Continuous and size-control synthesis of lipopolyplex nanoparticles enabled by controlled micromixing performance for mRNA delivery

Accurate control of core–shell lipopolyplex nanoparticles (LPP NPs) size is crucial for finely adjusting their biomedical performance. However, the synthesis of LPP NPs encounters challenges as two mixing-sensitive processes are involved in the synthesis, rendering precise control over particle size difficult using conventional batch methods. In this study, the formation of the nucleic acid/cationic polymer cores through electrostatic complexation and the subsequent encapsulation by lipid shells via self-assembly were conducted in microreactors, with polyadenylic acid (poly A) and branched polyethylenimine (bPEI) employed as the model system. By assessing the micromixing performance of the microreactors using the Villermaux-Dushman method, the characteristic time scale for electrostatic complexation between poly A and bPEI, as well as the self-assembly of lipids, was determined to be below 1 ms. The Reynolds number, governing micromixing performance, emerged as a crucial factor influencing the sizes of poly A/bPEI cores and LPP NPs. In the kinetic control region, characterized by rapid mixing, the size of poly A/bPEI remained slightly influenced by the N/P molar ratio and volumetric flow rate ratio, irrespective of concentration. The zeta potential, however, was primarily affected by the N/P molar ratio. In the case of LPP NPs, under optimized conditions of anionic lipid molar ratio, the size of LPP NPs was significantly influenced by the composition of lipid shells. This study establishes the foundation for elucidating the structure–activity relationship of LPP NPs.