Study On Protein Corona Formed By Amphiphilic Polymer Coated Gold Nanoparticles And Plasma Proteins

With the rise of nanotechnology,nanomaterials have been widely used in various fields,especially in biomedicine.It is well known that protein can bind to the surface of nanomaterials to form protein corona,which is an unavoidable phenomenon.After years of research,people have a preliminary understanding of protein corona.In fact,protein corona is the result of immune response when nanomaterials substance into the body as an exogenous.Protein is a biological macromolecule composed of amino acids,the arrangement of amino acid and folding of the peptide chain give protein a space structure and functions.Protein's conformation changes may lead to functional loses;while nanomaterials' surface properties and functions can be great different due to the discrepancy on material,size,shape,charge,surface modification and so on.So the interaction between different nanomaterials and proteins can be influenced by a variety of factors.Some studies have shown that protein adsorbed on the surface of nanomaterials undergoes a conformational change.Proteins can be affected by nanomaterials while it can also affect nanomaterials.Many studies have shown that protein corona changes the biological properties,biodistribution,cytotoxicity of nanomaterials in body.That may alter the function of nanomaterials in body,so protein corona becomes a major research hotspot in biomedical applications of nanomaterials.Under normal circumstances,the first entry of nanomaterials into the body is the blood circulation system,a various of proteins in the plasma can form protein corona with nanomaterials.Therefore,most of the researches are mainly focused on the interaction between plasma protein and nanomaterials.In this paper,protein corona formed by proteins of high abundance in plasma(bovine serum albumin,fibrinogen,and transferrin)and amphiphilic polymer coated gold nanoparticles were studied by optical technique(absorption spectroscopy,fluorescence spectroscopy,dynamic light scattering),centrifugation,size exclusion chromatography,mass spectrometry and gel electrophoresis.The mainly works carried out are as follows:1.The study of discrete protein corona formed by bovine serum albumin and amphiphilic polymer coated gold nanoparticles.The different protein-nanoparticles complex(adsorbed with one,two or several bovine serum albumin)can be isolated by gel electrophoresis method.The exact protein number in protein corona is difficult to calculate,generally only the relative content of protein can be got.Combining the method of absorption spectroscopy and centrifugation,we calculated the number of adsorbed proteins on the surface of nanoparticles.The results show that the number of proteins adsorbed by amphiphilic polymer coated gold nanoparticles is limited,which is related to the surface properties and size of nanoparticles.We also use this method to calculate the number of protein(chymotrypsin)on nanoparticles which cannot form discrete protein corona,and deduce an equation suitable for calculating the number of proteins in protein corona that formed on amphiphilic polymer coated nanomaterials.This method can lay the foundation for controlling protein number on the nanomaterials in the future.2.Determine the size of the protein corona formed by bovine serum albumin and amphiphilic polymer coated gold nanoparticles.The principles of gel electrophoresis,dynamic light scattering,size exclusion chromatography are all related to the size of measured molecules.We used these three methods to determine the size of discrete protein corona formed by bovine serum albumin and amphiphilic polymer coated gold nanoparticles.The result of molecular simulation show that bovine serum albumin is embedded in amphiphilic polymer layer,so the size of amphiphilic polymer coated gold nanoparticles doesn't show obvious change after adsorption of bovine serum albumin.Compare the results of the three methods,size exclusion chromatography is the most accurate,followed by dynamic light scattering,gel electrophoresis has the worst results.But each method has their own suitable application system,which method should be chosen is according to the actual situation.3.Study the interaction force between bovine serum albumin and amphiphilic polymer coated gold nanoparticles.A series of previous results indicate that bovine serum albumin and amphiphilic polymer-coated gold nanoparticles form a very stable protein corona.By polyacrylamide gel electrophoresis and mass spectrometry,we first get three fragments with 13,13 and 15 amino acids,respectively.Each of them have 7 hydrophobic amino acids.However,the result of polyacrylamide gel electrophoresis showed that the molecular weight of the peptide should be?13 kDa.So after comparison with the bovine serum albumin sequence,we think the full length of the peptide in bovine serum albumin which directly binding with nanoparticlesshould contain 105 amino acids.There are 46 hydrophobic amino acids in the peptide and centrally located that can induce the hydrophobicity of the peptide is relatively strong.The binding constant between bovine serum albumin and amphiphilic polymer coated gold nanoparticles obtained by fluorescence quenching method was calculated to be much larger than the general limit value.The binding constant was negatively correlated with the temperature change,indicating that the fluorescence quenching model between bovine serum albumin and amphiphilic polymer coated gold nanoparticles is static quenching,and the thermodynamic parameters of the whole reaction system are also in accord with the second law of thermodynamics.These data reflect the binding force between them is very strong.There are many driving force for adsorption,but the hydrophobicity of the peptide and the hydrophobic chain on the amphiphilic polymer make us believe the main driving force is hydrophobic force.4.Study the protein corona formed by transferrin and amphiphilic polymer coated gold nanoparticles.The protein corona formed by transferrin and amphiphilic polymer coated gold nanoparticles was also analyzed by gel electrophoresis and fluorescence quenching method.The molecular weight of transferrin is?77 kDa,and its size is?3.91 nm that similar to bovine serum albumin,so the gel electrophoresis result of transferrin is also similar to bovine serum albumin.It turns out that transferrin can also form discrete protein coronas with nanoparticles.The binding constant calculated by fluorescence quenching method shows a stable binding between transferrin and amphiphilic polymer coated gold nanoparticles.Transferrin is a commonly used target molecule,so it is important to study its protein corona formation with nanoparticles for its further targeted application.5.Study the protein corona formed by fibrinogen and amphiphilic polymer coated gold nanoparticles.Fibrinogen is also a high abundance plasma protein in plasma,the conformation change of fibrinogen is connected with the activation of some pathways.We don't see any changes when fibrinogen binds to the amphiphilic polymer coated gold nanoparticles.The significant lag in gel electrophoresis indicates that the protein corona formed by fibrinogen and amphiphilic polymer coated gold nanoparticles is larger than both bovine serum albumin and transferrin due to its large molecular weight and size.Fluorescence quenching data also show that fibrinogen formed a very stable hard protein corona with nanoparticles.Nanoparticles binding molecule will certainly have an effect on the nanoparticles and this will be further studied in the future.