Cytotoxicity of 2D engineered nanomaterials in pulmonary and corneal epithelium

Abstract

Two-dimensional (2D) engineered nanomaterials are widely used in consumer and industrial goods due to their unique chemical and physical characteristics. Engineered nanomaterials are incredibly small and capable of being aerosolized during manufacturing, with the potential for biological interaction at first-contact sites such as the eye and lung. The unique properties of 2D nanomaterials that make them of interest to many industries may also cause toxicity towards epithelial cells. Using murine and human respiratory epithelial cell culture models, we tested the cytotoxicity of eight 2D engineered nanomaterials: graphene (110 nm), graphene oxide (2 um), graphene oxide (400 nm), reduced graphene oxide (2 um), reduced graphene oxide (400 nm), partially reduced graphene oxide (400 nm), molybdenum disulfide (400 nm), and hexagonal boron nitride (150 nm). Non-graphene nanomaterials were also tested in human corneal epithelial cells for ocular epithelial cytotoxicity. Hexagonal boron nitride was found to be cytotoxic in mouse tracheal, human alveolar, and human corneal epithelial cells. Hexagonal boron nitride was also tested for inhibition of wound healing in alveolar epithelial cells; no inhibition was seen at sub-cytotoxic doses. Nanomaterials should be considered with care before use, due to specific regional cytotoxicity that also varies by cell type. Supported by U01ES027288 and T32HL007013 and T32ES007059.

Keywords: 2D engineered nanomaterials; Airway epithelial cells; Corneal epithelial cells; Hexagonal boron nitride; Nanotoxicity.

Fig. 1.

Cytotoxicity of eight 2D engineered nanomaterials (ENMs) in primary mouse tracheal epithelial cell culture as percent cell density. Nanomaterials tested included graphene oxide of two sizes (A & C), reduced graphene oxide of two sizes (B & E), graphene (D), partially reduced graphene oxide (F), hexagonal boron nitride (G), and molybdenum disulfide (H). All density values were normalized to the media control of the respective plate. Cytotoxicity was induced in three ENMs: reduced graphene oxide 2 um (250 μg/mL), partially reduced graphene oxide 400 um (250 μg/mL), and hexagonal boron nitride (200 μg/mL). Hydrogen peroxide (H₂O₂) was used as a positive control for cytotoxicity. An unpaired student’s t-test was performed to compare each treated group to the media control group: *P < 0.05, **P < 0.01. N = 3 (F), N = 4 (C, E), or N = 5 (A, B, D, G, H) for all treatment groups.

Fig. 2.

Sodium cholate, the dispersant for six of the engineered nanomaterials (ENMs), was tested for cytotoxicity (percent cell density) in primary mouse tracheal epithelial cell culture at 1 mg/kg (A). Comparable sodium cholate concentrations of tested nanomaterials are also provided (B). All density values were normalized to the media control of the respective plate. Hydrogen peroxide (H₂O₂) was used as a positive control for cytotoxicity. An unpaired student’s t-test was performed to compare each treated group to the media control group: *P < 0.05, **P < 0.01. N = 3 for all conditions.

Fig. 3.

A diagram depicting the sizes of the nanomaterials used, and where in the lung these particles would be capable of depositing based on size. Created with BioRender.com.

Fig. 4.

Cytotoxicity, measured as percent cell density, following a dose response of hexagonal boron nitride (hBN) in (A) primary mouse tracheal epithelial cell culture (mTEC) and (B) human alveolar epithelial cell culture (A549). All density values were normalized to the media control (0 mg/kg hBN) of the respective plate. Cytotoxicity was induced in A549 cells at 40 μg/mL and 80 μg/mL of hBN. Hydrogen peroxide (H₂O₂) was used as a positive control for cytotoxicity. An unpaired student’s t-test was performed to compare each treated group to the media control group: *P < 0.05, **P < 0.01, ****p < 0.0001. N = 5 for all groups.

Fig. 5.

Cell migration (wound healing) following a dose response of hexagonal boron nitride (hBN) in human alveolar cell culture (A549) for either (A) 24-h continuous or (B) the initial 2 h of a 24-h time period. Sub-cytotoxic doses of hBN did not significantly inhibit wound healing at either exposure duration. Hydrogen peroxide (H₂O₂) and V₂O₅, a nanomaterial known to inhibit cellular migration, were used as positive controls. Kruskal-Wallis and Dunn’s multiple comparisons tests were conducted: **P < 0.01, ***P < 0.001, and ****P < 0.0001. For the 24-h exposure, N = 3 replicates (each an average of technical triplicates) for all conditions. For the 2-h exposure, N = 6 media control, N = 3 for each positive control, and N = 6 (each an average of technical triplicates), for the hBN exposures.

Fig. 6.

Cytotoxicity following a dose response of hexagonal boron nitride (hBN; A) and molybdenum disulfide (MoS₂; B) in human telomerase reverse transcriptase-immortalized corneal epithelial (hTCEpi) cells. Percent viability was measured by Calcein AM assay. Cytotoxicity of MoS₂ was induced from doses of 2.5 to 50 μg/mL, and cytotoxicity of hBN was induced from doses 4 to 40 μg/mL. Saponin and citrate-capped gold nanoparticles (AuNP) were used as a positive and negative control for cytotoxicity, respectively. An unpaired student’s t-test with Welch’s correction was performed to compare each treated group to the media control group: *P < 0.05, **P < 0.01, ***p < 0.001, and ****P < 0.0001. N = 3 (hBN) or 4 (MoS₂) for all groups of that analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)