Abstract
The efficacy of antimicrobial peptides (AMPs) is limited by challenges of delivery and potency. We enhance AMP performance in the lung by converting AMPs to a peptibody format that fuses AMPs with fragment crystallizable domains to activate innate immunity and cathelin domains for infection-responsive activation, with their mRNA constructs delivered by anti-inflammatory lipid nanoparticles. The highest-scoring design outperforms antibiotic therapy approved by the US Food and Drug Administration in multidrug-resistant pneumonia models, eradicating representative MDR bacteria while mitigating inflammation.
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Acknowledgements
Y.D. acknowledges support from the Maximizing Investigators’ Research Award (R35GM144117) from the National Institute of General Medical Sciences and institutional funds from the Icahn School of Medicine at Mount Sinai. We thank the Biorepository and Pathology CoRE Laboratory at the Icahn School of Medicine at Mount Sinai for their support. Cryo-electron microscopy was performed at the Center for Electron Microscopy and Analysis at The Ohio State University. We are grateful to E. Purisic and J. Dai for sharing the vibratome and providing instructions. Certain figures were created with BioRender.com.
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Y.X., X.H. and S.W. conceptualized the work, performed the experiments, analyzed the data and wrote the paper. Y. Zhang., C.W. and C.Y. contributed to the lipid synthesis. Y. Zhong., D.D.K. and Y.Y.Z. contributed to the mRNA synthesis. H.L., Z.L., M.T. and D.C. contributed to the animal studies. B.D. contributed to the cryo-electron microscopy imaging. P.H. and M.M. contributed to the collection of human lung tissues. Y.D. conceptualized and supervised the project and wrote the paper. The final paper was edited and approved by all authors.
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Y.D. is a cofounder and holds equity in Immunanoengineering Therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Characterizations of TS LNPs for pulmonary mRNA delivery.
a, Relative luminescence intensity of TS LNP-FLuc mRNA in MLG cells. Intensity was normalized to SM-102 LNP group. b, Relative luminescence intensity of TS LNP-FLuc mRNA in A549 cells. Intensity was normalized to SM-102 LNP group. c, Luminescence intensity in the lungs of mice intratracheally injected with TS LNPs-FLuc mRNA. d, Representative IVIS images of lung tissues from c. e, Luminescence intensity in the lungs of mice following IT injection of SM-102 LNP-FLuc mRNA, TS41 LNP-FLuc mRNA, or TS41S LNP-FLuc mRNA. f, Representative IVIS images of lung tissues from e. g, Schematic illustration showing that delivery of Cre recombinase mRNA induces tdTomato expression in Ai14 reporter mice through Cre-mediated recombination. h, Flow cytometry analysis of tdTomato expression in various lung cell types following IT injection of SM-102 LNP-Cre mRNA or TS41S LNP-Cre mRNA. All data are from n = 3 biologically independent samples and are presented as mean values ± s.d. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Fig. 2 Optimization of TS41 LNP formulation using orthogonal assay.
a, Table for two rounds of TS41 LNP optimization. Chol, cholesterol. b, Orthogonal assay to evaluate the impact trends of individual lipid component in TS41 LNP formulation at four levels in MLG cells. c, Luminescence intensity fold changes of the two rounds of optimization in MLG cells. d, Orthogonal assay to evaluate the impact trends of individual lipid component in TS41 LNP formulation at four levels in A549 cells. e, Luminescence intensity fold changes of the two rounds of optimization in A549 cells. f, Hydrodynamic diameter and PDI of TS41S LNP. PDI, polydispersity index. g, Zeta potential and encapsulation efficiency of TS41S LNP. h, Representative Cryo-TEM image of TS41S LNP. Scale bar = 100 nm. Data in b-g are from n = 3 biologically independent samples. Data in b and d are plotted as floating bar charts, where each bar represents the range of luminescence intensity from a start value (minimum intensity) to an end value (maximum intensity), with a line indicating the mean intensity. Data in c and e-g are presented as mean values ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. **P < 0.01, ****P < 0.0001.
Extended Data Fig. 3 Anti-inflammatory ability of TS41S LNP in pneumonia lung.
a, Flow cytometry analysis of ROS levels in neutrophils, alveolar macrophages and monocyte-derived macrophages in bronchoalveolar lavage fluid (BALF) from pneumonia lung treated with different LNPs. b, Percentage and cell number of infiltrating neutrophils among total immune cells in BALF. c, Expression level of inducible nitric oxide synthase (iNOS) in neutrophils. d, e, f, g, Expression level of IL-1β (d), IL-6 (e), TNF-α (f), and IFN-γ (g) in BALF. All data are from n = 5 biologically independent samples and are presented as mean values ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. n.s. not significant P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Fig. 4 Therapeutic effects of TS41S LNP-PB9 mRNA in chronic pneumonia models.
a, Treatment scheme for chronic pneumonia models induced by S. aureus infection or co-infection of S. aureus and P. aeruginosa. b, Relative body weight for each group in chronic pneumonia model of S. aureus infection. c, Quantification of bacteria load in the lung tissues of each group in chronic pneumonia model of S. aureus infection. d, Relative body weight for each group in chronic pneumonia model of S. aureus and P. aeruginosa co-infection. e, Quantification of bacteria load in the lung tissues of each group in chronic pneumonia model of S. aureus and P. aeruginosa co-infection. All data are from n = 5 (b and c) or 6 (d and e) biologically independent samples and are presented as mean values ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Panel a created using BioRender.com.
Supplementary information
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Unprocessed western blots and statistical source data.
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Unprocessed western blots and statistical source data.
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Source Data Extended Data Figs. 1–4
Statistical source data.
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Xue, Y., Hou, X., Wang, S. et al. Antimicrobial peptide delivery to lung as peptibody mRNA in anti-inflammatory lipids treats multidrug-resistant bacterial pneumonia. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02928-x
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DOI: https://doi.org/10.1038/s41587-025-02928-x
