Development of novel multifunctional metal-oxide-based nanozymes and their toxicological characterisation (NaNoZymSafe)

Natural enzymes are ubiquitous biomolecules that regulate the rates of biochemical reactions in living organisms, as an essential part of cell metabolic pathways. They have either extreme specificity to catalyze a reaction for a single molecule or flexibility to catalyze a whole group of biochemical reactions. Due to their high catalytic activity, substrate specificity and good biocompatibility, they are widely used in various fields like food processing, agriculture, medicine, chemical industry and environmental protection. However, natural enzymes have some limitations, as they are usually not available at large scale, and have labile catalytical activity and environmental sensitivity. Nanomaterials serving as artificial enzymes of the new generation called nanozymes have recently attracted significant attention due to their unique properties compared to natural enzymes. They exhibit enhanced catalytic performance, excellent stability, low production cost, and very importantly enzyme-like catalytic properties (e.g. oxidase, peroxidase, catalase, superoxide dismutase, phosphatase). The emerging field of nanozymes has a broad range of potential applications (e.g., sensing, imaging, tissue engineering, diseases diagnosis and therapeutics, waste scavenging and environmental protection) in bridging the areas of nanotechnology, biomedicine and environmental science. The NaNoZymSafe project aims to develop and synthesize novel multifunctional metal-oxide-based nanozymes with potential applications in medical, analytical, biotechnology, food preservation, and environment fields. First, we will perform structural and physicochemical, and further toxicologically characterisation of nanozymes to assess their safety for humans and the environment. As experimental model advanced in vitro 3D-cell models developed from human hepatocellular (HepG2) and normal adult zebrafish (Danio rerio) liver (ZFL) cells will be used. Their potential adverse effects will be studied by assessing primary DNA damage (comet, γH2AX, H3- histone assays) and chromosomal (micronucleus assay) damage, and the influence on cell cycle, proliferation, oxidative stress, and apoptosis. To clarify the underlying mechanisms of toxicity, which is essential information for the contemporary risk assessment, we will perform toxicogenomic analyses to identify the most sensitive molecular pathways associated with the observed cellular effects. This will enable better understanding of the possible risks to human health and the environment. Further, the impact of nanozymes on environmental organisms will be assessed on different trophic levels including primary producers and invertebrates, while the influence on embryonic development will be studied in zebrafish (Danio rerio) embryo model. Combining structural, physicochemical and toxicological characterisation of novel multifunctional metal-oxide-based nanozymes will allow for a more complete understanding of associations between nanozyme characteristics and biological features. To understand the mechanisms underlying the biological effects is particularly important to prevent the use of hazardous nanomaterials for human applications. Altogether, this will enable more safe-by-design approaches that will significantly improve nanozyme safety and most likely also foster public trust toward these materials and technologies utilizing them. The innovative project will be realized in the frame of a collaboration of Slovenian scientists from the National Institute of Biology and the Institute Jozef Stefan with the collaboration of foreign experts from University of Zaragoza and Spanish National Research Council, Spain, the National Institute of Health dr. Ricardo Jorge, Portugal and Institute for Medical Research and Occupational Health, Croatia though their complementary expertise in the fields of material science, nanotechnology, nanotoxicology, genetic toxicology and ecotoxicology, molecular biology, and “omics” technologies.