Ionizable lipid nanoparticles (LNPs) have emerged as a highly effective platform for nucleic acid delivery, necessitating high-throughput formulation (HTF) strategies to identify top-performing LNPs from a large number of formulations. However, conventional LNP formulation methods, such as manual pipetting and microfluidic mixing, pose challenges for HTF, such as batch-to-batch variability and difficulty in parallelizing formulations. Herein, we present HTF using the fast lipid nanoparticle assembly via simple thermomixing (HTF-FLASH) technique with a thermomixer. Using a 96-deep-well plate and a multichannel pipette, this approach enables rapid and uniform production of multiple LNPs within a minute, accelerating LNP screening workflows. We demonstrate that LNPs formulated via HTF-FLASH exhibit physicochemical properties and mRNA transfection efficiencies, bothand, comparable to those of microfluidic-formulated LNPs. Our findings highlight HTF-FLASH as a high-throughput and efficient alternative to traditional formulation methods, offering a rapid, reproducible, and cost-effective strategy for high-throughput LNP screening. in vitro in vivo
