Perfluoroalkylated polymeric constructs bypass endosomal entrapment to stimulate transnuclear delivery in proliferating and non-proliferating cells.

A synthetic gene-delivery construct from the multi-functionalized polyethyleneimine derivatives, was engineered by conjugating sparse perfluoroalkyl chains and a tripartite peptide cassette (RGD-TAT-NLS consisting of Arg-Gly-Asp, trans-activator of transcription and nuclear localization signal peptide) onto 25 kDa branched polyethylenimine (PF-RNT). Sparse perfluoroalkylation generates amphiphobic nanointerfaces that enable direct, energy-independent transbilayer translocation, bypassing clathrin- and caveolae-mediated endocytosis. Pharmacologic interrogation with metabolic poisons and pathway-specific inhibitors-chlorpromazine, genistein, dynasore, methyl-β-cyclodextrin-corroborates this non-endocytic, non-dynamin-dependent mechanism. The RGD-TAT-NLS cassette orchestrates deterministic intracellular trafficking: RGD prolongs membrane engagement via αvβ3/αvβ5 integrin binding and receptor clustering, TAT facilitates cytosolic dissemination via guanidinium-mediated bidentate hydrogen bonding with anionic phospholipids, and NLS drives importin-α/β-mediated, RanGTP-directed nuclear pore translocation. This achieves approximately 18.7% nuclear accumulation of total internalized pDNA- > 180-fold enhancement over unmodified bPEI (<0.1%). PF-RNT delivers appreciable transfection efficiencies across HeLa, AGS, THP-1 and 293T cells, with mean fluorescence intensities remarkably surpassing the commercial golden standard of Lipofectamine™ 3000. Notably, NLS-mediated active import enables approximately 41% transfection in terminally differentiated, post-mitotic primary neurons-conventionally refractory targets where the intact nuclear envelope excludes passive diffusion and mitotic bypass is impossible. The modular, click-chemistry-compatible architecture, coupled with negligible hemolysis and minimal cytotoxicity establishes a versatile, translation-ready platform for nucleic acid vaccines, ex vivo CAR-T engineering, in situ genome editing, and neural gene therapy.