Study Of Artificial Affinity Reagent Based On Self-Assembled Cyclodextrin Polymer Nanoparticles

Protein affinity reagents,such as antibodies and antibody-like scaffold proteins,have been widely used in basic biomedical research,clinical diagnosis and treatment.However,there are some problems in this kind of affinity reagent,such as the high cost of research,the difficulty of chemical modification,the great difference between batches and the difficulty of preservation.As a result,more and more synthetic affinity reagents have been developed,including molecularly imprinted polymers,linear polymers,dendritic polymers and polymer nanoparticles,which show high affinity for peptides,proteins,polysaccharides and other targets.Self-assembly technology has shown many advantages in the preparation of functional materials,such as flexible design,mild reaction conditions,simple operation and so on.Therefore,we utilized the host-guest interaction between the cavity of cyclodextrin and different adamantane derivatives(Ad-D),so as to construct a selfassembled nanoparticle(SNP)library and screen out the particles with affinity for the target.This method had the advantages of flexible design,simple operation and low cost.Taking polypeptides,enzymes and lipopolysaccharides as model targets,we have carried out relevant research,which includes the following three parts:1.Screening of self-assembled nanoparticles for neutralizing hemolytic toxicity of melittinFirstly,we constructed a SNP library based on host-guest recognition of ?-CDP and Ad-D.The library consisted of five Ad-D with different groups and ?-CDP,and eighty-two SNPs were obtained by adjusting the type and proportion of Ad-D.Different Ad-D had different hydrophobic and charged properties,thus providing different sites for binding of target compounds.By investigating the changes of melittin hemolytic toxicity in the presence of nanoparticles,we screened eight SNPs from the library that could neutralize the hemolytic toxicity of melittin.At the same time,we found that these SNPs not only had strong binding ability to melittin,but also could neutralized the toxicity of melittin to cultured cells.In addition,the phenol soluble polypeptide PSM?3 secreted by staphylococcus aureus was used as the target to screen SNP that could neutralize the hemolytic toxicity of PSM?3 by the same method.If the diversity of functional monomer Ad-D can be further increased,it is hopeful to construct a universal screening platform for artificial affinity reagents.2.Screening of self-assembled nanoparticles for enhancing the catalytic activity of horseradish peroxidaseIn the above work,we obtained SNPs that bound to melittin and PSM?3,respectively.However,these targets are polypeptides with similar structures and properties,so it is necessary to further investigate the effects of SNP on other biomolecules.Horseradish peroxidase(HRP)is an enzyme commonly used in basic research and clinical testing,and its catalytic activity may be affected by nanomaterials.In view of this,we continued to screen with HRP as the target,and obtained four SNPs from the library constructed in the previous research work.The selected SNP not only effectively enhanced the catalytic activity of HRP,but also prolonged the service life of HRP.3.Screening of self-assembled nanoparticles for binding lipopolysaccharideIn the above research,the polymer library that we constructed contained eightytwo SNPs,and the screening process was time consuming and labor intensive.Since the SNP bound to target molecule through weak interactions such as static electricity and hydrophobicity,the affinity reagent can be designed by targeted selection of functional monomers.For this we chose lipopolysaccharide(LPS)as a target for investigation.Since LPS was a negatively charged and hydrophobic biomacromolecule,four Ad-D(one positively charged Ad-D and three hydrophobic groups Ad-D)were selected in the construction of SNP library.A library of 50 SNPs was constructed by adjusting the type and proportion of Ad-D.From this library we obtained a SNP that bound to lipopolysaccharide and distinguished lipopolysaccharide from other proteins.It was worth noting that the SNP only included two Ad-D with hydrophobic groups,and not all functional monomers contributed to the binding of SNP to lipopolysaccharides.This may be because affinity was not just the result of a binding site but the sum of noncovalent interactions on the surface of molecules.