Nanostructured Zinc Carbonate Hydroxide Microflakes: Assessing the Toxicity Against Erythrocytes and L929 Cells in Vitro

Abstract

Introduction. Nanostructured materials have been suggested to be used as a source of dietary zinc for livestock animals. In this study, we assessed the cytotoxicity of newly synthesized nanostructured zinc carbonate hydroxide (ZnCH) Zn5(CO3)(OH)6 microflakes. Materials and methods. Cytotoxicity of the microflakes was assessed against murine L929 cell line and rat mature erythrocytes. MTT and neutral red uptake assays, scratch assay, Annexin V-FITC/7-aminoactinomycin D staining, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining, Fura 2-AM staining, staining for reactive nitrogen species (RNS), detection of caspases, and lipid order-sensitive NR12S probe staining were employed to evaluate cell viability, motility, cell death pathways, implication of Ca2+, reactive oxygen species (ROS) and RNS signaling, caspases, and alterations of cell membranes following exposure to the microflakes, respectively. To assess hemocompatibility of the Zn-containing microflakes, osmotic fragility assay and hemolysis assay were performed, as well as multiple eryptosis parameters were evaluated. Results. Our findings indicate a dose-response cytotoxicity of ZnCH microflakes against L929 cells with no toxicity observed for low concentrations (10 mg/L and below). At high concentrations, ZnCH microflakes promoted oxidative stress, Ca2+- and caspase-dependent apoptosis and altered lipid membranes. Surprisingly, toxicity to enucleated mature erythrocytes was found to be lower compared to L929 cells. ZnCH microflakes induced eryptosis associated with oxidative stress, Ca2+ signaling and recruitment of caspases. Eryptosis assays were found to be more sensitive than evaluation of hemolysis. Conclusions. Zn5(CO3)(OH)6 microflakes show no cytotoxicity at low concentrations indicating their potential as a source of zinc for livestock animals.