*3.2. Lipopolysaccharide/Endotoxin Content in SiNP*

dispersion.

Lipopolysaccharide (LPS) induces the expression of pro-inflammatory cytokines and surface activation markers at a concentration as low as 20 pg/mL in primary human monocytes [38]. A potential contamination of LPS can cause a health threat in pharmaceutical products or compromise experimental results. Thus, it is necessary to determine the LPS contents in the particle systems, proteins and reagents involved in the in vitro experiments. These were tested by the most sensitive monocyte activation test [39,40] and the HEK BlueTM LPS Detection Kit 2. The results from both the tests indicate that the LPS content in the samples is below 20 pg/mL (Figure 2). Bet v 1 was previously tested after the original preparation and showed LPS values of less than 0.044 pg/µg [41]. *3.2. Lipopolysaccharide/Endotoxin Content in SiNP*  Lipopolysaccharide (LPS) induces the expression of pro-inflammatory cytokines and surface activation markers at a concentration as low as 20 pg/mL in primary human monocytes [38]. A potential contamination of LPS can cause a health threat in pharmaceutical products or compromise experimental results. Thus, it is necessary to determine the LPS contents in the particle systems, proteins and reagents involved in the in vitro experiments. These were tested by the most sensitive monocyte activation test [39,40] and the HEK BlueTM LPS Detection Kit 2. The results from both the tests indicate that the LPS content in the samples is below 20 pg/mL (Figure 2). Bet v 1 was previously tested after the original preparation and showed LPS values of less than 0.044 pg/µg [41].

**Figure 2.** Quantification of LPS content by (**A**) HEK blue assay and (**B**) monocyte activation test (MAT) to analyze LPS contamination in the SiNP samples and Bet v 1. The LPS content was quantified based on LPS standard curves. The data are presented as mean ± SD (*n* = 4). **Figure 2.** Quantification of LPS content by (**A**) HEK blue assay and (**B**) monocyte activation test (MAT) to analyze LPS contamination in the SiNP samples and Bet v 1. The LPS content was quantified based on LPS standard curves. The data are presented as mean ± SD (*n* = 4).

#### *3.3. Impact of Particle Functionalization on the Binding Efficiency* Nanoparticle surfaces spontaneously adsorb proteins and form a nanoparticle pro-

*Pharmaceutics* **2022**, *13*, x FOR PEER REVIEW 10 of 19

*3.3. Impact of Particle Functionalization on the Binding Efficiency* 

Nanoparticle surfaces spontaneously adsorb proteins and form a nanoparticle protein corona [42,43]. Allergens are known to form a stable corona on the particle interface [43]. We have previously demonstrated that silica nanoparticles both mesoporous and nonporous adsorb Bet v 1, effectively forming a biocorona [22,44]. The efficiency of binding was determined by SDS-PAGE and BCA assay. From the SDS-PAGE analysis, 21.75 ± 6.50% of Bet v 1 was bound to SiNPs at pH 7.4, whereas the SiNP\_M and SiNP\_A showed binding efficiencies of 11.41 ± 4.55% and 0.1 ± 0.1%, respectively (Figures 3A and S6). The BCA assay displayed similar yet statistically significant results (Figure 3B). Both SiNP\_M and SiNP\_A functionalization decreased the binding efficiency when compared to the SiNPs. A similar decrease in serum protein adsorption to COOH-modified mesoporous SiNPs was previously reported by Lin et al. [45] and Beck et al. [46]. While a decreased protein binding was reported to be due to the increasing negative charge on the particles surface, the influence of the particles' charge on protein binding was not evident in this study. Furthermore, SiNP\_A showed almost negligible binding capacity. The pKa of the used SiNP\_A is 7.6, based on the components' ratios during functionalization [47]. Consequently, Bet v 1 displays an isoelectric point (IEP) in the similar range [48]. This would result in hindered binding due to the similarities in their isoelectric points. Here, it is clear that functionalization of SiNPs is altering the binding efficiency. tein corona [42,43]. Allergens are known to form a stable corona on the particle interface [43]. We have previously demonstrated that silica nanoparticles both mesoporous and nonporous adsorb Bet v 1, effectively forming a biocorona [22,44]. The efficiency of binding was determined by SDS-PAGE and BCA assay. From the SDS-PAGE analysis, 21.75 ± 6.50% of Bet v 1 was bound to SiNPs at pH 7.4, whereas the SiNP\_M and SiNP\_A showed binding efficiencies of 11.41 ± 4.55% and 0.1 ± 0.1%, respectively (Figures 3A and S6). The BCA assay displayed similar yet statistically significant results (Figure 3B). Both SiNP\_M and SiNP\_A functionalization decreased the binding efficiency when compared to the SiNPs. A similar decrease in serum protein adsorption to COOH-modified mesoporous SiNPs was previously reported by Lin et al. [45] and Beck et al. [46]. While a decreased protein binding was reported to be due to the increasing negative charge on the particles surface, the influence of the particles' charge on protein binding was not evident in this study. Furthermore, SiNP\_A showed almost negligible binding capacity. The pKa of the used SiNP\_A is 7.6, based on the components' ratios during functionalization [47]. Consequently, Bet v 1 displays an isoelectric point (IEP) in the similar range [48]. This would result in hindered binding due to the similarities in their isoelectric points. Here, it is clear that functionalization of SiNPs is altering the binding efficiency.

**Figure 3.** The binding efficiencies of differently functionalized SiNP samples were determined (**A**) directly from the pellet by SDS-PAGE and (**B**) indirectly from the supernatant by BCA assay. The data are presented as mean ± SD (*n* = 4) \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001. **Figure 3.** The binding efficiencies of differently functionalized SiNP samples were determined (**A**) directly from the pellet by SDS-PAGE and (**B**) indirectly from the supernatant by BCA assay. The data are presented as mean ± SD (*n* = 4) \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001.
