Morphology evolution of lipid nanoparticle discovered by small angle neutron scattering

The structure of mRNA lipid nanoparticles (LNPs) is still under debate, with different studies presenting varying morphological characteristics, significantly hindering their biomedical potential. A typical formulation process of mRNA LNPs involves three steps: initial rapid mixing of lipids in an ethanol phase and mRNA in an acidic aqueous phase, followed by the swift removal of ethanol, and finally adjusting the solution to a neutral environment. In this study, we utilize Small Angle Neutron Scattering (SANS) with contrast matching to reveal the kinetic pathway-dependent of mRNA LNPs morphology. We find that the formulation process of the Moderna COVID-19 vaccine is controlled by a competition between aggregation and microphase separation, dictating the diverse morphologies observed in mRNA LNPs. The first step leads to the formation of polydisperse spherical droplets with an average diameter of 42 +/- 6.0 nm in an acidic ethanol aqueous solution. Ethanol removal initiates both aggregation and internal microphase separation, resulting in a polydisperse core-shell structure with an average diameter of 48 +/- 3.7 nm. Heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM102) binds to mRNA via electrostatic interaction to form a reverse-wormlike micelle structure inside. The 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and PEG-lipid are just in the shell and cholesterol acting as a filler throughout the core-shell structure. Upon transitioning to a neutral environment, SM102 loses its charge and neither the periphery nor the reverse-wormlike micelle can maintain their stabilities, leading to further aggregation and microphase separation. The average diameter of core-shell structure turns to be 66 +/- 5.2 nm. In the actual formulation process of the Moderna COVID-19 vaccine, steps 2 and 3 occur simultaneously, and the competition between aggregation and microphase separation determines the final morphology. These findings offer crucial insights into optimizing the morphology of mRNA LNPs, thereby facilitating advancements in vaccine development and mRNA vaccine delivery technologies.