Synthesis, Characterization, and Bioactivity of Mid-Functional PolyHPMA−Lysozyme Bioconjugates

A thiazolidine-2-thione mid-functionalized chain transfer agent (CTA) was synthesized and used as a reversible addition−fragmentation chain transfer (RAFT) polymerization agent to prepare poly(N-(2-hydroxypropyl)methacrylamide) (polyHPMA) with mid-chain thiazolidine-2-thione functionality. The synthesized polymers were fully analyzed by 1H NMR and GPC, confirming well-defined structures (predesigned molecular weights, narrow polydispersities, and high functionalization efficiencies). A subsequent hydrolysis/analysis of the polymers was performed to verify their mid-functional structures. These mid-functionalized polymers were then incubated with a model protein (lysozyme) to generate branched polymer−protein bioconjugates. The bioactivity of the branched polymer−protein conjugate was tested and compared to similar molecular weight linear polyHPMA−protein bioconjugate; the branched polymer−protein conjugate remained much more protein activity, indicating the mid-chain-functional polyHPMA was more selective in its conjugation reaction on the lysozyme surface when compared with conjugation reactions involving terminal-functional polyHPMA. This straightforward methodology, described herein, for the synthesis of branched polymer−protein bioconjugates strikes a balance between protein protection by the attachment of polymer chains and the subsequent bioactivity retention of the bioconjugate.