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Abstract
Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) Device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL/min range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL/min) of LNPs than commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (> 90%). At a significantly lower cost (US $1.5) compared to commercial instruments, the OSEM device provides an unprecedented all-in-one solution to LNP production from lab to market.
Supplementary materials
Title
Supplementary Information
Description
Plasmid insert sequence encoding SARS-CoV-2 spike protein and PCR primers sequence. Gel electrophoresis results for PCR and transcription products from pDNA encoding SARS-CoV-2 spike protein. Photographs of 3D-printed microfluidic devices featuring a 2-way sheath flow channel followed by a downstream SHM and a 4-way sheath flow channel without downstream mixers. COMSOL Multiphysics simulation results showing ethanol/water hydrodynamic focusing at 5 μL/min total water inlet flow rate. DLS size measurement results of LNPs on the day they were produced vs 30 days after storage at 4°C. Gel electrophoresis results showing RT-PCR of mRNA in LNP after 70 days (stored at 4°C) vs mRNA (stored at −20°C). The OSEM device design in drawings showing detailed design parameters. Figure S8 Dynamic viscosity of ethanol and buffer with and without lipids or mRNA. Experimental results for LNPs produced using 1 vs 4 OSEM devices in parallel.
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