Robust, Scalable Microfluidic Manufacturing of RNA-Lipid Nanoparticles Using Immobilized Antifouling Lubricant...

Despite the numerous advantages demonstrated by microfluidic mixing for RNA-loaded lipid nanoparticle (RNA-LNP) production over bulk methods, such as precise size control, homogeneous distributions, higher encapsulation efficiencies, and improved reproducibility, their translation from research to commercial manufacturing remains elusive. A persistent challenge hindering the adoption of microfluidics for LNP production is the fouling of device surfaces during prolonged operation, which significantly diminishes performance and reliability. The complexity of LNP constituents, including lipids, cholesterol, RNA, and solvent mixtures, makes it difficult to find a single coating that can prevent fouling. To address this challenge, we propose using an immobilized liquid lubricant layer of perfluorodecalin (PFD) to create an antifouling surface that can repel the multiple LNP constituents. We apply this technology to a staggered herringbone microfluidic (SHM) mixing chip and achieve >3 h of stable operation, a >15× increase relative to gold standard approaches. We also demonstrate the compatibility of this approach with a parallelized microfluidic platform that incorporates 256 SHM mixers, with which we demonstrate scale up, stable production at L/h production rates suitable for commercial scale applications. We verify that the LNPs produced on our chip match both the physiochemical properties and performance for both in vitro and in vivo mRNA delivery as those made on chips without the coating. By suppressing surface fouling with an immobilized liquid lubricant layer, this technology not only enhances RNA-LNP production but also promises to transform the microfluidic manufacturing of diverse materials, ensuring more reliable and robust processes.