pH-responsive folate-targeted catalase conjugate for enhanced cellular uptake and oxidative stress protection in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) remains one of the most aggressive breast cancer subtypes, with limited targeted treatment options. This study investigates the efficacy of folic acid (FOL) as a targeting molecule for TNBC. It also examines whether the cellular uptake of FOL is influenced by acidic pH, a major feature of tumor tissue. A multifunctional FITC-Catalase-Folate (FITC-CAT-FOL) conjugate was developed and optimized to combine fluorescence tracking, enzymatic activity, and receptor targeting. Two conjugation strategies were compared to evaluate how reaction order affects labeling efficiency and catalase (CAT) stability. The optimized FITC-CAT-FOL configuration showed higher fluorescence, efficient FOL incorporation, and greater retention of catalytic activity compared to the reverse order (FOL-CAT-FITC). The conjugate maintained stable enzymatic function across different pH levels and demonstrated enhanced cellular uptake and viability at mildly acidic to neutral conditions (pH 6-7.4) through folate receptor-mediated endocytosis in TNBC cells. Fluorescence microscopy further confirmed enhanced intracellular localization of the conjugate under mildly acidic to neutral conditions. Moreover, cells treated with FITC-CAT-FOL showed higher viability and improved tolerance to oxidative stress, supporting the conjugate's biocompatibility and protective antioxidant function. These results confirm that folic acid exhibits strong affinity toward TNBC cells under physiological and tumor-relevant pH, supporting its use as an effective targeting molecule in enzyme-based drug delivery systems.