The mixing method used to formulate lipid nanoparticles affects mRNA delivery efficacy and organ tropism.

mRNA is a versatile drug molecule with therapeutic applications ranging from protein replacement therapies to in vivo gene engineering. mRNA delivery is often accomplished using lipid nanoparticles, which are formulated via mixing of aqueous and organic solutions. Although this has historically been accomplished by manual mixing for bench scale science, microfluidic mixing is required for scalable continuous manufacturing and batch to batch control. Currently, there is limited understanding on how the mixing process affects mRNA delivery efficacy, particularly in regard to tropism. To address this knowledge gap, we examined the influence of the type of mixing and microfluidic mixing parameters on the performance of lipid nanoparticles in mice. This was accomplished with a Design of Experiment approach using four nanoparticle formulations with varied ionizable lipid chemistry. We found that each formulation required unique optimization of mixing parameters, with the total delivery efficacy of each lipid nanoparticle generated with microfluidics ranging from 100-fold less to 4-fold more than manually mixed LNPs. Further, mixing parameters influenced organ tropism, with the most efficacious formulations disproportionately increasing liver delivery compared to other organs. These data suggest that mixing parameters for lipid nanoparticle production may require optimization for each unique chemical formulation, complicating translational efforts. Further, microfluidic parameters must be chosen carefully to balance overall mRNA delivery efficacy with application-specific tropism requirements.