A single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and T-cell reactivity in ovalbumin-sensitized BALB/c mice

Figures & data

Figure 1 Protocol of iron oxide nanoparticle administration and OVA sensitization. BALB/c mice were randomly divided into the following groups (3–5 animals/group): naïve (NA), ovalbumin-sensitized (OVA), vehicle-treated and OVA-sensitized (VH), and iron oxide nanoparticle-treated and OVA-sensitized. Except for NA and OVA groups, the mice were administered intravenously with a single dose of iron oxide nanoparticles (10, 30 and 60 mg Fe/kg; 0.2 mL/mouse) and/or vehicle (VH; saline; 0.2 mL/mouse). Except for the NA group, the mice were sensitized 1 hour after drug administration with ovalbumin (OVA) by intraperitoneal injection using a sensitization solution containing 100 μg OVA plus 1 mg alum (as adjuvant) in saline (250 μL/mouse). Mice were sacrificed 7 days after OVA sensitization, and the spleen and blood were harvested for further experimentation.

Figure 2 Attenuation by iron oxide nanoparticles of the serum production of OVA-specific IgG₁ and IgG_2a. Mice were treated with iron oxide nanoparticles and sensitized with OVA as depicted in Figure 1. The serum levels of OVA-specific IgG₁ and IgG_2a were measured by ELISA.

Note: Data are expressed as the mean ± SE of 9–15 samples pooled from 3 independent experiments. *P < 0.05 compared to the VH group.

Abbreviations: OVA, ovalbumin; ELISA, enzyme-linked immunosorbent assay; SE, standard error; VH, vehicle treated group; OD, optical density.

Table 1 Exposure to iron oxide nanoparticles did not affect splenocyte cellularity

	NA	OVA	VH	Iron oxide nanoparticles (mg Fe/kg)
				10	30	60
CD4^+ (%)	25.4 ± 0.8	25.4 ± 1.1	24.8 ± 1.1	25.5 ± 0.6	24.5 ± 0.5	24.6 ± 0.6
CD8^+ (%)	10.1 ± 0.5	9.1 ± 0.6	9.2 ± 0.5	10.0 ± 0.5	9.5 ± 0.8	8.6 ± 0.2
B220^+ (%)	51.0 ± 1.0	46.1 ± 0.3	46.1 ± 1.0	47.3 ± 1.0	48.3 ± 0.6	47.8 ± 0.5

Notes: Splenocytes were prepared as described in the materials and methods. The percentage of CD4⁺, CD8⁺ and B220⁺ cells was determined by flow cytometry. The data are expressed as the mean ± SE of 3 experiments.

Abbreviations: NA, naive; OVA, ovalbumin; VH, vehicle.

Figure 3 Attenuation by iron oxide nanoparticles of antigen-induced production of IFN-γ and IL-4 by splenocytes. Mice were treated with iron oxide nanoparticles and sensitized with OVA as depicted in Figure 1. The splenocytes (5 × 10⁶ cells/mL) isolated from each group of mice were re-stimulated with OVA (50 μg/mL) in culture for 72 hours to induce cytokine production. The levels of (A) IFN-γ and (B) IL-4 in the supernatants were measured by ELISA.

Notes: Data are expressed as the mean ± SE of quadruplicate cultures. *P < 0.05 compared to the VH control. Results are a representative of 3 independent experiments.

Abbreviations: OVA, ovalbumin; ELISA, enzyme-linked immunosorbent assay; SE, standard error; VH, vehicle treated group.

Figure 4 Effect of iron oxide nanoparticles on the viability of splenocytes. Mice were treated with iron oxide nanoparticles and sensitized with OVA as depicted in Figure 1. The splenocytes (5 × 10⁶ cells/mL) isolated from each group of mice were cultured in the absence or presence of OVA (50 μg/mL) for 72 hours or ConA (5 μg/mL) for 48 hours, and the viability of splenocytes was determined by an MTT assay.

Notes: Data are expressed as the mean ± SE of quadruplicate cultures. *P < 0.05 as compared with the matched VH group. Results are representative of 3 independent experiments.

Abbreviations: OVA, ovalbumin; ConA, concanavalin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; SE, standard error; VH, vehicle treated group; OD, optical density.