The Interaction Of Nanomaterials With Different Hardness With Blood Proteins And Their Effect On Endocytosis Of Nanoparticlese

The application of nanomaterials in organisms has attracted more and more attention from scientists.For example,nanomaterials were expected to be used as multifunctional diagnostic and therapeutic agents.However,according to the survey of the literature in the past ten years,only 0.7%of the dose given to nanoparticles has been deliver to malignant tissues.The reason for this situation was that nanomaterials could be quickly adsorbed by proteins after entering biological fluids.The mononuclear phagocytic system(MPS)treated nanomaterials as foreign bodies.Nanoparticles would be isolated,degraded and eliminated by MPS,resulting in nanomaterial delivery.Reduced efficiency and increased liver accumulation affected the pharmacokinetics,biological applications and clinical trials of nanomaterials.When nanomaterials entered a biological system,the bio-nano interaction between the protein in the biological system and the nanomaterial was the first step in the interaction between the nanomaterial and the biological system.Therefore,it was of great significance to study the influence of protein corona on nanomaterials in biological applications,especially in the process of entering cells.In response to the above problems,this project designed two kinds of nanoparticles with different hardness and hardness on the surface.Through in vitro simulation,when the nanoparticles entered the blood,they quickly adsorbed plasma proteins to form a protein crown.Subsequently,through proteomics analysis,the difference in the quantity and type of protein in the protein crown adsorbed by the two nanomaterials with different surface softness and hardness was understood.And through the extraction of cell membrane proteins,the effect of protein corona on the membrane proteins of nanomaterials in the process of penetrating the membrane was studied.Furthermore,through the changes of membrane proteins,we explored the influence of protein corona on the endocytosis pathway.The specific content is as follows:1.The formulation study of nanomaterials with different surface softness and hardness.In this study,gold nanoparticles(AuNPs)were synthesized by the Frens-Turkevich method,liposomes(LipoICG)were synthesized by phacoemulsification,and AuNPs and LipoICG were simulated in vitro to adsorb mouse plasma proteins to form AuNPs@PC and LipoICG@PC.The particle size and zeta potential of the above four materials were characterized,and the results showed that the average particle size of the nanomaterials increased by about 50-80nm after the protein crown was wrapped.As the synthesized AuNPs and LipoICG were both negatively charged materials,they were passed Electrostatic adsorption could form a thicker protein crown on the surface of the material,resulting in a decrease in absolute charge.A better simulation effect was achieved by optimizing the concentration of plasma protein.ICG was wrapped in liposomes,and the method of fluorescence quantification was used to determine the concentration of liposomes and used for quantification in subsequent experiments.It was observed by transmission electron microscope(TEM)that AuNPs were uniformly spherical and easier to agglomerate,while AuNPs@PC was more uniform because it wraped the protein crown;LipoICG was a shell-like structure with solid outside and virtual inside,and it was easier to form after covering the protein crown group.Through the CCK-8 experiment,the AuNPs,LipoICG,AuNPs@PC and LipoICG@PC were incubated with macrophages for 12 hours,and the effect on the activity of macrophages was detected.The results showed that the safety of the material was higher after the protein corona was wrapped.,The damage to cells was less.Lactate dehydrogenase(LDH)test results showed that under certain conditions,AuNPs,LipoICG,AuNPs@PC and LipoICG@PC had less damage to cell membranes and higher cell membrane integrity.2.Proteomics analysis of nanomaterials with different softness and hardness of coating protein corona and cell membrane proteins.The proteomics analysis of the number and types of proteins in the surface protein corona of AuNPs@PC and LipoICG@PC showed that the softer nanomaterial LipoICG was easier to adsorb complement-related proteins and coagulation-related proteins.The proteins related to membrane protein transport were 14-3-3 protein and cell membrane marker protein Na+-K+-ATPase.The endocytosis of AuNPs and AuNPs@PC by macrophages was determined by ICP-MS and biological transmission electron microscopy.The experimental results showed,the phagocytosis of AuNPs and AuNPs@PC increased,and the presence of protein corona inhibited the endocytosis of gold nanoparticles by macrophages.Flow cytometry was used to determine the endocytosis of LipoICG and LipoICG@PC by macrophages.Similar to the results of gold nanoparticles,the presence of protein corona inhibited the endocytosis of liposomes by macrophages.In response to the above phenomenon,after incubating the material with the cells,we extracted membrane proteins and performed proteomic analysis of the cell membrane proteins to explore the influence of the protein corona or the endocytic pathway.According to the results of proteomics,the proteins AP-2 and clathrin related to endocytosis were selected.3.The effect of protein crown on the endocytic pathway of nanomaterials with different surface softness and hardness.According to the selected proteins AP-2,clathrin and Na~+-K~+-ATPase,through Western-Blotting and immunofluorescence detection,the results showed that as the incubation time increases,the more material the macrophages consume,the more material was needed.There were more GPCRs and clathrins,and the expression of?-arrestin and AP-2 also increased,and there was no significant change in Dynamin-2.On the contrary,the presence of protein corona leaded to a decrease in the amount of phagocytosis of the material by macrophages,and the protein expression of GPCR,clathrin,?-arrestin and AP-2decreases accordingly.The presence or absence of protein crown had no significant effect on the protein expression of Dynamin-2.In summary,the endocytosis pathway of materials could be introduced:the first step was to identify the GPCR receptor on the cell surface,which leads to phosphorylation of?-arrestin and activates the GPCR receptor to exert its effect;The second step is to stimulate related proteins in the body,recruit a large amount of clathrin to the cell membrane,and combine with AP-2 to form invaginated grooves and gradually form vesicles;in the third step,Dynamin-2 acts as a“scissor”to remove the formed depression from the cell membrane After cutting it down,vesicles were completely formed and entered the cells to exert their effects.