Amine headgroups in ionizable lipids drive immune responses to lipid nanoparticles by binding to the receptors TLR4 and CD1d | Nature Biomedical Engineering

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Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicle for RNA therapeutics, partly because of established lipid structure–activity relationships focused on formulation potency. Yet such knowledge has not extended to LNP immunogenicity. Here we show that the innate and adaptive immune responses elicited by LNPs are linked to their ionizable lipid chemistry. Specifically, we show that the amine headgroups in ionizable lipids drive LNP immunogenicity by binding to Toll-like receptor 4 and CD1d and by promoting lipid-raft formation. Immunogenic LNPs favour a type-1 T-helper-cell-biased immune response marked by increases in the immunoglobulins IgG2c and IgG1 and in the pro-inflammatory cytokines tumour necrosis factor, interferon γ and the interleukins IL-6 and IL-2. Notably, the inflammatory signals originating from these receptors inhibit the production of anti-poly(ethylene glycol) IgM antibodies, preventing the often-observed loss of efficacy in the LNP-mediated delivery of siRNA and mRNA. Moreover, we identified computational methods for the prediction of the structure-dependent innate and adaptive responses of LNPs. Our findings may help accelerate the discovery of well-tolerated ionizable lipids suitable for repeated dosing.

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The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets and simulation data are available for research purposes from the corresponding author on reasonable request. Source data for the figures are provided with this paper.

Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 1078–1094 (2021).

Article CAS PubMed PubMed Central Google Scholar

Chaudhary, N., Weissman, D. & Whitehead, K. A. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat. Rev. Drug Discov. 20, 817–838 (2021).

Article CAS PubMed PubMed Central Google Scholar

Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017).

Article CAS Google Scholar

Melo, M. et al. Immunogenicity of RNA replicons encoding HIV Env immunogens designed for self-assembly into nanoparticles. Mol. Ther. 27, 2080–2090 (2019).

Article CAS PubMed PubMed Central Google Scholar

McKay, P. F. et al. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat. Commun. 11, 3523 (2020).

Article CAS PubMed PubMed Central Google Scholar

Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520.e4 (2019).

Article CAS PubMed PubMed Central Google Scholar

Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018).

Article PubMed PubMed Central Google Scholar

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

Article CAS PubMed PubMed Central Google Scholar

August, A. et al. A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus. Nat. Med. 27, 2224–2233 (2021).

Article CAS PubMed PubMed Central Google Scholar

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

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

Tahtinen, S. et al. IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines. Nat. Immunol. 23, 532–542 (2022).

Article CAS PubMed Google Scholar

Kranz, L. M. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396–401 (2016).

Article PubMed Google Scholar

Sahin, U. et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 586, 594–599 (2020).

Article CAS PubMed Google Scholar

Ferraresso, F. et al. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA delivery to hepatocytes and hepatic stellate cells. Mol. Pharm. 19, 2175–2182 (2022).

Article CAS PubMed PubMed Central Google Scholar

Kozma, G. T., Shimizu, T., Ishida, T. & Szebeni, J. Anti-PEG antibodies: properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals. Adv. Drug Deliv. Rev. 154–155, 163–175 (2020).

Article PubMed Google Scholar

Ju, Y. et al. Anti-PEG antibodies boosted in humans by SARS-CoV-2 lipid nanoparticle mRNA vaccine. ACS Nano 16, 11769–11780 (2022).

Article CAS PubMed Google Scholar

Burris, H. A. et al. A phase I multicenter study to assess the safety, tolerability and immunogenicity of mRNA-4157 alone in patients with resected solid tumors and in combination with pembrolizumab in patients with unresectable solid tumors. J. Clin. Oncol. 37, 2523 (2019).

Article Google Scholar

Walsh, E. E. et al. Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates. N. Engl. J. Med. 383, 2439–2450 (2020).

Article CAS PubMed Google Scholar

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

Ndeupen, S. et al. The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience 24, 103479 (2021).

Article CAS PubMed PubMed Central Google Scholar

Kleinman, M. E. et al. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452, 591–597 (2008).

Article PubMed PubMed Central Google Scholar

Hu, B. et al. Therapeutic siRNA: state of the art. Signal Transduct. Target. Ther. 5, 101 (2020).

Article CAS PubMed PubMed Central Google Scholar

Besin, G. et al. Accelerated blood clearance of lipid nanoparticles entails a biphasic humoral response of B-1 followed by B-2 lymphocytes to distinct antigenic moieties. Immunohorizons 3, 282–293 (2019).

Article CAS PubMed Google Scholar

Abu Lila, A. S., Kiwada, H. & Ishida, T. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J. Control. Release 172, 38–47 (2013).

Article CAS PubMed Google Scholar

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

Article CAS PubMed Google Scholar

Jayaraman, M. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. 51, 8529–8533 (2012).

Article CAS Google Scholar

Li, C. et al. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nat. Immunol. 23, 543–555 (2022).

Article CAS PubMed PubMed Central Google Scholar

Arunachalam, P. S. et al. Systems vaccinology of the BNT162b2 mRNA vaccine in humans. Nature 596, 410–416 (2021).

Article CAS PubMed PubMed Central Google Scholar

de Groot, A. M. et al. Immunogenicity testing of lipidoids in vitro and in silico: modulating lipidoid-mediated TLR4 activation by nanoparticle design. Mol. Ther. Nucleic Acids 11, 159–169 (2018).

Article PubMed PubMed Central Google Scholar

Pizzuto, M. et al. Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers. J. Control. Release 247, 182–193 (2017).

Article CAS PubMed PubMed Central Google Scholar

Kedmi, R., Ben-Arie, N. & Peer, D. The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Biomaterials 31, 6867–6875 (2010).

Article CAS PubMed Google Scholar

Ozinsky, A. et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc. Natl Acad. Sci. USA 97, 13766–13771 (2000).

Article CAS PubMed PubMed Central Google Scholar

Park, B. S. & Lee, J. O. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp. Mol. Med. 45, e66 (2013).

Article PubMed PubMed Central Google Scholar

Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

Article CAS PubMed Google Scholar

Okusawa, T. et al. Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by Toll-like receptors 2 and 6. Infect. Immun. 72, 1657–1665 (2004).

Article PubMed PubMed Central Google Scholar

Irvine, K. L., Hopkins, L. J., Gangloff, M. & Bryant, C. E. The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6. Vet. Res. 44, 50 (2013).

Article CAS PubMed PubMed Central Google Scholar

Jin, M. S. et al. Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130, 1071–1082 (2007).

Article CAS PubMed Google Scholar

Ii, M. et al. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling. Mol. Pharmacol. 69, 1288–1295 (2006).

Article CAS PubMed Google Scholar

Park, B. S. et al. The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex. Nature 458, 1191–1195 (2009).

Article CAS PubMed Google Scholar

Patra, M. C., Kwon, H. K., Batool, M. & Choi, S. Computational insight into the structural organization of full-length toll-like receptor 4 dimer in a model phospholipid bilayer. Front. Immunol. 9, 489 (2018).

Article PubMed PubMed Central Google Scholar

Kim, H. M. et al. Crystal structure of the TLR4–MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130, 906–917 (2007).

Article CAS PubMed Google Scholar

Tafazzol, A. & Duan, Y. Key residues in TLR4–MD2 tetramer formation identified by free energy simulations. PLoS Comput. Biol. 15, e1007228 (2019).

Article CAS PubMed PubMed Central Google Scholar

Chen, D. et al. Regulation of protein-ligand binding affinity by hydrogen bond pairing. Sci. Adv. 2, e1501240 (2016).

Article PubMed PubMed Central Google Scholar

Shimazu, R. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J. Exp. Med. 189, 1777–1782 (1999).

Article CAS PubMed PubMed Central Google Scholar

Triantafilou, M., Miyake, K., Golenbock, D. T. & Triantafilou, K. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J. Cell Sci. 115, 2603–2611 (2002).

Article CAS PubMed Google Scholar

Wong, S. W. et al. Fatty acids modulate toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner. J. Biol. Chem. 284, 27384–27392 (2009).

Article CAS PubMed PubMed Central Google Scholar

Lee, J. Y. et al. Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids. J. Lipid Res. 44, 479–486 (2003).

Article CAS PubMed Google Scholar

Hwang, D. H., Kim, J. A. & Lee, J. Y. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur. J. Pharmacol. 785, 24–35 (2016).

Article CAS PubMed PubMed Central Google Scholar

Shaikh, S. R. et al. Oleic and docosahexaenoic acid differentially phase separate from lipid raft molecules: a comparative NMR, DSC, AFM and detergent extraction study. Biophys. J. 87, 1752–1766 (2004).

Article CAS PubMed PubMed Central Google Scholar

Tan, Y., Zanoni, I., Cullen, T. W., Goodman, A. L. & Kagan, J. C. Mechanisms of Toll-like receptor 4 endocytosis reveal a common immune-evasion strategy used by pathogenic and commensal bacteria. Immunity 43, 909–922 (2015).

Article PubMed PubMed Central Google Scholar

Kagan, J. C. et al. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-β. Nat. Immunol. 9, 361–368 (2008).

Article CAS PubMed PubMed Central Google Scholar

Zanoni, I. et al. CD14 controls the LPS-induced endocytosis of Toll-like receptor 4. Cell 147, 868–880 (2011).

Article CAS PubMed PubMed Central Google Scholar

Schappe, M. S. & Desai, B. N. Measurement of TLR4 and CD14 receptor endocytosis using flow cytometry. Bio Protoc. 8, e2926 (2018).

Article CAS PubMed PubMed Central Google Scholar

Marrink, S. J., Risselada, H. J., Yefimov, S., Tieleman, D. P. & de Vries, A. H. The MARTINI force field: coarse grained model for biomolecular simulations. J. Phys. Chem. B 111, 7812–7824 (2007).

Article CAS PubMed Google Scholar

Monticelli, L. et al. The MARTINI coarse-grained force field: extension to proteins. J. Chem. Theory Comput. 4, 819–834 (2008).

Article CAS PubMed Google Scholar

Barnoud, J., Rossi, G., Marrink, S. J. & Monticelli, L. Hydrophobic compounds reshape membrane domains. PLoS Comput. Biol. 10, e1003873 (2014).

Article PubMed PubMed Central Google Scholar

Raphael, I., Nalawade, S., Eagar, T. N. & Forsthuber, T. G. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine 74, 5–17 (2015).

Article PubMed Google Scholar

Ross, S. H. & Cantrell, D. A. Signaling and function of interleukin-2 in T lymphocytes. Annu. Rev. Immunol. 36, 411–433 (2018).

Article PubMed PubMed Central Google Scholar

Saito, H. et al. Expression and self-regulatory function of cardiac interleukin-6 during endotoxemia. Am. J. Physiol. Hear. Circ. Physiol. 279, 2241–2248 (2000).

Article Google Scholar

Severgnini, M. et al. Activation of the STAT pathway in acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, 1282–1292 (2004).

Article Google Scholar

Godfrey, D. I. & Rossjohn, J. New ways to turn on NKT cells. J. Exp. Med. 208, 1121–1125 (2011).

Article PubMed PubMed Central Google Scholar

Singh, A. Eliciting B cell immunity against infectious diseases using nanovaccines. Nat. Nanotechnol. 16, 16–24 (2021).

Article CAS PubMed Google Scholar

Lee, M. S. J. et al. B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant. Eur. J. Immunol. 49, 1433–1440 (2019).

Article CAS PubMed Google Scholar

Vidarsson, G., Dekkers, G. & Rispens, T. IgG subclasses and allotypes: from structure to effector functions. Front. Immunol. 5, 520 (2014).

Article PubMed PubMed Central Google Scholar

Stevens, T. L. et al. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334, 255–258 (1988).

Article CAS PubMed Google Scholar

Snapper, C. M. & Paul, W. E. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236, 944–947 (1987).

Article CAS PubMed Google Scholar

Nimmerjahn, F. Divergent immunoglobulin G subclass activity through selective Fc receptor binding. Science 310, 1510–1512 (2005).

Article CAS PubMed Google Scholar

Dams, E. T. M. et al. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J. Pharmacol. Exp. Ther. 292, 1071–1079 (2000).

CAS PubMed Google Scholar

Ishida, T. et al. Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes. J. Control. Release 105, 305–317 (2005).

Article CAS PubMed Google Scholar

Ishida, T. et al. Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J. Control. Release 112, 15–25 (2006).

Article CAS PubMed Google Scholar

Wang, X. Y., Ishida, T. & Kiwada, H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control. Release 119, 236–244 (2007).

Article CAS PubMed Google Scholar

Shimizu, T., Ishida, T. & Kiwada, H. Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon. Immunobiology 218, 725–732 (2013).

Article CAS PubMed Google Scholar

Alabi, C. A. et al. Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery. Proc. Natl Acad. Sci. USA 110, 12881–12886 (2013).

Article CAS PubMed PubMed Central Google Scholar

Estapé Senti, M. et al. Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement. J. Control. Release 341, 475–486 (2022).

Article PubMed Google Scholar

Sharp, T. H. et al. Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b. Proc. Natl Acad. Sci. USA 116, 11900–11905 (2019).

Article CAS PubMed PubMed Central Google Scholar

Diebolder, C. A. et al. Complement is activated by IgG hexamers assembled at the cell surface. Science 343, 1260–1263 (2014).

Article CAS PubMed PubMed Central Google Scholar

Abed, N. S., Chace, J. H., Fleming, A. L. & Cowdery, J. S. Interferon-γ regulation of B lymphocyte differentiation: activation of B cells is a prerequisite for IFN-γ-mediated inhibition of B cell differentiation. Cell. Immunol. 153, 356–366 (1994).

Article CAS PubMed Google Scholar

Finkelman, F. D., Katona, I. M., Mosmann, T. R. & Coffman, R. L. IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. 140, 1022–1027 (1988).

Article CAS PubMed Google Scholar

Chen, B.-M., Cheng, T.-L. & Roffler, S. R. Polyethylene glycol immunogenicity: theoretical, clinical and practical aspects of anti-polyethylene glycol antibodies. ACS Nano. https://doi.org/10.1021/acsnano.1c05922 (2021).

Mima, Y. et al. Ganglioside inserted into PEGylated liposome attenuates anti-PEG immunity. J. Control. Release 250, 20–26 (2017).

Article CAS PubMed Google Scholar

Hajj, K. A. et al. A potent branched-tail lipid nanoparticle enables multiplexed mRNA delivery and gene editing in vivo. Nano Lett. 20, 5167–5175 (2020).

Article CAS PubMed PubMed Central Google Scholar

Landrum, G. RDKit: Open-Source Cheminformatics (Open-Source Chemoinformatics, 2006); http://rdkit.org

Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

Article CAS PubMed PubMed Central Google Scholar

Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

Article CAS PubMed PubMed Central Google Scholar

Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

Article CAS PubMed PubMed Central Google Scholar

Schrödinger, L. The PyMOL Molecular Graphics System, Version 2.5. (Schrödinger LLC, 2021); http://www.pymol.org/pymol

Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

Article CAS PubMed Google Scholar

Lindahl, E., Abraham, M. J, Hess, B. & van der Spoel, D. GROMACS 2020.2 Source code. Zenodo https://doi.org/10.5281/zenodo.3773801 (2020).

Wassenaar, T. A., Ingólfsson, H. I., Böckmann, R. A., Tieleman, D. P. & Marrink, S. J. Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations. J. Chem. Theory Comput. 11, 2144–2155 (2015).

Article CAS PubMed Google Scholar

Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

Article CAS PubMed Google Scholar

Reitman, S. & Frankel, S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56–63 (1957).

Article CAS PubMed Google Scholar

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Funding for this research was provided by the NIH (grant number DP2-HD098860), the Wadhwani Foundation, and generous support from Jon Saxe and Myrna Marshall. M.L.A. discloses support for the research described in this study from the NSF Graduate Research Fellowship Program (award number DGE1745016). J.R.M. discloses support for the research described in this study from an NIH F32 fellowship (number 1F32EB029345).

Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA

Namit Chaudhary, Lisa N. Kasiewicz, Alexandra N. Newby, Mariah L. Arral, Saigopalakrishna S. Yerneni, Jilian R. Melamed, Samuel T. LoPresti, Katherine C. Fein & Kathryn A. Whitehead

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA

Daria M. Strelkova Petersen & Kathryn A. Whitehead

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

Sushant Kumar & Rahul Purwar

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N.C., L.N.K., R.P. and K.A.W designed the research. N.C., L.N.K., A.N.N., M.L.A., S.S.Y, J.R.M., S.T.L., K.C.F., D.M.S. P. and S.K. performed research. N.C., L.N.K., A.N.N., M.L.A., S.S.Y., J.R.M., S.T.L. and S.K. analysed data. K.A.W. secured funding and provided oversight of the project. N.C. and K.A.W. wrote the paper.

Correspondence to Kathryn A. Whitehead.

K.A.W. is an inventor on US patents 9,227,917 (2016) and 9,439,968 (2016) related to the materials described here, and is a consultant for several companies dealing with non-viral RNA delivery.

Nature Biomedical Engineering thanks Dan Peer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Mice were dosed twice, 30 days apart, with 1 mg/kg siGFP-LNPs. Proinflammatory cytokine levels were measured in serum (a, b) four hours after each dose, (c-f) two and seven days after the second dose, and (g-j) in stimulated splenocyte supernatant seven days after the second dose (n = 3–4). Error bars represent s.e.m. Significance was determined according to according to two-way ANOVA with Tukey’s post-hoc analysis.

Source data

Mice were dosed twice, 30 days apart, with 1 mg/kg siGFP-LNPs. Spleens were collected seven days after the second LNP dose. Isolated splenocytes were stimulated with either PMA/ionomycin or LPS for 24 hours, and (a) TNFα, (b) IL-6, (c) IFNγ, and (d) IL-2 were measured from cell culture supernatant. PBS was used as a negative control (n = 3–4). Error bars represent s.e.m. Significance was determined according to two-way ANOVA with Tukey’s post-hoc analysis.

Source data

Mice were injected with 306Oi10 LNPs along with CD1d and TLR4 inhibitors and corresponding isotype and solvent controls 30 days apart, and (a) anti-PEG IgM and (b) anti-PEG IgG levels were measured weekly (n = 5). Significance was determined according to two-way ANOVA with Tukey’s post-hoc analysis.

Source data

(a) Mice were IV-injected twice with SM-102, 306Oi10, or 304Oi10 LNPs containing anti-Factor VII siRNA at a dose of 0.5 mg/kg one month apart, and Factor VII levels were measured two days after each injection relative to PBS negative control (n = 3). In separate experiments, mice were IV-injected twice with LNPs containing anti-GFP siRNA one month apart at a dose of 1 mg/kg. Blood was collected four hours after each dose, and (b) TNFα and (c) IL-6 levels were measured using ELISA. Two and seven days after the second dose, counts of (d) germinal center cells, (e) plasma cells, (f) and memory B cells were assessed via flow cytometry, and (g) TNFα and (h) IL-6 levels were measured using ELISA (n = 4). Mice were IV-injected twice with LNPs containing anti-GFP siRNA one month apart at a dose of 1 mg/kg. Blood was collected weekly for two months, and (i) anti-PEG IgM and (j) anti-PEG IgG levels were measured using ELISA (n = 4). Error bars represent s.e.m. Significance was determined according to two-way ANOVA with Šidák’s post-hoc analysis (A-H) or Tukey’s post-hoc analysis (i, j).

Source data

Supplementary Figs. 1–13 and Table 1.

Statistical source data.

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Chaudhary, N., Kasiewicz, L.N., Newby, A.N. et al. Amine headgroups in ionizable lipids drive immune responses to lipid nanoparticles by binding to the receptors TLR4 and CD1d. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01256-w

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Received: 04 November 2022

Accepted: 05 September 2024

Published: 03 October 2024

DOI: https://doi.org/10.1038/s41551-024-01256-w

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