HSAbased nanobioconjugate combining human serum albumin (HSA)-encapsulated lapatinib, an EGFR1/2 inhibitor, with the VEGFR1/2blocking peptide VGB3 to achieve coordinated HER and VEGFR inhibition. Molecular docking predicted stable hydrogenbonding and hydrophobic interactions between Lapatinib (free and HSAbound) and EGFR1/2, and between VGB3 and VEGFR1/2. HSA encapsulation enabled pHresponsive, sustained drug release with faster kinetics under mildly acidic tumor conditions. Thermal denaturation assays confirmed an increase in the conformational stability of HSA after binding, indicating structural integrity and biocompatibility. In vitro studies revealed that HSALapatinib exhibited a ~55 % reduction in IC₅₀ (from ≈ 71 µM to ≈ 32 µM) compared with free Lapatinib. The combination of 17 µM HSALapatinib and 0.18 µM VGB3 reduced cell viability to 25 % (CI = 0.29), confirming a strong synergistic effect. Flow cytometric analysis demonstrated a significant increase in apoptosis-from 40.8 % (HSA-Lapatinib) to 46.8 % (combination) (P < 0.05). RTPCR further showed a fourfold upregulation of VEGFR2 (P < 0.0001), supporting that HSALapatinib triggers compensatory VEGFR2 activation which is neutralized by VGB3. Collectively, these findings substantiate a mechanistic and quantitative synergy between HSAmediated HER inhibition and VGB3based VEGFR blockade. This dualtarget HSALapatinib/VGB3 system offers enhanced potency, reduced resistance, and a promising platform for precisionguided TNBC therapy.