Cellular Autophagy Induced By The Interaction Of Silica Nanoparticles And Proteins

In the past few decades,nanotechnology obtained rapid development due to the unique property of nanomaterials.Nanomaterials have been extensively applied in biomedicine,food,cosmetics,industry,etc.,which raises the attention of their potential risks on the environment and the human body.Consequently,the biological safety of nanoparticles（NPs）requires comprehensively and deeply evaluation.When NPs entered the living organism,the surface of NPs will be quickly covered by protein in body fluid to form the nanoparticle-protein complex,which is termed as protein corona.Protein corona remodels the biological identity of NPs and decides the biological effect of NPs in vivo.Accordingly,the formation rules and biological effect of the protein corona can be better understood via invesigating the interaction of NPs and proteins in the body fluid.Autophagy is a self circulation process of cells with strictly conservative evolution,which is important for maintaining the cell homostasis.The abnormal autophagy level typically causes the dysfunction of cells.A variety of diseases are closely related to autophagy,such as cancer,neurodegenerative diseases and metabolic diseases.So far,as exogenous substances,NPs were reported to elevate the autophagy level of cells after entering cells,while few studies focused on how protein corona affects the cell autophagy induced by NPs.Typically,NPs would not directly interact with living cells after getting into the biological fluids owing to the interaction between NPs and biomolecules.Proteins in the body fluid were absorbed on NPs to form protein corona,causing subsequent biological effects after interacting with cells.Besides,NPs may also change the biological functions of the essential enzymes through absorbing them in the organism,thereby generating cytotoxicity.Consequently,it is imperative to proceed an in-depth investigation of the biological properties of the nanoparticle-protein complexes and autophagy effect mediated by protein corona.The study will help us to more comprehensively and deeply evaluate the application of SiO₂ NPs in biomedical field,and also enables us to better understand the effect of nanoparticles on enzyme biological activity.In this work,the rules of the interaction between silica nanoparticles（SiO₂ NPs）and different blood proteins were systemically analyzed.It was found that SiO₂ NPs possessed the highest adsorption ability to fibrinogen（Fg）.Fg could prevent the sedimentation of SiO₂ NPs,reducing the interaction between cells and NPs.In addition,Fg significantly relieve the autophagy effect of non-phagocytes induced by SiO₂ NPs.It was also found that the secondary structure and biological activity of the catalase were changed by SiO₂ NPs via protein adsorption.The main research contents are divided into the following parts.PartⅠ:This section aims to explore the rules and characteristics of the interaction between SiO₂ NPs and different blood proteins.In this chapter,we selected bovine plasma FBP,bovine serum FBS,bovine albumin（BSA）,fibrinogen（Fg）and hemoglobin（Hb）as model proteins for in vitro study.We found that the main proteins adsorbed by SiO₂ NPs after incubation with FBP contain BSA,Fg and Hb by Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）analysis.BSA and Hb are the main parts of proteins in FBS adsorbed by SiO₂ NPs.With the increasing of FBS concentration,the amount and species of proteins detected in the protein corona on the surface of SiO₂ NPs improved in turn.At the same time,the results showed that the adsorption capacity of SiO₂ NPs to the three proteins was different,and the adsorption capacity of SiO₂ NPs to BSA was significantly lower than other proteins,indicating that the more adsorption capacity proved that the affinity between them is higher,on the contrary,it indicates that there is only a relatively weak affinity between SiO₂ NPs and proteins.The following BCA protein quantitative experiment showed that the order of adsorption capacity of SiO₂ NPs to three proteins was Fg>Hb>BSA.Next,we also observed morphology and particle size of the SiO₂ NPs and different protein coated SiO₂ NPs（Protein@SiO₂ NPs）by TEM.We found that protein corona was formed after SiO₂ NPs were incubated with FBS and individual protein.Compared to pristine SiO₂NPs,the particle size of protein coated NPs increased owing to protein molecules adsorbed on the surface of NPs,and the diameter of Fg@SiO₂ increased most significantly.We further measured the hydrodynamic size and zeta potential of SiO₂NPs and protein-SiO₂ NPs complex by dynamic light scattering（DLS）.The results showed that the hydrodynamic size of BSA@SiO₂ NPs and Hb@SiO₂ increased obviously compared with pristine SiO₂ NPs group,and the absolute value of zeta potential of the of BSA@SiO₂ and Hb@SiO₂ decreased significantly,indicating that the stability of NPs suspension system was significantly reduced.Nevertheless,there was no significant change in Fg@SiO₂ NPs and FBS@SiO₂ groups.We also found that the protein corona could be formed in a relatively short period of time.Temperature and p H value of solution systems were also important factor influencing the adsorption of proteins by SiO₂ NPs.In addition,we also found that the protein corona formed by SiO₂NPs adsorbed three proteins was very firmly.Even when boiling at high temperature,only a small amount of protein would dissociate from the surface of SiO₂ NPs.Only in the presence of protein denaturant sodium dodecyl sulfate（SDS）could the protein dissociate from the surface of SiO₂ NPs.Next,we analyzed the changes of the secondary structure of each protein before and after adsorbed on SiO₂ NPs using circular dichroic spectrum（CD）.The results revealed that with the continuous increasing of SiO₂ NPs dosages,theα-helix andβ-sheet structure of BSA and Hb protein increased obviously,and random coil had not dramatically changed.Theα-helix structure of Fg protein decreased from 19.2%to 14.4%,while the other three secondary structures have changed little.These results demonstrated that BSA and Hb adsorbed to the pristine SiO₂ NPs led to loosening and unfolding of the protein skeleton,while the secondary structure of Fg was intact upon SiO₂ NPs adsorption.We also analyzed the system stability of different nanoparticle-protein complex suspensions by multiple light scattering technique.Our results showed that within 48 h,the order of stability of different protein corona systems was:FBS@SiO₂<SiO₂<Fg@SiO₂<BSA@SiO₂<Hb@SiO₂.The findings revealed that FBS and Fg protein could prevent the sedimentation of SiO₂ NPs and stabilize SiO₂ NPs suspension,yet BSA and Hb proteins could not.In summary,it was proved that the particle size of SiO₂ NPs would increase after SiO₂ NPs absorbed BSA,Fg,and Hb,and it was found that SiO₂ NPs had the highest adsorption ability for Fg.The size of Fg coated SiO₂ NPs increased significantly.The temperature and p H value of the system will affect the adsorption capacity of SiO₂ NPs to proteins.In addition,SiO₂ NPs had a greater impact on the secondary structure of BSA and Hb proteins and less impact on Fg proteins.At the same time,Fg protein could stabilize the SiO₂ NPs suspension.PartⅡ:This section aims to explore the effects of different protein corona on autophagy induced by SiO₂ NPs.In this chapter,we found that SiO₂ NPs could induce significant autophagy in HUVEC and RAW264.7 cells in a concentration dependent manner.At the same time,we also detected the toxic effect of SiO₂ NPs on the two cells.The results showed that the survival rate of HUVEC and RAW264.7 cells treated with SiO₂ NPs decreased significantly in a concentration dependent manner.We observed the accumulation of autophagosomes in cells by transmission electron microscopy（TEM）to judge the level of autophagy.The results showed that Fg protein could significantly reduce the number of autophagosomes induced by SiO₂ NPs and the uptake of SiO₂ NPs by HUVEC cells,whereas BSA protein did not.Based on the results of this experiment,it can be considered that we can regulate autophagy by using different proteins to form protein corona on the surface of SiO₂ NPs.Next,we further evaluated the effects of BSA and Fg proteins on autophagy induced by SiO₂ NPs by detecting the expression level of autophagy marker protein LC3.We selected two kinds of non phagocytic cells A549 and HUVEC,as well as two kinds of phagocytic cells RAW264.7 and J774A.1.The results showed that whether it was cancer cell A549 or non cancer cell HUVEC,protein Fg coated SiO₂ NPs obviously reduced the expression of autophagy protein LC3B-Ⅱ,while BSA protein did not.The results showed that Fg does play a key role in alleviating the non phagocytic autophagy induced by SiO₂ NPs.However,interestingly,we found that Fg and BSA did not visibly lower the autophagy level induced by SiO₂ NPs in RAW264.7 or J774A.1 cells.The results of the experiment revealed that Fg protein alleviated the autophagy level induced by SiO₂ NPs,which was closely relevant to cell type.We also found that Fg just only delayed the autophagy induced by SiO₂ NPs.Because the effect of Fg protein on alleviating autophagy induced by SiO₂ NPs would be eliminated with the prolonging of time.Next,we continued to explore whether Fg protein will enter the cell with SiO₂NPs.In order to facilitate observation,we first labeled Fg protein with red fluorescent dyes cyanine 3monosuccinimidyl ester（Fg-Cy3）,then incubated SiO₂ NPs to form fluorescent protein corona,and finally observed its interaction with cells.We found that the fluorescent protein corona in HUVEC cells gradually increased with increasing of time,indicating that the uptake of Fg-Cy3 protein coated SiO₂ NPs（Fg-Cy3@SiO₂）by cells was time-dependent.To summarize,the above experimental results showed that Fg protein could significantly alleviate the autophagy level of non phagocytes induced by SiO₂ NPs within 12 hours,but could not alleviate the autophagy effect of phagocytes.BSA protein could neither alleviate the autophagy effect of phagocytic nor the non phagocytic induced by SiO₂ NPs.PartⅢ:This section aims to explore the effect of Fg and Hb protein on the contact of NPs with cell membrane and their cellular uptake by cells.In this chapter,to better visualize the uptake processes of NPs or NPs-protein complexes,Zn Cd Se/Zn S quantum dots（QDs）was enclosed in SiO₂ NPs to obtain a fluorescent SiO₂ entity（QDs@SiO₂）.Similarly,we found that Fg protein had inhibitory effect on autophagy of nonphagocytic HUVEC induced by QDs@SiO₂.And this effect was not observed in phagocytic RAW264.7 cells.So,we could use QDs@SiO₂ instead of SiO₂ NPs to study on process of cellular uptake.The first step of cellular uptake of NPs is that NPs adhere to cell membrane through sedimentation,and then NPs would be subsequently internalized by cells via energy dependent endocytosis.We detected surface fluorescence intensity of cell membrane to assess the deposition velocity of QDs@SiO₂NPs and different protein coated QDs@SiO₂ by high content analysis system.In order to prevent NPs and protein coated NPs were internalized into cells,we only analyzed the first step that the process of NPs and protein corona adhering to cell membrane by placing cells at 4℃for 12 h.The results showed that Fg protein could be significantly reduced the sedimentation rate of NPs,while BSA protein did not.That was,Fg protein decreased the settling velocity of NPs by increasing the stability of NPs in suspension,and then reduced the adhesion between NPs and cell membrane.Next,we analyzed the second process of the cellular uptake of NPs and protein corona by confocal and flow cytometry.In order to eliminate the above differences in the sedimentation rates of NPs and NPs-protein complexes,we exposed the cells to minimal medium of NPs or NPs protein complex suspension respectively,made them adhere to the cell membrane immediately.We found that Fg protein could significantly reduce the cellular uptake of NPs in HUVEC and RAW264.7,while BSA protein did not.Similarly,the results of ICP-OES also proved that Fg protein could evidently decrease the uptake of SiO₂ NPs by HUVEC and RAW264.7,and BSA protein had little effect.The comparable results were proved by live cell imaging system.Furthermore,we also analyzed the endocytosis pathway of QDs@SiO₂ NPs and Fg@QDs@SiO₂ by cells.To explore whether Fg protein would change the main endocytosis pathway of NPs.The results showed that the internalization pathways of the NPs and Fg protein coated NPs were both largely through caveolin mediated endocytosis,which indicated that Fg protein had no effect on the main endocytic pathway of NPs by HUVEC and RAW264.7 cells.To conclude,the work of this chapter revealed that Fg protein could be significantly reduced the sedimentation velocity of NPs,which was almost not affected by BSA protein.Meanwhile,Fg protein could obviously decrease the uptake of SiO₂NPs by HUVEC and RAW264.7,and BSA protein had little effect.In addition,Fg protein did not change the main endocytic pathway of QDs@SiO₂ NPs by cells.PartⅣ:The purpose of this section is to investigate the interaction between NPs and catalase（CAT）and superoxide dismutase（SOD）,and the effect of NPs on the structure and activity of the antioxidant enzymes.In this chapter,we chose SiO₂ and Ti O₂ NPs for research,firstly,we compared the adsorption capacity of NPs to CAT and SOD.The results showed that the adsorption capacity of SiO₂ NPs to CAT is significantly greater than SOD.The similar results had been shown in Ti O₂ NPs.At the same time,we also found that the adsorption capacity of SiO₂ NPs-NH₂ to CAT was obviously lower than that of unmodified SiO₂ NPs.So,it was suggested that different type of modification on SiO₂ NPs would affect the adsorption capacity of SiO₂NPs to enzymes.Moreover,the adsorption of CAT by SiO₂ NPs was almost independent of temperature.In addition,we also found that the adsorption capacity of SiO₂ NPs was significantly higher than that of Ti O₂ NPs for the adsorption of CAT and SOD.Therefore,we selected SiO₂ NPs to do the following experiments.Subsequently,we continued to explore whether the secondary structure of two antioxidant enzymes would be altered after adsorbed by SiO₂ NPs.The results demonstrated the secondary structure of CAT enzyme changed significantly,while the secondary structure of SOD only changed slightly after adsorbed on the surface of SiO₂ NPs.We further assessed the impact of SiO₂ NPs on the biological activity of the two antioxidant enzymes.The results showed that the adsorption of SiO₂ NPs could lead to the change of CAT enzyme activity,but it hardly affected the activity of SOD.Meanwhile,we found that the adsorption amount of SiO₂ NPs was linear with CAT enzyme activity.The more CAT adsorbed by SiO₂ NPs,the more enzyme activity in the system would decrease.Finally,we investigated that how many molecules of CAT enzyme could bind by a molecule SiO₂ NPs.The binding constants and binding sites of SiO₂NPs and CAT were obtained by fluorescence quenching method.The binding constant Ka was 3.0605 M^-1 and the binding constant n was 0.6257.The results indicated that one molecule of SiO₂ NPs bound 2 molecules of CAT enzyme.In conclusion,the research of this chapter demonstrated that SiO₂ and Ti O₂ NPs could adsorb the antioxidant enzyme CAT and SOD to form NPs-protein complexes,and the adsorption capacity of SiO₂ NPs to CAT and SOD was significantly higher than that of Ti O₂ NPs.Simultaneously,the adsorption capacity of SiO₂ NPs to CAT was significantly greater than SOD.Furthermore,it was found that SiO₂ NPs could change the secondary structure and enzyme activity of CAT,but had little effect on SOD.