“Zip-to-Cytosol”: Glutathione-Cleavable Fluorinated Polyplexes Deliver siRNA at Single-Digit Nanomolar Dose with >90% Gene Silencing

A fluorinated, disulfide-cross-linked polyplex platform (PFND) was developed for safe and potent cytosolic delivery of siRNA. Branched PEI (25 kDa) was first perfluoro-acylated to yield a membrane-zipper PF backbone, followed by orthogonal installation of azide (PF-N3) and strained alkyne (PF-DBCO) handles that undergo in situ copper-free click cross-linking in the presence of siRNA. The resulting 60 nm polyplexes (PDI < 0.1, ζ potential of approximately +22 mV) are stable in 10 mg mL–1 heparin (<5% siRNA leakage) yet quantitatively disassemble within 60 min in 10 mM glutathione, liberating the siRNA payloads. Compared with the commercial gold standard of Lipofectamine 3000, PFND delivers 2- to 3-fold more Cy5-siRNA into HeLa, HepG2, and MDA-MB-468 cells without detectable hemolysis or cytotoxicity. Consequently, 10 nM siGAPDH delivered by PFND silences approximately 93–98% of GAPDH mRNA across the three lines, remarkably outperforming gold-standard transfection reagents. The proposed reversible “locked-outside/labile-inside” design reconciles extracellular stability with rapid intracellular release, offering a valid tool for utilities of high-throughput siRNA screening or subject to be developed further for potential clinical translation of RNAi-based therapeutics.