Hydrophobic scaffolds of pH-sensitive cationic lipids contribute to miscibility with phospholipids and improve the efficiency of delivering short interfering RNA by small-sized lipid nanoparticles

Purpose: Small-sized lipid nanoparticles (LNPs) are attractive for tissue penetration but often lose potency because lipid components diffuse out and serum proteins adsorb onto poorly packed surfaces. The study asked how the hydrophobic scaffold structure of pH-sensitive cationic lipids affects small-LNP stability, lipid miscibility, endosomal escape, and siRNA delivery.

Hypothesis: If pH-sensitive cationic lipids have long, linear hydrophobic scaffolds, then small-sized LNPs will show reduced lipid diffusion and altered miscibility with phospholipids, leading to improved endosomal escape and more efficient siRNA-mediated gene silencing.

Aims: Compare pH-sensitive cationic lipids with different hydrophobic scaffold lengths and shapes (CL15A6, CL15H6, CL4H6, YSK05). - Optimize small-sized LNPs by replacing cholesterol with egg sphingomyelin (ESM). - Characterize LNP structure, siRNA encapsulation, stability, and lipid distribution. - Determine how scaffold length affects miscibility with phosphocholine-containing lipids and endosomal escape.

Delivery system: Platform: Small-sized pH-sensitive cationic lipid nanoparticles (LNPs). - Cationic lipids: CL15A6, CL15H6, CL4H6, YSK05. - Helper lipids: Cholesterol or egg sphingomyelin (ESM); other phospholipids tested include DOPC, DPPC, EPC, DSPC, POPC, BSM, MSM. - PEG lipid: PEG-DMG. - Payload: siRNA (siGL4; also fluorescently labeled AF647-siGL4). - Optimized formulation: CL15H6/ESM/PEG-DMG at 40/60/3 molar ratio; diameter ~22 nm. - Preparation: Microfluidic mixing; no targeting ligand.

Approach: In vitro model: HeLa cells stably expressing firefly and Renilla luciferase (HeLa-dluc). - Readouts: Gene silencing, cellular uptake, cell viability. - Biophysical characterization: ³¹P NMR, SAXS, TEM, RNase protection assay, DLS/Zetasizer. - No in vivo animal studies were performed in this paper.

Key methods: Dual-Glo luciferase assay for siRNA silencing. - Flow cytometry for cellular uptake of AF647-siRNA and DiO-labeled LNPs. - CCK-8 assay for cytotoxicity. - ³¹P NMR with MnCl₂ and detergent to locate ESM and siRNA. - SAXS for lipid phase determination. - TEM for LNP morphology. - RNase A protection assay for siRNA encapsulation.

Key results: CL15H6-LNPs with 3 mol% PEG-DMG induced clear gene silencing (IC₅₀ ≈ 20 nM siRNA), whereas CL15A6-LNPs failed; cellular uptake of CL15H6-LNPs was about 2-fold higher. - Replacing cholesterol with ESM produced smaller LNPs (~24 nm) and improved silencing: CL15H6/ESM-LNPs IC₅₀ ≈ 7 nM vs cholesterol counterpart. - Optimized CL15H6/ESM/PEG-DMG (40/60/3) had diameter ~22 nm, >90% siRNA encapsulation, IC₅₀ ~10 nM, no toxicity up to 50 nM, and stability for at least 4 weeks. - ³¹P NMR showed most ESM located in the outer layer and siRNA physically entrapped; MnCl₂ reduced ESM signal by ~80%. - ESM stabilized LNPs even without PEG-DMG, unlike cholesterol-based LNPs. - Long, linear scaffolds were immiscible with phosphocholine lipids (C16–C18); LNPs with BSM/MSM (C22–C24) showed no gene silencing. - SAXS/³¹P NMR indicated POPC segregated from H_II structures formed by CL14H6/DSPS, preventing aggregation and forming small vesicles.

Interpretation: Long, linear hydrophobic scaffolds in pH-sensitive cationic lipids improve small-sized LNP potency by reducing lipid diffusion and creating immiscibility with phosphocholine-containing helper lipids, thereby avoiding inhibition of membrane fusion-mediated endosomal escape. These findings provide design rules for cationic lipids and helper lipid selection for potent small LNPs.

Limitations: Only in vitro cell culture; no in vivo biodistribution, efficacy, or toxicity. - No disease model or therapeutic endpoint beyond reporter gene silencing. - Mechanism studied with simplified lipid suspensions; may not fully represent intact LNP behavior in vivo. - No active targeting ligand. - Long-term stability and serum stability in vivo not fully assessed. - No large-animal validation.