Studies On The Interaction Mechanisms Between Biomimetic Phosphorylcholine-based Polymers And C-reactive Protein

C-reactive protein(CRP)plays an important protective role within the innate immune system,but there are some controversies surrounding its physiological functions such as pro-inflammatory,cytokine stimulating,pro-atherogenic and pro-thrombotic etc.It has been shown that these controversies were mainly caused by contaminants,such as endotoxin and sodium azide,in commercial CRP preparations.Therefore,in order to resolve these controversies to verify the physiological functions of CRP and broaden its clinical application range,it is very urgently to obtain highly purified,non-polluted,structurally intact and fully functional human CRP.Phosphorylcholine(PC)has been widely used in biomedical fields due to its excellent biocompatibility,and has attracted much attention as a key ligand of CRP.However,the traditional PC-based materials for CRP purification present certain shortcomings such as cumbersome preparation,poor mechanical strength,mass transfer limitations,and high non-specific adsorption.So,it is very difficult to meet the urgent need for efficient and rapid purification of CRP in complex systems.Therefore,on the basis of our previous work,the present research developed a series of biomimetic PC-based polymers with different crosslinkers,spacer lengths/numbers,and PC ligand arrangements to study their effects on the adsorption performance for CRP and explore the differences in the interaction mechanisms between theses materials and CRP.The results of this study would provide a scientific basis for the development of novel materials and methods for CRP affinity purification in the later period.The main contents and conclusions of this study are as follows:In the first chapter,the clinical applications,structure and functions of CRP were systematically introduced.Focused on the binding mechanism of CRP with PC and the controversies over the physiological functions of CRP,and then the existing methods for CRP purification were briefly summarized.Moreover,the applications of biomimetic PC-based polymers were reviewed in detail,which were mainly related to its biocompatibility and artificial receptors for CRP.On this basis,the research ideas and innovations of this study were put forward.In the second chapter,the biomimetic PC-based polymers with different crosslinkers,spacer lengths/numbers,and PC ligand arrangements were prepared to study their effects on the adsorption performance(specificity and selectivity,adsorption capacity,binding affinity and CRP purification from spiked human serum)for CRP.Firstly,the surface charge,ligand density,specific surface area and pore size of these polymers were characterized.The results showed that the prepared biomimetic PC-based polymers were not only suitable for the separation and analysis of biomacromolecules,but also had the potential to resist non-specific protein adsorption.In the third chapter,the effects of the crosslinkers on the adsorption performance for CRP were studied.Good specificity and selectivity,similar adsorption capacity and binding affinity could be observed on the biomimetic PC-based polymers with three different crosslinkers,which indicated that the crosslinkers had no obvious effect on the above adsorption performance for CRP.However,only Poly(MPC-co-MBA)and Poly(MPC-co-PDA)could effectively purify CRP from spiked human serum,and the purity of the resulting CRP was significantly better than that of Poly(MPC-co-EDMA).This phenomenon showed that the increased hydrophilicity of the biomimetic PC-based polymers caused by the increased polarity of the crosslinkers could effectively avoid the non-specific adsorption of proteins.In the fourth chapter,the effects of the spacer lengths/numbers on the adsorption performance for CRP were studied.It was found that the biomimetic PC-based polymers with three different spacer lengths/numbers had good specificity and selectivity for CRP,but they would affect the adsorption capacity and binding affinity of biomimetic PC-based polymers for CRP.Our results showed that appropriate spacer length and number could reduce the steric hindrance in the process of CRP and PC binding,increase the effective binding probability between CRP and PC ligand,and then obtained a higher adsorption capacity for CRP on the biomimetic PC-based polymers.However,excessive adsorption capacity might enhance the steric repulsion between CRPs,resulting in poor stability of the CRP-PC complex and reduced binding affinity.In the experiment for CRP purification,only long-chain Poly(MDPC-co-EDMA)could effectively purify CRP from spiked human serum,and the purity of the resulting CRP was significantly better than that of short-chain Poly(MPC-co-EDMA),indicating that spacer with appropriate length could effectively avoid the non-specific adsorption of other proteins on the surface of polymer materials.However,double-chain Poly(MDSPC-co-EDMA)was slightly better than that of short-chain Poly(MPC-co-EDMA),but obviously inferior to that of long-chain Poly(MDPC-co-EDMA).It could be attributed to the non-specific adsorption between another hydrophobic alkyl chain of Poly(MDSPC-co-EDMA)and proteins in human serum.In the fifth chapter,the effects of the PC ligand arrangements on the adsorption performance for CRP were studied.We observed that compared with Poly(MPC-co-MBA),when the PC ligand was only arranged with phosphate moiety(Poly(EGMP-co-MBA)),choline moiety(Poly(METAM-co-MBA)),or phosphate moiety and choline moiety were equimolar laterally arranged(Poly(E1M1-co-MBA)),these polymer materials didn't recognize CRP.It revealed that the integrity of the PC ligand and its inherent spatial arrangement are prerequisites for CRP binding.Surprisingly,Poly(MMPC)(The PC group analogous moiety in MMPC is located in the middle of the molecule.)not only specifically and selectively capture CRP,but also had a larger adsorption capacity,in addition to a slightly lower binding affinity than Poly(MPC-co-MBA)(The PC group is at the free end in the side chain of MPC.).This might be due to the different recognition behaviors between CRP and PC ligand in this two polymers.Furthermore,Poly(MMPC)could effectively resist non-specific protein adsorption because of its stronger hydrophilicity,thus exhibiting excellent efficiency for CRP purification with a purity as high as 98%,which demonstrated that Poly(MMPC)is very promising to be developed for the purification of CRP.In the sixth chapter,the conclusions and prospects of this research were described.