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Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy

Nature Nanotechnology (2024)Cite this article

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Abstract

Protein adsorption onto nanomaterials often results in denaturation and loss of bioactivity. Controlling the adsorption process to maintain the protein structure and function has potential for a range of applications. Here we report that self-assembled poly(propylene sulfone) (PPSU) nanoparticles support the controlled formation of multicomponent enzyme and antibody coatings and maintain their bioactivity. Simulations indicate that hydrophobic patches on protein surfaces induce a site-specific dipole relaxation of PPSU assemblies to non-covalently anchor the proteins without disrupting the protein hydrogen bonding or structure. As a proof of concept, a nanotherapy employing multiple mast-cell-targeted antibodies for preventing anaphylaxis is demonstrated in a humanized mouse model. PPSU nanoparticles displaying an optimized ratio of co-adsorbed anti-Siglec-6 and anti-FcεRIα antibodies effectively inhibit mast cell activation and degranulation, preventing anaphylaxis. Protein immobilization on PPSU surfaces provides a simple and rapid platform for the development of targeted protein nanomedicines.

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Fig. 1: Protein-induced dipole rotation of interfacial PS leads to heterogeneous PPSU surfaces that mimic protein surfaces.
Fig. 2: Atomistic explicit solvent simulations confirm the interfacial hydrophilic/hydrophobic transition within PPSU surface on trypsin adsorption.
Fig. 3: PPSU NPs preserve protein function within stable adlayers.
Fig. 4: Optimization of a nanotherapy to inhibit anaphylaxis in a humanized mouse engraftment model.

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Data availability

The main data supporting the results of this study are available within the Article and its Supplementary Information. Other raw and analysed datasets generated during this study are available for research purposes from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the National Institute of Biomedical Imaging and Bioengineering (NIH grant no. 1R01EB030629-01A1) (to E.A.S.) and the National Institute of Allergy and Infectious Disease (NIH grant no. R21AI159586) (to E.A.S. and B.S.B.). We are grateful to E. W. Roth for the cryo-STEM observation. We acknowledge support from the BioCryo facility of Northwestern University’s NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-2025633), the IIN and Northwestern’s MRSEC program (NSF DMR-1720139). SAXS analysis benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement no. 654000. We are also grateful for the donation of chimeric human IgE from Allakos, Inc. used in our hapten experiments.

Author information

Author notes

  1. These authors contributed equally: Fanfan Du, Clayton H. Rische, Yang Li.

Authors and Affiliations

  1. Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA

    Fanfan Du, Clayton H. Rische, Michael P. Vincent, Yuan Qian, Simseok A. Yuk, Sultan Almunif & Evan A. Scott

  2. Department of Medicine, Division of Allergy and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Clayton H. Rische, Rebecca A. Krier-Burris & Bruce S. Bochner

  3. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Yang Li

  4. Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA

    Baofu Qiao

  5. Department of Natural Sciences, Baruch College, City University of New York, New York, NY, USA

    Baofu Qiao

  6. Simpson Querrey Institute, Northwestern University, Chicago, IL, USA

    Evan A. Scott

  7. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Evan A. Scott

  8. Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, USA

    Evan A. Scott

  9. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

    Evan A. Scott

  10. Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Evan A. Scott

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Contributions

F.D. designed and contributed to all the experiments, analysed the data and wrote the manuscript. C.H.R. performed the MC experiments, completed the in vivo validation, analysed the data and wrote the manuscript. Y.L. carried out the simulations, analysed the data and wrote the manuscript. M.P.V. contributed to the trypsin proteolysis assay. R.A.K.-B. performed the MC experiments and completed the in vivo validation. Y.Q. performed the cellular uptake studies. S.A.Y. completed the SEM experiments. S.A. contributed to the SAXS measurements. B.S.B. supervised the validation research and wrote the manuscript. B.Q. supervised the simulations and wrote the manuscript. E.A.S. supervised all the research and wrote the manuscript.

Corresponding author

Correspondence to Evan A. Scott.

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Competing interests

B.S.B. receives remuneration for serving on the scientific advisory board of Allakos, Inc., a biotechnology company developing Siglec-based therapies. He also owns stock in Allakos, Inc. He receives consulting fees from Third Harmonic Bio. He receives publication-related royalty payments from Elsevier and UpToDate. He is a co-inventor on existing Siglec-8-related patents and thus may be entitled to a share of royalties received by Johns Hopkins University during the development and potential sales of such products. B.S.B. is also a co-founder of Allakos, Inc., which makes him subject to certain restrictions under university policy. The terms of this arrangement are managed by Johns Hopkins University and Northwestern University in accordance with their conflict-of-interest policies. E.A.S., B.S.B. and C.H.R. are inventors on a patent application submitted by Northwestern University that covers the developed nanomedicine to inhibit anaphylaxis. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Simulation results of IgG adsorption on to the 600-chain PPSU NP.

(a) Atomistic simulation snapshots showing the adsorption of IgG (cyan) onto the surface of the 600-chain NP in three parallel simulations. Only one IgG molecule can be simulated in each system due to the limit set by space constraints. (b) Orientations of the 3 IgG molecules in the three parallel simulations are non-specific upon adsorption. The adsorption sites are colored red. (c) The Lennard-Jones IgG-NP interactions dominated over the IgG-NP Coulombic interactions, supporting the hydrophobicity-driven feature of IgG adsorption. (d) Percentage of sulfone groups at the IgG-NP contact region revealing enhanced NP surface hydrophobicity after IgG adsorption. Significant P values relative to water-NP interface are displayed on the graph. (e) No significant (ns) differences in IgG hydration were detected between the adsorbed IgG and unbound IgG. The numbers of IgG-water H-bonds and of water neighbors of IgG were calculated. The data in (d) and (e) are presented as mean values ± standard error. Statistical significance was determined by two-sample t-test from 52 ns to 196 ns (calculated every two nanoseconds; n = 73).

Extended Data Fig. 2 Representative gating strategy for in vitro experiments.

Primary human skin cell activation was analyzed by first gating singlets and live cells, followed by verification of mast cell-specific markers Siglec-6 and KIT receptor. This population was then analyzed for degranulation marker expression using the relevant antibodies. Here, we show the gating strategy for CD107a, along with an example of FMO control used to authenticate CD107a+ stained cells. This strategy was used for CD63, and CD107a flow cytometry data seen in Fig. 4d, e and Extended Data Fig. 3b.

Extended Data Fig. 3 Effects of Siglec-6-targeting nanotherapy in a humanized mouse model of IgE-mediated anaphylaxis.

(a) Activation of mast cells (left) is achieved via cross-linking of FcεRIα (IgE-sensitized) by nBSA@NP, whereas anti-Siglec-6/nBSA@NP inhibits mast cell degranulation (right) via co-localized engagement. (b) Optimizing nanotherapy formulation via adjusting the surface density of anti-Siglec-6. Data are represented as mean values +/− standard error. Results were from two independent experiments (n = 2). Statistical significance was determined by one-way ANOVA with Tukey-post hoc test. (c) Humanized mice (n = 5 biologically independent animals combined from 2 independent experiments) were IgE-sensitized for the 4-hydroxy-3-nitrophenylacetyl hapten-BSA (nBSA) allergen followed by intravenous injections of formulations containing different combinations of PPSU NP, nBSA, and anti-Siglec-6. Mice injected with solubilized or NP-bound nBSA experienced a decrease of 2–2.5 °C in body temperature, indicating the onset of anaphylaxis. In contrast, the presence of anti-Siglec-6 in conjunction with nBSA on the surface of NPs resulted in inhibition of anaphylaxis. Data are represented as mean values +/− standard error. Statistical significance was determined by two-way ANOVA with Tukey-post hoc test.

Supplementary information

Supplementary Information

Supplementary Figs. 1–20, methods and references.

Reporting Summary

Supplementary Video 1

AAMD simulation of protein adsorption onto a 600-chain PPSU NP.

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Du, F., Rische, C.H., Li, Y. et al. Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-023-01584-z

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