**4. Conclusions**

In this study, we have investigated whether different types of surface functionalization of SiNP impact immune effects by differential activation, i.e., the maturation, of APCs. In addition to first-line safety assessment, early insight on efficacy (second-line) is crucial for decision making in pharmaceutical development; in the context of immunotherapy, this represents the investigation of the immune activating and modulating capacities of administered substances, including nanomaterial platforms. Both the immune activating as well as modulating potentials involve APCs as a crucial player. As a suitable surrogate for human APCs, monocyte-derived dendritic cells were used here. We observed negligible changes induced by the differently functionalized SiNPs in the viability and maturation state of APCs, which is an indication of their immunological inertness. Nevertheless, a significant impact on the binding of proteins to the differently functionalized SiNPs was evident, and could not be simply explained by considering the isoelectric point of protein the net charge of particles upon functionalization. Enhanced uptake was noted for the particle system used here, irrespective of functionalization, while surface functionalization even optimized their immunological inertness, suggesting that SiNPs may be a suitable vehicle for the delivery of biopharmaceutical products, in particular for allergen-specific immunotherapy. Inert (nano)carriers afford the opportunity to intentionally load additional immune modifiers for fine-tuning of desired immunological outcome (e.g., immunomodulation towards a regulatory T helper cell response in allergy or immunosuppression in autoimmunity). As protein binding can be modulated by various types of chemical functionalization, functionalized SiNPs could be useful to deliver a range of biopharmaceutical drugs and lessen the potential immune response. Nevertheless, in vivo studies about the safety and efficacy profiles are required.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14051103/s1, Reference [56] is cited in the supplementary materials. Figure S1: Schiff base reaction for testing amino functionalization of SiNP; Figure S2. Gating strategy for moDC experiments; Figure S3. Fluorescence minus one (FMO) controls of moDCs; Figure S4. FTIR spectra of differently functionalized SiNP; Figure S5. NTA and DLS size distribution graphs; Figure S6. SDS-PAGE for estimation of protein binding; Figure S7. Suspension stability test; Table S1. Uptake inhibitors; Figure S8. SDS-PAGE analysis with pHrodo-labelled Bet v 1; Figure S9. Viability of moDCs on exposure to labelled allergen; Figure S10. Viability of moDCs on exposure to selected inhibitors.

**Author Contributions:** Conceptualization: L.J. and M.H. Methodology: B.P., L.J., J.H.-H., M.G. and M.H. Investigation: B.P., L.J., M.G. and H.-H.D. Visualization: B.P. and L.J. Writing—original draft: B.P., L.J. and M.H. Writing—review & editing: B.P., L.J., M.G., J.H.-H., A.D. and M.H. Supervision: L.J. and M.H. Funding acquisition: A.D. and M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the international PhD program "Immunity in Cancer and Allergy" of the Austrian Science Fund (Open Access Funding by the Austrian Science Fund (FWF), grant number W01213), the EU H2020 NanoCommons project (grant number 731032), the SmartCERIALS project of the Austrian Research Promotion Agency (FFG, grant number 890610) and the Allergy Cancer Bio-Nano Research Centre (ACBN) of the University of Salzburg.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in this manuscript and its supplementary information files. Raw data of the cyto-/chemokine secretion multiplex assay are available at https://doi.org/10.5281/zenodo.6473305 (accessed on 20 April 2022).

**Acknowledgments:** The authors thank Robert Mills-Goodlet; Ingrid Hasenkopf; Alexandra Fux; Angela Schmiedlechner; Sabine Hofer; Norbert Hofstätter; and Nanett Mokran for provision of materials for experiments, conduct of supporting experiments and scientific inputs.
