Therapeutic Copper-based Nanoparticles Release Labile Copper(II) and Trigger Cellular Responses in Glutathione and NRF2 Redox Pathways and Metal Homeostasis

Metal-based nanoparticles are a promising class of materials for diagnosis and treatment of cancer and other diseases. However, mechanisms of action of these nanomedicines remain insufficiently understood due in large part to our limited understanding of the dynamic equilibria between solid metal nanoparticles and labile metal ions generated from these nanoparticles within complex biological milieus. Here we apply activity-based sensing to directly identify and investigate the fate of labile copper pools with metal and oxidation state-specificity generated by anticancer copper nanomedicines. We found that treatment of cells with copper-releasing CuFe2O4 and Cu2Fe(CN)6 nanoparticles alter labile Cu(I)/Cu(II) ratios through an increase in labile Cu(II), while overall labile copper levels decrease. Labile copper release triggers compensatory responses in two major antioxidant pathways, glutathione (GSH) and nuclear factor erythroid 2–related factor 2 (NRF2), as well as in metal homeostasis to limit copper availability via synergistic upregulation of copper export (ATP7B) and downregulation of copper import (CTR1) proteins. These findings establish the value of activity-based sensing as a generalizable approach for labile metal imaging to help decipher molecular mechanisms of bioactive metal nanoparticles and guide the development of more effective nanomedicine diagnostics and therapies to target metal-dependent disease vulnerabilities.