Controlled preparation of lipid nanoparticles in microreactors: Mixing time, morphology and mRNA delivery

Microreactors are essential tools for the production of lipid nanoparticles (LNPs), offering precise control over particle size and monodispersity. However, the effect of microchannel geometry on the physicochemical and biological characteristics of LNPs remains unclear. Here, we systematically studied mRNA-loaded LNPs produced using two representative microreactors including a T-junction microreactor and a ring microreactor. Under optimized mixing conditions, both microreactors yielded mRNA-loaded LNPs with comparable sizes, mRNA encapsulation efficiencies, and proportions of empty LNPs. Notably, significant differences emerged in the prevalence of LNPs with bleb structures (bleb LNPs). The T-junction microreactor produced a higher proportion of bleb LNPs, associated with slightly enhanced mRNA integrity, while the ring microreactor predominantly formed spherical LNPs. Based on experimental and simulation results, a mechanism for the formation of the bleb structure was proposed. In vitro and in vivo studies demonstrated that spherical and small LNPs exhibit more efficient transfection, whereas bleb LNPs sustain prolonged mRNA expression in vivo. Fluorescent labeling experiments further highlighted enhanced cellular uptake and rapid liver accumulation of spherical LNPs. These findings provide valuable insights for designing and optimizing microreactors tailored to specific therapeutic and developmental needs.