Endocytosis-independent cytosolic entry of messenger RNA via fluorous bilayer zippering attenuating Toll-like receptor signaling and enables ischemic tissue salvage.

A fundamental constraint of conventional messenger RNA (mRNA) delivery systems is their obligatory trafficking through endosomal-lysosomal compartments, wherein cargo degradation and activation of endosomal Toll-like receptors precipitate substantial translational attrition and deleterious inflammatory cascades. We herein report a chemically engineered platform that circumvents these limitations ab initio. Through strategic perfluoro-acylation of branched polyethyleneimine (PEI, 25 kDa) with pentafluoropropionic anhydride, we install approximately 26 fluoro-amide "zipper" moieties per polymer chain that orchestrate direct, energy-independent trans-bilayer translocation without recruitment of clathrin, caveolae, or lipid raft microdomains-thereby precluding lysosomal entrapment and catabolism. Bio-orthogonal copper-free click chemistry between azide- and dibenzocyclooctyne (DBCO)-terminated PEI-F derivatives, coupled with redox-labile disulfide crosslinkers, engenders polyplexes of exceptional extracellular stability that undergo quantitative glutathione-triggered disassembly within the cytosolic milieu. This endosome-evasive entry mechanism effectively sequesters single-stranded mRNA from Toll-like receptor 3, TLR7, and TLR8 surveillance, establishing a "TLR-attenuated" delivery paradigm characterized by undetectable interferon-α, interferon-β, TNF-α, and IL-6 induction. In human umbilical vein endothelial cells, GFP-mRNA transfection exceeds 90% fluorescent positivity with 4.8-fold superior luciferase expression relative to Lipofectamine™ 3000, whilst maintaining >95% viability. Therapeutic translatability is demonstrated in a murine hindlimb ischemia model, wherein a single 10 μg intramuscular dose of mVEGF-A polyplexes restores blood perfusion to 118% of baseline within 28 days-representing marked superiority over the commercial gold standard and effectuating complete tissue salvage without necrosis. Comprehensive hematological and immunological profiling corroborates the absence of hematotoxicity, systemic inflammation, or innate immune activation. This modular, purely synthetic platform resolves the classical stability-availability paradox whilst eliminating the immunogenic liabilities inherent to endocytic delivery, furnishing a readily translatable scaffold for precision regenerative medicine.