Self-assembly of RNA lipid nanoparticles studied using isothermal titration calorimetry, dynamic light scattering and microscopy

Abstract The self-assembly of lipid nanoparticle (LNP) was achieved using slow pulsed addition of the ionizable lipid SM-102 into a solution containing RNA. The reaction was observed using isothermal titration calorimetry (ITC), dynamic light scattering (DLS), light and transmission electron microscopy. The LNP self-assembly was a thermodynamically controlled process and different from kinetically controlled LNP production in microfluidic devices. LNP self-assembly has three discrete stages, the first two were clearly illustrated in the ITC curve. The first stage of the interaction was exothermic and agreed with the electrostatic interaction between ionizable lipid SM-102 and negatively charged RNA. Microscopic observation showed that this was followed by a liquid–liquid phase separation. The second stage of the interaction was endothermic and entropy driven. This was consistent with hydrophobic interaction between the alkyl tails of SM-102 and the alkyl tails in the SM-102/tRNA complex/coacervate. The third stage was not detected by ITC but DLS was able to measure the reduction in the nanoparticle hydrodynamic diameter ( D h ) and the polydispersity index. The D h of the coacervate was around 1,000 nm which decreased until the LNPs have a D h around 150 nm. The reduction in D h reflected spontaneous emulsification, driven by negative surface tension caused by the addition of SM-102 at the coacervate–water interface.