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

Protein-protected copper nanoclusters are promising candidates for bioimaging and therapeutic applications. However, harsh reducing conditions required for their synthesis, compromising the proteins 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 -helical stabilization as revealed by circular dichroism. Notably, atomistic simulation studies reveal preferential binding of the copper core in the proteins active site, enhancing the -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 Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/719779v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1ca9292org.highwire.dtl.DTLVardef@5cd82aorg.highwire.dtl.DTLVardef@173ed2forg.highwire.dtl.DTLVardef@1d8c30c_HPS_FORMAT_FIGEXP M_FIG C_FIG NADH-mediated physiological redox synthesis of papain-stabilized copper nanoclusters with enhanced -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.