Precise pH-triggered cleavage of PEG-lipid conjugates drives endosomal escape of siRNA from fusogenic lipid-based nanoparticles.

Lipid-based nanoparticles (LNPs) are leading synthetic delivery systems for the functional delivery of small interfering RNAs (siRNAs) and mRNA vaccines in vivo. However, current state-of-the-art LNPs, although designed with near optimal properties of fusogenicity for target cell entry and release of RNA APIs, possess suboptimal colloidal stability and biocompatibility due to limited uses of polyethylene glycol-lipids (PEG-lipids) in formulation. We overcome this shortcoming here with novel pH-triggered RNA-LNPs (ZK2 LNPs) that comprise PEG-lipid oxime conjugates (5 mol%), prepared in situ using pH-reversible aminoxy-aldehyde click chemistry. Using mass spectrometry and electron microscopy, ZK2 LNPs are shown colloidally stable in PBS at neutral pH but subject (between pH 7 and 6) to pH-triggered PEG-shedding, from hydrolytic cleavage of PEG-lipid oxime conjugates, and pH-sensitive lipid phase changes. These same mild acid-induced physicochemical changes appear to promote increasingly efficient fusion of ZK2 LNPs with endosome model extracellular vesicles (EVs) at pH values in tune with early endosome pH changes (pH 6.8 to 6), hence explaining in confocal microscopy studies how ZK2 LNPs drive endosome lysis then RNA release into the cytoplasm of HepG2 cells in vitro, following cell entry by endocytosis. Finally, ZK2 LNPs mediate effective functional hepatitis B virus (HBV) gene silencing in the HepG2.2.15 model of chronic HBV (CHB) infection and demonstrate excellent LNP tolerability in vivo, too. Therefore, ZK2 LNPs appear to possess unique physicochemical characteristics for colloidal stability, biocompatibility, potential stealth, and efficient RNA API delivery to cytoplasm in target cells, hence could represent a powerful next generation LNP technology.