Tunable PEGylated albumin nanocarriers enhance 5-FU cytotoxic selectivity and modulate oxidative and immune stress in colorectal cancer model

5-Fluorouracil (5-FU) is a first-line chemotherapeutic for colorectal cancer (CRC), yet its clinical application is limited by low bioavailability, rapid systemic clearance, and off-target toxicity. We developed tunable PEGylated bovine serum albumin (BSA) nanoparticles as a nanocarrier platform for 5-FU delivery, aiming to improve drug encapsulation, enhance cytotoxic selectivity toward CRC cells, and modulate oxidative and immune-related responses. Using PEG of varying molecular weights (2, 4, and 10 kDa), monodisperse nanoparticles (71-111 nm) were synthesized via a desolvation method and characterized by multi-angle dynamic light scattering, transmission electron microscopy, microscale thermophoresis and HPLC. PEGylation improved colloidal stability and increased 5-FU loading efficiency, particularly at higher drug input concentrations. Binding studies revealed molecular weight- and concentration-dependent interactions of PEG and 5-FU with albumin, influencing nanoparticle architecture and physicochemical properties. Biological evaluation was performed across CRC cell lines (HT-29, Caco-2), non-cancerous fibroblasts (L929), and THP-1-derived macrophages. PEGylated BSA-FU nanoparticles exhibited selective, long-term cytotoxicity toward colorectal cancer cells. In macrophages, PEGylated formulations modulated phagocytic activity and oxidative stress responses in a formulation-dependent manner, indicating that PEGylation alters-rather than uniformly suppresses-immune cell interactions. These findings highlight the importance of rational PEG architecture optimization and provide a strong foundation for future in vivo studies addressing repeated dosing, immune interactions, and translational potential in colorectal cancer therapy.