Assessment of the toxicity and inflammatory effects of different-sized zinc oxide nanoparticles in 2D and 3D cell cultures

Two-dimensional (2D) monolayer cell cultures are the most common in vitro models for mechanistic studies on the toxicity of engineered nanoparticles (NPs). However, 2D cell cultures may not accurately model the functions of three-dimensional (3D) tissues that have extensive cell-cell and cell-matrix interactions, and thus may lead to wrong experimental results. Hence, to obtain more adequate and detailed information about ZnO NP-tissue interactions, we here assess the toxicity and inflammatory effects of different-sized ZnO NPs at various concentrations on Caco-2 cells which are three-dimensionally cultured in agarose gels, measuring ROS expression, pro-inflammatory cytokines, cell proliferation and death. The results showed that the significant effects of cell dimensionality to cellular responses such as inflammatory response, cytotoxicity and different modes of cell death in response to ZnO NPs treatment. Moreover, ZnO NPs induced the toxicity of 2D and 3D cells in different size-dependent manners. This study revealed that the nanotoxicity conclusions obtained from the 2D cell model might overestimate the toxic effects of ZnO NPs. There is a more realistic mechanism study of nanoparticle-induced toxicity in the 3D cell model, which acts as an intermediate stage bridging in vitro 2D and in vivo models.