Study On The Toxicity And Mechanisms Of Absorption And Transportation Of Nanomaterials In Caco-2 Cell Models

Objective:Through the characterization,three kinds of nano Zinc Oxide on different sizes of nano Zinc Oxide toxic effects on the differentiation of human colonic adenocarcinoma cells and establish Caco-2 cell monolayer model,clarify nanomaterials key influence on the cytotoxicity of Caco-2 cells and nano membrane transport characteristics of Zinc Oxide and its mechanism.Method: 1.Characterization of nanoscale Zinc Oxide Three nanoparticleZinc Oxide particles were characterized by transmission electron microscopy and nanoparticle size,potentiometric analysis and X ray diffraction,and the dissolution rate of nano Zinc Oxide particles in culture medium was detected.2.toxic effects of different particle sizes of nano Zinc Oxide on Caco-2 cellsCaco-2 cells were inoculated into 96 well culture plates for 24h(undifferentiated cells)or 21 days(cell differentiation),using three nanometer Zinc Oxide,respectively ZnO-20(25.3±5.8 nm),ZnO-L(25.6±5.7 nm)and ZnO-100(96.7±42.7 nm),set different doses(6.25,12.5,25,50 and 10 0mg/ml)were cocultured with undifferentiated or differentiated Caco-2 cells in24 h,48 h or 72 h after the cell activity was detected by CCK-8 assay,detection of lactat e dehydrogenase assay(LDH)and the release rate of alkaline phosphatase(ALP)activity.3.The establishment and evaluation of Caco-2 cell modelCaco-2 cells were seeded on Transwell plates at a concentration of 4×10 5 /ml and cultured for 21 days.TEER and Papp values and ALP expression on both sides of the model were measured on different culture days.The c ell differentiation was observed by transmission electron microscopy and evaluated comprehensively.Whether the Caco-2 cell monolayer model was established successfully.4.Absorption,Transport and Mechanism of Nanometer Zinc Oxide on Caco-2 Cell ModelThe concentrations of the three nano-zinc oxides were set to 50 mg/ml,25 mg/ml,12.5 mg/ml,and 6.25 mg/ml.Two duplicate wells were set for each concentration,and 0.5 ml each of three kinds of nano zinc was added to each.The Caco-2 monolayer cell model was in the chamber side and the culture fluid was drawn from the basal side at different incubation times.The zinc content was detected by ICP-MS.Set to control group,low-temperature inhibition group,amiloride group(2.5mmol/L),methyl ?-cyclodextrin group,low-density lipoprotein group(100 ?g/m L),chlorpromazine group(10 ?g /m L).The above inhibitors were added to a Caco-2 cell model chamber and three kinds of nano-zinc oxide(6.25 ug/ml,0.5 ml)were added to the chamber side.The culture solution wa s taken from the substrate side at different times of culture,and the zinc content was detected by ICP-MS.Result:1.The particle sizes of the three nano zinc oxides were 25.3±5.8 nm,96.7±42.7 nm,and 25.6±5.7 nm,respectively,and they were in three different shapes.The detection of crystal structure suggests that zinc oxide is a hexagonal wurtzite structure with d ifferent specific surface areas.Under different conditions,the particle size distribution in the culture fluid is different,the agglomeration state is different,the potential is different,and the dissolution rate of nano-zinc oxide in the culture flu id is27%?2.Under different concentrations,the three kinds of nano-zinc oxide can inhibit the activity of differentiated or undifferentiated cells,increase the release rate of LDH by affecting the integrity of cell membrane structure,and inhibit the expression of ALP.There is a dose-response relationship.Under the same concentration conditions,the smaller the particle size,the greater the toxicity,while the similar particle size,the toxicity may still be different and may be related to other properties such as the shape of the particles.At the same time,undifferentiated cells are more sensitive than differentiated cells for the toxic effects of the same nanomaterial.3.The Caco-2 cell model was established.The cells were inoculated with Transwell plate at a concentration of 4×10 5 /ml and cultured for 21 days.The cells differentiated into small intestinal epithelial cells in a tightly connected state.The transmittance of the osmotic substance decreased to 16% after 21 days of culture,and the activity of the ALP enz yme in the side of the intestinal cavity(AP side)was higher than that in the basal side(BL side),and the activity ratio was 4.2 times.All indicators in this experiment reached the standard for establishing a monolayer cell model.4.The transport experiments of ZnO-20,ZnO-100,and ZnO-L nano zinc oxides on a Caco-2 monolayer cell model revealed that nano zinc oxide was transported by active transport,with the addition of concentration and time.With the increase,the transshipme nt volume increases.By adding different endocytosis pathway inhibitors to study the endocytic pathway of nano-zinc oxide,it was found that the nano-zinc oxide cell pathway is diverse and energy-dependent.conclusion: 1.The toxicity of nano zinc oxide on Caco-2 cells was related to its physi cal and chemical properties such as particle size,shape,and cell differentiat ion.2.The verification parameters of the Caco-2 monolayer cell model establ ished under the experimental conditions meet the requirements of its integrit y,cell polarity,and permeability,and can be used for nano zinc oxide trans port experiments.3.Nano-zinc oxide is transported by active transport,and the cell ular p athways are diverse and energy-dependent.