Design, optimization, and evaluation of lyophilized lipid nanoparticles for mRNA-based pulmonary mucosal vaccination

Lipid nanoparticles (LNP) have emerged at the forefront of the delivery of RNA molecules during the COVID-19 pandemic, leading to a giant leap in RNA therapies. Despite their great success, the long-term storage and transportation of mRNA vaccines without ultra-low temperatures is still challenging due to their poor stability. Here, we demonstrated that LNP-mRNA could be lyophilized via a simple freeze‒drying process. This process produced a dry powder formulation that could maintain the physicochemical properties of LNP-mRNA after storage at 4 °C for at least two months. However, the shear forces generated during the lyophilization process may disrupt the structure of the LNP, affecting the efficacy of the vaccine. Therefore, a cholesterol analogue, β-sitosterol, and a type of phospholipid, DOPE, were utilized instead of cholesterol and DSPC to improve the transfection efficiency after freeze-drying. The optimized formulation of LNP exhibited an enhanced transfection effect both <i>in vitro</i> and in vivo. Additionally, intratracheal administration of reconstituted lyophilized LNPs could induce innate cellular, humoral and mucosal immunity in vivo, indicating that our LNP-mRNA may serve as an effective vaccine against COVID-19. In summary, our study revealed that lyophilization of LNPs could increase their stability and maintain their ability to be transfected both <i>in vitro</i> and in vivo, inducing strong immune responses.