2.8.1. Cytokine Multiplexing

The 45-Plex Human Procarta-PlexTM (ThermoFisher, Waltham, MA, USA) was used to measure the release of cytokines and chemokines from moDCs activated with SiNPs following manufacturer's instructions. Shortly, beads were rinsed (PBS, 0.05% Tween-20) and resuspended in the assay buffer (PBS, 0.05% Tween-20, 1% heat-inactivated FCS) (Biowest) before adding 8.34 µL per well into a 96 V-bottom well plate. Afterwards, 15 µL of samples were added, and the plate was incubated on a 500 rpm shaking orbital shaker at 4 ◦C overnight. The following day, samples were washed three times and resuspended in 15 µL of detection antibody solution before being incubated at room temperature for 30 min. After three further washes, 20 µL of Streptavidin-PE solution (1:1 in assay buffer) was added to each well and incubated at room temperature for 30 min. Lastly, the samples were rinsed three times before being resuspended in drive fluid for testing. The data were processed using Procarta Plex Analyst Software (ThermoFisher, Waltham, MA, USA) and measured on a Luminex Magpix device.

#### 2.8.2. ELISA *Pharmaceutics* **2022**, *13*, x FOR PEER REVIEW 8 of 19

ELISA was performed in accordance with the manufacturer's protocol (Peprotech, London, UK).

### *2.9. Statistical Analysis 2.9. Statistical Analysis*

Statistical analyses were accomplished with GraphPad Prism 9. For multiple comparisons, one-way ANOVA (Analysis of Variance) followed by a Tukey's post hoc test was performed; *p*-values ≤ 0.05 were considered as statistically significant (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001). Statistical analyses were accomplished with GraphPad Prism 9. For multiple comparisons, one-way ANOVA (Analysis of Variance) followed by a Tukey's post hoc test was performed; *p*-values ≤ 0.05 were considered as statistically significant (\* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; \*\*\*\* *p* ≤ 0.0001).

### **3. Results and Discussion 3. Results and Discussion**

#### *3.1. Characterization of SiNPs 3.1. Characterization of SiNPs*

SiNPs synthesized wibyth the Stöber method were chemically functionalized with negatively charged COOH (SiNP\_M) and positively charged NH<sup>2</sup> (SiNP\_A) functional groups. As the initial step, the SiNPs and functionalized SiNPs were characterized for their primary size and morphology. The TEM images of the particles displayed a uniform spherical shape with a primary size of 51.02 ± 3.80 nm (SiNP), 47.31 ± 4.72 nm (SiNP\_A) and 50.42 ± 4.57 nm (SiNP\_M) (Figure 1) [22]. SiNPs synthesized wibyth the Stöber method were chemically functionalized with negatively charged COOH (SiNP\_M) and positively charged NH2 (SiNP\_A) functional groups. As the initial step, the SiNPs and functionalized SiNPs were characterized for their primary size and morphology. The TEM images of the particles displayed a uniform spherical shape with a primary size of 51.02 ± 3.80 nm (SiNP), 47.31 ± 4.72 nm (SiNP\_A) and 50.42 ± 4.57 nm (SiNP\_M) (Figure 1) [22].

**Figure 1.** TEM images of the differently functionalized SiNPs for determination of primary particle size (**A**) SiNP, (**B**) SiNP\_A, (**C**) SiNP\_M, Size bar: 100 nm. **Figure 1.** TEM images of the differently functionalized SiNPs for determination of primary particle size (**A**) SiNP, (**B**) SiNP\_A, (**C**) SiNP\_M, Size bar: 100 nm.

Thereafter, the efficiency of functionalization was tested by FTIR, zeta potential measurements, and the salicylaldehyde test. The FTIR spectra of all the samples exhibited the characteristic peak of silica at 1070 cm−1 [34] (Figure S4A–C). SiNP\_A showed an additional peak at 1547 cm−1 due to NH2 bending, which indicated that the functionalization was effective [35] (Figure S4D). For SiNP\_M we observed the "C=O" stretch vibration at a wavelength of 1730–1700 cm−1; however, the "C-O" stretch and "O-H" bend vibrations at 1320–1310 and 960–900 cm−1 were not evident [36] (Figure S4C,D). This might be due to some partial degree of esterification of the carboxyl group when washed with ethanol [37]. Thereafter, the efficiency of functionalization was tested by FTIR, zeta potential measurements, and the salicylaldehyde test. The FTIR spectra of all the samples exhibited the characteristic peak of silica at 1070 cm−<sup>1</sup> [34] (Figure S4A–C). SiNP\_A showed an additional peak at 1547 cm−<sup>1</sup> due to NH<sup>2</sup> bending, which indicated that the functionalization was effective [35] (Figure S4D). For SiNP\_M we observed the "C=O" stretch vibration at awavelength of 1730–1700 cm−<sup>1</sup> ; however, the "C–O" stretch and "O–H" bend vibrations at 1320–1310 and 960–900 cm−<sup>1</sup> were not evident [36] (Figure S4C,D). This might be due tosome partial degree of esterification of the carboxyl group when washed with ethanol [37].

To further confirm effective particle functionalization, we determined the pH values of their aqueous suspensions and the surface charge of the particles with the ZetaSizer Nano ZS. SiNP\_A exhibited a pH of 8.5 and positive zeta potential (33.4 ± 1.1 mV, whereas SiNP\_M exhibited a pH of 4.5 and a negative zeta potential (−26.2 ± 1.8 mV) when compared to the SiNPs that displayed a pH of 7.0 and a negative zeta potential (−25.7 ± 0.5 mV) (Table 1). Moreover, the amino functionalization was tested by a Schiff base reaction. The addition of salicylaldehyde to SiNP\_A led to the formation of a bright yellow-colored To further confirm effective particle functionalization, we determined the pH valuesof their aqueous suspensions and the surface charge of the particles with the ZetaSizer Nano ZS. SiNP\_A exhibited a pH of 8.5 and positive zeta potential (33.4 <sup>±</sup> 1.1 mV, whereasSiNP\_M exhibited a pH of 4.5 and a negative zeta potential (−26.2 <sup>±</sup> 1.8 mV) when compared to the SiNPs that displayed a pH of 7.0 and a negative zeta potential (−25.7 ± 0.5 mV) (Table 1). Moreover, the amino functionalization was tested by a Schiff base reaction. The addition of salicylaldehyde to SiNP\_A led to the formation of a bright yellow-colored Schiff

Schiff base. Centrifugation of the sample resulted in the formation of a yellow-colored pellet and a clear supernatant (Figure S1). This further proved effective amino functional-

ization.

base. Centrifugation of the sample resulted in the formation of a yellow-colored pellet and a clear supernatant (Figure S1). This further proved effective amino functionalization.

**Table 1.** Physicochemical properties of SiNP, SiNP\_A and SiNP\_M. Size (NTA): Average ± SD of the hydrodynamic diameter mode values of five measurements. Size (DLS): Average ± SD of mean values from intensity- and number-weighted distribution analyses of three measurements. Zeta potential: average ± SD in mV determined when resuspended in buffer (HEPES). PDI: Polydispersity index ranging from 0 (perfectly uniform sample with respect to particle size) to 1 (highly polydisperse sample with multiple particle size populations). *Pharmaceutics* **2022**, *13*, x FOR PEER REVIEW 9 of 19 **Table 1.** Physicochemical properties of SiNP, SiNP\_A and SiNP\_M. Size (NTA): Average ± SD of the hydrodynamic diameter mode values of five measurements. Size (DLS): Average ± SD of mean


Finally, the hydrodynamic sizes and distributions of the particles were determined by NTA and DLS (weighted for number and intensity, Figure S5). The SiNPs displayed an intensity-weighted mean hydrodynamic size of 99.0 ± 42.4 nm (SiNPs), 114.5 ± 58.9 nm (SiNP\_M), and 108.0 ± 46.4 nm (SiNP\_A) by DLS, which were in good agreement with NTA (Table 1). The intensity-weighted values and distributions (Figure S5A–F) show that for most of the particles, small agglomerates were abundant in suspension, whereas the number-weighted distributions (Figure S5G–I) indicate the presence of still non-agglomerated particles, since their sizes match the values obtained from TEM (Figure 1). From all the characterization data, it can be concluded that the functionalization worked efficiently and both the functionalized and SiNPs exhibited a hydrodynamic size of about 100 nm in dispersion. SiNP\_M 136.8 ± 62.1 114.5 ± 58.9 50.2 ± 7.4 −26.2 ± 1.8 0.38 Finally, the hydrodynamic sizes and distributions of the particles were determined by NTA and DLS (weighted for number and intensity, Figure S5). The SiNPs displayed an intensity-weighted mean hydrodynamic size of 99.0 ± 42.4 nm (SiNPs), 114.5 ± 58.9 nm (SiNP\_M), and 108.0 ± 46.4 nm (SiNP\_A) by DLS, which were in good agreement with NTA (Table 1). The intensity-weighted values and distributions (Figure S5A–F) show that for most of the particles, small agglomerates were abundant in suspension, whereas the number-weighted distributions (Figure S5G–I) indicate the presence of still non-agglomerated particles, since their sizes match the values obtained from TEM (Figure 1). From all the characterization data, it can be concluded that the functionalization worked efficiently and both the functionalized and SiNPs exhibited a hydrodynamic size of about 100 nm in
