Application Of Thermoresponsive Polymer Poly(N-Isopropylacrylamide)in Regulating Protein Activity

Stimuli- responsive polymers are the polymers that can undergo dramatic physical or chemical changes(conformation, solubility, wettability) upon specific environmental stimuli like temperature and p H change. Thermoresponsive polymers which can undergo a change of comformation and solubility at specific temperature have been applied in bio medical engineering and other fields. And the interactions with proteins is of great significance for the application of the thermoresponsive polymer Poly N-Isopropylacrylamide(PNIPAAm). This research was conducted fro m the control of protein adsorption on a PNIPAAm modified surface and the regulation of protein activity by construction of protein-PNIPAAm conjugate to study the funcations of PNIPAAm.First, a modified surface PU-P(NIPAAm-co-Lys) with the qualities of thermosensitivity and fibrinolysis was prepared through surface grafting of the copolymer of N-Isopropylacrylamide(NIPAAm) and ε- lysine(Lys MA) to polyurethane(PU) surface. The PU surfaces modified by copolymer with different monomer ratios were characterized by 1H NMR, GPC and Lysine Density Assay. It was found that the composition of the modified copolymer could be adjusted by change of monomer feed ratio, and the lysine density on the surface increased as LysMA. The static water contact angles measurement indicated that Lower Critical Solution Temperature(LCST) of PU-P(NIPAAm-co- Lys) surfaces were about 28 oC, which determined the protein adsorption of the surfaces below and above LCST was studied at 23 oC and 37 oC respectively. Copolymer modified surfaces showed a higher level of plasminogen(Plg) adsorption with high activity than the PU-PNIPAAm surface, due to specific binding of plasminogen to ε- lysine residues, and the quantity adsorbed increased with surface lysine density. It is particularly interesting that much more plasminogen was adsorbed on PU-P(NIPAAm-co- Lys) at 37 oC than 23 oC. Thanks to PNIPAAm resistant to nonspecfic protein adsorption and the preferential surface uptake of plasminogen, the quantity of the nonspecific protein Fibrinogen(Fg) adsorbed on PU-P(NIPAAm-co-Lys) was quite low at both temperarure. In conclusion,the copolymer modified surface PU-P(NIPAAm-co-Lys) possessed the capability of resisting nonspecific protein adsorption and combining the specific protein plasminogen to construct fibrinolytic system.The second section was the preparation of the protein conjugate PPa-PNIPAAm with high bioactivity and thermosensitivity through modifying a pyridine chain transfer agent(CTA-Py) to mutant phosphatase(PPa) at the sites close to the active sites, which followed by surface- initiated RAFT polymerization of N-Isopropylacrylamide(NIPAAm). The molecular weight change and surface thiol group chang of the protein were characterized by MALDI-TOF-MS and Ellman’s Assay respectively to indicate CTA-Py modified to PPa successfully. The result of molecular dynamics simulation showed that the pyridine chain transfer agent(CTA-Py) selected in this study had no apparent side effect to the structure after modified to PPa, while the protein comformation was changed and the protein was inaccessible for its substrate after maleimide chain transfer agent(CTA-Ma) modification. Besides, the activity assay also indicated that the protein activity of PPa-CTA-Py was well retained compared to unmodified PPa which was different from protein activity drop after CTA-Ma modification. For the PPa-PNIPAAm conjugate prepared from PPa-CTA-Py, the protein activity was maintained well and could be regulated by temperature change. It is worth mentioning that PNIPAAm in the conjugate could enhance the ability of the protein to maitain its activity in high temperature.In conclusion, through the application of Poly N-Isopropylacrylamide(PNIPAAm), protein adsorption on the copolymer grafted surface was effectively controlled and the protein activity could be well regulated by construction of protein-PNIPAAm conjugate.