Formation of RNA lipid nanoparticles: an equilibrium process with a liquid intermediate stage

Abstract

This paper presents evidence that RNA–lipid nanoparticles (LNPs) can be assembled using a slow, thermodynamically controlled process. The lipid mixture used in the Spikevax vaccine was dissolved in 52% (v/v) ethanol and titrated into a solution containing tRNA. The titration was conducted using an isothermal titration calorimeter (ITC) and consisted of 40 injections with enough time between each injection so that equilibrium was reached before the next injection. Liquid phase separation occurred where the RNA–lipid complex had a lipid amine to RNA phosphate (N/P) ratio around 1 : 1. The liquid droplets had a mean hydrodynamic diameter of 1020 ± 150 nm measured using dynamic light scattering (DLS) and were clearly observable by light microscopy. At a N/P ratio of 6 : 1 the LNPs were 109 ± 0 nm and LNPs were observed using transmission electron microscopy. The mechanism for conversion of the liquid intermediate into LNPs is likely to be spontaneous emulsification driven by negative surface tension. This equilibrium process for making RNA–LNPs is very different from current manufacture strategies using rapid single step mixing in a microfluidic device. This approach provides a low shear alternative process that could be adapted to a stirred tank.