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Interest in mRNA vaccines and therapeutic drugs continues to grow. While effective for infectious disease immunity, lipid nanoparticle (LNP) formulations for other mRNA delivery applications face limitations such as non-targeted accumulation, poor immunotransfection, and reactogenicity, restricting their use in immunoengineering. The development of new low-density mRNA nucleases has historically had low throughput due to reliance on low-frequency measurements, severely limiting the development of new mRNAs. We have developed a high-throughput in vivo mRNA LNP screening platform based on barcode mRNA (b-mRNA). Using this b-mRNA screening platform, we simultaneously evaluated 122 LNPs and identified novel LNP formulations with efficient extrahepatic transfection capabilities. We assessed a lead LNP candidate for in situ immune modulation, which performed exceptionally in a syngeneic mouse model of melanoma, showing significantly reduced tumor burden and prolonged survival compared to mice treated with the gold-standard mRNA LNP formulation. We employed a novel biochemical characterization technique to analyze nanoparticle protein corona formation at single-particle resolution, providing insights into how protein adsorption affects transfection in the liver and spleen. Together, our results demonstrate the value of advanced LNP screening and characterization technologies in developing next-generation mRNA LNPs for immunoengineering.
