Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries

Abstract Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and in vivo screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds. Each formulation is defined by varying the reagent flow ratios into one of eight microscale mixers using litho-graphically encoded fluidic resistors and dynamically controlled external pressure supplies. The microfluidic chip is integrated with custom frobotic plate handling for the rapid collection of each distinct formulation. Using this platform, we produce a library of 96 formulations, which we profile physicochemically and evaluate in terms of both in vitro and in vivo transfection. Competing Interest Statement A.R.H., S.J.S., M.J.M., and D.A.I. are inventors on a patent filed by the Trustees of the University of Pennsylvania (US Provisional Patent Application No. 63/717,568, filed November 7, 2024) describing the parallelized microfluidic generation of many nanoparticle formulations described herein. D.A.I. is a founder and holds equity in InfiniFluidics.