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Updated: Feb 12, 2026

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing
Published on: January 20, 2023
Lipid nanoparticles containing zwitterionic lipids versatilely enhance the efficiency of mRNA delivery
Yuichi Suzuki1, Yuma Yamada2, Hideyoshi Harashima3
1Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Abstract:
Ionizable lipids enable lipid nanoparticles (LNPs) to deliver nucleic acids, including mRNA, to target cells. However, the efficiency of the endosomal escape of conventional LNPs remains limited to a small percentage. Lipid components that actively interact with net-neutral phospholipids in endosomal membranes and promote membrane fusion potentially enhance the efficiency of endosomal escape and further improve functional mRNA delivery. In this study, we developed a zwitterionic tri-oleoyl-Tris (zTOT) library and used it to enhance mRNA delivery by replacing the widely used helper lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in LNPs with zTOT. LNPs containing TOT-15, an example of zTOT, demonstrated a 5-fold higher gene expression compared with that of DSPC-LNPs. TOT-15-LNPs composed of commercially available ionizable lipids showed enhanced gene expression compared with that of DSPC-LNPs. The TOT-15-LNPs also showed almost 100% gene knockout efficiency with no toxicity. The TOT-15 system interacts with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and induces a non-lamellar structure. Pharmacokinetic calculation revealed that endosomal escape efficiency of the TOT-15-LNPs was more than 9.6%. In summary, the results of this study demonstrate that the zTOT system enhances the efficiency of both endosomal escape and functional mRNA delivery.
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