Mass production system for RNA-loaded lipid nanoparticles using piling up microfluidic devices

Microfluidic devices are widely used in lipid nanoparticle (LNP)-based vaccines and nanomedicine research. These devices should be stiff enough to withstand the high flow rate for the mass production of LNPs, and malleable enough to use when fabricating complicated microchannel or micromixer structures, such as staggering herringbone micromixers. Due to the limitations of the available fabrication methods, optimal microfluidic devices have not yet been developed. In this study, we report the development of a glass-based microfluidic device based on the invasive Lipid Nanoparticle Production (iLiNP) device® reported previously. The LNP size controllability of glass-based iLiNP device was similar to that of the poly(dimethylsiloxane) (PDMS)-based iLiNP device, and the glass-iLiNP device was used for mRNA-loaded LNP production with ionizable lipids used for COVID-19 mRNA vaccines. We also demonstrate a piling- and numbering-up strategy based on glass-iLiNP device. The iLiNP unit composed of five-layered microchannels was fabricated by piling-up each glass-iLiNP device followed by parallelization (numbering-up) for the mass production of LNPs. This iLiNP system can produce LNPs with sizes ranging between 20 and 60 nm at a flow rate of 20–50 mL/min, and its performance is comparable to that of the commercially available microfluidic system like NanoAssemblr®.