Effects Of Titanium Dioxide Nanoparticles On Biological Behaviors Of Lung Epithelial Cells Under Simulated Microgravity

Background and ObjectiveIn recent years,all countries are actively promoting the development of nanotechnology in the field of space with a focus on the research of novel exploitable nanomaterials.However,with the gradual penetration of nanomaterials into all aspects of the space field,the opportunities for astronauts to be exposed to nanomaterials in a confined environment are increasing.Therefore,nanomaterials in the space environment pose a potential health threat to astronauts that cannot be ignored.It is known that the microgravity environment in space has a significant impact on astronauts’physiological functions.Titanium dioxide nanoparticles（TiO₂ NPs）（diameter～20 nm）,which have a wide application prospect in aerospace engineering,can enter the lungs through the respiratory system and deposit in the alveoli in large quantities,which cannot be metabolized and discharged,thus causing harm to astronauts.Therefore,it is necessary to systematically explore the potential effects of astronaut exposure to nanomaterials under microgravity.To more effectively evaluate the potential effects mentioned above,we constructed a cell culture system using a random positioning machine（RPM）to simulate the microgravity environment on the ground and explore the effects of TiO₂ NPs on the biological behavior of lung epithelial cells A549 under simulated microgravity.It provides a preliminary theoretical basis for the establishment of safety evaluation standards for the respiratory system by TiO₂ NPs in microgravity and also provides a preliminary clue for the application and protection of nanomaterials in aerospace.It has a certain reference value for the potential exposure risk assessment of space flight.Methods1.RPM was used to simulate the microgravity environment,and a set of cell culture equipment was constructed based on this.The cell culture device was optimized by making a cell culture dish fixator and combining a microporous filter membrane,sealing ring,air permeability hole,gasket,and other modules.The rotary speed was set at 6 rpm and the mode was random,which can better simulate the microgravity effect.2.For the physical and chemical characterization of TiO₂ NPs,a transmission electron microscope（TEM）was used to analyze their morphology and size,and energy-dispersive X-ray spectrometer（EDX）was used to analyze their element composition,and a dynamic light scattering method（DLS）was used to determine their average hydration particle size（HD）,dispersion coefficient（PDI）and Zeta potential.The crystal structure was determined by X-ray powder diffraction（XRD）and the surface chemical structure was determined by Fourier transform infrared spectroscopy（FT-IR）.3.For the study of cell-related biological behavior,the cell proliferation/viability,cell cycle,and apoptosis were detected by trypan blue staining/CCK-8,flow cytometry PI staining,and Annexin V-FITC/PI double staining,respectively.The cytoskeleton F-actin of A549 cells was observed by staining with TRITC-Phalloidin.The cell migration was tested by wound-healing assay,The cell uptake of TiO₂ NPs was analyzed by using a fluorescence microscope and an inductively coupled plasma-optical emission spectrometer（ICP-OES）.Cell adhesion-related proteins(vinculin,E-cadherin,integrinβ1,FAK,and phosphorylation of FAK at Tyr397（p-FAK（Tyr397））were detected by immunofluorescence and western blot（WB）.ResultsPartⅠ.Construction of cell culture system under simulated microgravityThe cell culture device could meet the requirements of good gas exchange,no leakage,and low cell shear force in the flow state when rotating.In addition,the device has the advantages of good repeatability,safety,and reliability,economic and practical.The acceleration of gravity in normal gravity is 1 g,while the acceleration of gravity in microgravity simulated by this device can reach 10^-3g.PartⅡ.Effects of simulated microgravity on cell-related biological behaviors1.Effects of simulated microgravity on cell proliferation,cell cycle,and apoptosisThe results showed that there was no significant difference in the proliferation of A549cells under simulated microgravity and normal gravity for 24,48,and 72 h,respectively.However,the simulated microgravity significantly inhibited the proliferation of HUVEC cells.In addition,it was found that simulated microgravity had no effect on the cell cycle and did not induce apoptosis of A549 cells.2.Effects of simulated microgravity on cytoskeleton F-actin,cell migration,and adhesion-related molecules in A549 cellsThe results showed that the cytoskeleton F-actin of A549 cells could shorten and bend under simulated microgravity for 48 h.The wound-healing assay showed that the migration of A549 cells was significantly inhibited under simulated microgravity for 48 h.The protein expression of cell adhesion-related molecules was further detected,and the results showed that simulated microgravity had no significant effect on the distribution and expression of vinculin,E-cadherin and integrinβ1 in A549 cells.PartⅢ.Effects of titanium dioxide nanoparticles on the biological behavior of lung epithelial cells A549 under simulated microgravity1.Characterization of physical and chemical properties of titanium dioxide nanoparticlesThe TEM showed that TiO₂ NPs were spherical with a particle size of 20.49±5.15 nm.HD was 122.11±10.33 nm,PDI was 0.15±0.087,and Zeta potential was-21.97±2.57 m V.The crystal structure of TiO₂ NPs is anatase type.The FT-IR showed that the water adsorbed on the surface of TiO₂ NPs is easily polarized to form hydroxyl groups.2.Effects of titanium dioxide nanoparticles on cell viability,apoptosis,cell cycle,and migration under simulated microgravity,and the effect of simulated microgravity on cell uptake of nano titanium dioxideA549 cells were exposed to different concentrations of TiO₂ NPs（0,25,and 100μg/mL）under normal gravity and simulated microgravity,respectively,to explore the changes in cell-related biological behaviors.The results showed that 25μg/mL TiO₂ NPs had no significant effect on cell viability,apoptosis,and cell cycle of A549 cells under simulated microgravity.However,exposure to 100μg/mL TiO₂ NPs resulted in a slight decrease in cell viability,a slight increase in apoptosis rate,and a slight effect on the cell cycle.In addition,simulated microgravity significantly reduced A549 cell migration,and that concurrent exposure to TiO₂ NPs further inhibited cell migration.By microscope observation and ICP-OES experiments,we found that cell uptake of TiO₂ NPs in the simulated microgravity group was slightly lower than that in the normal gravity group when exposed to 25μg/mL TiO₂ NPs.However,when the concentration of TiO₂ NPs was 100μg/mL,the cell uptake of TiO₂ NPs in the simulated microgravity group was much lower than that in the normal gravity group.3.Effects of titanium dioxide nanoparticles on cytoskeleton F-actin and adhesion-related molecules under simulated microgravityThe results showed that the F-actin microfilaments were shortened and the stress fibers decreased under simulated microgravity,while the cells showed a well-organized F-actin network under normal gravity.When cells were exposed to TiO₂ NPs,actin filaments were significantly disturbed.The higher concentration of TiO₂ NPs exposure,the more damage to the F-actin network.Both immunofluorescence and WB results showed no significant change in the distribution and expression of the adhesion molecules（vinculin,E-cadherin,and integrinβ1）in A549 cells with or without exposure to TiO₂ NPs under normal gravity or simulated microgravity.In addition,the expression of focal adhesion kinase（FAK）in A549cells was significantly decreased when exposed to 100μg/mL TiO₂ NPs,especially under normal gravity.P-FAK（Tyr397）was decreased in A549 cells under simulated microgravity,and p-FAK（Tyr397）expression was also decreased when TiO₂ NPs were exposed.When exposed to 100μg/mL TiO₂ NPs,the expression levels of FAK and p-FAK proteins in A549cells were lower,and the normal gravity group decreased more,which was consistent with the cell migration experiment results.Conclusion1.Simulated microgravity did not significantly affect the proliferation,cycle,and apoptosis of A549 cells,but inhibited cell migration,which was related to the disturbance of the cytoskeleton F-actin network.2.A549 cells internalized less TiO₂ NPs under simulated microgravity than under normal gravity,which may be one of the reasons for lower cytotoxicity and less inhibition of cell migration induced by simulated microgravity under the same exposure concentration of TiO₂ NPs.3.The disruption of the cytoskeletal F-actin network and decreased phosphorylation of FAK at Tyr397 may explain the reduced migration of A549 cells exposed to or absence of TiO₂ NPs under simulated microgravity from a biological mechanism perspective.