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Nanomedicine platform for targeting activated neutrophils and neutrophil–platelet complexes using an α1-antitrypsin-derived peptide motif

Nature Nanotechnology volume 17, pages 1004–1014 (2022)Cite this article

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Abstract

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α1-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet–neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

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Fig. 1: Design and characterization of NPs for selective targeting of activated neutrophils.
Fig. 2: NT-NPs selectively bind to activated neutrophils in vitro and in vivo.
Fig. 3: NT-NPs selectively bind to activated neutrophils and NETs.
Fig. 4: Quantitation of NP trafficking by neutrophils.
Fig. 5: Heteromultivalent NPs selectively bind to activated platelet–neutrophil complexes in vitro.
Fig. 6: PNT-NPs bind to thromboinflammatory sites and effectively deliver therapeutic cargo to reduce thrombus size.

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Data availability

Modelling of docking complex with highlighted reactive centre loop and NEBP was performed using native AAT (Protein Data Bank code 1QLP)20,21. The authors declare that data supporting the findings of this study are available in their entirety within the article and its Supplementary Information. Relevant data can be provided by the corresponding authors upon reasonable request.

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Acknowledgements

We thank the Hematopoietic Biorepository Core of Case Western Reserve University for human blood sample provision; M. Sramkoski at the Case Western Reserve University Cancer Center Flow Cytometry core facility; S. Bandyopadhyay at the Cleveland Clinic for assistance with BiaCore studies; and G. Deshpande and the Cleveland Clinic Lerner Research Institute Imaging Core for expert histologic analysis. This work was supported by the National Institutes of Health R01 HL137695 (E.X.S.); R01 HL129179, R01 HL137695, R01 HL141080, R01 HL121212 (A.S.G.); R33HL141794, R01HL120728, R01HL151984 (K.B.N.); R35 GM119526, R01 HL141080 (M.D.N.); R01 HL098217 (M.T.N.); EY022938, R24 EY024864 (T.S.K.); F32 HL149207 (J.A.); the Clinical and Translational Science Collaborative of Cleveland [UL1TR002548 from the National Center for Advancing Translational Sciences component of the National Institutes of Health and National Institutes of Health roadmap for Medical Research (E.X.S., A.S.G.)]; Merit Review Awards (BX003851 (E.X.S.) and BX003604 (T.S.K.) from the Department of Veterans Affairs); a Case Coulter Translational Research Partnership Award (RES514649 (E.X.S., A.S.G.)); a University of Pittsburgh Physician-Scientist Award from the Burroughs Wellcome Fund (E.A.A., J.A.); a T-32 PostDoctoral Training award [NIH T32HL098036 (E.A.A.)]; an American Heart Association Scientist Development Award (E.X.S.); and the Oscar D. Ratnoff Endowed Professorship (E.X.S.). We acknowledge an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness (New York, NY; T.S.K.). S.d.M. gratefully acknowledges the Toegepaste en Technische Wetenschappen (TTW) section of the Netherlands Organization for Scientific Research (NWO, 2019/TTW/00704802). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health, the US Department of Veterans Affairs or the United States Government.

Author information

Author notes

  1. These authors contributed equally: Michelle A. Cruz, Dillon Bohinc, Elizabeth A. Andraska.

Authors and Affiliations

  1. Department of Pathology, Immunology Training Program, CWRU School of Medicine, Cleveland, OH, USA

    Michelle A. Cruz & Anirban Sen Gupta

  2. Department of Medicine, Hematology and Oncology Division, CWRU School of Medicine, Cleveland, OH, USA

    Dillon Bohinc, Kara L. Bane, Agharnan Gandhi & Evi X. Stavrou

  3. Department of Surgery, Division of Vascular Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

    Elizabeth A. Andraska

  4. Department of Surgery, Pittsburgh Trauma Research Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

    Jurgis Alvikas, Shannon Haldeman & Matthew D. Neal

  5. Department of Biomedical Engineering, Case Western Reserve University (CWRU), Cleveland, OH, USA

    Shruti Raghunathan, Kathryn Hart, Kathryn Medrow, Michael Sun, Ankush Banerjee, Kara Hageman & Anirban Sen Gupta

  6. Department of Civil and Environmental Engineering, Colorado School of Mines, Golden, CO, USA

    Nicole A. Masters

  7. CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

    Nadine D. van Kleef, Coen Maas & Steven de Maat

  8. Department of Ophthalmology, Children’s Hospital of University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Haitao Liu

  9. Center for Translational Vision Research, Gavin Herbert Eye Institute, University of California Irvine, Irvine, CA, USA

    Emma M. Lessieur & Timothy S. Kern

  10. Department of Pharmacology, CWRU School of Medicine, Cleveland, OH, USA

    Maria de la Fuente, Marvin T. Nieman & Anirban Sen Gupta

  11. Veterans Administration Medical Center Research Service, Long Beach, CA, USA

    Timothy S. Kern

  12. Department of Bioengineering and Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplant Hemophilia and Thrombosis Center, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA

    Keith B. Neeves

  13. Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA

    Anirban Sen Gupta & Evi X. Stavrou

  14. Department of Medicine, Section of Hematology-Oncology, Louis Stokes Veterans Administration Medical Center, Cleveland, OH, USA

    Evi X. Stavrou

Authors
  1. Michelle A. Cruz
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Contributions

M.A.C., D.B., E.A.A., M.T.N., T.S.K., K.B.N., C.M., S.d.M., M.D.N., A.S.G. and E.X.S. conceptualized and planned the experiments. M.A.C., D.B., E.A.A., J.A., S.R., N.A.M., N.D.v.K., K.L.B., S.H., K. Hageman, K.M., M.S., H.L., A.B., E.M.L., K. Hart, A.G., M.d.l.F. and E.X.S. performed the experiments. M.A.C., S.d.M., A.S.G. and E.X.S. prepared the figures. M.A.C. and E.X.S. wrote the manuscript, and all authors reviewed and edited the manuscript before submission.

Corresponding authors

Correspondence to Anirban Sen Gupta or Evi X. Stavrou.

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Competing interests

A.S.G. is a coinventor on issued patent US 9107845 (Synthetic Platelets) that is licensed from Case Western Reserve University to Haima Therapeutics. A.S.G. is a cofounder and equity stakeholder of Haima Therapeutics. The patent is on the design of a heteromultivalent NP system that can mimic the haemostatic functions of a platelet. A.S.G. is also a coinventor on issued patent US 9107963 (Heteromultivalent Nanoparticle Compositions). The patent is on the design of heteromultivalently decorated NPs for clot targeting. Although the specific NP systems described in these two patents have no direct relevance to any specific aspect of the manuscript, the context of ‘heteromultivalent NP design’ is a central aspect of the NT-NP and PNT-NP systems described in the manuscript. M.D.N. serves on the scientific advisory board of Haima Therapeutics and holds equity stake. E.X.S. is coinventor of intellectual property that has been licensed by Case Western Reserve University to XaTek and receives royalties. The patent PCT/US2017/013797 is on dielectric spectroscopy for whole blood assessment of haemostasis. This patent bears no relevance to any of the work presented in the manuscript. C.M. has been a speaker for Shire-Takeda. C.M. and S.d.M. are cofounders of TargED BV, a biotech spinout company of University Medical Center Utrecht (based upon the WO2019185723 A1 patent). C.M. and S.d.M. participate in revenue sharing as inventors through the commercialization arm of the University Medical Center Utrecht. The remaining authors declare no competing interests.

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Nature Nanotechnology thanks Benoit Ho-Tin-Noe’, Zoltan Jakus and Guillermo Ruiz-Esparza for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10, Videos 1 and 2 and Methods.

Reporting Summary

Supplementary Video 1

Binding of PNT-NPs to thrombus in DVT microfluidic model.

Supplementary Video 2

Binding of U-NPs to a developing thrombus in vitro.

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Cruz, M.A., Bohinc, D., Andraska, E.A. et al. Nanomedicine platform for targeting activated neutrophils and neutrophil–platelet complexes using an α1-antitrypsin-derived peptide motif. Nat. Nanotechnol. 17, 1004–1014 (2022). https://doi.org/10.1038/s41565-022-01161-w

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