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 copolymers was synthesized via aqueous RAFT polymerization, incorporating varying amounts of three functional monomers: IPMA (imidazole, pH-responsive), GPMA(guanidinium, cationic), and IEMA (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 polymers 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.