Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration

Commun Biol. 2021 Aug 11;4(1):956. doi: 10.1038/s42003-021-02441-2.


Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

MeSH terms

  • Administration, Intravenous
  • Animals
  • Drug Compounding
  • Gene Expression*
  • Humans
  • Hydrogen-Ion Concentration
  • Injections, Intramuscular
  • Ions / chemistry*
  • Lipids / chemistry*
  • Mice
  • Molecular Structure
  • Nanoparticles / chemistry*
  • Nanoparticles / ultrastructure
  • RNA, Messenger / administration & dosage
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics*
  • RNA, Messenger / pharmacokinetics
  • Spectrum Analysis
  • Tissue Distribution
  • Transfection


  • Ions
  • Lipids
  • RNA, Messenger