Piperazine-Derived Diamine Lipid Nanoparticles Targeting to the Liver for Delivering Clustered Regularly Interspaced Short Palindromic Repeat Editing of PCSK9 to Durably Maintain Plasmatic Low-Density Lipoprotein Cholesterol in Low Levels

Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, and durable suppression of low-density lipoprotein cholesterol (LDL-C) via genome editing represents a promising therapeutic strategy. Here, we report the rational design of a piperazine-derived bis-tertiary amine ionizable lipid (M10) and its optimized lipid nanoparticle formulation (M10-F4) for efficient and liver-targeted CRISPR/Cas9 delivery. Compared with benchmark lipids such as SM-102, M10 enables a reduced molar ratio of ionizable lipid while maintaining high nucleic acid encapsulation efficiency (>80%) and forming stable, spherical nanoparticles. The piperazine-based multi-cationic core confers an optimized apparent pKa of 6.56, facilitating endosomal escape through enhanced protonation under acidic conditions. Confocal microscopy in HepG2 and Huh-7 cells reveals efficient cellular uptake and enhanced cytosolic release of RNA cargo with minimal lysosomal entrapment. In vivo, M10-F4 exhibits strong liver tropism following systemic administration. A single intravenous dose mediates robust PCSK9 gene editing in C57BL/6 mice, resulting in sustained reductions of circulating PCSK9 and LDL-C levels under both normal and high-fat diet conditions for up to 48 days, accompanied by decreased hepatic PCSK9 expression. Importantly, acute safety evaluation in BALB/c mice showed no obvious signs of short-term systemic toxicity, including stable body weight, minimal induction of inflammatory cytokines (IL-6, TNF-α, and CXCL-10), no significant elevation of liver enzymes, and normal gross organ morphology. Collectively, this work establishes M10-F4 as a molecularly engineered, liver-targeted LNP platform in which ionizable lipid architecture and formulation composition enable effective in vivo genome editing with favorable tolerability, highlighting the importance of rational materials design at the materials−bio interface for cardiometabolic gene-editing applications.