PEG-OligoRNA Hybridization of mRNA for Developing Sterically Stable Lipid Nanoparticles toward In Vivo Administration
Shota Kurimoto, Naoto Yoshinaga, Kazunori Igarashi, Yu Matsumoto, Horacio Cabral, and Satoshi Uchida|Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan, Department of Otorhinolaryngology and Head and Neck Surgery, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan|2019|Molecules|24:1303 doi:10.3390/molecules24071303
Lipid nanoparticles (LNPs) are widely used to prevent mRNA degradation for in vivo mRNA delivery to target cells. One drawback for clinical use of LNPs is aggregation in the buffer due to the highly charged anionic nature of mRNA. In this study, a simple and robust strategy of LNP preparation was developed. Polyethylene glycol (PEG) was installed onto mRNA molecules to prevent LNP aggregation as PEG has steric repulsive forces between them. This allows mixing of two aqueous solutions of LNPs and mRNA without the need of special devices or buffer replacements. Hybridization of mRNA with PEG-OligoRNA (17 nucleotide sequence containing a 12 kDa PEG chain on the 5’ end) was performed and the efficiency was measured using gel permeation chromatography (GPC). The PEG-OligoRNAs/mRNA was then mixed by pipetting with lipofectamine LTX, a lipid-based transfection reagent. The efficiency of mRNA cellular uptake with PEG-OligoRNA mRNAs vs unPEGlayted mRNAs was then evaluated by measuring GLuc mRNA amount in the cells by qRT-PCR performed on the Mic Cycler. The study found that there was minimal influence on translation activity whilst exhibiting high structural stability in blood when hybridizing mRNA with PEGylated short RNA oligonucleotides.