Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo

Purpose: Endosomal escape remains a major rate-limiting step for siRNA delivery. The structure–activity relationships (SAR) of pH-sensitive cationic lipids were incompletely understood, limiting rational design of lipid nanoparticles (LNPs) for efficient in vivo siRNA delivery to hepatocytes.

Hypothesis: Systematic derivatization of both the hydrophilic head group and hydrophobic tails of a benchmark pH-sensitive cationic lipid (YSK12-C4) will reveal separable SAR rules: the head group will mainly control apparent LNP pKa, while the hydrophobic tail will mainly control intrahepatic distribution. Combining optimized head and tail structures will yield a potent, biodegradable, and well-tolerated LNP for hepatocyte siRNA delivery.

Aims: Synthesize a library of pH-sensitive cationic lipids by varying hydrophilic head groups and hydrophobic tails. - Measure apparent pKa, in vitro gene silencing, in vivo Factor VII silencing, intrahepatic distribution, and biodegradability. - Identify the optimal head/tail combination for hepatocyte targeting. - Compare lead CL4H6-LNPs with prior YSK05- and YSK13-C3-LNPs and assess toxicity.

Delivery system: Platform: pH-sensitive cationic lipid nanoparticles (LNPs) for siRNA delivery. - Key lipid identified: CL4H6 (CL4 head group + H6 hydrophobic tails). - Optimized LNP composition: CL4H6:cholesterol:mPEG2k-DMG = 60:40:1 mol%; siRNA/lipid weight ratio ~0.024. - Payload: Chemically modified siRNA against Factor VII (siFVII) or PLK1 (siPLK1); fluorescently labeled siRNA for imaging. - Targeting: Hepatocyte targeting via ApoE/LDLR pathway; no exogenous targeting ligand. - Preparation: Self-assembly in citrate buffer, ultrafiltration. - Physicochemical properties: CL4H6-LNP apparent pKa 6.35; spherical droplet-like morphology; size generally controlled to 80–120 nm.

Approach: In vitro: HeLa-dluc cells for luciferase silencing; hemolysis assay for fusogenicity. - In vivo: ICR mice for FVII silencing, intrahepatic distribution, RISC loading, lipid quantification; ApoE-deficient mice to test ApoE/LDLR dependence; repeated-dose toxicity in ICR mice. - Doses: FVII silencing 0.001–0.1 mg siRNA/kg; intrahepatic imaging 0.5 mg/kg; lipid quantification 1 mg/kg; single-dose toxicity 7 mg/kg; repeated dosing 0.3 or 1 mg/kg twice weekly for 4 weeks. - Controls: Prior LNPs YSK05, YSK13-C3; MC3-LNPs; untreated; ApoE-deficient vs wild-type.

Key methods: TNS assay for apparent pKa. - FVII chromogenic activity assay for hepatic gene silencing. - Confocal microscopy for intrahepatic distribution and cytosolic siRNA delivery. - HIT qRT-PCR for siRNA quantification in liver. - Ago2 immunoprecipitation + qRT-PCR for RISC-loaded siRNA. - LC/MS for lipid and metabolite quantification in liver/spleen. - ALT/AST, hematology, and histopathology for toxicity.

Key results: Hydrophilic head group structure strongly affected apparent LNP pKa (range ~4.5–8.2); clogP of the head group correlated with apparent pKa. Optimal in vivo FVII silencing occurred at pKa ~6.3. - Hydrophobic tail structure strongly affected intrahepatic distribution and in vivo silencing, independent of pKa. Unsaturated or branched tails (A6, F6, G6, H6) promoted hepatocyte accumulation; saturated linear tails (C6, D6) did not. - Lead CL4H6-LNPs achieved FVII silencing ED50 = 0.0025 mg siRNA/kg, superior to YSK05 (0.06 mg/kg) and YSK13-C3 (0.015 mg/kg). - CL4H6-LNPs delivered siRNA into cytoplasm; RISC-loaded siRNA was 4.58 ng/g liver (~4.2% of accumulated siRNA), significantly higher than YSK05 (~0.28 ng) and YSK13-C3 (~1.09 ng). - CL4H6 degraded rapidly in liver/spleen; at 24 h only 2.85 nmol/liver remained. Single-dose 7 mg/kg caused minimal ALT/AST elevation, unlike YSK13-C3 (>10,000 IU/L). Repeated dosing up to 1 mg/kg twice weekly for 4 weeks showed no significant toxicity.

Interpretation: The study establishes separable SAR rules for pH-sensitive cationic lipids: head groups tune apparent pKa, while tails tune intrahepatic distribution. CL4H6 is a potent, biodegradable, and well-tolerated lipid for hepatocyte siRNA delivery, with high endosomal escape and RISC loading. This enables rational design of next-generation siRNA-LNPs.

Limitations: Mouse studies only; no nonhuman primate or human validation. - Efficacy mainly shown with FVII silencing, not a disease model. - Toxicity studies used healthy mice and limited endpoints; chronic toxicity and immunogenicity not fully assessed. - The mechanism by which hydrophobic tails alter biodistribution is not fully resolved; protein corona involvement is proposed but not directly demonstrated. - No formal Design of Experiments (DoE) to statistically test head–tail interactions. - CL4H6-LNP formulation may require further optimization for clinical translation.