Process intensification of lipid nanoparticle manufacturing via vortex-enhanced flow synthesis: from design to functional mRNA delivery

Lipid nanoparticles (LNPs) are widely used as delivery systems for nucleic acid therapeutics. However, reproducible manufacturing at elevated throughput remains challenging. Microfluidic platforms can be scaled through higher-flow operation or numbering-up of parallel channels. Alternative reactor geometries that intensify mixing at larger channel dimensions are therefore of complementary interest. In this study, a vortex tube reactor (VTR)–based flow chemistry platform was developed for the production of nucleic acid–loaded LNPs. A design of experiments (DoE) approach was applied to systematically investigate the effects of total flow rate and organic-to-aqueous flow rate ratio on key physicochemical properties, including hydrodynamic diameter, polydispersity index, zeta potential, entrapment efficiency, and productivity. The optimized formulation produced LNPs with controlled particle size and high entrapment efficiency (>90%), while substantially improving production throughput relative to a conventional batch synthesis. Under the single condition tested, the optimized parameters proved partially transferable from DNA to mRNA. Particle size, polydispersity, zeta potential, loading capacity and productivity were comparable, although entrapment efficiency was significantly lower for mRNA. The optimized conditions were further evaluated using eGFP mRNA in HEK293T cells. Biological evaluation demonstrated measurable intracellular delivery and eGFP protein expression, confirming delivery of translationally competent mRNA. Taken together, the findings demonstrate that the VTR-based platform produces LNPs with tunable properties and functional biological activity at substantially higher throughput than the batch synthesis. The integration of vortex-induced mixing with statistical optimization provides a systematic strategy for advancing the manufacturing of nucleic acid therapeutics and vaccine delivery systems.