Cell Autophagy Induction By Two-dimensional Transition Metal Dichalcogenides Via Perturbing Cell Surface Proteins

Due to the ultra high specific surface area and unique physicochemical properties,nanoparticles have been widely applied in multiple fields,such as biomedicine,electronic devices,energy and aerospace.Multiple nanoparticle-based products are accelerating into people's life.About 3036 nanoparticle-based products have been put into market by 2019.The wider and wider application increase the chance for nanoparticle of exposure to human.It is necessary to investigate the biological safety of nanoparticles.Autophagy is the degradation process of cellular components through lysosome pathway.As a highly conserved cellular behavior,autophagy exists from yeast to mammalian cells.In normal conditions,cells maintain basal level autophagy to degrade the old and damaged organelles,aggregated or mis-folded proteins.Under stress conditions,such as starvation or hypoxia,autophagy will be regulated to higher level to degrade cellular components and provide energy and nutrients for cell use.In a word,under both normal and stressful conditions autophagy play important role in maintaining the healthy status of cell.In addition,autophagy disturbance has closely relationship with multiple diseases,such as tumor,neurodegeneration,type II diabetes,and cardiopathy.With the various applications of nanomaterials and increased opportunities of exposure to human,the effects of nanoparticles on cell autophagy are indispensable in their biological safety evaluation.However related investigations are still at the initial stage and further research is urgently needed.Unique physicochemical properties and ultrathin structure made two-dimensional transition metal dichalcogenides(2D TMDCs)promising in multiple fields,such as catalysis,energy storage,photoelectronic devices,carriers for drug and gene delivery,photothermal therapy,photodynamic therapy,and biosensing.The increasing usage of 2D TMDCs increases the possibility of human exposures to TMDCs.For example,biomedical applications of 2D TMDCs require their direct injection into circulation and lung is one of the major target organs for the deposition of 2D TMDCs.In addition,the lung may be directly exposed to low concentrations of 2D TMDCs as air-borne particles generated during the production,transportation,usage and disposal of 2D TMDCs-based products.Therefore,exposure of human lung to these nanoparticles and associated health effects need to be addressed.As one of the important physicochemical properties of 2D TMDCs,the role of thickness on the interaction between 2D TMDCs and cells for the overall understanding biological effects of 2D TMDCs will be of great importance.In this study,we selected(molybdenum disulfide nanosheet)MoS2 NSand(tungsten dilsulfidenanosheet)WS2 NS with different thickness as representatives of 2D TMDCs and human bronchial epithelial cells 16HBE and non-small cell lung cancer cell line A549 as cell models.Under different cellular locations,cellular perturbations especially cell autophagy induced by 2D TMDCs with different thickness,was evaluated systematically.The morphology of MoS2 NS and WS2 NS with different thickness were first characterized by AFM.MoS2 NS and WS2 NS with different thickness respectively showed similar size however different thickness.Next,we investigated the cellular locations of MoS2 NS and WS2 NS with different thickness.FITC-BSA labeled MoS2 NS or WS2 NS were incubated with 16HBE cells or A549 cell first and then cells were analyzed by confocal laser scanning microscope(CLSM).The results showed that the thinner nanosheets were adsorbed to the cell membranes,while thicker nanosheets were mostly internalized by cells.The qualitative observation based on transmission electron microscopy(TEM)was consistent with results from CLSM.In addition to qualitative observation and semi-quantitative statistic,we determined the cellular locations of MoS2 NS with different thickness by quantifying amount of Mo using(inductively coupled plasma mass spectrometry)ICP-MS analysis.The quantitative statistic results indicated that more than 80%of thinner MoS2 NS were outside of cells,however about 80%of thicker MoS2 NS were internalized by cells.The quantitative analysis showed similar trend to results of CLSM and TEM.We also performed computational simulation to investigate the interaction between nanosheets with different thickness and cells at the molecular level.The results showed the thin nanosheets were found to keep adhering on the membrane,the thick nanosheets can be finally wrapped by the membrane to achieve the internalization.These experimental data and simulation clarified the effects of thickness on cell locations.Cell fate relies heavily on the cell localization of nanoparticles.We analyzed the cell localizations of MoS2 NS and WS2 NS by qualitative and quantitative methods.The results indicated that the cell localization trend of MoS2 NS and WS2 NS was consistent.The thinner nanosheets were found to keep adhering on the membrane,however the thicker nanosheets were more liable to enter cells.We also performed computational simulation to investigate the interaction between TMDCs nanosheets with different thickness and cells at the molecular level and clarified the effects of thickness on cellular locations and provided some clues for the cellular perturbations by 2D TMDCs.The investigation of cellular perturbations by 2D TMDCs is supplementary for their biological safety evaluation.We found that under dfferent cell locations,these two kinds of 2D TMDCs did not induce obvious cell death,membrane physical damage,cell apoptosis or cell cycle arrest,however cell autophagy was enhanced.To screen cell autophagy induced by these two kinds of 2D TMDCs preliminarily,an autophagy-reporting cell line LC3-GFP-U87 and 16HBE was used firstly.With the help of CLSM and TEM,we observed autophagosomes formation in LC3-GFP-U87 and 16HBE cells after treatment with MoS2 NS and WS2NS with different thickness.Immunoblot assay and autophagy flux assay against autophagy marker LC3-?showed that both MoS2 NS and WS2 NS with different thickness induced LC3-II formation in dose-dependent manner,rather than blocked the degradation of autophagosomes.To clarify the molecular mechanism of autophagy induced by MoS2 NS and WS2 NS with different thickness,we carried out related studies in protein and gene levels.As the central regulatory molecule,mTOR play important role in autophagy signaling pathway.We found that phosphorylation of mTOR was inhibited by MoS2 NS and WS2 NS in dose-dependent manner,indicating the mTOR-dependent autophagy induction.Further we analyzed the expression of 84 autophagic genes in 16HBE cells after treatment with MoS2 NS with different thickness,the analyses showed that two nanosheets chaged the expression levels of some common(APP and TMEM74)and some different(IGF-1,DAPK1,ULK1 etc.)autophagy-related genes.To make clear the relationship between mTOR-dependent autophagy and the obviously changed genes by MoS2 NS,we constructed molecular interaction network based KEGG database by Cytoscape software.The analysis results indicated IGF-1,mTOR and APP for the thinner MoS2 NS,while APP and mTOR for the thicker MoS2 NS showed higher interdependence among all the obviously changed genes,suggesting the central roles in regulating autophagy.The immunoblotting assay showed the thinner MoS2 NS downregulated APP and IGF-1 in protein level,the thicker MoS2 NS downregulated APP protein level.These results indicated that the thinner MoS2 NS might interact with cell surface proteins,APP and IGF-1 receptors,while thicker MoS2 NS may also interact with APP,thus inducing mTOR-dependent autophagy.For WS2NS with different thickness,we also analyzed the expression of 84 autophagic genes and carried out the molecular interaction network construction in 16HBE cells.We found that the thinner WS2NS might interact with CXCR-4 and IGF-1 receptor and inhibited the expression of CXCR-4 and IGF-1,thus inducing mTOR-dependent autophagy.The thicker WS2NS might bound to cell surface protein APP during the endocytosis process and triggered autophagy in mTOR-dependent manner.In this study,we systematically investigated cellular perturbations induced by 2D TMDCs and clarified the effects of thickness on the cell localization and signaling pathway perturbation.We found the significant influence of thickness of 2D nanoparticles on their cellular locations and perturbations of signaling pathway.Both these two kinds of 2D TMDCs regulated cell autophagy,which indicating the biological safety evaluation of 2D TMDCs needed high attention.In addition,the unique findings from this work point out possibly a general cell activation feature for 2D nanoparticles.