Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

TLDR

Lipid nanoparticles are the leading siRNA delivery systems, yet conventional macroscopic mixing yields 70‑nm or larger particles with variable encapsulation, homogeneity, and reproducibility. The study proposes microfluidic mixing as a scalable, precisely controlled method to formulate LNP siRNA delivery systems. Microfluidic mixing produced 20‑nm LNP with near‑complete siRNA encapsulation, polydispersity as low as 0.02, and achieved 50 % target gene silencing in mouse hepatocytes at 10 µg/kg.

Abstract

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.