The Study Of Specific And Non-Specific Nanoparticle-Protein Interactions

With a large specific surface,nanoparticles usually adsorb a variety of proteins in the biological fluid,forming a layer of protein coating on the surface,termed‘protein corona’.Protein corona changes the surface properties of nanoparticles,affecting the recognition of nanoparticles by cells and determining the fate of nanoparticles in the organisms.At the same time,as an ideal platform of drug delivery,nanoparticles usually are functionalized to produce targeted drug delivery systems,which are very important for developing nanomedicines.The formation of protein corona on nanoparticles and the interaction of targeted nanoparticles with their target proteins are quite different.In general,the former is formed from the non-specific adsorption of proteins by the non-specific nanoparticle-protein interaction;this non-specific interaction may shade the targeting groups on nanoparticles,and then reduce the targeting effect.The interaction between nanoparticles and their targeted proteins is the specific interaction,which is a specific mutual recognition and is of great significance for the development of targeted nanomedicines.In order to realize the specific nanoparticle-protein interaction and reduce the non-specific nanoparticle-protein interaction,it is indispensable to study the characteristics and differences between the two kinds of interactions.Two kind of gold nanoparticles which specifically（Au-P1）and non-specifically（Au-P1s）bind to hen egg white lysozyme（HEWL）have been synthesized in our laboratory.They are as the ideal objects for the study of the specific and non-specific nanoparticle-protein interaction.We have obtained the following findings:In the thermodynamic study,by using polyacrylamide gel electrophoresis,fluorescence spectrometer and stopped-flow spectrometer,we have quantified the number of HEWL adsorbed on the nanoparticles.Au-P1 combined with more HEWL than Au-P1s.In addition,the dissociation constant K_d between Au-P1 with HEWL was 7.5×10^-8 M,which was much higher than that between Au-P1s and HEWL.We also found that the structure of HEWL was changed on Au-P1/Au-P1s,while the change formed quickly,less than 0.5 h.In the kinetics study,the stopped-flow spectrometer was used to determine the binding process between Au-P1/Au-P1s and HEWL.It was found there was a rapid binding phase in the combination of Au-P1 with HEWL.The k_on at this phase was 3.6×10⁶（M·s）^-1 and k_off was 0.27s^-1.But there was no such phase in the non-specific Au-P1s-HEWL interaction.In addition,the exchange kinetics between the HEWL absorbed on Au-P1/Au-P1s and the free HEWL was studied by the stopped-flow spectrometer.The results showed that the exchange existed in both interactions,both of the exchange processes were composed of a fast and a slow exchange stages,and the exchange rates of both interactions reached 30%.In order to explore the key amino acid site in the specific interaction of Au-P1 and HEWL,the amino acid site of P1 was mutated one by one.And then,mutant P1 was grafted onto gold nanoparticles and used to study the binding kinetics with HEWL.The results indicated that the sites 4,6,7 and 8 in P1 were the key sites to maintain the specific AuP1-HEWL interaction.By studying the characteristics,similarities and differences between the specific and non-specific nanoparticle-protein interactions,we hope to explore the interaction between nanoparticles and proteins from a new perspective,which helps guiding the research and development of nanomedicines.