*3.6. SiNP Adsorption and Functionalization Do Not Alter the Maturation of Antigen-Presenting Cells*

In the final part of the study, we investigated the impact of functionalization on the maturation of APCs, which is characterized by the expression of co-stimulatory molecules like HLA-DR, CD86, CD83, and more, along with the release of soluble mediators such as cytokines. These co-stimulatory molecules and cytokines mediate the presentation of antigen to the T cells to modulate an immune response. To study the maturation of APCs, immature moDCs were stimulated with the samples for 24 h and the expression of CD40, CD80, CD83, CD86, and HLA-DR was measured by flow cytometry. The gating strategy is presented in Figure S2. The results obtained clearly showed negligible deviations in the expression of co-stimulatory molecules for Bet v 1 when compared to the untreated moDCs (Figure 6). This is in line with the study of Aglas et al. [53]. Furthermore, as reported before, the positive control (100 ng/mL LPS) showed a significant upregulation of all measured costimulatory molecules [54]. SiNPs showed the tendency to upregulate CD40, CD86, and HLA-DR, particularly when protein was conjugated onto them. In contrast, SiNP\_M and SiNP\_A caused no upregulation of costimulatory molecules and can thus be considered as a more inert particle system than silica without further chemical functionalization (Figure 6). The release of soluble mediators upon stimulation of moDCs was investigated using the human Procarta Plex Multiplex assay, where 45 different cytokines, chemokines, and growth factors were analyzed; raw data for the multiplex assay are available at https://doi.org/10 .5281/zenodo.6473305 (accessed on 20 April 2022). For the control, LPS was used; it induced the release of the cyto-/chemokines and growth factors (IFN-gamma, IL12p70, IL-13, IL-2,

IL-4, IL-6, TNF-alpha, IL-18, IL-10, IL-1alpha, IL-1RA, Eetaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1alpha, MIP-1beta, SDF-1alpha, RANTES, HGF, and VEGF-A) in agreement with previous results [55]. When testing the SiNP samples and Bet v 1, we observed the release of the cyto-/chemokines IL-4, IL-6, IL-8, TNF-α, IL-1RA, MCP-1, MIP-1α, and MIP-1β (Figure 7). Notably, we found statistical significance between the Bet v 1-conjugated SiNP samples in IL-8, IL1-RA, and MIP-1α release, suggesting that functionalized particles are immunologically more inert compared to non-functionalized SiNPs when they are used as biopharmaceutical carrier system. To sum up, SiNPs tended to cause the upregulation of costimulatory molecules and increased release of pro-inflammatory cytokines, while these tendencies were not present in the functionalized samples. This indicates that surface functionalization of SiNPs can further decrease the maturation potential of moDCs and can thus be instrumental in preventing the activation and proliferation of T cells, rendering them safer in the context of inducing of an unwanted immune response. It further implies that functionalized SiNPs may remain undetectable as they showed no propensity to activate the innate immune system. *Pharmaceutics* **2022**, *13*, x FOR PEER REVIEW 13 of 19

**Figure 5.** Impact on the uptake of allergen by moDCs. (**A**) Kinetics of uptake represented as mean fluorescence intensity (MFI) over the course of 24 h with SiNP-Bet, SiNP\_M-Bet, and Bet. (**B**) Inhibition of uptake with the endocytosis inhibitors chlorpromazine hydrochloride (CPZ), cytochalasin D (CytoD), filipin, and rottlerin after stimulation with moDCs for 24 h (*n* = 4 individual donors). The data are presented as mean ± SD (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001). **Figure 5.** Impact on the uptake of allergen by moDCs. (**A**) Kinetics of uptake represented as mean fluorescence intensity (MFI) over the course of 24 h with SiNP-Bet, SiNP\_M-Bet, and Bet. (**B**) Inhibition of uptake with the endocytosis inhibitors chlorpromazine hydrochloride (CPZ), cytochalasin D (CytoD), filipin, and rottlerin after stimulation with moDCs for 24 h (*n* = 4 individual donors). The data are presented as mean ± SD (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001).

*3.6. SiNP Adsorption and Functionalization Do Not Alter the Maturation* 

In the final part of the study, we investigated the impact of functionalization on the maturation of APCs, which is characterized by the expression of co-stimulatory molecules like HLA-DR, CD86, CD83, and more, along with the release of soluble mediators such as

immature moDCs were stimulated with the samples for 24 h and the expression of CD40, CD80, CD83, CD86, and HLA-DR was measured by flow cytometry. The gating strategy is presented in Figure S2. The results obtained clearly showed negligible deviations in the expression of co-stimulatory molecules for Bet v 1 when compared to the untreated moDCs (Figure 6). This is in line with the study of Aglas et al. [53]. Furthermore, as reported before, the positive control (100 ng/mL LPS) showed a significant upregulation of

*of Antigen-Presenting Cells* 

all measured costimulatory molecules [54]. SiNPs showed the tendency to upregulate CD40, CD86, and HLA-DR, particularly when protein was conjugated onto them. In contrast, SiNP\_M and SiNP\_A caused no upregulation of costimulatory molecules and can thus be considered as a more inert particle system than silica without further chemical functionalization (Figure 6). The release of soluble mediators upon stimulation of moDCs was investigated using the human Procarta Plex Multiplex assay, where 45 different cytokines, chemokines, and growth factors were analyzed; raw data for the multiplex assay are available at https://doi.org/10.5281/zenodo.6473305 (accessed on 20 April 2022). For the control, LPS was used; it induced the release of the cyto-/chemokines and growth factors (IFN-gamma, IL12p70, IL-13, IL-2, IL-4, IL-6, TNF-alpha, IL-18, IL-10, IL-1alpha, IL-

1RA, Eetaxin, GRO-alpha, IL-8, IP-10, MCP-1, MIP-1alpha, MIP-1beta, SDF-1alpha, RANTES, HGF, and VEGF-A) in agreement with previous results [55]. When testing the SiNP samples and Bet v 1, we observed the release of the cyto-/chemokines IL-4, IL-6, IL-8, TNF-α, IL-1RA, MCP-1, MIP-1α, and MIP-1β (Figure 7). Notably, we found statistical significance between the Bet v 1-conjugated SiNP samples in IL-8, IL1-RA, and MIP-1α release, suggesting that functionalized particles are immunologically more inert compared to non-functionalized SiNPs when they are used as biopharmaceutical carrier system. To sum up, SiNPs tended to cause the upregulation of costimulatory molecules and increased release of pro-inflammatory cytokines, while these tendencies were not present in the functionalized samples. This indicates that surface functionalization of SiNPs can

further decrease the maturation potential of moDCs and can thus be instrumental in pre-

venting the activation and proliferation of T cells, rendering them safer in the context of inducing of an unwanted immune response. It further implies that functionalized SiNPs

**Figure 6.** Co-stimulatory molecule expression by stimulated moDCs with functionalized SiNPs and Bet. (**A**) CD40, (**B**) CD86, (**C**) CD83, (**D**) HLA-DR, and (**E**) CD80 were analyzed. LPS was used as the **Figure 6.** Co-stimulatory molecule expression by stimulated moDCs with functionalized SiNPs and Bet. (**A**) CD40, (**B**) CD86, (**C**) CD83, (**D**) HLA-DR, and (**E**) CD80 were analyzed. LPS was used as the positive control and a statistical significance was observed in comparison to the cell control indicating a successful stimulation. All particles and conjugates were compared to Bet v 1 for statistical analysis (*n* = 6 individual donors) (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\*\* *p* ≤ 0.0001).

positive control and a statistical significance was observed in comparison to the cell control indicating a successful stimulation. All particles and conjugates were compared to Bet v 1 for statistical

analysis (*n* = 6 individual donors) (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\*\* *p* ≤ 0.0001).

**Figure 7.** Cyto-/chemokine release of APCs. (**A**) IL8, (**B**) MCP-1, (**C**) MIP-1α, (**D**) MIP-1 β, (**E**) IL-4, (**F**) IL-6, (**G**) TNFα, (**H**) IL1-RA. LPS was used as the positive control and a statistical significance **Figure 7.** Cyto-/chemokine release of APCs. (**A**) IL8, (**B**) MCP-1, (**C**) MIP-1α, (**D**) MIP-1 β, (**E**) IL-4, (**F**) IL-6, (**G**) TNFα, (**H**) IL1-RA. LPS was used as the positive control and a statistical significance was observed in comparison to the cell control, indicating successful stimulation. The particles were compared to Bet v 1 for statistical analysis (*n* = 5 individual donors) (\* *p* ≤ 0.05, \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001).
