A Post-Encapsulation Method for the Preparation of mRNA-LNPs via the Nucleic Acid-Bridged Fusion of mRNA-Free LNPs
Hiroki Tanaka, Yuka Sato, Tomoya Nakabayashi, Akira Tanaka, Kazuma Nishio, Chika Matsumoto, Atsuya Matsumaru, Takuma Yamakawa, Kota Ishizaki, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Yuta Nakai, Kota Tange, Hidetaka Akita
Abstract
Lipid nanoparticles with encapsulated mRNA (mRNA-LNPs) have become key modalities for personalized medicines and RNA vaccines. Once the platform technology is established, the mRNA-LNPs could be applicable to a variety of protein-based therapeutic strategies. A post-encapsulation method, in which the mRNA solution is incubated with preformed mRNA-free LNPs to prepare the mRNA-LNPs, would accelerate the development of RNA-based therapeutics since even nonexperts could manufacture the mRNA-LNPs. In this study, we describe that the post-encapsulation of mRNA into mRNA-free LNPs is accompanied by "nucleic acid-bridged fusion" of them. The adsorption of mRNA onto mRNA-free LNPs via electrostatic interactions and the internalization of mRNA into the LNPs via particle-to-particle fusion are two steps that occur at different levels of pH. To complete post-encapsulation using only one-step mixing, the pH must be controlled within a limited region where both processes occur simultaneously. The size of the mRNA-free LNPs determines the effectiveness of mRNA loading.