Click grafting of alkyne-containing vinyl polymers onto biosynthesized extracellular matrix protein containing azide functionality and adhesion control of human umbilical vein endothelial cells

Abstract

In vivo incorporation of a phenylalanine (Phe) analogue, p-azidophenylalanine (p-N₃Phe) into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket, in which the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) was expressed under the control of T7 promoters. In this study, biosynthesized aECM-CS5-ELF containing p-N₃Phe (aECM-CS5-ELF-N₃) was coupled with alkyne-containing vinyl polymers prepared via controlled radical polymerization of three vinyl monomers, (styrene, acrylamide, and N-isopropylacrylamide) using a trithiocarbonate as the RAFT agent. Grafting of the vinyl polymers onto the aECM was accomplished via a copper-catalyzed alkyne–azide click reaction. The lower critical transition temperature (LCST) was evaluated, as well as the solubility in aqueous and organic media, which are dependent on the incorporation ratio of p-N₃Phe and species of graft chains, in which the LCST behavior was altered remarkably when poly(N-isopropylacrylamide) moieties were attached as side chains. Circular dichroism measurements indicate conformational change was not induced by the grafting. Specific adhesion of human umbilical vein endothelial cells (HUVECs) onto the (aECM-CS5-ELF-N₃)-graft-poly(N-isopropylacrylamide) composite surface and subsequent temperature-sensitive detachment were also demonstrated.