Optimization of formulation and atomization of lipid nanoparticles for the inhalation of mRNA

Lipid nanoparticles (LNPs) have demonstrated efficacy and safety for mRNA vaccine administration by intramuscular injection; however, the pulmonary delivery of mRNA encapsulated LNPs remains challenging. The atomization process of LNPs will cause shear stress due to dispersed air, air jets, ultrasonication, vibrating mesh etc., leading to the agglomeration or leakage of LNPs, which can be detrimental to transcellular transport and endosomal escape. In this study, the LNP formulation, atomization methods and buffer system were optimized to maintain the LNP stability and mRNA efficiency during the atomization process. Firstly, a suitable LNP formulation for atomization was optimized based on the in vitro results, and the optimized LNP formulation was AX4, DSPC, cholesterol and DMG-PEG_2K at a 35/16/46.5/2.5 (%) molar ratio. Subsequently, different atomization methods were compared to find the most suitable method to deliver mRNA-LNP solution. Soft mist inhaler (SMI) was found to be the best for pulmonary delivery of mRNA encapsulated LNPs. The physico-chemical properties such as size and entrapment efficiency (EE) of the LNPs were further improved by adjusting the buffer system with trehalose. Lastly, the in vivo fluorescence imaging of mice demonstrated that SMI with proper LNPs design and buffer system hold promise for inhaled mRNA-LNP therapies.