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-É) 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.
Subject(s)
COVID-19 , Nanoparticles , Animals , Anti-Inflammatory Agents/pharmacology , Liposomes , Mice , Nanoparticles/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.