Rational design of anti-inflammatory lipid nanoparticles for mRNA delivery

Purpose: LNPs can trigger innate immune responses and inflammation, which can suppress mRNA translation and cause adverse effects. There is a need for LNP formulations that reduce LNP-induced inflammation while maintaining or improving mRNA delivery.

Hypothesis: Partially substituting cholesterol with dexamethasone (Dex), a corticosteroid structurally similar to cholesterol, in MC3 LNPs will reduce LNP-triggered inflammation and improve in vivo mRNA expression.

Aims: Develop Dex-incorporated LNPs by partially replacing cholesterol with Dex. - Characterize LNP size, PDI, zeta potential, and mRNA encapsulation. - Evaluate in vitro transfection, cytotoxicity, and TNF-α suppression in macrophages. - Assess in vivo TNF-α reduction and luciferase mRNA expression in mice.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs) based on DLin-MC3-DMA (MC3). - Components: MC3, DSPC, C14-PEG2000, cholesterol, and dexamethasone. - Lead formulation: C9D1 — cholesterol:dexamethasone molar ratio 9:1; C:D molar percentage 34.65:3.85. - Payload: Luciferase mRNA; N1-methylpseudouridine-modified and purified. - Targeting ligand: None; hepatic delivery via endogenous LNP tropism. - Physicochemical properties: C9D1: ~76.84 nm, PDI ~0.124, zeta potential ~ −0.12 mV, encapsulation efficiency >90%.

Approach: In vitro: HepG2 human hepatoma cells for transfection/viability; RAW264.7 murine macrophages for TNF-α response. - In vivo: C57BL/6 female mice, n = 3 per group: PBS, C10D0 (original MC3 LNP), C9D1 (Dex-incorporated LNP); IV injection of 4 µg luciferase mRNA; serum TNF-α at 20 h; IVIS imaging at 20 h. - Dex dose in vivo: 0.62 µg per mouse for C9D1. - Controls: Untreated cells/mice and original C10D0 LNP.

Key methods: Dynamic light scattering (DLS) for size, PDI, zeta potential. - RiboGreen assay for mRNA encapsulation efficiency. - Luciferase assay for transfection. - CellTiter-Glo for viability. - ELISA for TNF-α in RAW264.7 supernatant and mouse serum. - IVIS imaging for in vivo luciferase expression.

Key results: C9D1 and C10D0 had similar size, PDI, and >90% mRNA encapsulation; Dex substitution did not impair in vitro transfection or increase cytotoxicity. - Higher Dex substitution (C7D3, C5D5, C3D7, C0D10) reduced encapsulation efficiency and transfection, so C9D1 was selected as optimal. - In RAW264.7 cells, C10D0 increased TNF-α ~2.6-fold, while C9D1 only increased it ~1.2-fold, near untreated levels. - In mice, C10D0 significantly increased serum TNF-α vs untreated; C9D1 significantly reduced TNF-α compared with C10D0. - In vivo luciferase expression was 1.5-fold higher with C9D1 than C10D0.

Interpretation: Dex-incorporated LNPs (C9D1) suppress LNP-induced TNF-α both in vitro and in vivo and improve hepatic mRNA expression by 1.5-fold. The authors propose this as a promising strategy to reduce inflammation-related adverse effects and enhance mRNA therapeutic potency, potentially avoiding systemic anti-inflammatory premedication.

Limitations: Only one ionizable lipid (MC3) and one mRNA cargo (luciferase) were tested. - In vivo studies used small groups (n = 3) and a short 20 h endpoint. - No disease model, repeated dosing, long-term safety, or therapeutic efficacy endpoint. - No large-animal validation. - The mechanism by which Dex reduces inflammation and improves transfection was not deeply dissected. - Effects on other immune cells, tissues, or off-target inflammation were not comprehensively assessed.