Novel Gemini surfactant-polyglutamic acid composite system enhances DNA delivery via a "Dual-Engine" uptake strategy

Biophysical barriers, including limited cell uptake routes and inefficient intracellular trafficking, critically restrict the efficacy of cationic gene vectors. Herein, we engineered a ternary plasmid DNA delivery platform based on a newly synthesized sulfonyl-functionalized Gemini surfactant (NGS) via electrostatic assembly with anionic γ-polyglutamic acid (γ-PGA). The optimized ternary complexes NGS-pDNA-PGA (N/P/C ratio of 5:1:1), particularly those incorporating low-molecular-weight γ-PGA (10 kDa), exhibited favorable transfection efficiency and biocompatibility both in vitro and in vivo. Unlike conventional binary NGS-pDNA complexes, which were restricted to caveolae-mediated endocytosis (CvME) and subsequent endoplasmic reticulum (ER) trafficking, γ-PGA modification introduced a "dual-engine" uptake profile, with uptake-pathway inhibition studies supporting the involvement of both caveolae-mediated and clathrin-mediated endocytosis. Further investigation revealed that this multi-route internalization led to programmed intracellular routing, utilizing both ER-dependent trafficking and lysosomal escape pathways to maximize nuclear transport. Transcriptomic analysis provided potential regulating molecules in this process. This study not only presented a high transfection efficiency, biocompatible nanocarrier but also offered a proposed framework of uptake-trafficking regulation dependent routing for next-generation nucleic acid delivery. This study provided valuable insights into the mechanisms driving the enhanced efficacy of NGS nanoparticles, offering a promising platform for transporting-regulated gene delivery.