Macrophages as carriers of boron carbide nanoparticles dedicated to boron neutron capture therapy | Journal of Nanobiotechnology

Compounds

Two boron carbide preparations were synthesized, differing in physicochemical properties such as nanoparticle size (32 ± 10 nm for preparation 1 – B₄C 1, and 80 ± 30 nm for preparation 2 – B₄C 2), zeta potential and hydrodynamic diameter, as already described in our previous work [18].

Preparation and characterization of bone marrow-derived macrophages

To obtain bone marrow cells, femurs and tibias were harvested from healthy 8-10-week-old female C57BL/6 mice. Subsequently, the marrow cavities in cleaned bones were flushed with RPMI-1640 medium (Gibco) supplemented with 3% heat-inactivated fetal bovine serum (FBS; Biowest) and centrifuged for 7 min at 192 × g in 4 °C. The isolated bone marrow cells were cultured in RPMI-1640 medium with the addition of 10% FBS ( Sigma-Aldrich) and 50 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF; ImmunoTools) with the medium change every two days. Cells were cultured in this condition for 8 days to obtain unpolarized bone marrow-derived macrophages (M0 BMDM), while to obtain M1 and M2 macrophages, after 7 days of culture with M-CSF, 20 ng/ml interferon-γ (IFN-γ; ImmunoTools) and 100 ng/ml lipopolysaccharide (LPS, from E. coli O111:B4; Sigma Aldrich) to polarize into M1 and 20 ng/ml interleukin 4 (IL-4; ImmunoTools) to polarize into M2 were added for 24 h.

The phenotypic characterization was assessed after 8 days of bone marrow culture to determine the percentage of cells differentiated into macrophages and their activation state. For this purpose, BMDM were stained with anti-F4/80 Alexa Fluor 700, anti-CD11b PerCP-Cy5.5, anti-CD86 PE-Cy7, anti-CD206 APC (all from BioLegend) and anti-CD40 PE (Becton Dickinson). The analysis was performed using flow cytometer LSRFortessa with Diva Software (Becton Dickinson) and NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

Cell culture

RAW264.7 and J774A.1 cells of the mouse macrophages line obtained from American Type Culture Collection (ATCC; TIB-71™ and TIB-67™, respectively) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; ATCC) containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Sigma-Aldrich).

JAWS II cells of immature mouse dendritic cell line received from ATCC (CRL-11,904™) were cultured on a 1:1 mixture of MEM-α (Gibco) and RPMI-1640 (Gibco) supplemented with 10% FBS, 0.5 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Sigma-Aldrich) and 5 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; ImmunoTools).

All cell cultures were maintained in a NUAIRE CO₂ incubator (37 °C, 5% CO₂, 95% humidity) at the Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.

MTT cell viability assay

The bone marrow-derived macrophages (M0, M1 and M2) were placed in 96-well plates at a density of 0.15 × 10⁵ cells/well. After 24 h, boron carbide preparations (B₄C 1 and B₄C 2) were added at concentrations in the range from 0.1 to 200 µg/ml and incubated for 72 h. Next, cells were incubated with MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5 mg/ml)(Sigma-Aldrich) for 4 h and then lysed overnight in lysis buffer (N,N-dimethylmethanamide, sodium dodecyl sulfate, and water) at 37 °C. The absorbance of the solubilized crystal of formazan was measured at 570 nm using a Thermo Labsystems Multiskan RC microplate reader (Thermo Fisher Scientific Inc.) with Genesis Lite 3.05 Software (Thermo Life Sciences). The half maximal inhibitory concentration (IC₅₀) value was calculated using GraphPad Prism 8 software (GraphPad Software). MTT cell viability assay for lines: RAW264.7, J774A.1 and JAWS II was performed and described in our previous work by Kozień et al. [18]. MTT assays were performed in two independent experiments, each in triplicate. The IC₅₀ was calculated for each experiment individually and averaged on the graph for two independent experiments.

Annexin V/ propidium iodide apoptosis assay

The RAW264.7 and J774A.1 cells (0.5 × 10⁵ cells/well for 24 h incubation and 0.25 × 10⁵ cells/well for 72 h in 24-well plates), JAWS II cells (1 × 10⁵ cells/well for 24 h incubation and 0.5 × 10⁵ cells/well for 72 h in 24-well plates) were incubated with boron carbide preparations at concentrations 10, 50, 100 and 200 µg/ml, and BMDM (1.2 × 10⁵ cells/well for both times) at concentrations 10, 50 and 100 µg/ml for 24 and 72 h. After this time, cells were harvested, suspended in a binding buffer and centrifuged (10 min, 100 × g, 4 °C). Next, cells were stained with Annexin V protein conjugated with APC fluorochrome (Becton Dickinson) for 15 min at room temperature. BMDM were additionally stained with anti-F4/80 BV421 (BioLegend) for 45 min at 4 °C. To assess the percentage of dead cells, 10 µg/ml propidium iodide (PI; Invitrogen) was added. The cells were analyzed using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). Scheme of flow cytometry analysis was performed using NovoExpress software 1.3.0 (ACEA Biosciences, Inc.). Apoptosis assays were performed in two independent experiments, each in triplicate.

BrdU cell cycle assay

The RAW264.7, J774A.1 and JAWS II cells (1 × 10⁵ cells/well in 24-well plates) were incubated with boron carbide preparations at concentrations 10, 50, 100 and 200 µg/ml, and BMDM (1.2 × 10⁵ cells/well) at concentrations 10, 50 and 100 µg/ml for 24 h. After this time, 32 µM bromodeoxyuridine (BrdU; Becton Dickinson) solution was added for 1 h, then collected and centrifuged for 10 min at 500 × g. The pellets were suspended in 70% ethanol and stored at -20 °C.

For BrdU staining, fixed cells were centrifuged (5 min, 500 × g, 4 °C) and incubated with 2 M HCl, 0.5% Triton X-100 for 30 min at room temperature. Next, the suspension was centrifuged (10 min, 500 × g, 4 °C), resuspended in 0.1 M Na Borate pH 8.5 and centrifuged again (10 min, 500 × g, 4 °C). Pellets were resuspended in PBS with 1% FBS (Biowest), 0.5% Tween20 (Sigma-Aldrich) and 20 µg/ml ribonuclease A (RNase; Sigma-Aldrich) and stained with anti-BrdU-FITC antibody (Becton Dickinson) for 30 min at room temperature. After this time, cells were centrifuged (10 min, 500 × g, 4 °C) and resuspended in PBS with 5 µg/ml propidium iodide (Invitrogen). Samples were analyzed using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). The scheme of flow cytometry analysis was performed using NovoExpress software 1.3.0 (ACEA Biosciences, Inc.). Cell cycle assays were conducted in three repetitions.

Interaction of boron carbide preparations with phagocytic cells

Boron carbide preparations at a concentration of 100 µg/ml were incubated with the cultured RAW264.7, J774A.1, JAWS II cells and BMDM (0.1 × 10⁶ cells/well) in 24-well plates for 24 h. After this time, the changes in size and granularity of cells were analyzed in triplicate for each sample based on forward scatter (FSC) versus side scatter (SSC) using flow cytometer LSRFortessa with Diva Software (Becton Dickinson). Flow cytometric dot plots were prepared using the NovoExpress software 1.3.0 (ACEA Biosciences, Inc.).

Transmission electron microscopy with EDS analysis

The RAW264.7, J774A.1, JAWS II cells and BMDM (0.1 × 10⁶ cells/well in 24-well plates) were incubated with boron carbide preparations at concentrations of 100 µg/ml for 24 h. Next, cells were harvested, fixed in 2.5% buffered glutaraldehyde at pH 7.2, and contrasted with 2% osmium tetroxide in the dark for 2 h. After this time, cells were washed with buffer and contrasted with 2% uranyl acetate for 12 h. The cell samples were then passed through an ascending alcohol series of 30%, 50%, 70%, 90%, 96% and 99.8%, and embedded in a medium-hard epoxy resin. After polymerization, ultra-thin sections were prepared on an ultramicrotome (Leica). Sections of 60 nm were prepared from the resin blocks and placed on copper grids (400 Mesh) with formvar film and carbon coating. Imaging was performed using a JEOL F-200 transmission electron microscope. The elemental composition analysis was also conducted by energy-dispersive X-ray spectroscopy (EDS) using a JEOL microscope. Elemental analyses were performed in the STEM mode of the microscope and spectra analysis was carried out in the Analysis program JED Series.

Determination of cytokine production

Cytokine production by cells after 24 and 72 h of exposure to boron carbide preparations was assessed using commercially available ELISA kits (IL-1β – eBioscience; IL-6, TNF-α – Invitrogen; IL-10 – Becton Dickinson) in triplicate according to the manufacturer’s instructions. The values on the heat map were calculated according to the formula log₁₀(concentration (pg/ml) + 1).

Scratch assay

The RAW264.7 macrophages were plated at a density of 0.5 × 10⁶ cells/well in 6-well plates in the complete growth medium. After 24 h, boron carbide preparations were added for 24 h at a concentration of 100 µg/ml. Next, when 100% cell confluency was achieved, a gentle scratch was made with a 200 µl tip in the center of the cell monolayer in the wells. The floating cells were removed by washing with phosphate-buffered saline and then the complete medium was added. The macrophages were incubated at 37 °C and imaged over time for 24 h using an Olympus CKX53 light microscope. The experiment was performed in three repetitions, but the images show a selected part of the well for each sample at 0, 4 and 24 h.

Transwell assay

The RAW264.7 macrophages were incubated with boron carbide preparations at a concentration of 100 µg/ml for 24 h. After this time, macrophages alone (0.4 × 10⁵ cells) and loaded with B₄C nanoparticles were suspended in 150 µl of medium without FBS and placed in Transwell inserts with a pore size of 8 μm (Corning Costar). 500 µl RPMI-1640 with 5% FBS as a control medium and 72-hour supernatant from MC38 mouse colorectal cancer cells (1.5 × 10⁵ cells/well in 1 ml of RPMI-1640 with 5% FBS in a 24-well plate) were added to the lower chambers. After 16 h of incubation at 37 °C and 5% CO₂, the upper part of the inserts was wiped using a cotton swab to remove cells that did not migrate. Then, the cells on the inserts were stained with the RAL 555 kit (RAL Diagnostics) according to the manufacturer’s instructions. Migrated cells were observed by an Olympus CKX53 light microscope and counted using ImageJ software from 7 images of the central part of each insert.

Statistical analysis

All data were analyzed using the GraphPad Prism 8 software (GraphPad Software, Inc.). The type of statistical analysis used is described in the captions under the figures. All statistically significant differences are presented on the graphs; otherwise, the differences were not significant.