Dual-Functional Polyphosphoesters for Gene Delivery: Synergistic Effects of Guanidinium and Hydrophobic Side Chains in Degradable Polymers

Polyphosphoesters (PPEs) have emerged as promising degradable carriers for drug and gene delivery, yet fine-tuning their physicochemical properties for optimized gene transfection remains a key challenge. Here, we introduce guanidinium- and indole-functionalized PPEs synthesized via living anionic ring-opening polymerization and thiol-ene post-polymerization modification, enabling precise control over charge density and hydrophobicity. Variants with 66–91 mol% guanidinium and 7 mol% indole form stable polyplexes with plasmid DNA, yielding nanoparticles < 200 nm with high zeta potentials (+34 to +43 mV), strong DNA binding, and cytocompatibility comparable to linear poly(ethylene imine) (LPEI). Despite similar molar masses and charge densities, incorporation of indole or increasing the guanidinium content dramatically enhances transfection-up to 200-fold relative to lower-charged variants-underscoring the synergistic role of charge distribution and hydrophobic balance. The PPEs also exhibit pH-responsive degradation, degrading slowly at physiological pH and more rapidly under mildly basic conditions, supporting extracellular stability with potential for cytosolic DNA release. These results demonstrate the potential of side-chain-engineered PPEs as a modular, degradable platform for gene delivery, and highlight the critical influence of chemical structure on transfection performance.