Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air–liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.
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This work was supported by the NIH (grant numbers UG3HL147367 and R01 HL162564-02 to A.Y.J., F.E., C.M. and D.G.A.) and Sanofi (formerly Translate Bio, to I.O.R., Y.H., R.S.M. and D.G.A.). J.W. was supported by the Cystic Fibrosis Foundation under award WITTEN19XX0. S.M. and F.A.O. were supported by the MIT Undergraduate Research Opportunities Program. We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically the Animal Imaging & Preclinical Testing, Histology, Nanotechnology Materials, BioMicro Center and Microscopy core facilities. This work was also supported in part by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute.
A.Y.J., J.W., I.O.R. and D.G.A. have filed a patent for the biodegradable lipid library described herein (US Patent Application No. 18080299). D.G.A. receives research funding from Sanofi/Translate Bio, and is a founder of oRNA Tx. R.L. is co-founder and a director of Moderna. He also serves on the board and has equity in Particles For Humanity. For a list of entities with which R.L. is, or has been recently involved, compensated or uncompensated, see https://www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0. The other authors declare no competing interests.
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Jiang, A.Y., Witten, J., Raji, I.O. et al. Combinatorial development of nebulized mRNA delivery formulations for the lungs. Nat. Nanotechnol. (2023). https://doi.org/10.1038/s41565-023-01548-3