Thermoresponsive block copolymers containing reactive azlactone groups and their bioconjugation with lysozyme

Abstract

Thermoresponsive block copolymers based on poly(ethylene oxide) (PEO) and poly(N-isopropyl acrylamide) (PNIPAM) containing azlactone groups along the backbone and at the chain-end of the macromolecular chain were synthesized by statistically reversible addition–fragmentation chain transfer (RAFT) copolymerization and by using a combination of RAFT polymerization and thiol–ene Michael addition. Well-defined poly(ethylene oxide)-b-poly(2-vinyl-4,4-dimethylazlactone-co-N-isopropyl acrylamide) (PEO-b-P(VDM-co-NIPAM)) block copolymers and azlactone-terminated poly(ethylene oxide)-b-poly(N-isopropyl acrylamide) (PEO-b-PNIPAM-VDM) diblock copolymers with low polydispersity indices (PDIs ≤ 1.10) were prepared and fully characterized by ¹H NMR spectroscopy, FT-IR spectroscopy, and SEC. Such PEO-b-P(VDM-co-NIPAM) block copolymers and azlactone-terminated PEO-b-PNIPAM block copolymers present tunable lower critical solution temperature (LCST) depending on PEO, PNIPAM, and PVDM molar ratios. The reactivity of the PEO₄₄-b-P(VDM₂₀-co-NIPAM₈₀) copolymer (M_n,NMR = 14 200 g mol⁻¹, PDI = 1.08) and of the PEO₄₄-b-PNIPAM₁₀₁-VDM copolymer (M_n,NMR = 13 700 g mol⁻¹, PDI = 1.08) was studied with lysozyme as a model protein. A bioconjugate with a higher apparent molecular weight was obtained with the PEO₄₄-b-P(VDM₂₀-co-NIPAM₈₀) copolymer in comparison with the one obtained using the PEO₄₄-b-PNIPAM₁₀₁-VDM copolymer as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results suggest promising applications of azlactone-functionalized polymers within the field of bioconjugation.