Influence of Impurities from Manufacturing Process on the Toxicity Profile of Boron Nitride Nanotubes

Abstract

The toxicity of boron nitride nanotubes (BNNTs) has been the subject of conflicting reports, likely due to differences in the residuals and impurities that can make up to 30-60% of the material produced based on the manufacturing processes and purification employed. Four BNNTs manufactured by induction thermal plasma process with a gradient of BNNT purity levels achieved through sequential gas purification, water and solvent washing, allowed assessing the influence of these residuals/impurities on the toxicity profile of BNNTs. Extensive characterization including infrared and X-ray spectroscopy, thermogravimetric analysis, size, charge, surface area, and density captured the alteration in physicochemical properties as the material went through sequential purification. The material from each step is screened using acellular and in vitro assays for evaluating general toxicity, mechanisms of toxicity, and macrophage function. As the material increased in purity, there are more high-aspect-ratio particulates and a corresponding distinct increase in cytotoxicity, nuclear factor-κB transcription, and inflammasome activation. There is no alteration in macrophage function after BNNT exposure with all purity grades. The cytotoxicity and mechanism of screening clustered with the purity grade of BNNTs, illustrating that greater purity of BNNT corresponds to greater toxicity.

Keywords: NALP3 inflammasome; aspect ratio; boron nitride nanotubes; mechanism-based screening; nuclear factor-κB; purity; residuals; toxicity.

Figure 1.

A) Comparison of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectra of as-produced BNNTs with BNNTs from various sequential purification steps including gas-phase chlorine etching and water/solvent washing. B) Thermogravimetric analysis coupled to Fourier transform infrared spectroscopy (TGA-FTIR) data of various purity grade BNNTs showing FTIR absorption spectra of desorbed species as a function of temperature during TGA heating under argon.

Figure 2.

Scanning electron microscopy (SEM) images of various purity grade BNNTs. A decrease in the number of low aspect ratio and an increase in high aspect ratio particulate can be observed as the material goes through sequential purification process.

Figure 3.

A) SEM-EDX showing peaks at energies (eV) assigned to B, N, O, and C as well as other elements present at <1 at%. Chlorine, used in the boron-removal step, is observed more clearly for BR-BNNTs (0.12 at%) relative to AP-BNNTs (0.02 at%). B) High resolution XPS spectra in the B1s, N1s, C1s, and O1s regions for AP- (red), BR- (green), and W- (blue). C) Component fitting for the B1s region for (top to bottom): W2-, W1-, BR-, and AP-BNNT.

Figure 4.

A) Representative electron paramagnetic resonance spectroscopy (EPR) spectra and B) EPR peak height with negative control (DMPO only), positive control (Cr (VI)), hBN, and various purity grade BNNTs AP, BR, W1, and W2. Data analyzed by one way ANOVA showed hBN and all purity grade BNNTs had a significant change (p < 0.05) compared to positive or negative control. Within the test group, hBN (*) and AP-BNNT (#) induced significant (p < 0.05) free radicals compared to other BNNTs.

Figure 5.

A) Cytotoxicity measured by evaluating the change in WST-1 metabolism in macrophages exposed to 0–100 μg mL⁻¹ of either hBN or various purity grade BNNTs AP, BR, W1, and W2. B) Hierarchal clustergram of the cytotoxic response showing grouping of the material based on their response. C) Dose-response in membrane damage induced due to exposure of hBN or various purity grade BNNTs for 24 h at 0–100 μg mL⁻¹. D) Benchmark dose (BMD), the lower (BMDL), and higher limit (BMDU) based on the cytotoxicity response. Model selection for BMD was performed based on the lowest Akaike Information Criterion (AIC). The bars above the charts represent significant response (p < 0.05) from control cells with no exposure determined by two-way ANOVA analysis followed by pairwise comparison using Tukey’s multiple comparisons test.

Figure 6.

Mechanism-based screening of hBN and various purity grade BNNTs. A) NF-kB activation and inflammasome markers B) IL-18 and C) IL-1β secretion after exposure to 0–100 μg mL⁻¹ of various test particulate. * highlights statistical significance (p < 0.05) from control cells with no exposure determined by two-way ANOVA analysis followed by pairwise comparison using Tukey’s multiple comparisons test. D) Integrative visualization of the mechanism-based screening using toxicological priority index. Similarity in the response to mechanism-based screening was realized by unsupervised hierarchical clustering.

Figure 7.

A) Heat map visualization of fold change in protein secretions in supernatant of differentiated THP-1 macrophages after challenge of 6.25, 25, and 100 μg mL⁻¹ of hBN and various purity grade BNNTs compared to control cells with no exposure. * Highlights statistical significance (p < 0.05) by pairwise comparison to control cells identified by Student’s t-test. B) Proteins significantly upregulated (Red) and downregulated (Green) after exposure to 6.25, 25, and 100 μg mL⁻¹ of hBN and various purity grade BNNTs compared to control cells with no exposure. C) Constellation plot of the ward-based hierarchical cluster analysis of the protein expression at exposure of 6.25, 25, and 100 μg mL⁻¹ of hBN and various purity grade BNNTs. D) Unsupervised hierarchical two-way clustering across exposure type and protein expression at 25 μg mL⁻¹.

Figure 8.

Change in GFP E. coli uptake after pre-challenge with 6.25, 25, and 100 μg mL⁻¹ of hBN and various purity grade BNNTs. * Highlights statistical significance (p < 0.05) by pairwise comparison to control cells with no exposure.