Nanopore-Mediated Assembly Enables Precise and Continuous Synthesis of mRNA-Encapsulated Lipid Nanoparticles for...

RNA therapeutics are reshaping modern medicine, as exemplified by the rapid deployment of lipid nanoparticle (LNP)-based mRNA vaccines during the COVID-19 pandemic. However, the ability to reproducibly generate LNPs with precisely defined physicochemical properties at scale remains a critical challenge, particularly as the particle size and size distribution strongly influence biodistribution, cellular uptake, and therapeutic efficacy. Conventional mixing technologies ensure reproducibility but offer limited control over interfacial mixing and nanoparticle assembly, constraining both tunability and scalability. We present a hollow fiber membrane (HFM)-based platform that leverages dense arrays of nanoscale pores to mediate uniform, highly localized interfacial mixing, enabling controlled lipid self-assembly and RNA encapsulation. This nanopore-mediated architecture allows continuous, high-throughput synthesis of LNPs with tunable particle sizes, narrow size distributions, and high encapsulation efficiencies, with particle characteristics directly correlated to the membrane pore size. HFM-derived LNPs exhibit robust in vitro transfection and potent in vivo immune responses and are compatible with multiple lipid chemistries and nucleic acid payloads, including mRNA vaccine constructs. Together, this work establishes HFM-based nanopore assembly as a versatile and scalable approach for producing well-defined LNPs, with direct relevance to the current and future mRNA vaccine and therapeutic development.