2.10. Cellular Uptake

In order to study the cellular uptake of NPs, fluorescent DOX was loaded in the MSNs instead of CP and CQ, following the same protocol as detailed above. To check the influence of the His-tag on uptake, control nanoparticles in which B3int was conjugated to the NP surface were also prepared. These were produced by replacing HtB with B3int in the MSN synthesis procedure, and then loading with DOX. The cellular uptake of DOX@MSN−HtB/Cu²⁺ NPs was evaluated by confocal laser scanning microscopy (CLSM, Nikon C1-si, Tokyo, Japan). B16 and L-929 cells were separately plated on glass-bottomed culture dishes (1 × 10⁵ cells·mL⁻¹, 2 mL) for 24 h at 37 °C. The media was aspirated, and then replaced with fresh media containing the test formulations. The cells were divided into 4 groups—(1) control; (2) free DOX (0.5 μg·mL⁻¹); (3) DOX@MSN−B3int NPs (DOX: 0.5 μg·mL⁻¹); and (4) DOX@MSN−HtB/Cu²⁺ NPs (DOX: 0.5 μg·mL⁻¹)—and incubated in complete medium for another 4 h. After removal of the medium and washing with PBS, the cells were stained with a lysosome staining kit for 45 min, fixed with aqueous formaldehyde (4%, v/v) at room temperature, stained with DAPI for 3 min, and then imaged by CLSM.