*2.7. Kinetics and Mechanism of Uptake*

To assess the impact of functionalization on the uptake by moDCs, the protein Bet v 1 was fluorescently labelled with pHrodo™ Red, succinimidyl ester (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction. The detailed protocol for labelling can be found in the supplementary materials file. The labelled protein was bound to SiNPs as described in 2.4. moDCs were seeded in a 24-well plate at a density of <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells per mL, stimulated with 1 µg/mL of Bet v 1 bound to 100 µg/mL of different SiNPs and incubated for different time points (1, 2, 4, 6, 8, 24 h) to assess the kinetics of uptake. The mechanism of uptake was investigated using four different inhibitors, i.e., 2 µM Cytochalasin D (macropinocytosis and phagocytosis) (Sigma); 20 µM chlorpromazinehydrochloride (clathrin-mediated endocytosis) (Sigma); 1 µM filipin (caveolin-dependent endocytosis) (Sigma); and 10 µM rottlerin (macropinocytosis) (Merck, Darmstadt, Germany) [30–33].

The concentration of inhibitors for the study was determined by testing its effect on the viability of moDCs. The inhibitors were preincubated with the moDCs for the desired time (cytochalasin D for 90 min and chlorpromazine hydrochloride, filipin, and rottlerin for 30 min). The samples were incubated with the cells for a period of 24 h. After the desired incubation time, the amount of pHrodo inside the cells was quantitatively determined by flow cytometry. The gating strategy presented is mean fluorescence intensity (MFI) and this has been obtained by excluding the dead cells, nanoparticles, and doublets. For the inhibition experiments, the Bet v 1 control was considered as 100% uptake control and all other samples were placed in relation to that. The gating strategy is shown in Figure S2 in Supplementary Materials.
