Unravelling the Relationship Between Degradation and Toxicity of Copper Chalcogenide Nanocrystals via Ligand Control and Compositional Changes

Copper chalcogenide nanocrystals with a doped composition of Cu_xM_yS_z (M = doping element), are promising agents for biomedical imaging and therapeutic applications. These nanocrystals include direct bandgap semiconductors such as copper indium sulfide (CIS) and stoichiometric Cu₂S quantum dots (QDs) exhibiting photoluminescence and indirect bandgap materials like vacancy-doped copper sulfide (Cu_2-xS) and iron-doped copper iron sulfide (CuFeS₂) that exhibit plasmonic features. Compositionally, copper chalcogenide nanomaterials like CIS were thought to have better translational potential than the prototypical QDs that contain the heavy metals cadmium and lead. However, we found that CIS QDs are only biostable and biocompatible when coated with a protective zinc sulfide (ZnS) outer shell; bare CIS QDs degrade rapidly causing acute toxicity <i>in vivo</i>. These findings link the nanotoxicity of CIS nanocrystals to their degradation; expanding on these results will provide a framework for the concerted design of biocompatible and optically active copper chalcogenides by controlling the degradation products and rate.<br/><br/>In this study, we quantitatively analyze the toxicity and biodegradation of similarly sized copper chalcogenide nanocrystals with compositional differences (CIS, Cu_2-xS, CuFeS₂) as well as the impact of coating and ligand differences (lipid-PEG micelle, poly-<i>iso</i>-maleic anhydride polymer, etc.) on CIS. We first observe the degradation of different chalcogenide species coated with the same DSPE-PEG_2k micelle encapsulation in biological media to examine the impact of composition on degradation rate. The rate of copper release into the solution is gradual and comparable between compositions. Release of indium and iron mimics that of copper in neutral and chelator-free biological media, but both indium and iron rapidly dissociate from the particles at very early time points upon exposure to the artificial lysosomal fluid containing citric acid, which is prevalent in the lysosomal compartment of cells. When testing the cellular toxicity of these particles on a HepG₂ cell line following 24-hour incubation, CIS exhibits severe toxicity with an estimated IC50 of 124.1 mg/mL total cation concentration. Cu_2-xS shows moderate toxicity with IC50 cation concentration of 401.3 mg/mL, and CuFeS₂ demonstrates the least cytotoxicity with over 75% cell viability at 1 mg/mL cation concentration. We hypothesize that the chelation effect of citric acid in the artificial lysosomal fluid rapidly leaches the indium and iron from the particles. This high local concentration of indium ions appears to be detrimental to the cells, while the strong homeostasis mechanisms used to buffer iron bioavailability may prove protective even in the face of burst release. We found that the surface coating (lipid-PEG micelle encapsulation vs polymer wrapping) alters the rate of oxidation of the surface and particle degradation, inspiring further study into the effect of ligand coating on the degradation and toxicity of CIS QDs. We hypothesize that a covalently bonded ligand will slow the rate of CIS degradation in biological environments and will therefore lead to reduced acute nanotoxicity. This study informs the design of biocompatible nanocrystals by elucidating the relationship between nanoparticle surface ligands, particle degradation, and cytotoxicity for copper chalcogenide nanomaterials.