The Intracellular Fate Of Plasma Proteins Adsorbed By Fe₃O₄ Nanoparticles

With the rapid development of nanotechnology in recent decades,nanomaterials have been widely used in people’s daily work and life,including food additives,cosmetics,coating products,medical imaging,targeted drug delivery,and disease diagnosis.At the same time,products containing nanomaterials will enter the organism in various ways,seriously threatening the ecological environment and human health.When nanoparticles（NPs）entered into the biological environment,NPs could absorb proteins rapidly in biological fluids to form nanoparticle-protein complexes,which named"protein corona".The formation of protein corona could alter the properties of NPs,thereby affecting their biological properties.The formation of the protein corona is dynamic,which can be internalized and transported within the cell with the nanoparticles.The protein corona around nanoparticles evolves with changes in the biological environment during internalization.Magnetic nanoparticles,as a new type of biomedical material,have been widely used in magnetic resonance imaging,drug targeting,gene therapy,and bioseparation technologies.Most of these biological applications require an in-depth understanding of the interactions between magnetic nanoparticles,biological fluids,and cells.Currently,there is limited research on the protein corona formed by magnetic nanoparticles in the biological environment and their effects on the biological effects of living cells.Therefore,in this study,the composition of the protein corona formed by magnetic nanoparticles in the blood circulation was analyzed,and an in-depth analysis of the internalization of magnetic nanoparticles in the blood circulation and the evolution of protein corona,and the composition of the protein corona and its influence on the biological effects of NPs were explored.These insights into the properties of intracellular protein corona will aid the development of nanoparticles for specific therapeutic applications,and evaluating the biological effects of magnetic nanoparticles in biological fluids has important implications for biomedicine.PartⅠ:The composition of the protein corona formed around magnetic nanoparticles in the blood circulation and the effect on the biological effect were analyzed.Therefore,in this chapter,Fe₃O₄ magnetic nanoparticles（Fe₃O₄ NPs）was selected as the NPs model.The mouse plasma was selected to simulate the biological environment during blood circulation.The interaction of Fe₃O₄ NPs with plasma protein and related biological effects were analyzed.Firstly,The results of SDS-PAGE and LC-MS/MS showed that Fe₃O₄ NPs could adsorb different molecular weights and different types of proteins in plasma after incubation with plasma to form protein corona.The most abundantly adsorbed proteins were Fibrinogen.The results of GO enrichment indicated that the plasma proteins adsorbed on Fe₃O₄ NPs were mainly involved in biological processes including regulation of wound response,protein activation cascade,regulation of blood coagulation and regulation of wound healing.PPI further analyzes the relationship between biological processes and related proteins.Further explore the relationship between nanoparticles-protein corona and cell interaction.The uptake of Fe₃O₄ NPs by RAW264.7 cells is the primary factor determining their cellular biological effects.Therefore,we first analyzed the uptake of:Fe₃O₄ NPs by RAW264.7 cells,the cells were observed in real time,the results showed that Fe₃O₄NPs were gradually taken up into RAW264.7 cells when exposed to RAW264.7 cells for 2 h.The further study found that the side scatter angle of RAW264.7 cells was increased in a concentration dependent manner after Fe₃O₄ NPs exposure.Notably,compared with the bare Fe₃O₄ NPs,Fe₃O₄NPs@10%MP complex treated cells significantly reduced the side scatter angle and total iron content of RAW264.7 cells.These results indicated that Fe₃O₄ NPs could be taken by RAW264.7 cells,and plasma protein adsorption could significantly reduce the uptake of Fe₃O₄ NPs by RAW264.7 cells.Next,the biological effect of the corona formation around NPs were analyzed.The results of CCK-8 showed that Fe₃O₄ NPs reduced the viability of RAW264.7 cells in a concentration-and time-dependent manner.Compared with bare Fe₃O₄ NPs,the cell viability was higher after treatment with the same concentration of Fe₃O₄ NPs@10%MP.These results indicated that the formation of plasma protein corona significantly reduced the cytotoxicity of Fe₃O₄ NPs.Western blot and RT-q PCR were used to detect the expression levels of inflammation-related proteins and nucleic acids in cells after the interaction of Fe₃O₄ NPs with RAW264.7.The experimental results indicated that the exposure of Fe₃O₄ NPs could induce the secretion of inflammatory factors and the formation of plasma protein corona in RAW264.7 cells.It can significantly reduce the inflammatory response of RAW264.7 cells induced by Fe₃O₄ NPs.Above all,the results showed that Fe₃O₄ NPs in the blood circulation adsorbed a large amount of protein to form the plasma protein corona,and the main component of the plasma protein corona was fibrinogen.Meanwhile,the formation of protein corona reduces the uptake of Fe₃O₄ NPs by RAW264.7 cells and alleviates the cytotoxic and inflammatory responses induced by Fe₃O₄ NPs.PartⅡ:This part aims to investigate the composition of the adsorbed protein corona after Fe₃O₄ NPs in the blood circulation were internalized by RAW264.7 cells and related biological processes.Firstly,the proteins components adsorbed on Fe₃O₄ NPs in different cellular protein environments were explored,and it was found that Fe₃O₄ NPs could adsorb a large number of intracellular proteins in both RAW264.7 cell lysates and living cells,but there are obvious differences in the type,quantity and biological function of proteins.Next,we further investigated the changes in plasma protein corona after being internalized by RAW264.7 cells.By interacting Fe₃O₄ NPs@10%MP with RAW264.7cell lysate and live cells,respectively,the total concentration of proteins adsorbed by Fe₃O₄ NPs detected by BCA remained consistent among different groups.The results of LC-MS/MS showed that after the interaction of Fe₃O₄ NPs@10%MP with cell lysate,the relative abundance of plasma proteins decreased sharply,and the most significantly decreased protein was Fg,and emerged new cellular proteins.In addition,the protein corona isolated after Fe₃O₄ NPs@10%MP was internalized by cells,a considerable part of the proteins around Fe₃O₄ NPs were intracellular proteins,and Fg,which was the most abundant in the original plasma corona,was also absent in the cellular corona.The results showed that the composition of the surface protein corona of Fe₃O₄ NPs changed with the change of biological environment.Notably,Fg protein was not detected in the living cell protein environment,but remained in cell lysates,suggesting that Fg protein can dynamically exchange with intracellular proteins but cannot enter cytogenesis in the living cell state to replace accordingly.Different from the protein environment of cell lysate,the changes of the protein environment in living cells require NPs to interact with the cell membrane and be internalized and then contact and interact with intracellular proteins through specific pathways in the cell.This suggests that the cell membrane has specific selectivity for the components of the NPs protein corona.Next,we selected Fg protein to specifically explore the interaction between Fe₃O₄NPs@Fg and RAW264.7 cells.The side scatter angle results of cells indicated that the adsorption of Fg could significantly reduce the uptake of Fe₃O₄ NPs by RAW264.7 cells.Further analysis by confocal microscopy showed that Fg-F green fluorescence only appeared on the cell membrane,but there was no green fluorescence in the cells.Besides,the continuous shooting of the live cell imaging system also observed that Fe₃O₄ NPs entered cells while Fg-F stayed on the cell membrane with time.Next,we further investigated the reason that Fg protein was"left behind"by the cell membrane.After inhibiting of MAC-1 expression on RAW264.7 membranes by MAC-1 siRNA,the Fg-F protein in the cell was observed by confocal microscopy.These results indicate that due to the existence of the MAC-1 receptors on the cell membrane,which can bind to Fg,thereby"intercepting"the Fg from entering the cell for further dynamic changes.BSA was another abundant protein adsorbed by Fe₃O₄ NPs,the existence of BSA-F green fluorescent was observed in the cells after Fe₃O₄ NPs@BSA-F interacted with RAW264.7cells.But the absence of BSA in the intracellular protein corona around Fe₃O₄ NPs suggests that part of the protein taken up into the cell was replaced by a higher-affinity intracellular protein.These results above suggested that not all plasma proteins along with Fe₃O₄ NPs could be internalized by cells.The above results indicate that Fe₃O₄ NPs in the blood circulation were internalized by cells,the composition of the surface protein corona changes dynamically with the change of biological environment,but not all plasma proteins can be internalized by cells with Fe₃O₄ NPs.The cell membrane surface receptor MAC-1 will specifically select internalized proteins,among which the Fg protein interacts with MAC-1 and is"Intercepted"outside the cell,and other plasma proteins are internalized into the cell and will be intracellular with higher affinity.The proteins are exchanged down to form new intracellular protein corona,which play different molecular function and biological process.