ABSTRACT 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

structure-dependent innate and adaptive responses of LNPs. Our findings may help accelerate the discovery of well-tolerated ionizable lipids suitable for repeated dosing. Access through your

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OTHERS IONIZATION AND STRUCTURAL PROPERTIES OF MRNA LIPID NANOPARTICLES INFLUENCE EXPRESSION IN INTRAMUSCULAR AND INTRAVASCULAR ADMINISTRATION Article Open access 11 August 2021

IMMUNOGENICITY OF LIPID NANOPARTICLES AND ITS IMPACT ON THE EFFICACY OF MRNA VACCINES AND THERAPEUTICS Article Open access 02 October 2023 POLY(CARBOXYBETAINE) LIPIDS ENHANCE MRNA

THERAPEUTICS EFFICACY AND REDUCE THEIR IMMUNOGENICITY Article 29 May 2025 DATA AVAILABILITY 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

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ACKNOWLEDGEMENTS 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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 Authors * Namit Chaudhary View author publications You can also search for this author inPubMed Google

Scholar * Lisa N. Kasiewicz View author publications You can also search for this author inPubMed Google Scholar * Alexandra N. Newby View author publications You can also search for this

author inPubMed Google Scholar * Mariah L. Arral View author publications You can also search for this author inPubMed Google Scholar * Saigopalakrishna S. Yerneni View author publications

You can also search for this author inPubMed Google Scholar * Jilian R. Melamed View author publications You can also search for this author inPubMed Google Scholar * Samuel T. LoPresti View

author publications You can also search for this author inPubMed Google Scholar * Katherine C. Fein View author publications You can also search for this author inPubMed Google Scholar *

Daria M. Strelkova Petersen View author publications You can also search for this author inPubMed Google Scholar * Sushant Kumar View author publications You can also search for this author

inPubMed Google Scholar * Rahul Purwar View author publications You can also search for this author inPubMed Google Scholar * Kathryn A. Whitehead View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to Kathryn A. Whitehead. ETHICS DECLARATIONS COMPETING INTERESTS 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. PEER REVIEW PEER REVIEW INFORMATION _Nature Biomedical Engineering_ thanks Dan

Peer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 IMMUNE RESPONSE OF TAIL GROUPS VARIES WITH SAMPLING TIME, TYPE OF SAMPLE, AND

MEASURED ANALYTE. 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 EXTENDED DATA FIG. 2 313 LNPS ELICIT HIGHER LEVELS OF INFLAMMATORY CYTOKINES IN STIMULATED SPLENOCYTES. 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 EXTENDED DATA FIG. 3 CD1D AND TLR4 BLOCKADE INCREASES ANTI-PEG RESPONSES FOR 306OI10. 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 EXTENDED DATA FIG. 4 SM-102 ELICITS SIMILAR EFFICACY AND IMMUNE RESPONSE AS 306OI10.

(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 INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Figs. 1–13 and Table 1. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIGS. 1–6 Statistical source data. SOURCE DATA EXTENDED DATA FIGS. 1–4 Statistical

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chaudhary, N., Kasiewicz, L.N., Newby, A.N. _et al._ Amine headgroups in ionizable lipids drive immune responses to lipid nanoparticles

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