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®.

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery.

In 2021, mRNA vaccines against COVID-19 were approved by the Food and Drug Administration. mRNA vaccines are important for preventing severe COVID-19 and returning to normal life. The development of RNA-delivery technology, including mRNA vaccines, has been investigated worldwide for ~30 years. Lipid nanoparticles (LNPs) are a breakthrough technology that stably delivers RNA to target organs, and RNA-loaded LNP-based nanomedicines have been studied for the development of vaccines and nanomedicines for RNA-, gene-, and cell-based therapies. Recently, microfluidic devices and technologies have attracted attention for the production of LNPs, particularly RNA-loaded LNPs. Microfluidics provides many advantages for RNA-loaded LNP production, including precise LNP size controllability, high reproducibility, high-throughput optimization of LNP formulation, and continuous LNP-production processes. In this review, we summarize microfluidic-based RNA-loaded LNP production and its applications in RNA-based therapy and genome editing.