Polymeric Gene Delivery at Sub-Stoichiometric N/P Ratios

Efficient cytosolic delivery remains a major challenge for the clinical translation of nucleic acid therapeutics. While cationic polymers are promising gene carriers, they suffer from an inherent trade-off between transfection efficiency and cytotoxicity: high nitrogen-to-phosphorus (N/P) ratios can enhance gene delivery efficacy but inevitably increase cellular toxicity due to excess free cationic polymers. Here, we report a fluorinated polymer that achieves potent gene transfection at an unprecedentedly low N/P ratio below 1. Mechanistic studies demonstrate that the obtained polymer completely condenses DNA into stable nanoparticles at an N/P ratio around 0.4, with negligible unbound polymers remaining in the polyplexes. Detailed intracellular trafficking analysis revealed that the polymer/DNA complexes are primarily internalized via scavenger-receptor-mediated endocytosis, followed by rapid endosomal escape and efficient nuclear entry within 4 h. The polymer successfully delivered multiple protein-expressing plasmids across diverse cell lines, demonstrating broad applicability. Our findings establish this fluorinated polymer as a novel class of highly efficient gene vectors that function without requiring excess free cationic polymers. This study provides valuable insights for the rational design of next-generation gene delivery systems with enhanced efficiency and minimized cytotoxicity.