One-Pot NADH-Mediated Physiological Redox-Controlled Synthesis of Papain-Stabilized Copper Nanoclusters with Preserved Bioactivity for Efficient Drug Delivery

Abstract Protein-protected copper nanoclusters are promising candidates for bioimaging and therapeutic applications. However, harsh reducing conditions required for their synthesis, compromising the protein’s structural integrity and bioactivity. Here, we report a one-pot, physiologically compatible aqueous synthesis for papain-protected copper nanoclusters (Pap-CuNCs) with blue photoluminescence and excellent photostability using nicotinamide adenine dinucleotide (NADH) as a biological reducing agent. The mild conditions (ambient temperature, neutral pH) enable the simultaneous formation of a metallic Cu0 core while stabilizing the helical content of the protein. This approach introduces a physiologically redox-controlled strategy for nanocluster formation, establishing physiological redox chemistry as a governing principle for controlling nanoscale structure and protein conformational stability. Spectroscopic and microscopic studies have demonstrated the presence of crystalline nanoclusters with a protein corona that undergoes α-helical stabilization as revealed by circular dichroism. Notably, atomistic simulation studies reveal preferential binding of the copper core in the protein’s active site, enhancing the α-helical content of papain, consistent with experimental observations. Functionally, the Pap–CuNCs possess biocompatibility and serve as an effective delivery platform for 5-fluorouracil, leading to a 50-fold decrease in IC50 for HeLa cells without causing cytotoxicity to normal cells. This establishes a generalizable framework for bio-integrated nanocluster design under biologically compatible conditions. TOC TextNADH-mediated physiological redox synthesis of papain-stabilized copper nanoclusters with enhanced α-helical stability. This bio-integrated nanoplatform facilitates drug loading and delivery, resulting in a ∼50-fold increase in cytotoxicity in cancer cells while maintaining excellent biocompatibility toward normal cells. Competing Interest Statement The authors have declared no competing interest.