F ova

Fluorescein conjugates of ovalbumin (F-OVA, Life Technologies, Carlsbad, CA, USA) were used as the cancer antigen to test its in vivo cellular uptake. First, F-OVA (100 μg) was simply mixed with HMS nanoparticles or HPS microparticles (0.9 mg/100 μL) in saline and subcutaneously injected into the flank of a mouse (C57BL/6J, female, 6 weeks old, CLEA Inc., Tokyo, Japan). The mice were divided into four groups: (1) F-OVA, (2) HMS-F-OVA, (3) HPS-F-OVA, and (4) HMS-HPS-F-OVA. Cells around the injection site were collected on d3 to prepare a single-cell suspension. Non-specific staining was inhibited by anti-CD16/CD32 antibody (2.4G2, BD Pharmingen, San Jose, CA, USA). Then, the cells were stained using anti-mouse CD11c and anti-mouse CD86 antibodies (Biolegend, San Diego, CA, USA) for 30 min. Flow cytometry was performed using FACSAria (BD Bioscience, Franklin Lakes, NJ, USA).

Bone marrow-derived dendritic cells (BMDCs) collected in accordance with a previous report [24 (link)] were used for in vitro testing. At first, Gd₂O₃ nanotubes were mixed with a green fluorescent fluorescein ovalbumin conjugate (F-OVA, Life Technologies, Carlsbad, CA, USA) at 4 °C overnight. Then, BMDCs were cultured with F-OVA and F-OVA-loaded Gd₂O₃ nanotubes (25 μg/mL for Gd₂O₃ nanotubes; 5 μg/mL for F-OVA). After culture for 1 d, the BMDCs were washed with calcium- and magnesium-free phosphate buffered saline [PBS(-)] and tested using a fluorescent microplate reader (MTP-900, Hitachi). In addition, the cells were stained with LysoTracker red DND-99 (Invitrogen, Waltham, MA, USA) and Hoechst (Thermo Fisher, Waltham, MA, USA), and analyzed using a confocal laser scanning microscope (Leica, Wetzlar, Germany, TCS SP5). The media were also collected after culture for 2 days and tested using mouse TNFα and IL1β enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Jose, CA, USA).