Rational design of anti-inflammatory lipid nanoparticles for mRNA delivery

Abstract

Lipid nanoparticles (LNPs) play a crucial role in delivering messenger RNA (mRNA) therapeutics for clinical applications, including COVID-19 mRNA vaccines. While mRNA can be chemically modified to become immune-silent and increase protein expression, LNPs can still trigger innate immune responses and cause inflammation-related adverse effects. Inflammation can in turn suppress mRNA translation and reduce the therapeutic effect. Dexamethasone (Dex) is a widely used anti-inflammatory corticosteroid medication that is structurally similar to cholesterol, a key component of LNPs. Here, we developed LNP formulations with anti-inflammatory properties by partially substituting cholesterol with Dex as a means to reduce inflammation. We demonstrated that Dex-incorporated LNPs effectively abrogated the induction of tumor necrosis factor alpha (TNF-ɑ) in vitro and significantly reduced its expression in vivo. Reduction of inflammation using this strategy improved in vivo mRNA expression in mice by 1.5-fold. Thus, we envision that our Dex-incorporated LNPs could potentially be used to broadly to reduce the inflammatory responses of LNPs and enhance protein expression of a range of mRNA therapeutics.

Keywords: anti-inflammation; dexamethasone; gene delivery; lipid nanoparticles; mRNA.

Figure 1.. Chemical structures of cholesterol and dexamethasone and schematic illustration of anti-inflammatory LNP to reduce adverse effects and improve mRNA transfection.

a) Chemical structures of cholesterol (left, MW: 386.65 g/mol) and dexamethasone (right, MW: 392.47 g/mol. b) Anti-inflammatory LNPs suppress the local inflammation caused by LNPs in immune cells leading to reduced adverse effects and enhanced hepatic mRNA transfection. LNPs are proposed to stimulate immune cells such as macrophages. Dex can reduce the release of pro-inflammatory cytokines (e.g., TNF-ɑ), and thus improve hepatic transfection and minimize the adverse effects of LNPs.

Figure 2.. Formulation of Dex-incorporated LNPs via microfluidic mixing.

mRNA is dissolved in the aqueous phase while PEG conjugated lipid (C14PEG-2000), MC3, DSPC, cholesterol, and dexamethasone are dissolved in the organic phase. The two solutions are rapidly mixed in a microfluidic device to form mRNA-LNPs.

Figure 3.. Hydrodynamic size of LNPs measured by Dynamic Light Scattering (DLS)

a) Intensity-based size distribution of C10D0 LNP. b) Intensity-based size distribution of C9D1 LNP. Three representative technical replicates result for each LNP are shown.

Figure 4.. In vitro luciferase expression and cell viability in HepG2 cells, and TNF-ɑ levels in RAW246.7 cells, following treatment with mRNA-LNPs incorporating Dex.

a) In vitro luciferase mRNA transfection in HepG2 cells 24 h after treatment. b) Cell viability of HepG2 cells 24 h after treatment. c) TNF-ɑ production in RAW246.7 cells 24 h after treatment. Data are presented as mean ± SD (n = 3). n.s., non-significant, ***P<0.001.

Figure 5.. In vitro luciferase expression and cell viability in HepG2 cells following treatment with LNPs formulated at different C:D ratios.

a) In vitro luciferase mRNA transfection in HepG2 cells 24 h post-treatment. b) Cell viability of HepG2 cells 24 h post-treatment. Data are presented as mean ± SD (n = 3).

Figure 6.. In vivo TNF-ɑ levels and mRNA delivery following i.v. treatment with C10D0 and C9D1 LNPs.

a) Serum TNF-α levels following treatment with C10D0 or C9D1 LNPs in mice. Serum was collected 20 h after treatment. Data are presented as mean ± SD (n = 3). *P<0.05. b) In vivo luciferase expression. For each mouse, 4 μg of LNP-formulated luciferase mRNA was i.v. injected. Data are presented as mean ± SD (n = 3).