*3.4. SiNPs Do Not Affect the Viability of Antigen-Presenting Cells*

*3.4. SiNPs Do Not Affect the Viability of Antigen-Presenting Cells*  Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by adherence method and artificially differentiated into immature monocyte-derived dendritic cells (moDCs) by the addition of IL-4 and GM-CSF. The immature moDCs were then stimulated with the samples (100 µg/mL of SiNP, 1 µg/mL of Bet v 1 bound to SiNP, and 1 µg/mL of Bet v 1) for 24 h. The impact of surface functionalization on the viability of moDCs was assessed by LDH assay and flow cytometry after staining the cells with Fixable Viability Dye eFluor506 (live/dead staining). The data from the LDH assay show that Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by adherence method and artificially differentiated into immature monocyte-derived dendritic cells (moDCs) by the addition of IL-4 and GM-CSF. The immature moDCs were then stimulated with the samples (100 µg/mL of SiNP, 1 µg/mL of Bet v 1 bound to SiNP, and 1 µg/mL of Bet v 1) for 24 h. The impact of surface functionalization on the viability of moDCs was assessed by LDH assay and flow cytometry after staining the cells with Fixable Viability Dye eFluor506 (live/dead staining). The data from the LDH assay show that the functionalization did not alter the viability of the cells significantly, although we observed a small decrease in viability of moDCs when incubated with SiNP-Bet (Figure 4A). Still, the moDCs proved to be more than 80% viable. The negligible impact of functionalization

on the viability was further proved by live/dead staining using flow cytometry analysis (Figure 4B). The gating strategy used for the analysis of the flow cytometry data is represented in Figure S2. analysis (Figure 4B). The gating strategy used for the analysis of the flow cytometry data is represented in Figure S2.

the functionalization did not alter the viability of the cells significantly, although we observed a small decrease in viability of moDCs when incubated with SiNP-Bet (Figure 4A). Still, the moDCs proved to be more than 80% viable. The negligible impact of functionalization on the viability was further proved by live/dead staining using flow cytometry

*Pharmaceutics* **2022**, *13*, x FOR PEER REVIEW 11 of 19

**Figure 4.** Viability of the moDCs tested with (**A**) LDH assay based on the release of LDH and (**B**) Live/dead staining via flow cytometry after stimulation of moDCs with the samples after 24 h. The **Figure 4.** Viability of the moDCs tested with (**A**) LDH assay based on the release of LDH and (**B**) Live/dead staining via flow cytometry after stimulation of moDCs with the samples after 24 h. The negative control in the experiment was dead cells (incubated at 95 ◦C for 10 min) and the positive control (viable cells) was unstimulated moDCs.

### negative control in the experiment was dead cells (incubated at 95 °C for 10 min) and the positive control (viable cells) was unstimulated moDCs. *3.5. SiNP Adsorption Induces Enhanced Allergen Uptake into Antigen-Presenting Cells Preferentially by Macropinocytosis*

The first and foremost mechanism contributing to an immune response involves the recognition and internalization of antigens. The internalization of nanoparticles can change based on their physicochemical properties such as size, shape and surface chemistry [6]. We studied the kinetics of uptake using pHrodo-labeled Bet v 1. The labeled Bet v 1 was bound to differently functionalized SiNPs and the samples (Bet v 1, SiNP\_M-Bet, and SiNP-Bet) were incubated with moDCs for 1, 2, 4, 6, 8, and 24 h with a concentration of 1 µg/mL of Bet v 1. All particle systems were stable in the suspension for 24 h as controlled by

sedimentation analysis employing the silicomolybdic assay (Figure S7) and the protein labeling did not interfere with SiNP adsorption of Bet v 1, as confirmed by SDS-PAGE (Figure S8). Furthermore, the viability of moDCs was not affected when stimulated with the samples (Figure S9). We observed a significant increase in the uptake of allergen when bound to SiNPs at almost all time points (Figure 5A). This increased tendency for uptake was previously reported by Lu et al. (2009) and Heller et al. (2009) where it was shown that uptake is mostly dependent on size, and the maximum uptake by cells occurred when the size of NPs are about 50 nm [21,49]. Similar to Holzapfel et al. (2006), we found an enhanced uptake of protein bound to COOH-functionalized nanoparticles at early time points (up to 8 h) [50]. To clarify further the potential differences in the uptake behavior, we investigated the mechanism of uptake by using specific inhibitors of the major endocytosis mechanism of antigen uptake. The moDCs were pre-incubated with the inhibitors for their required times (Table S1), stimulated with the samples for 24 h and uptake was monitored by flow cytometry. The viability of moDCs remained 90 to 95% when incubated with the inhibitors (Figure S10). Chloropormazine hydrochoride (CPZ) inhibits clathrin-mediated endocytosis, and cytochalasin D (CytoD) inhibits both phagocytosis and macropinocytosis. Filipin inhibits caveolin-dependent endocytosis and rottlerin inhibits macropinocytosis [30–33]. The results revealed that there was a significant decrease in uptake of SiNP-Bet when incubated with rottlerin (around 75% inhibition), cytochalasin D (around 50% inhibition), and chlorpromazine HCl (40% inhibition). Thus, Bet v 1 molecules adsorbed to SiNPs are taken up mostly by macropinocytosis and Clathrin mediated endocytosis. It has previously been reported by Sahay et al. (2010) [51] that silica-based nanoparticles are internalized with clathrin-mediated endocytosis mechanisms. However, in SiNP\_M-Bet we observed inhibition with rottlerin (around 70%) and cytochalasin D (around 50%). Macropinocytosis was thus revealed as the dominant mechanism of uptake for SiNP\_M-Bet. Similarly, in Bet, about 40% uptake was inhibited with rottlerin and cytochalasin D, suggesting macropinocytosis as the preferred mechanism of uptake (Figure 5B) [32,52]. Overall, we observed an efficient uptake of Bet v 1 when adsorbed to SiNPs and that macropinocytosis is the preferred endocytosis mechanism for the internalization of Bet v 1. However, we did not observe any significant differences in either the kinetics or mechanisms of uptake between the functionalized SiNP samples, putatively due to negligible differences in size.
