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A sonosensitive diphenylalanine-based broad-spectrum antimicrobial peptide

Nature Biomedical Engineering volume 9, pages 1349–1365 (2025)Cite this article

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Abstract

The antimicrobial effect of antimicrobial peptides is typically slow; they can be rapidly biodegraded and often have non-selective toxicity and elaborate sequences. Here we report a short peptide that is activated by ultrasound, that shows high broad-spectrum antibacterial efficiency (>99%) against clinically isolated methicillin-resistant bacteria (specifically, Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Enterobacter cancerogenus and Pseudomonas aeruginosa) with 15 min of ultrasound irradiation, and that has negligible toxicity and low self-antibacterial activity. We selected the peptide, FFRKSKEK (a segment from the human host-defence LL-37 peptide), from a library of peptides with piezoelectric diphenylalanine (FF) sequences, low toxicity, hydrophobicity and net positive charge. We show via all-atom molecular dynamics simulations that ultrasound amplifies the membrane-penetrating ability of peptides with FF sequences and that its piezoelectric polarization generates reactive-oxygen species and disturbs bacterial electron-transport chains. In a goat model of hard-to-treat intervertebral infection, the sonosensitive peptide led to better outcomes than vancomycin. Antimicrobial peptides activated by ultrasound may offer a clinically relevant strategy for combating antibiotic-resistant infections.

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Fig. 1: Exploration of multiple FF-based peptide sonosensitizers’ properties.
Fig. 2: Molecular dynamics simulations and experiments verification of peptide/membrane interaction.
Fig. 3: Multiple explorations on the sonosensitive mechanism of peptides.
Fig. 4: Antibacterial and biocompatible properties of FFRK8 + US in vitro and in vivo.
Fig. 5: Anti-infection of sonosensitive peptide for MRSA-infected IVD in goats.
Fig. 6: Histological analysis of IVD and related adjacent tissues in goats.

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Data availability

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

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Acknowledgements

This work was supported by the Natural Science Foundation of China (numbers 82372380, 22175058, 82130072, 82272459, 82072505, 51801056), Natural Science Foundation of Hubei Province (2023AFB770) and the National Key Research and Development Plan of China (number 2020YFC2006000).

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  1. These authors contributed equally: Xiaoguang Zhang, Xiaobo Feng, Liang Ma.

Authors and Affiliations

  1. Department of Orthopaedics, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Xiaoguang Zhang, Xiaobo Feng, Liang Ma, Jie Lei, Gaocai Li, Weifeng Zhang, Huaizhen Liang, Bide Tong, Di Wu, Cao Yang & Lei Tan

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Contributions

L.T., X.Z. and C.Y. conceived and designed the study. X.Z., X.F., L.M., J.L., G.L., W.Z. and H.L. performed the experiments. X.Z., L.T., D.W. and B.T. analysed and interpreted the data. The manuscript was written by L.T. and X.Z.

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Correspondence to Cao Yang or Lei Tan.

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Nature Biomedical Engineering thanks Jianfeng Cai, Francesca Cavalieri, Ebru Oral, Tzanko Tzanov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Spread plate assay after antimicrobial activity of 15 peptides against MRSA and then performed analysis.

The antibacterial activities of 15 peptides against MRSA under US or without US for 15 min. The data are presented as means ± s.d., with n = 5 biological replicates. Statistical analysis was performed using one-way ANOVA. ns: no significant.

Source data

Extended Data Fig. 2 Spread plate assay for self-antibacterial efficiency of the peptides.

Antibacterial activities of 15 peptides (without US) incubated with MRSA at 37 °C for 18 h. Data are means ± s.d., with n = 5 biological replicates. Statistical analysis was performed using one-way ANOVA.

Source data

Extended Data Fig. 3 The toxicity of the peptides to the NP-derived cells was calculated by MTT and LDH assays.

a, The effect of 15 peptides with gradient concentrations on the viability of NP-derived cells. The IC50 is extracted in curve according to MTT assay; n = 6. b, The effect of FFRK8’s gradient concentration on the viability of NP-derived cells. The IC50 is extracted in curve according to LDH assay. n = 6. Data and error bars in (a,b) represent the mean ± s.d., with n = 6 biological replicates.

Source data

Extended Data Fig. 4 Investigation of optimal ultrasonic antimicrobial conditions for FFRK8 peptide.

Antibacterial experiments on MRSA using different concentrations of FFRK8 under US. The experimental results of spread-plate assay are respectively presented in the form of antibacterial efficiency (a) and counting of CFU/mL (b). c, After undergoing 15 min of antibacterial treatment with FFRK8+US, the supernatant was either removed by centrifugation or left intact, and then the treated MRSA were cultured in a 96-well plate. After 24 h of incubation, the absorbance at 600 nm was measured. n = 6. d, Antibacterial ability of FFRK8 with US treatment for 15 and 30 min, respectively. Panels b and d are shown as mean ± s.d. n = 5 biological replicates. Statistical significance was calculated using one- (b) or two-way (d) ANOVA.

Source data

Extended Data Fig. 5 Biocompatibility testing of peptides.

a, Representative live/dead fluorescence staining of HAMA hydrogel loaded BMSCs and paravertebral fibroblasts treated with FFRK8, US, and FFRK8+US for 15 min. Green fluorescence stained by Calcein dye indicates live BMSCs and paravertebral fibroblasts cells, and red fluorescence stained by PI dye represents dead cells. b,c, Mean fluorescence intensity (MFI) visually quantifies the green and red fluorescence intensity (arbitrary units; a.u.) of BMSCs and paravertebral fibroblasts. ns: no significant. The values are presented as mean ± s.d. of n = 3 biological replicates, and were calculated using two-way ANOVA. d,e, Testing of IC50 of FFRK8 peptide on BMSCs and paravertebral fibroblasts. Cell viability of BMSCs and paravertebral fibroblasts treated by FFRK8. Data and error bars represent the mean ± s.d., with n = 3 biological replicates.

Source data

Extended Data Fig. 6 Ultrasonic antimicrobial broad-spectrum testing of the FFRK8 peptide.

Antibacterial activities of the peptides against S. aureus, MRSA, E. coli, S. epidermidis, E. cancerogenus, and P. aeruginosa. The values are presented as mean ± s.d. of n = 5 biological replicates, and were calculated using two-way ANOVA.

Source data

Extended Data Fig. 7 Imaging and histology of intervertebral anti-infection experiments in rats.

a, Reconstructed 3D images according to micro-CT. b, Nuclear magnetic resonance imaging (MRI) of intervertebral disk (IVD) of rats and corresponding X-ray images as background. c, Histological staining of Masson. d, Immunohistochemistry staining of Col2a1 and Mmp13.

Supplementary information

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Supplementary Figs. 1–15 and Tables 1–5.

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Zhang, X., Feng, X., Ma, L. et al. A sonosensitive diphenylalanine-based broad-spectrum antimicrobial peptide. Nat. Biomed. Eng 9, 1349–1365 (2025). https://doi.org/10.1038/s41551-025-01377-w

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