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[Preprint]. 2023 Jan 26:rs.3.rs-2468299.
doi: 10.21203/rs.3.rs-2468299/v1.

Bioactive multi-protein adsorption enables targeted mast cell nanotherapy

Fanfan Du  1 , Clayton H Rische  1   2 , Yang Li  3 , Michael P Vincent  1 , Rebecca A Krier-Burris  2 , Yuan Qian  1 , Simseok A Yuk  1 , Sultan Almunif  1 , Bruce S Bochner  2 , Baofu Qiao  4   5 , Evan A Scott  1   6   7   8   9
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Bioactive multi-protein adsorption enables targeted mast cell nanotherapy

Fanfan Du et al. Res Sq. .

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Conflict of interest statement

Competing interests The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Protein surfaces induce site-to-site dipole rotation of interfacial propylene sulfone.
(a) Simultaneously recorded cryo-STEM and cryo-SEM images showing a typical PPSU hollow nanoparticle and its surface, respectively. (b) Schematic illustration of the dipole moment of amphiphilic propylene sulfone, the repeating unit of PPSU homopolymer (blue indicates hydrophilic and grey indicates hydrophobic for all the schemes in the work, 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 inducing 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 weak enough not to outcompete the forces governing protein folding despite irreversible adsorption. Fast spreading of protein on a hydrophobic surface is included for comparison.
Fig. 2
Fig. 2. Atomistic explicit solvent simulations confirm the interfacial hydrophilic-hydrophobic transition within PPSU surfaces upon trypsin adsorption.
(a) Simulation snapshot of equilibrated 600-chain NP adsorbed with all the 6 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 PPSU chains and trypsin via H-bonds. (b) The Lennard-Jones trypsin-NP interactions dominated over the trypsin-NP Coulombic interactions, supporting the hydrophobicity-driven feature of trypsin adsorption. (c) Orientations of the 6 trypsin molecules are non-specific upon adsorption. The adsorption sites and active sites on trypsin are colored in red and blue, respectively. (d) Percentage of sulfone groups at the trypsin-NP contact region revealing enhanced NP surface hydrophobicity after trypsin adsorption. (e) No significant (ns) differences in trypsin hydration were detected between the adsorbed trypsin and unbound trypsin. The numbers of trypsin-water H-bonds and of water neighbors of trypsin were calculated. Error bars represent standard deviations. Statistical significance is determined by Tukey-post hoc test.
Fig. 3
Fig. 3. PPSU NPs preserve protein function within stable adlayers.
(a) Schematic illustration of the rapid and facile process of coating PPSU NPs with protein adlayers. After incubation, unbound proteins are removed by thorough washing. (b) The zeta potential of protein-coated NPs is dependent on the adsorbed proteins. (c) SAXS data supporting the increase of shell thicknesses from 5.3 nm to 7.1 nm after BSA adsorption. (d) Stability of trypsin@NP. Desorption of trypsin from the nanozymes is undetectable during storage at 4 °C for 48 h by MALDI-TOF mass spectrometry. (e) Kinetic assay demonstrating 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. 10 nm colloidal gold–secondary antibody, indicated by the arrows. (h) Targeting of irreversibly adsorbed anti-CD3 towards Jurkat T cells is confirmed by flow cytometry as assessed by percentage of NP positive cells and median fluorescence intensity (MFI). Statistical significance is determined by Tukey-post hoc test: * p < 0.005, ** p < 0.001, *** p < 0.0005, **** p < 0.0001.
Fig. 4
Fig. 4. Optimization of multi-antibody coatings allows mast cell targeting for anaphylaxis nanotherapy.
(a) PPSU-based nanomedicines consisting of co-adsorbed anti-Siglec-6 and anti-FcεRIα with controlled surface density (as wt% of PPSU) of anti-Siglec-6. (b) Imaging the nanomedicines (blue, indicated by red arrows) on the surface of a mast cell (grey) by SEM. (c) Activation of mast cells (left) is achieved via cross-linking of FcεRIα by anti-FcεRIα/BSA@NP, whereas the nanomedicine inhibits mast cell degranulation (right) via co-localized engagement. (d) Optimizing nanomedicine formulation via adjusting the surface density of anti-Siglec-6. In vitro results showing that 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 FcεRI in suppressing CD107a and CD63 expression by mast cells. The optimized formulation (0.01 wt% of anti-Siglec-6) is used for 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. (d-e) Inhibition is normalized from mast cells receiving only anti-FcεRIα and expressing a positive population mean of 52.7 ± 5% for CD63 and 71.2 ± 8% for CD107a (n = 3). (f) The optimized nanomedicine (0.01 wt% of anti-Siglec-6) succeeds in administering allergen immunotherapy without triggering anaphylaxis in a humanized mouse model. Results were from 2 combined datasets (total n = 6). Statistical significance is determined by Tukey-post hoc test: * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
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