Analysis Of Protein Corona Formation And Aging On The Biological Effects Of Polystyrene Nanoplastics

Micro?nano?plastics have posed potential threat to the ecological environment and human life and health for their special properties such as small particle size,high surface activity and difficulty in degradation,and have been highly regarded as a new type of environmental pollutant by researchers.Numerous studies have reported the accumulation and toxic effects of micro?nano?plastics in aquatic organisms.In addition,engineered nanomaterials?ENMs?have been shown to quickly absorb large molecules such as proteins in body fluids to form"protein corona"after entering the biological environment,which not only changes the surface properties of ENMs,but also further affects their biological effects.However,the effect of plastic debris mediated protein corona is still unclear,especially from the perspective of nano-characteristics of nanoplastics,so it is urgent to carry out relevant work.Plastics exposed to the environment will gradually age under the influence of external factors such as ultraviolet exposure,weathering,mechanical stress and oxygen in the air.And aging will significantly change the physical and chemical properties of plastics,making them exhibit different surface properties from those of the original plastics.Therefore,when studying the environmental behavior and biological effects of micro?nano?plastics,the related effects of aging should not be ignored.Part?:Polystyrene is one of the most widely used commercial plastics in production and daily life.Therefore,in this chapter,polystyrene nanoplastics?PS NPs?was selected as the nanoplastic model,and its interaction with fetal bovine serum?FBS?protein and subsequent toxic effect on mouse mononuclear macrophage leukemia cells?RAW264.7?were analyzed.SDS-PAGE and LC-MS/MS results showed that after incubation with FBS,PS NPs could adsorb different molecular weights and different types of proteins in FBS to form protein coronas,and their interaction was a dynamic process related to concentration and time.TEM result also observed that FBS proteins were coated on the surface of PS NPs,forming a protein corona with a thickness of 46nm.Next,the effects of the corona formation on the cytotoxicity of PS NPs were analyzed.CCK-8 analysis of cell viability showed that exposure of PS NPs caused a concentration-dependent decrease in RAW264.7 cell viability.But compared with bare PS NPs,the exposure of PS@FBS complexes at the same concentration resulted in an increase in cell survival rate from 72.7%to 88.2%,a decrease in the percentage of PI-positive cells from 26.4%to 12.4%and also a relative decrease in LDH release.These results suggested that coating of FBS protein significantly reduced the cytotoxicity of PS NPs.Then the expression levels of autophagy-related proteins in cells upon PS NPs exposure were detected by WB experiments,and the result showed that the expression of LC3B-?and P62 proteins were up-regulated in a time and concentration-dependent manner.For further confirmation,GFP-LC3 plasmid was transfected into RAW264.7 cells to mark autophagosomes,and it was indeed found that remarkable of GFP-LC3 positive punctuate were observed in cells of PS NPs treatment,we also confirmed that this effect is time-dependent.These results demonstrated that the accumulation of intracellular autophagosomes was induced by the exposure of PS NPs.While compared with the bare PS NPs,PS@FBS complexes reduced LC3B-II and P62expression levels,the number of GFP-LC3 positive punctuate and the net autophagic flux level in RAW264.7 cells,indicating that coating of FBS alleviated the autophagic flux blocking and the accumulation of autophagosomes caused by PS NPs.Then,the intracellular localization of the material was evaluated,and the results showed that both of PS NPs and PS@FBS complexes were co-located with lysosomes.However,lysosomes were co-located with bare PS NPs more common than PS@FBS complexes.The structure and function of lysosomes were further tested,and it was found that the exposure of PS NPs resulted in increased permeability of lysosomal membrane,leakage of cathepsin B,increased lysosomal pH and decreased acid phosphatase activity,while coating of FBS reduced the lysosomal dysfunction caused by PS NPs.In addition,the role of oxidative stress in the cytotoxicity induced by PS NPs and PS@FBS complexes was evaluated by the detection of oxidative stress-related indicators in cells.Similarly,compared with bare PS NPs,FBS coatings reduced intracellular ROS level,G-PX activity and GSH consumption.However,the combination of antioxidant VE resulted in a partial decrease in the percentage of AO^low cells and PI-positive cells caused by PS NPs and PS@FBS complexes.This proved that oxidative stress was partly involved in the cell damage caused by PS NPs and PS@FBS complexes.The internalization of materials was detected by flow cytometry and confocal microscopy,and the results showed that the cellular uptake of PS@FBS complexes was less than that of PS NPs.In summary,the experiments in this chapter demonstrated that PS NPs could adsorb different molecular weights and different types of proteins in FBS to form protein coronas.Moreover,the formation of corona reduced the cytotoxicity,blocking of autophagic flux,damage of lysosomal function as well as oxidative stress level caused by PS NPs,and the reduction of such cytotoxicity and damage might be related to its reduced cellular uptake.Part?:This section aims to explore the evolution of the protein corona with the uptake and the intracellular transport of PS NPs in RAW264.7 cells.In this chapter,bovine serum albumin?BSA?with high abundance in FBS and high adsorption on the surface of PS NPs was selected for subsequent experiments.First,PS NPs and BSA were incubated in vitro,and the adsorption of BSA on the surface of PS NPs was verified by SDS-PAGE and silver staining experiments.Then the internalization of materials was detected by flow cytometry and confocal microscopy and the results showed that both of PS NPs and PS@BSA complexes could be internalized by RAW264.7 cells and without significant difference in uptake capacity.The fate of the protein corona in the cell was further observed by living cell workstation and confocal microscope.According to the result,when the PS@BSA complexes was endocytosis and transported in RAW264.7 cells,the BSA protein remained at least partially on the surface of PS NPs,but gradually degraded over time.The localization of PS@BSA complexes in cells was observed by confocal microscopy and it was found that the BSA protein corona entered the lysosome with PS NPs and was completely degraded.In order to investigate the influence of the formation of the BSA protein corona and its degradation in lysosomes on the cytotoxicity of PS NPs,we tested relevant indicators.The results of CCK-8 assay on cell viability confirmed that with the degradation of the BSA protein corona,the cytotoxicity of PS@BSA complexes gradually recovered,resulting in the decrease of cell viability,and the difference than that of PS NPs group gradually reduced.Besides,PI experiment result found that bare PS NPs rapidly induced cell membrane damage in RAW264.7 cells,which caused the percentage of PI-positive cells to increase from 2.2%in the control group to 8.7%after2 h.However,no such membrane damage was observed within 10 h after exposure of PS@BSA complexes,indicating that the BSA corona had a protective effect.However,at 12 h,treatment of PS@BSA complexes led to an increase in the percentage of PI positive cells to 7.4%,indicating the recovery of their membrane damage ability.In summary,the experiments in this chapter demonstrated that when the PS@BSA complexes was endocytosis and transported in RAW264.7 cells,the BSA protein remained at least partially on the surface of PS NPs and alleviated the cell membrane damage of PS NPs during early exposure.However,with the degradation of the protein corona in lysosomes,its toxicity gradually recovered.Part?:This section aims to explore the effects of aging on the biological effects and the protein adsorption behavior of PS NPs.We artificially accelerated the aging process of PS NPs by ultraviolet irradiation to prepared Aged PS NPs and then characterized.FTIR spectrum analysis results showed that,compared with PS NPs,the enhanced-OH and C=O characteristic peaks were observed in the FTIR spectrum of Aged PS NPs.This proved that surface oxidation of PS NPs occured after ultraviolet irradiation.Moreover,the hydrodynamic size and Zeta potential of Aged PS NPs were reduced than that of PS NPs.According to the above studies,we proved that aging significantly changed the physical and chemical properties of PS NPs.In order to investigate the effect of aging on cell damage caused by PS NPs,PS NPs and Aged PS NPs were exposed to RAW264.7 cells and relevant indicators were tested.WB detection of autophagy-related proteins revealed that compared with the original PS NPs,treatment of Aged PS NPs reduced the expression of LC3B-II and P62 proteins,indicating reduced accumulation of autophagosomes in RAW264.7 cells.In addition,Aged PS NPs reduced the percentage of AO^low cells from 63.5%to 16.6%compared with original PS NPs,which demonstrated that the lysosomal damage of the aged PS NPs was reduced than that of original PS NPs.Similarly,PI staining experiments also showed that compared with original PS NPs,Aged PS NPs decreased the percentage of PI-positive cells?from 26.5%to 11.3%?,indicating the decreased cell membrane damage.We further explored the effect of aging on the adsorption characteristics of PS NPs on FBS proteins.SDS-PAGE and silver staining experiments found that both of PS NPs and Aged PS NPs were able to adsorb proteins of different molecular weight in FBS to form protein coronas and there were no differences in protein molecular weight.After changing the action time and concentration of FBS,the interaction between Aged PS NPs and FBS was further observed.It was found that there were some defference between PS NPs and Aged PS NPs in the adsorption characteristics.With the increase of FBS concentration and the extension of the action time,the protein contents of different molecular weights adsorbed on the Aged PS NPs surface gradually increased.This proved that aging changes the adsorption characteristics of PS NPs to specific molecular weight?40-80 kD?proteins in FBS.In summary,the experiments in this chapter demonstrated that aging changed the physical and chemical properties of PS NPs and further reduced its damage to RAW264.7 cells.In addition,aging also changed the adsorption characteristics of PS NPs on FBS proteins.