Modification Of Protein And Regulation Of Protein Adsorption By Synthetic Polymers

Biomedical polymer is a biocompatible polymer that can be used in diagnosis andtreatment of diseases. As polymer stuctures are diverse and devisable, it is possible toachieve a variety of functions by biomedical polymers which can be used formodification of biomaterials and biomolecules to improve their biological properties.Firstly, for biomedical materials, whether they can be successfully applied or not isdetermined by their interfacial properties. When materials are in contact with biologicalenvironment, the first event is the protein adsorption on material surfaces. The adsorbedprotein layer will lead to a series of subsequent host responses, for example, plateletadhesion, thrombosis and immunological responses. If the protein adsorption on materialsurfaces can be regulated, then the subsequent biological reactions may happen in afavorable way. Regulation of protein adsorption can be achieved by surface modificationwith synthetic polymers. Strategies of regulation include repelling nonspecific proteinadsorption, selective binding of specific proteins and control of protein adsorption byexternal stimuli. Regulation of protein adsorption is an eternal topic in the field ofbiomedical materials. Secondly, biological molecules, particularly proteins, are oftenmodified to improve their functions or to endow them with controllable properties. Whena protein is used as drug, it is desirable to maintain its activity and to prolong itscirculation time. For enzymes, the on/off switchable property is usually required. Themodification of proteins with functional polymers, i.e. polymer-protein conjugates, is aneffective way to accomplish the aims above, which has aroused a great deal of interests inrecent years. In view of the applications of synthetic polymers in the fields of materialmodification and the protein modification, the content of this thesis was divided into twoparts: modificaiton of blood contact materials using lysine-containing polymers toregulate plasminogen adsorption; regulation of enzyme activity by graftingthermo-responsive polymer on the enzyme. The details are as follows.1. The conjugates of poly(N-isopropyl acrylamide) and lysozyme(LYZ-PNIPAAm) were successfully prepared. Firstly, an N-hydroxysuccinimide ester(NHS) functionalized water-soluble bromine-containing initiator (NHS-Br) wassynthesized, which was then immobilized on LYZ through reaction with NH2. Then,PNIPAAm was successfully grafted form LYZ to prepare LYZ-PNIPAAm conjugates byusing single electron transfer living radical polymerization (SET-LRP) method. Theproducts were characterized by1H NMR, TNBS assay, SDS-PAGE, UV-Vis spectroscopyand enzyme activity assay. It was shown that the number of grafted initiators on LYZ canbe adjusted by the feed ratio of bromine-containing initiator to amino groups on LYZ.The molecular weight of LYZ-PNIPAAm conjugates can be controlled by the feed ratioof NIPAAm to initiator during the polymerization. The results of enzyme activity testshowed that the relative activity of LYZ-PNIPAAm conjugates can be tuned bytemperature, which exhibited a higher activity at temperature above the LCST ofPNIPAAm and a lower activity at temperature below the LCST. Compared with theoriginal LYZ, PNIPAAm-conjugated LYZ showed a reduced protein activity, and proteinactivity decreased with the increase of the number of PNIPAAm chains grafted andmolecular weight of PNIPAAm.2. Lysine-functionalized polyurethane (PU) surfaces were successfully prepared byphysical blending method. Lysine-containing ternary copolymers P(E-O-L), which wassynthesized via the random copolymerization of2-ethylhexyl methacrylate (EHMA, E),oligo (ethylene glycol) methyl ether methacrylate (OEGMA, O) and6-tert-butoxycarbonyl amino-2-(2-methyl-acryloylamino)-hexanoic acid tert-butyl ester (Lys(P)MA, Lp) and followed by the deprotection of–COOH and ε-NH2groups onlysine residues in the copolymer, were subsequently blended with PU. The chemicalcomposition and molecular weight of the copolymer were measured by1H NMR andGPC, respectively. The composition of the copolymer, as well as the surface lysinedensity, could be adjusted by varying the feed ratios of monomers. Copolymer-blendedPU membranes became hydrophilic as indicated by static water contact anglemeasurements. Protein adsorption on blend membranes was investigated using isotopelabeled protein adsorption. It was shown that fibrinogen (Fg) adsorption on the blendmembranes decreased by about95%compared with pure PU surface, indicating anefficicent repellence to nonspecific protein adsorption. In addition, the blend membranescould selectively adsorb plasminogen (Plg) from plasma, and the amount adsorbedincreased with the increase of lysine content in the copolymer.