Controlled design of guanidinium-functionalized methacrylamides: tuning polyplex formation and transfection efficiency through hydrophobic and pH-responsive motifs

Efficient polymeric gene delivery systems require a fine balance of positive charges for nucleic acid binding, hydrophobic interactions for complex stability, and stimuli-responsive elements for intracellular release. Imidazole-containing groups, inspired by the natural amino acid histidine, offer unique pH-buffering and membrane-interacting properties that can enhance endosomal escape and transfection efficiency. A library of eleven poly(methacrylamide)-based statistical copolymers was synthesized via aqueous RAFT polymerization, incorporating varying amounts of three functional monomers: IPMA(imidazole, pH-responsive),GPMA (guanidinium, cationic), andIEMA(indole, hydrophobic). Polyplex formation was strongly dependent on the presence of GPMA, leading to the formation of stable nanosized polyplexes (90-130 nm) with high positive surface charges, providing stable DNA binding through electrostatic and hydrogen bonding interactions. Heparin displacement and DNase I digestion assays identified a high GPMA content and incorporation of the hydrophobic IEMA to be responsible for improved polyplex stability and enzymatic plasmid protection. Replacing GPMA in HPMA- and IEMA-based copolymers with pH-sensitive imidazole (IPMA) increased plasmid release and transfection efficiency in HEK293T cells and correlated with membrane destabilization as shown in hemolysis experiments. The most efficient transfection (∼55 % EGFP-positive cells in HEK293T) was achieved with a copolymer combining high IPMA and IEMA content. Overall, the results highlight how tuning the balance between cationic, hydrophobic, and pH-responsive components enables the design of gene carriers with optimized delivery and expression efficiency.