2.1 Materials and animals
OVA, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT), fluorescein 5(6)-isothiocyanate (FITC), and MSNs were purchased from Sigma-Aldrich (St. Louis, MO, USA). CpG-ODN1826 (5′-TCCATGACGTTCCTGACGTT-3′–phosphonothioate) was synthesized by Genewiz (Suzhou, China). Bicinchoninic acid protein assay kits were purchased from Applygen (Beijing, China). Anti-mouse monoclonal antibodies (mAb) for flow cytometry (CD45, CD3, F4/80, B226, CD19, CD11b, CD11c, CD80, CD86, MHC-II, and CD8α) and APC-conjugated anti-mouse interferon γ (IFNγ) antibodies were purchased from eBioscience (San Diego, CA, USA). Granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4 were purchased from PeproTech (Cranbury, NJ, USA). Hoechst 33342 and LysoTracker Red DND-99 were purchased from Invitrogen (Carlsbad, CA, USA) and Solarbio (Beijing, China), respectively. The EasySep mouse CD8+ T cell isolation kit (negative selection) was purchased from STEMCELL Technologies (Vancouver, Canada). Enzyme-linked immunosorbent assays (ELISA) for TNFα and IFNγ were purchased from Invitrogen. The murine agonistic αCD40 mAb (FGK45) and mouse IgG2a isotype control were purchased from BioCell (Irvine, CA, USA). Red blood cell lysis solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA). OVA-specific T cell receptor (TCR) transgenic mice (OT-I mice) and B16-OVA cells were kindly provided by Prof. Shengdian Wang (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). Female C57BL/6 mice were purchased from Vital River Laboratory (Beijing, China). All mice were housed in a specific pathogen-free facility.
2.2 Synthesis of CD40 mAb-engineered MSNs
CD40 mAb was covalently attached to the surface of the MSN-NH2 particles using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling chemistry. CD40 mAb-bound EDC (120 μg, final concentration 2mM) and N-hydroxysuccinimide (final concentration 5 mM) were dissolved in 0.1 M MES buffer (pH 6.0) for 15 min at room temperature. Simultaneously, MSN-NH2 particles (3 mg) were dispersed in phosphate-buffered saline (PBS; 100 mL, pH 8.5). The two solutions were mixed together for 2 h at room temperature. The MSN-CD40 nanoparticles were washed three times with PBS (pH 7.4) at 12,000 rpm. The nanoparticles were collected in KBr pellets, and the morphology and size of the nanoparticles were analyzed by Fourier transform-infrared spectroscopy (FT-IR; Nicolet iS 50; Thermo Fisher Scientific) and transmission electron microscopy (TEM; JEOL JEM-200CX, Japan) at 200 kV, respectively. The zeta potentials and hydrodynamic sizes of MSN-CD40/OVA/CpG nanoparticles and the controls were measured using a nanoparticle analyzer (Nano-ZS90, Malvern Instruments Co., Ltd., Malvern, UK).
2.3 Evaluation of cytotoxicity
C57BL/6 mice (6–8 weeks old) were sacrificed by cervical dislocation, the tibiae of hind limbs and intact femurs were removed using surgical instruments. The bones were washed in PBS (pH 7.4) and bone marrow was flushed with PBS (pH 7.4) using a syringe. Clusters within the bone marrow were disaggregated using red blood cell lysis solution to yield a homogeneous cell suspension. The cells were seeded in a 100-mm bacteriological Petri dish in 20 mL of RPMI 1640 media containing 10% fetal bovine serum (FBS), 20 ng/mL GM-CSF, and 5 ng/mL IL-4 on day 0. Three days later, half of the media was replaced with fresh RPMI 1640 media containing the same concentrations of GM-CSF and IL-4. Cells were maintained in a humidified incubator at 37 °C and 5% CO2. Bone marrow-derived dendritic cell (BMDC) viability was measured by the MTT assay. Briefly, BMDCs (5 × 104 cells) were seeded in a 96-well plate and incubated with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG, MSN/OVA/CpG nanoparticles), diluted to a concentration of 12.5 μg/mL OVA, for 24 h. After removing the supernatant, 20 μL of MTT (0.5 mg/mL in fresh RPMI-1640) was added to each well for 4 h at 37 °C, after which the medium was removed and dimethyl sulfoxide was added to solubilize the formazan. The absorbance of each well was measured at 560 nm.
2.4 Evaluation of cellular uptake
The intracellular uptake of FITC-labeled OVA by mouse splenocytes was determined by flow cytometry. Splenocytes (2 × 106 cells/well) were seeded onto a 24-well plate and subsequently incubated with fluorescently labeled OVA, using MSN-CD40/OVA-FITC/CpG nanoparticles or appropriate controls (OVA-FITC/CpG and MSN-IgG/OVA-FITC/CpG and MSN/OVA-FITC/CpG nanoparticles) at a final OVA-FITC concentration of 12.5 μg/mL, and incubated for 4 h at 37 °C. The cells were washed with cold PBS (pH 7.2) and stained with murine anti-CD45, anti-B220, anti-CD19, anti-CD11c, anti-F4/80, and anti-CD11b mAb to gate B cells, DC, and macrophages. We also incubated immature BMDC (4 × 106 cells/wells on six-well plates) in the same way and, after washing with cold PBS (pH 7.2), the cells were collected and stained with anti-CD11c mAb to test their uptake ability by flow cytometry. To investigate the uptake mechanism, immature BMDC (4 × 106 cells/well) were planted in six-well plates at 37 °C in the presence of genistein (50 μg/mL) and chlorpromazine (10 μg/mL) for 1 h before incubation with fluorescently labeled OVA in MSN-CD40/OVA-FITC/CpG nanoparticles.
2.5 Confocal imaging
To explore the localization of nanoparticles in DC, immature BMDC (4 × 106 cells/well) were plated onto 24-well plates at 37 °C, stained with LysoTrackerRed and Hoechst 33342, and fixed with paraformaldehyde. The cells were then analyzed by confocal laser scanning microscopy (TCS SP8, Leica, Wetzlar, Germany).
2.6 Maturation and cytokine detection in vitro
Immature BMDCs (2 × 105 cells/well) were seeded onto a 96-well plate and incubated for 24 h with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG and MSN/OVA/CpG nanoparticles) at a final OVA concentration of 12.5 μg /mL. The culture supernatant was removed and stained with anti-CD11c, anti-CD80, and anti-CD86 mAbs for 30 min on ice. The fluorescence markers expressed on the surface of BMDCs were measured by flow cytometry. The culture medium was collected to measure TNFα levels using an ELISA kit.
2.7 In vitro cross-presentation and T cell priming
Immature BMDCs (1 × 106 cells/well) were seeded onto a 96-well U-bottom plate and incubated for 4 h with MSN-CD40/OVA/CpG nanoparticles and appropriate controls (OVA/CpG and MSN-IgG/OVA/CpG and MSN/OVA/CpG nanoparticles) at a final OVA concentration of 12.5 μg/mL. CD8+ T cells were negatively isolated from the lymphocytes of OT-I mice using a mouse CD8+ T cell isolation kit and stained with 2 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 min, after which cells were washed twice with culture medium. Next, CD8+ T cells (1 × 105 cells/well) were seeded onto a 96-well U-bottom plate and cocultured with BMDC (1 × 104 cells/well) for 72 h. The culture medium was collected and the mixed cells were stained with anti-CD8 mAb for flow cytometry. The IFNγ concentration was also measured using an ELISA kit.
2.8 Animal immunization and CTL priming
C57BL/6 mice (6–8 weeks old) were immunized three times at 1-week intervals (n = 5 per group) with saline, OVA/CpG, MSN/OVA/CpG, MSN-IgG/OVA/CpG, or MSN-CD40/OVA/CpG with final concentrations of OVA and CpG of 100 and 30 μg per mouse, respectively. Five days after the last immunization, the immunized mice were sacrificed and the splenocytes were re-stimulated with OVA257–264 peptide for 6 h or 5 days at 37 °C in vitro to acquire CTL effectors. The proportion of IFNγ-secreting activated cytotoxic CD8+ T cells of splenocytes was analyzed by flow cytometry. IFNγ production was measured using an ELISA kit. To analyze DC maturation markers in the lymphocytes after administration of MSN-CD40/OVA/CpG or appropriate controls, the lymphocytes were stained with murine anti-CD45, anti-CD3, anti-CD11c, anti-CD80, anti-CD86, and anti-MHC-II mAb for 30 min on ice, washed with cold PBS, and analyzed by flow cytometry.
2.9 In vivo tumor challenge
B16-OVA cells were cultured in 100 mm cell culture dishes containing RPMI 1640 media with 10% FBS, 100 U/mL penicillin G, and 100 µg/mL streptomycin sulfate. Next, 2 × 105 B16-OVA melanoma cells were injected subcutaneously into the tail base of C57BL/6 mice, which were then immunized three times at 1-week intervals (n = 5 per group). After 3 days, the mice were subcutaneously injected with saline, OVA/CpG, MSN/OVA/CpG, MSN-IgG/OVA/CpG, or MSN-CD40/OVA/CpG, as appropriate. The final doses of OVA and CpG were 100 and 30 μg per mouse, respectively. The tumor size and body weight were measured every 2 days. Five days after the last immunization, the mice were sacrificed to detect tumor-infiltrating T cells. Tumor tissues were cut into small pieces and stained with murine anti-CD45, anti-CD3, anti-CD4, and anti-CD8 mAb for flow cytometry. After the splenocytes and lymphocytes were cultured with brefeldin A and OVA257−264 peptide at a final concentration of 10 μg/mL, the cells were collected to measure intracellular IFNγ production by flow cytometry. The heart, liver, kidneys, and tumor were harvested and fixed in 4% paraformaldehyde for histopathological examination.
2.10 Statistical analysis
Data are presented as the mean ± standard deviation (SD) unless otherwise stated. Student’s t test was used to determine the statistical significance of differences among the groups. P < 0.05 was considered statistically significant.