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Combinatorial development of nebulized mRNA delivery formulations for the lungs

Nature Nanotechnology volume 19, pages 364–375 (2024)Cite this article

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Abstract

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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Fig. 1: Formulation optimization of LNPs for nebulized delivery using DOE.
Fig. 2: Excipients and buffer modifications for improved LNP stability and in vivo delivery.
Fig. 3: Synthesis and screening of a biodegradable lipid library.
Fig. 4: In vivo testing and in vitro–in vivo comparison of top lipids.
Fig. 5: Pharmacokinetics and pharmacodynamics of top-performing LNPs.
Fig. 6: Evaluating functional mRNA delivery to lung epithelium with IR-117-17 LNPs in the Ai14 mouse model.

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Data availability

All data that support the findings of this study are provided within the paper and its Supplementary Information. Source data are provided with this paper.

References

  1. Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 1–17 (2017).

    Article  Google Scholar 

  3. Han, X. et al. An ionizable lipid toolbox for RNA delivery. Nat. Commun. 12, 7233 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Qiu, M. et al. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc. Natl Acad. Sci. USA 118, e2020401118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Swingle, K. L., Hamilton, A. G. & Mitchell, M. J. Lipid nanoparticle-mediated delivery of mRNA therapeutics and vaccines. Trends Mol. Med. 27, 616–617 (2021).

    Article  CAS  PubMed  Google Scholar 

  6. Miao, L. et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat. Biotechnol. 37, 1174–1185 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Zhang, X. et al. Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing. Sci. Adv. 6, eabc2315 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Billingsley, M. M. et al. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Lett. 20, 1578–1589 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Riley, R. S. et al. Ionizable lipid nanoparticles for in utero mRNA delivery. Sci. Adv. 7, eaba1028 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sabnis, S. et al. A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 26, 1509–1519 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fenton, O. S. et al. Synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger RNA to B lymphocytes. Adv. Mater. 29, 1606944 (2017).

    Article  Google Scholar 

  12. Liu, J. et al. Fast and efficient CRISPR/Cas9 genome editing in vivo enabled by bioreducible lipid and messenger RNA nanoparticles. Adv. Mater. 31, 1902575 (2019).

    Article  Google Scholar 

  13. Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N. Engl. J. Med. 383, 2603–2615 (2020).

    Article  CAS  PubMed  Google Scholar 

  14. Baden, L. R. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 384, 403–416 (2021).

    Article  CAS  PubMed  Google Scholar 

  15. Gillmore, J. D. et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Cornebise, M. et al. Discovery of a novel amino lipid that improves lipid nanoparticle performance through specific interactions with mRNA. Adv. Funct. Mater. https://doi.org/10.1002/adfm.202106727 (2021).

  17. Barbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R. & Anderson, D. G. The clinical progress of mRNA vaccines and immunotherapies. Nat. Biotechnol. 40, 840–854 (2022).

    Article  CAS  PubMed  Google Scholar 

  18. Chakraborty, C., Sharma, A. R., Bhattacharya, M. & Lee, S.-S. From COVID-19 to cancer mRNA vaccines: moving from bench to clinic in the vaccine landscape. Front. Immunol. 12, 2648 (2021).

    Article  Google Scholar 

  19. Cafri, G. et al. mRNA vaccine-induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer. J. Clin. Invest. 130, 5976–5988 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Oberli, M. A. et al. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy. Nano Lett. 17, 1326–1335 (2017).

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Espeseth, A. S. et al. Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection. NPJ Vaccines 5, 1–14 (2020).

    Article  Google Scholar 

  22. Aliprantis, A. O. et al. A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults. Hum. Vaccines Immunother. 17, 1248–1261 (2021).

    Article  CAS  Google Scholar 

  23. Bahl, K. et al. Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses. Mol. Ther. 25, 1316–1327 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Feldman, R. A. et al. mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials. Vaccine 37, 3326–3334 (2019).

    Article  CAS  PubMed  Google Scholar 

  25. John, S. et al. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity. Vaccine 36, 1689–1699 (2018).

    Article  CAS  PubMed  Google Scholar 

  26. Medina-Magües, L. G. et al. mRNA vaccine protects against zika virus. Vaccines 9, 1464 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Mu, Z., Haynes, B. F. & Cain, D. W. HIV mRNA vaccines—progress and future paths. Vaccines 9, 134 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zabaleta, N., Torella, L., Weber, N. D. & Gonzalez-Aseguinolaza, G. mRNA and gene editing: late breaking therapies in liver diseases. Hepatology https://doi.org/10.1002/hep.32441 (2022).

  29. Robinson, E. et al. Lipid nanoparticle-delivered chemically modified mRNA restores chloride secretion in cystic fibrosis. Mol. Ther. 26, 2034–2046 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Da Silva Sanchez, A., Paunovska, K., Cristian, A. & Dahlman, J. E. Treating cystic fibrosis with mRNA and CRISPR. Hum. Gene Ther. 31, 940–955 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Lai, M. et al. Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia. J. Med. Genet. 53, 242–249 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Paff, T., Omran, H., Nielsen, K. G. & Haarman, E. G. Current and future treatments in primary ciliary dyskinesia. Int. J. Mol. Sci. 22, 9834 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Guan, S., Darmstädter, M., Xu, C. & Rosenecker, J. In vitro investigations on optimizing and nebulization of IVT-mRNA formulations for potential pulmonary-based α-1-antitrypsin deficiency treatment. Pharmaceutics 13, 1281 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zeyer, F. et al. mRNA-mediated gene supplementation of Toll-like receptors as treatment strategy for asthma in vivo. PLoS ONE 11, e0154001 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mays, L. E. et al. Modified Foxp3 mRNA protects against asthma through an IL-10–dependent mechanism. J. Clin. Invest. 123, 1216–1228 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rakhra, K. et al. Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells. Sci. Immunol. 6, eabd8003 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bivas-Benita, M. et al. Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of a DNA vaccine encoding HLA-A*0201-restricted T-cell epitopes of Mycobacterium tuberculosis. Vaccine 22, 1609–1615 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Rajapaksa, A. E. et al. Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization. Respir. Res. 15, 60 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Wu, M. et al. Intranasal vaccination with mannosylated chitosan formulated DNA vaccine enables robust IgA and cellular response induction in the lungs of mice and improves protection against pulmonary mycobacterial challenge. Front. Cell. Infect. Microbiol. 7, 445 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  40. King, R. G. et al. Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 and fully protects mice from lethal challenge. Vaccines 9, 881 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. An, X. et al. Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2. iScience 24, 103037 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kim, Y. C. et al. Strategy to enhance dendritic cell-mediated DNA vaccination in the lung. Adv. Ther. 3, 2000013 (2020).

    Article  CAS  Google Scholar 

  43. Lu, D. & Hickey, A. J. Pulmonary vaccine delivery. Expert Rev. Vaccines 6, 213–226 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Sou, T. et al. New developments in dry powder pulmonary vaccine delivery. Trends Biotechnol. 29, 191–198 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Huang, J. et al. A novel dry powder influenza vaccine and intranasal delivery technology: induction of systemic and mucosal immune responses in rats. Vaccine 23, 794–801 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Minne, A. et al. The delivery site of a monovalent influenza vaccine within the respiratory tract impacts on the immune response. Immunology 122, 316–325 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang, Z. et al. Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine. Nat. Biomed. Eng. 6, 791–805 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Patel, A. K. et al. Inhaled nanoformulated mRNA polyplexes for protein production in lung epithelium. Adv. Mater. 31, 1805116 (2019).

    Article  Google Scholar 

  49. Lokugamage, M. P. et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat. Biomed. Eng. 5, 1059–1068 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wilson, C. Future therapies for cystic fibrosis. Lancet Respir. Med. 10, e75–e76 (2022).

    Article  CAS  PubMed  Google Scholar 

  51. Witten, J., Samad, T. & Ribbeck, K. Selective permeability of mucus barriers. Curr. Opin. Biotechnol. 52, 124–133 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Witten, J. & Ribbeck, K. The particle in the spider’s web: transport through biological hydrogels. Nanoscale 9, 8080–8095 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Cone, R. A. Barrier properties of mucus. Adv. Drug Deliv. Rev. 61, 75–85 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. Lieleg, O. & Ribbeck, K. Biological hydrogels as selective diffusion barriers. Trends Cell Biol. 21, 543–551 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kim, N., Duncan, G. A., Hanes, J. & Suk, J. S. Barriers to inhaled gene therapy of obstructive lung diseases: a review. J. Controlled Release 240, 465–488 (2016).

    Article  CAS  Google Scholar 

  56. Coyne, C. B., Kelly, M. M., Boucher, R. C. & Johnson, L. G. Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am. J. Respir. Cell Mol. Biol. 23, 602–609 (2000).

    Article  CAS  PubMed  Google Scholar 

  57. Kauffman, K. J. et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 7300–7306 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  58. Billingsley, M. M. et al. Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells. Nano Lett. 22, 533–542 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  59. Li, S. et al. Payload distribution and capacity of mRNA lipid nanoparticles. Nat. Commun. 13, 5561 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kauffman, K. J. et al. Rapid, single-cell analysis and discovery of vectored mRNA transfection in vivo with a loxP-flanked tdTomato reporter mouse. Mol. Ther. Nucleic Acids 10, 55–63 (2018).

    Article  CAS  PubMed  Google Scholar 

  61. Ball, R. L., Bajaj, P. & Whitehead, K. A. Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization. Int. J. Nanomed. 12, 305–315 (2017).

    Article  CAS  Google Scholar 

  62. Zhao, P. et al. Long-term storage of lipid-like nanoparticles for mRNA delivery. Bioact. Mater. 5, 358–363 (2020).

    PubMed  PubMed Central  Google Scholar 

  63. Crowe, J. H., Oliver, A. E., Hoekstra, F. A. & Crowe, L. M. Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. Cryobiology 35, 20–30 (1997).

    Article  CAS  PubMed  Google Scholar 

  64. Ohtake, S., Schebor, C., Palecek, S. P. & de Pablo, J. J. Phase behavior of freeze-dried phospholipid–cholesterol mixtures stabilized with trehalose. Biochim. Biophys. Acta Biomembr. 1713, 57–64 (2005).

    Article  CAS  Google Scholar 

  65. Eastman, S. J. et al. Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes. Hum. Gene Ther. 8, 313–322 (1997).

    Article  CAS  PubMed  Google Scholar 

  66. Whitehead, K. A. et al. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 5, 4277 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  67. Liu, S. et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing. Nat. Mater. 20, 701–710 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  68. Pezzulo, A. A. et al. The air–liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. 300, L25–L31 (2011).

    Article  CAS  PubMed  Google Scholar 

  69. Hill, D. B. & Button, B. in Mucins: Methods and Protocols (eds McGuckin, M. A. & Thornton, D. J.) 245–258 (Humana Press, 2012); https://doi.org/10.1007/978-1-61779-513-8_15

  70. Ramachandran, S. et al. Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 305, L23–L32 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Krishnamurthy, S. et al. Manipulation of cell physiology enables gene silencing in well-differentiated airway epithelia. Mol. Ther. Nucleic Acids 1, e41 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Burgel, P.-R., Montani, D., Danel, C., Dusser, D. J. & Nadel, J. A. A morphometric study of mucins and small airway plugging in cystic fibrosis. Thorax 62, 153–161 (2007).

    Article  PubMed  Google Scholar 

  73. Ratjen, F. Cystic fibrosis: the role of the small airways. J. Aerosol Med. Pulm. Drug Deliv. 25, 261–264 (2012).

    Article  PubMed  Google Scholar 

  74. van den Berge, M., ten Hacken, N. H. T., Cohen, J., Douma, W. R. & Postma, D. S. Small airway disease in asthma and COPD: clinical implications. Chest 139, 412–423 (2011).

    Article  PubMed  Google Scholar 

  75. Tiddens, H. A. W. M., Donaldson, S. H., Rosenfeld, M. & Paré, P. D. Cystic fibrosis lung disease starts in the small airways: can we treat it more effectively? Pediatr. Pulmonol. 45, 107–117 (2010).

    Article  PubMed  Google Scholar 

  76. Tatsuta, M. et al. Effects of cigarette smoke on barrier function and tight junction proteins in the bronchial epithelium: protective role of cathelicidin LL-37. Respir. Res. 20, 251 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Maeki, M., Uno, S., Niwa, A., Okada, Y. & Tokeshi, M. Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery. J. Control. Release 344, 80–96 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Cheng, M. H. Y. et al. Induction of bleb structures in lipid nanoparticle formulations of mRNA leads to improved transfection potency. Adv. Mater. https://doi.org/10.1002/adma.202303370 (2023).

  79. Brader, M. L. et al. Encapsulation state of messenger RNA inside lipid nanoparticles. Biophys. J. 120, 2766–2770 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  80. Kulkarni, J. A. et al. On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA. ACS Nano 12, 4787–4795 (2018).

    Article  CAS  PubMed  Google Scholar 

  81. Kulkarni, J. A. et al. Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads. Nanoscale 11, 9023–9031 (2019).

    Article  CAS  PubMed  Google Scholar 

  82. Richardson, S. J., Bai, A., Kulkarni, A. A. & Moghaddam, M. F. Efficiency in drug discovery: liver S9 fraction assay as a screen for metabolic stability. Drug Metab. Lett. 10, 83–90 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Scholte, B. J., Davidson, D. J., Wilke, M. & de Jonge, H. R. Animal models of cystic fibrosis. J. Cyst. Fibros. 3, 183–190 (2004).

    Article  CAS  PubMed  Google Scholar 

  84. McCarron, A., Donnelley, M. & Parsons, D. Airway disease phenotypes in animal models of cystic fibrosis. Respir. Res. 19, 54 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Kim, N. et al. Inhaled gene therapy of preclinical muco-obstructive lung diseases by nanoparticles capable of breaching the airway mucus barrier. Thorax 77, 812–820 (2022).

    Article  PubMed  Google Scholar 

  86. Phillips, J. E., Zhang, X. & Johnston, J. A. Dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system. J. Vis. Exp. https://doi.org/10.3791/55454 (2017).

  87. Beck, S. E. et al. Deposition and expression of aerosolized rAAV vectors in the lungs of rhesus macaques. Mol. Ther. 6, 546–554 (2002).

    Article  CAS  PubMed  Google Scholar 

  88. Woo, C. J. et al. Inhaled delivery of a lipid nanoparticle encapsulated messenger RNA encoding a ciliary protein for the treatment of primary ciliary dyskinesia. Pulm. Pharmacol. Ther. 75, 102134 (2022).

    Article  CAS  PubMed  Google Scholar 

  89. Okuda, K. et al. Secretory cells dominate airway CFTR expression and function in human airway superficial epithelia. Am. J. Respir. Crit. Care Med. 203, 1275–1289 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Carraro, G. et al. Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition. Nat. Med. 27, 806–814 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Hodges, C. A. & Conlon, R. A. Delivering on the promise of gene editing for cystic fibrosis. Genes Dis. 6, 97–108 (2019).

    Article  CAS  PubMed  Google Scholar 

  92. Vanover, D. et al. Nebulized mRNA-encoded antibodies protect hamsters from SARS-CoV-2 infection. Adv. Sci. 9, 2202771 (2022).

    Article  CAS  Google Scholar 

  93. Rhym, L. H., Manan, R. S., Koller, A., Stephanie, G. & Anderson, D. G. Peptide-encoding mRNA barcodes for the high-throughput in vivo screening of libraries of lipid nanoparticles for mRNA delivery. Nat. Biomed. Eng. 7, 901–910 (2023).

    Article  CAS  PubMed  Google Scholar 

  94. Chen, D. et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 134, 6948–6951 (2012).

    Article  CAS  PubMed  Google Scholar 

  95. Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. J. Control. Release 107, 276–287 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Allen Y. Jiang, Jacob Witten, Idris O. Raji.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Allen Y. Jiang, Jacob Witten, Idris O. Raji, Rajith S. Manan, Robert Langer & Daniel G. Anderson

  2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Allen Y. Jiang, Jacob Witten, Idris O. Raji, Corina MacIsaac, Yizong Hu, Rajith S. Manan, Robert Langer & Daniel G. Anderson

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jacob Witten, Sabrina Meng, Favour A. Oladimeji, Robert Langer & Daniel G. Anderson

  4. Department of Anesthesiology, Boston Children’s Hospital, Boston, MA, USA

    Idris O. Raji, Robert Langer & Daniel G. Anderson

  5. Harvard and MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Feyisayo Eweje, Corina MacIsaac, Robert Langer & Daniel G. Anderson

  6. Harvard/MIT MD–PhD Program, Boston, MA, USA

    Feyisayo Eweje

  7. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Robert Langer & Daniel G. Anderson

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Contributions

A.Y.J., J.W. and I.O.R. conceived the project, contributed equally and all reserve the right to list themselves first on their CVs. A.Y.J., J.W., I.O.R., F.E., C.M., S.M., F.A.O., Y.H. and R.S.M. performed the experiments and analysed data. A.Y.J., J.W., R.L. and D.G.A. discussed the results and wrote the paper with input from all authors. R.L. and D.G.A. acquired funding and supervised the project.

Corresponding author

Correspondence to Daniel G. Anderson.

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Competing interests

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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Nature Nanotechnology thanks Dan Peer, Bruno Pitard and the other, anonymous, reviewers for their contribution to the peer review of this work.

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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. 19, 364–375 (2024). https://doi.org/10.1038/s41565-023-01548-3

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