Admixing of mRNA with Pre‐Formed Lipid Nanoparticles Containing a Slightly‐Cationic Ionizable Lipid Allows for Efficient mRNA Transfection In Vitro and In Vivo

Abstract Therapeutic mRNA has emerged as a powerful tool in medicine. However, due to its fragility and large size, mRNA requires a carrier for delivery into the cellular cytosol. Lipid nanoparticles (LNPs), produced by rapidly mixing an aqueous mRNA solution with an ethanolic solution containing lipids, are currently considered the most advanced carriers for this purpose. Electrostatic interactions between mRNA and the ionizable cationic lipid, combined with hydrophobic interactions among all lipids, lead to self‐assembly into LNPs that accommodate the mRNA in their core. In this study, whether mixing mRNA with pre‐formed, empty LNPs (eLNPs) in an aqueous medium can be a viable alternative for mRNA expression is investigated. It is confirmed that mRNA can associate with eLNPs via electrostatic interactions, with the effectiveness of this association depending on the surface charge of the eLNPs and the ionizable lipid component. Furthermore, post‐loading mRNA into eLNPs demonstrates mRNA expression levels comparable to conventional LNP(mRNA) formulations, both in vitro and in mice. This method of leveraging eLNPs offers a practical alternative to conventional LNP(mRNA) formulation for the rapid screening of multiple mRNAs. It can also enable straightforward use of LNPs for mRNA transfection by users who do not have the capacity to perform LNP formulation.