Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy

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.

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-posthoc 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.

Fig. 1 ∣. Protein-induced dipole rotation of interfacial PS leads to heterogeneous PPSU surfaces that mimic protein surfaces.

a, Simultaneous recording of the cryo-STEM and cryo-SEM images, showing a typical PPSU hollow NP and its surface, respectively. b, Schematic of the dipole moment of amphiphilic PS–the repeating unit of a PPSU homopolymer (blue indicates hydrophilic and grey indicates hydrophobic for all the schemes in this Article, unless stated otherwise). c, Atomistic simulations demonstrating enhanced surface hydrophilicity as the aggregation of PPSU chains become disordered. PEG is included for comparison. d, As seen in trypsin using glycine as the reference, the surfaces of water-soluble proteins are heterogeneous, with characteristic patch size distributions based on hydrophobicity. e, Protein induces the formation of locally heterogeneous PPSU surfaces that mimic protein surfaces. Hydrophobic interactions at the interface between this PPSU surface and the adsorbed protein can be sufficiently weak to not outcompete the forces governing protein folding despite irreversible adsorption. Fast spreading of protein on a hydrophobic surface is included for comparison.

Fig. 2 ∣. Atomistic explicit solvent simulations confirm the interfacial hydrophilic/hydrophobic transition within PPSU surface on trypsin adsorption.

a, Simulation snapshot of an equilibrated 600-chain NP adsorbed with six adsorbed trypsin molecules (in cyan). The protein–NP interfaces are lubricated by water molecules, leading to the preserved structure of trypsin. The green cylinders refer to the water bridges connecting the PPSU chains and trypsin via hydrogen bonds. b, Lennard–Jones trypsin–NP interactions dominated over the trypsin–NP Coulombic interactions, supporting the hydrophobicity-driven feature of trypsin adsorption. c, Orientations of the six trypsin molecules are non-specific on adsorption. The adsorption sites and active sites on trypsin are coloured in red and blue, respectively. d, Percentage of sulfone groups at the trypsin–NP contact region, revealing enhanced NP surface hydrophobicity after trypsin adsorption. Significant P values relative to the water–NP interface are displayed on the graph. e, No significant (ns) differences in trypsin hydration were detected between the adsorbed trypsin and unbound trypsin. The numbers of trypsin–water hydrogen bonds and those of the water neighbours of trypsin were calculated. The data in d and e are presented as mean values ± standard error. Statistical significance was determined by a two-sample t-test from 52 to 196 ns (calculated every two nanoseconds; n = 73).

Fig. 3 ∣. PPSU NPs preserve protein function within stable adlayers.

a, Schematic of the rapid and facile process of coating PPSU NPs with protein adlayers. After incubation, the unbound proteins are removed by thorough washing. b, Zeta potential of protein-coated NPs is dependent on the adsorbed protein type. Data are presented as mean values ± standard error. The results were obtained from three parallel samples (n = 3). c, SAXS data supporting the increase in shell thicknesses from 5.3 to 7.1 nm after BSA adsorption. d, Trypsin is stably adsorbed at the NP surfaces and free trypsin is undetectable by MALDI-TOF mass spectrometry in the supernatant after the centrifugation of trypsin@NP. Samples were stored at 4 °C for 48 h before centrifugation. e, Kinetic assay demonstrating the bioactivity of adsorbed trypsin. Trypsin is encapsulated within the NPs for comparison. f, Fluorescence of GFP was detectable for GFP@NP but not for GFP-encapsulated NPs. g, Immunogold labelling showing the binding of pre-adsorbed anti-CD4 antibodies to secondary antibodies. Here 10 nm colloidal gold–secondary antibody is indicated by the arrows. h, Targeting of irreversibly adsorbed anti-CD3 towards Jurkat T cells is confirmed by flow cytometry as assessed by the percentage of NP-positive cells and median fluorescence intensity (MFI). Data were obtained from six parallel experiments (n = 6) and are presented as mean values ± standard error. Significant P values are displayed on the graph. Statistical significance was determined by a two-way analysis of variance with Tukey’s multiple comparisons test.

Fig. 4 ∣. Optimization of a nanotherapy to inhibit anaphylaxis in a humanized mouse engraftment model.

a, PPSU-based nanomedicines consisting of co-adsorbed anti-Siglec-6 (light blue), anti-FcεRIα (pink) and BSA (blocking agent, yellow) with a controlled surface density (as wt% of PPSU) of anti-Siglec-6. b, Imaging the nanomedicines (blue, indicated by the red arrows) on the surface of an MC (grey) by SEM. Three independent test samples were prepared and used for imaging. c, Activation of MCs (left) is achieved via the cross-linking of FcεRIα by anti-FcεRIα/BSA@NP, whereas the nanomedicine inhibits MC degranulation (right) via co-localized engagement of FcεRIα and Siglec-6. d, Optimizing the nanomedicine formulation by adjusting the surface density of anti-Siglec-6. In vitro results show that a lower anti-Siglec-6 density is more effective in suppressing CD107a and CD63 expression. e, In vitro results demonstrating the importance of binding Siglec-6 in close proximity and time with engagement of FcεRIα in suppressing CD107a and CD63 expression by MCs. The optimized in vitro formulation (0.01 wt% of anti-Siglec-6) was used as the nanomedicine; ctr-a: anti-Siglec-6/BSA@NP + anti-FcεRIα; ctr-b, anti-Siglec-6 + anti-FcεRIα + BSA; ctr-c, BSA@NP + anti-FcεRIα. In d and e, inhibition is normalized from MCs receiving only anti-FcεRIα and expressing a positive population mean of 52.7 ± 5.0% for CD63 and 71.2 ± 8.0% for CD107a (n = 3). f, The in vivo optimized nanomedicine (2.5 wt% of anti-Siglec-6) was highly effective as an allergen immunotherapy without triggering anaphylaxis in a humanized mouse model. The results were from two independent datasets (n = 5 biologically independent animals for anti-FcεRIα + anti-Siglec-6/BSA@NP and the isotype test groups; n = 6 for all the other groups). Each mouse sample was only measured once. Statistical significance was determined by one-way analysis of variance (ANOVA) in d and e, and by two-way ANOVA in f, both with a Tukey’s post hoc test. All data are represented as mean values ± standard error.