Exposure to titanium dioxide nanomaterials provokes inflammation of an in vitro human immune construct

Abstract

Nanoparticle technology is undergoing significant expansion largely because of the potential of nanoparticles as biomaterials, drug delivery vehicles, cancer therapeutics, and immunopotentiators. Incorporation of nanoparticle technologies for in vivo applications increases the urgency to characterize nanomaterial immunogenicity. This study explores titanium dioxide, one of the most widely manufactured nanomaterials, synthesized into its three most common nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length). The fully human autologous MIMIC immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naive CD4(+) T cells in comparison to dendritic cells treated with micrometer-sized (>1 microm) titanium dioxide, characteristic of an in vivo inflammatory response.

Figures

Figure 1

HRTEM image of titania nanoparticles (A) anatase, (B) rutile, and (C) nanotubes. Partially amorphous character of particles is observed in case of anatase nanoparticles, while the aspect ratio of nanotubes was around 1:10 to 1:15.

Figure 2

Toxicity of TiO2 nanomaterials in HUVEC and PBMC primary culture models. (A) HUVEC cells cultured in medium 199 containing 20% serum and (B) PBMCs cultured in X-VIVO-15 were exposed to TiO2 nanoparticles at concentrations indicated in the plot. MTT dye reduction was assessed after incubating the cells with the nanomaterials for 24 h, and the absorbance collected was compared to untreated cells to determine the relative percent cell viability. The cells were prepared in the same manner as described above and measured for LDH release by loss of cell membrane integrity after 24 h nanoparticle treatment. Both the (C) HUVEC cells and (D) PBMC coculture did not have a sharp reduction in viability as measured by absorbance from the LDH reaction. Error bars represent mean ± SD for at least three independent experiments.

Figure 3

Nanoparticle treatment induces inflammatory cytokine production. The complete peripheral tissue equivalent module was treated with nanoparticles (1.56 μM) or LPS (10 ng/mL) for 24 h. Supernatant was harvested from each well and examined by cytokine luminex. Increased expression in proinflammatory cytokines in nanoparticle treatments over micrometer-sized titania was visualized across three donors with great consistency ((*) p < 0.05 as compared to micrometer titania). Error bars represent mean ± SD for at least three donors.

Figure 4

Nanoparticles induce ROS production in primary tissue culture models. HUVEC cells cultured in medium 199 containing 20% serum and PBMCs cultured in X-VIVO-15 were exposed to TiO2 nanoparticles or PWM at concentrations indicated in the histograms. ROS production was assessed after a 24 h exposure. To properly assess ROS generation, the cells were washed of culture and condition media and stained briefly with DCF in PBS and immediately acquired by flow cytometry. DCF fluorescence was measured in the FITC channel. Analysis is gated on live cells only. Plots are representative of several independent experiments.

Figure 5

DCs increase expression of maturation markers upon stimulation with nanoparticles. The PTE was loaded with PBMCs which were incubated for 90 min to allow migration of the APC population. Nonmigrated cells were then removed and the cultures were incubated for 24 h. Nanoparticles were then applied at a concentration of 1.56 μM and subsequently incubated for 24 h. The reverse transmigratory (RT) fraction was then harvested and labeled with specific antibody. Treatment with nanotubes resulted in approximately 15% increase in maturation marker expression as compared to the micrometer titania treated cells ((*) p < 0.05 as compared to micrometer titania). Bars indicate expression level (mean fluorescent intensity, MFI) of surface proteins from the migrated RT fraction. Analysis includes only live gated monocytes. Error bars represent mean ± SD for at least three donors.

Figure 6

Nanoparticle-treated DCs induced proliferation of allogeneic Naïve CD4+ T Cells. (A) Naïve CD4+ T cells were untreated or stimulated with PHA and PMA in the absence of DCs. Control (B) naïve CD4+ T cells, primed with HUVEC-derived DCs pulsed with nanomaterials for 48 h (C), were cultured for 5 days. Proliferative response and T-cell activation were determined by FACS as a measure of CFSE dilution and CD25+ expression, respectively. Data are representative of five donors.