Based On 2D And 3D Caco-2 Cell Cultures To Study The Safety Of Zinc Oxide Nanoparticles

Zinc oxide nanoparticles(ZnO NPs)are broad prospect in application multifunctional as inorganic functional materials,which have many characteristics of small particle size,high specific surface activety,stable chemical properties,widely used in the field of cosmetics,electronic products,drug carrier,medicine and food packaing materials.With the increasing contact between people and ZnO NPs,potential toxicity to human health has also aroused great concern.While two-dimensional(2D)monolayer cell cultures remain the predominant in vitro model for numerous nanotoxicity studies.Cells lose their original tissue organization,polarity,and protein–protein interactions when they are cultured on conventional 2D plastic petri dishes.In addition,cells in monolayer cultures often have an altered metabolism,phenotype,and gene expression compared to in vivo models.When these limitations of 2D cell culture models are extended to nanotoxicological studies,there are risks of obtaining misleading results and conclusions.Virtually all cells in the body reside in a 3D environment,the phenotype and function(s)of individual cells are highly dependent on sophisticated interactions with 3D-organized extracellular matrix(ECM)proteins and neighboring cells.Therefore,a more tissue-like,physiologically relevant model such as 3D models is imperative to provide more theoretical basis and reference value for toxicity of nanoparticles.In order to assessment of vitro toxicity of different-sized ZnO NPs on Caco-2 cells in 2D and 3D cell cultures,Firstly,particle shape,particle size and distribution of ZnO NPs were characterized by transmission electron microscopy and nano particle size analyzer.Next,the morphology of 2D and 3D cells was observed by an inverted microscope.In 2D and 3D cultures,to detect cell activity using MTT colorimetry,inflammatory cytokines(IL-8 and IL-1?),intracellular ROS levels and different modes of cell death by an flow cytometric.The death morphology of 2D cells was observed after AO/EB double staining by an fluorescence microscope.The experimental results were shown as follows:Firstly,the average particle size of three ZnO NPs samples were 24.53 ± 8.54 nm,56.18 ± 9.56 nm,87.26 ± 13.47 nm,respectively.Secondly,ZnO NPs caused a significant change in 2D cell morphology,but no change for 3D cells.24-nm ZnO NPs showed the maximum cytotoxicity on 2D cells,56-nm ZnO NPs is second and 87-nm ZnO NPs is at least,whilst there are no significant difference in 3D cell viability.These results showed that particle size of ZnO NPs on cytotoxicity effect has no significant difference in the 3D culture.3D cell activity was significantly higher than that of the 2D cells,which showed that the cytotoxicity of ZnO NPs on the 2D cells was significantly higher than that of the 3D cells.Thirdly,3D cells had higher endogenous ROS levels compared to 2D cells.The ROS levels of 2D cells were increased in a dose-dependent manner,but it did not cause a significant increase in ROS levels of 3D cells.24-nm ZnO NPs showed the maximum extent of oxidative stress on 2D cells,56-nm ZnO NPs is second and 87-nm ZnO NPs is at least,whilst the effect of particle size on oxidative stress in 3D cells was not significantly different.The high concentration of ZnO NPs(?75 ?g/m L)after 12 h exposure induced oxidative stress of 2D cells than 3D cells.Zn O NPs induced a strong inflammatory response in 3D cells compared to 2D cells.The expression of inflammatory factors in 3D cells increased in a dose-dependent manner,and 24-nm ZnO NPs induced the most intense inflammatory response.24-nm and 56-nm ZnO NPs induced similar inflammatory response in 2D cells,while 87-nm ZnO NPs were relatively low.ZnO NPs could induce an inflammatory response which is independent of ROS levels in 3D cultures.Fourthly,75 ?g/mL of 24-nm ZnO NPs caused maximum number of late apoptotic and necrotic cells in the 2D culture.The number of Caco-2 cell death induced by 24-nm ZnO NPs increased in a dose-dependent manner in the two cell cultures.Compared with 2D cells,the toxicity of 24-nm ZnO NPs on 3D cells was significantly lower.Necrosis is the main mode of cell death in 2D cultures,but apoptosis is the main mode in 3D cultures.