Automated and Parallelized Microfluidic Generation of Large and Precisely Defined Lipid Nanoparticle Libraries

Lipid
nanoparticles (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 remains the
primary rate-limiting step during early-stage discovery. In this work,
we present a parallelized, automated microfluidic platform capable
of generating large, precisely defined LNP libraries in parallel,
with throughput on the order of 1000 distinct formulations per hour.
Each formulation is defined by varying the reagent flow ratios into
one of eight microscale mixers using lithographically encoded fluidic
resistors and dynamically controlled external pressure supplies. The
microfluidic chip is integrated with custom robotic plate handling
for the rapid collection of each distinct formulation. To evaluate
this platform, we characterized 96 formulations generated on-chip
in terms of both physicochemical properties and transfection efficiency
in vitro. We further validated our lead candidate against the state
of the art in vivo. We demonstrate the ability to rapidly discover
a formulation and scale its production to liters per hour under identical
mixing conditions, bridging from early discovery to manufacturing
through microfluidic parallelization.