Rational Design of pH-Switchable 3α-Amino Lithocholate-Modified Lipids for Efficient siRNA Delivery

Lipid nanoparticles (LNPs) represent a state-of-the-art platform for RNA-based therapeutics, yet inefficient endosomal escape remains a critical barrier to cytosolic RNA delivery. We designed novel pH-switchable 3α-amino lithocholate-modified lipids (LMLs) with a unique mechanism of action. Under weakly basic conditions, the 3α-amino lithocholate moieties sequester within LNP membranes, while acidic environments (e.g., endosomal pH 5.0) trigger their reorientation to the lipid-water interface. The pH-driven flipping behavior amplified the surface charge of LNPs, yielding a much higher zeta potential (+4-7 mV) than LNPs incorporating the 3α-OH LMLs (0 mV) or the benchmark lipid Dlin-MC3-DMA (MC3) (-3 mV). In vitro studies found that 3α-amino LMLs-based LNPs had endosomal escape efficiencies comparable to LNP-MC3, whereas the 3α-OH LML had no activity. In a mouse model, the lead compound, LML4 (pKa = 6.3), showed an efficacy equivalent to MC3, reducing serum Factor VII protein levels by approximately 40% following siRNA treatment at 0.5 mg/kg. We propose that the dual advantages of 3α-amino LMLs, including acidity-induced charge amplification and membrane reorientation, synergize to promote endosomal membrane disruption. This dynamic flipping process could represent a revolutionary shift in lipid design for next-generation LNPs with high pH-reactivity, addressing the persistent challenge of inefficient intracellular RNA release.