Synthesis and bioactivity of readily hydrolysable novel cationic lipids for potential lung delivery application of mRNAs

Purpose: Systemic LNP delivery predominantly targets the liver, while extrahepatic delivery—especially to lung airway epithelium for diseases such as cystic fibrosis—remains challenging. DOTAP is a common cationic lipid for lung gene delivery but is racemic, pseudo-glyceryl, and slowly biodegradable. Novel readily hydrolysable DOTAP analogues are needed for safer and more effective inhaled/airway mRNA delivery.

Hypothesis: If DOTAP-like cationic lipids are redesigned with achiral scaffolds and ester groups positioned farther from the quaternary ammonium headgroup, then the resulting lipids L1–L4 will retain or improve LNP physicochemical properties, mRNA encapsulation, nebulization stability, and transfection efficiency while showing faster plasma biodegradation than DOTAP.

Aims: Synthesize four novel achiral cationic lipids (L1–L4) as DOTAP analogues. - Formulate cationic LNPs with a proprietary ionizable lipid and TdTomato mRNA. - Characterize LNPs pre-/post-freeze-thaw and pre-/post-nebulization for size, PDI, encapsulation, and mRNA integrity. - Evaluate plasma biodegradability, in vitro transfection/cytotoxicity in CFBE cells, and preliminary in vivo lung expression in mice.

Delivery system: Platform: Cationic lipid nanoparticles (cLNPs). - Components: Proprietary ionizable lipid, phospholipid, cholesterol, PEG2000-DMG, plus cationic lipid DOTAP or L1–L4. - Payload: TdTomato mRNA. - Cationic lipids: DOTAP and novel analogues L1–L4; L1 contains an ether linkage, L2–L4 vary in esterifying alcohol and spacer length. - Targeting ligand: None; lung/airway delivery via cationic lipid and local administration. - Administration: Nebulization in vitro with Aerogen Solo vibrating mesh nebulizer; intratracheal dosing in mice for preliminary in vivo study. - Physicochemical properties: LNPs <100 nm; DOTAP+ and L1–L4 showed >95% mRNA encapsulation; DOTAP− had ~71%.

Approach: In vitro: CFBE410 cells (ΔF508-CFTR) transfected with pre- and post-nebulized LNPs at 1, 0.5, and 0.25 µg/mL; quadruplicate. - Biodegradability: Mouse plasma incubation up to 120 min; LC-MS quantification; T1/2 calculated. - In vivo: Female Balb/C mice, intratracheal dose of LNPs carrying TdTomato mRNA; lungs collected 24 h post-dose for immunohistochemistry. - Controls: DOTAP+ LNP (DOTAP-containing), DOTAP− LNP (no DOTAP), vehicle, untreated, PBS.

Key methods: Dynamic light scattering for particle size and PDI. - RiboGreen assay for mRNA encapsulation efficiency. - Fragment Analyzer capillary electrophoresis for mRNA purity/integrity. - Nebulization and post-nebulization characterization. - LC-MS for plasma lipid degradation and half-life. - In-Cell Western and flow cytometry for TdTomato expression. - CellTag 700 for viability; immunohistochemistry for in vivo TdTomato expression.

Key results: DOTAP+ and L1–L4 LNPs: <100 nm, >95% mRNA encapsulation, high mRNA purity; DOTAP− had ~71% encapsulation. - Freeze-thaw: all formulations retained size, PDI, and encapsulation within ~10% of initial values. - Nebulization: DOTAP− showed decreased encapsulation; DOTAP+ and L1–L4 maintained encapsulation and mRNA integrity. - Plasma half-lives: DOTAP >120 min; L1 8.1 min, L2 11.5 min, L3 8.0 min, L4 9.0 min—substantially faster degradation. - In vitro: DOTAP+ and L1–L4 transfected CFBE cells more efficiently than DOTAP−; dose-dependent TdTomato expression. Post-nebulization retention was better for L3 and L4 than DOTAP+ by heat map. - In vivo IHC: DOTAP+, L2, and L4 showed highest TdTomato expression in mouse airways; L1 and L3 showed minimal expression; PBS control showed no staining.

Interpretation: L1–L4 are readily biodegradable, achiral DOTAP analogues that form stable mRNA-LNPs with comparable in vitro transfection and improved plasma degradation relative to DOTAP. L2 and L4 appear promising for lung airway mRNA delivery. The authors propose these lipids as candidates for inhaled therapies for respiratory diseases such as cystic fibrosis, while noting that further in vivo validation is needed.

Limitations: In vivo study was preliminary and limited to intratracheal dosing in mice; no nebulized in vivo efficacy. - No cystic fibrosis disease model or functional CFTR correction. - Only TdTomato reporter mRNA; no therapeutic mRNA. - Small-scale in vivo characterization mainly by immunohistochemistry. - No long-term toxicity, repeated-dose, biodistribution, or clearance studies. - No large-animal validation. - L1 and L3 showed poor in vivo airway expression; structure–activity relationships require further study. - The paper states that more extensive in vivo studies are needed to validate L1–L4 for inhaled therapies.