Haemoglobin-modified chitosan-coated magnetic nanoparticles for improved Chlorin e6 loading and photodynamic properties.

This study presents haemoglobin functionalised chitosan-coated magnetic nanoparticles (Fe₃O₄-Cs-Hb) engineered as advanced carriers for chlorin e6 (Ce6) in photodynamic therapy (PDT). Chitosan shells were modified with long-distance amino groups and used to immobilise haemoglobin via amino- or carboxyl-directed conjugation, yielding systems with distinct physicochemical profiles. Hb immobilisation substantially increased Ce6 loading depending on the conjugation route. Drug release kinetics also shifted markedly: Hb-modified systems reduced the initial burst effect by 20-35% and prolonged release over 24 h. ROS-generation studies showed that Hb conjugation enhanced singlet oxygen production by 40-70% relative to non-modified carriers, with Fe₃O₄-Cs-3-Hb(a) providing the highest ROS yield for physically adsorbed Ce6, while Fe₃O₄-Cs-3-Hb(c) maximised ROS output for chemically bound Ce6. These two formulations also displayed the strongest photodynamic activity in HeLa cells, reducing EC₅₀ values from ~0,25-0,35 mg/mL (Hb-free systems) to 0,09-0,12 mg/mL (physically bound Ce6) and to 0,05-0,10 mg/mL (chemically bound Ce6). Collectively, the results demonstrate that haemoglobin imparts synergistic benefits, improving Ce6 loading, stabilising release, and significantly increasing PDT efficiency, highlighting its value as a functional component for next-generation phototherapeutic nanocarriers.