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Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins

Nature Biotechnology (2025)Cite this article

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Abstract

Intracellular delivery of biomacromolecules is hampered by low efficiency and cytotoxicity. Here we report the development of elastin-based nanoparticles for therapeutic delivery (ENTER), a recombinant elastin-like polypeptide (ELP)-based delivery system for effective cytosolic delivery of biomacromolecules in vitro and in vivo. Through iterative design, we developed fourth-generation ELPs fused to cationic endosomal escape peptides (EEPs) that self-assemble into pH-responsive micellar nanoparticles and enable cytosolic entry of cargo following endocytic uptake. In silico screening of α-helical peptide libraries led to the discovery of an EEP (EEP13) with 48% improved protein delivery efficiency versus a benchmark peptide. Our lead ELP–EEP13 showed similar or superior performance compared to lipid-based transfection reagents in the delivery of mRNA-encoded, DNA-encoded and protein-form Cre recombinase and CRISPR gene editors as well as short interfering RNAs to multiple cell lines and primary cell types. Intranasal administration of ELP–EEP13 combined with Cre protein achieved efficient editing of lung epithelial cells in reporter mice.

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Fig. 1: Design of elastin-based nanoparticles for cytosolic delivery of therapeutic macromolecules.
Fig. 2: Discovery of enhanced endosomolytic peptides via in silico screening.
Fig. 3: ELP-mediated protein delivery to primary cells.
Fig. 4: Complexation and delivery of siRNA by ELP nanoparticles.
Fig. 5: Complexation and delivery of mRNA by ELP nanoparticles.
Fig. 6: ELP-mediated delivery of Cre protein to the bronchial epithelium in vivo.

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Data availability

Data used for training EEP predictive models and the compiled α-helical peptide database are available on GitHub (https://github.com/sayoeweje/elp-eep-discovery)89. Source data are provided with this paper. All additional data are available upon request.

Code availability

All scripts and data used for EEP design can be found on GitHub at https://github.com/sayoeweje/elp-eep-discovery (ref. 89).

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Acknowledgements

We thank members of the laboratory of E.L.C. for helpful discussions; B. Pinckney, G. Haskett and J. Tigges (BIDMC Flow Cytometry Core); A. Pauer and T. Ferrante (Wyss Institute for Biologically Inspired Engineering); S. White (BIDMC Histology Core); A. Black (BIDMC Precision RNA Medicine Core); R. Nair (Harvard Molecular Electron Microscopy Suite) and A. Berger and P. Hammond for HeLa-d2eGFP cells (via P. Jain, University of Florida). Research in the laboratory of E.L.C. was supported by the NIH (UG3AI15055 and UH3AI150551) as part of the Somatic Cell Genome Editing consortium. Research in the laboratory of D.R.L. was additionally supported by HHMI. F.E. is supported by the Harvard/MIT MD–PhD program (5T32GM007753-42) and a Ruth L. Kirschstein NRSA F31 Fellowship (F31HL167533). V.I., K.A. and A.A. acknowledge research fellowship support from the Harvard College Research Program. M.L.W. is supported by the Harvard/MIT MD–PhD program (5T32GM144273-02). Images in Figs. 1a and 4a and Supplementary Fig. 1e were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Feyisayo Eweje, Jiaxuan Chen.

Authors and Affiliations

  1. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Feyisayo Eweje, Vanessa Ibrahim, Aram Shajii, Michelle L. Walsh, Kiran Ahmad, Assma Alrefai, Dominie Miyasato, Hyunok Ham, Kaicheng Li, Carolyn A. Haller, Jiaxuan Chen & Elliot L. Chaikof

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

    Feyisayo Eweje & Michelle L. Walsh

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

    Feyisayo Eweje & Michelle L. Walsh

  4. Wyss Institute of Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Feyisayo Eweje, Hyunok Ham, Kaicheng Li, Carolyn A. Haller, Jiaxuan Chen & Elliot L. Chaikof

  5. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Jessie R. Davis & David R. Liu

  6. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Jessie R. Davis & David R. Liu

  7. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Jessie R. Davis & David R. Liu

  8. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Michael Roehrl

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Contributions

E.L.C., J.C., F.E., C.A.H. and D.R.L. designed the experiments and analyzed the results. F.E., V.I., A.S., K.A. and A.A. expressed and purified ELPs and cargo proteins. J.C. performed sortase conjugation studies. H.H. and K.L. designed and synthesized linkers for monoclonal antibody–ELP sortase conjugation. J.R.D. designed and cloned protein cargo plasmids. F.E., V.I., A.S. and J.C. performed nanoparticle size and zeta potential characterization. F.E., J.C., V.I., A.S., K.A., A.A., D.M. and M.L.W. conducted in vitro delivery studies. F.E. conducted predictive model development and EEP design. F.E. and J.C. performed in vivo delivery studies and analyzed efficacy. M.R. conducted histological evaluation of lung tissues. F.E., J.C. and E.L.C. wrote the paper with input from coauthors.

Corresponding authors

Correspondence to Jiaxuan Chen or Elliot L. Chaikof.

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Competing interests

F.E., J.C. and E.L.C. are inventors on a pending patent related to this work filed by the Beth Israel Deaconess Medical Center (PCT/US2024/038614). D.R.L. is a consultant and equity holder of Nvelop Medicine, Prime Medicine, Beam Therapeutics, Pairwise Plants and Chroma Medicine, companies that use or deliver gene editing or genome engineering agents. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Formation of ELP-protein nanoparticle.

a) PAGE gel image depicting result of sortase-mediated Cre-LPETG conjugation to V1-ELP at varying ratios. The reaction solution underwent centrifugal filtration against a 100 kDa filter to remove unreacted Cre. b) Quantification of Cre-V1-ELP conjugation efficiency via densitometry analysis, measured as percentage of total ELP conjugated to Cre. c) Diameter of Cre-V1-ELP nanoparticles (n = 3 technical replicates). d) PAGE gel image depicting result of sortase-mediated SpCas9-LPETG conjugation to V1-ELP at varying ratios. e) Diameter and f) Zeta potential of SpCas9-V1-ELP nanoparticles (n = 3 technical replicates). g) PAGE gel image and h) Densitometry analysis of CD117 mAb-V1-ELP conjugation efficiency. i) MFI and j) Fluorescent images of CD117 + P815 mast cells treated with CD117 mAb-functionalized V1-ELP nanoparticles compared to unfunctionalized controls (n = 3). Scalebars = 10 μm. For all experiments, [V1-ELP] = 8 μM unless otherwise noted. Significance assessed via one-way ANOVA with Dunnett’s correction for multiple comparisons. Data represented by mean ± SEM.

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Supplementary information

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Supplementary Methods, Figs. 1–16, Tables 1 and 2 and uncropped supplementary gel images.

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Supplementary Data 1

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Supplementary Table 3

Sequences and catalog numbers of the oligonucleotides used.

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Eweje, F., Ibrahim, V., Shajii, A. et al. Self-assembling protein nanoparticles for cytosolic delivery of nucleic acids and proteins. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02664-2

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