Multi-walled carbon nanotube oxidation dependent keratinocyte cytotoxicity and skin inflammation

Abstract

Background: The effects of carbon nanotubes on skin toxicity have not been extensively studied; however, our lab has previously shown that a carboxylated multi-walled carbon nanotube (MWCNT) exacerbates the 2, 4-dinitrofluorobenzene induced contact hypersensitivity response in mice. Here we examine the role of carboxylation in MWCNT skin toxicity.

Results: MWCNTs were analyzed by transmission electron microscopy, zetasizer, and x-ray photoelectron spectroscopy to fully characterize the physical properties. Two MWCNTs with different levels of surface carboxylation were chosen for further testing. The MWCNTs with a high level of carboxylation displayed increased cytotoxicity in a HaCaT keratinocyte cell line, compared to the MWCNTs with intermediate levels of carboxylation. However, neither functionalized MWCNT increased the level of in vitro reactive oxygen species suggesting an alternative mechanism of cytotoxicity. Each MWCNT was tested in the contact hypersensitivity model, and only the MWCNTs with greater than 20% surface carboxylation exacerbated the ear swelling responses. Analysis of the skin after MWCNT exposure reveals that the same MWCNTs with a high level of carboxylation increase epidermal thickness, mast cell and basophil degranulation, and lead to increases in polymorphonuclear cell recruitment when co-administered with 2, 4-dinitrofluorobenzene.

Conclusions: The data presented here suggest that acute, topical application of low doses of MWCNTs can induce keratinocyte cytotoxicity and exacerbation of allergic skin conditions in a carboxylation dependent manner.

Keywords: Allergy; Carbon nanotube; Dermatitis; Nanoparticle; Skin.

Fig. 1

Pristine and carboxylated MWCNT TEM images and representative XPS plots. The 1 mg/mL suspensions of each nanoparticle were dried onto TEM grids for 30 s, and representative TEM images display the general structure of the pristine MWCNT (a), MWCNT Low-COOH (Lot #2) (b) and MWCNT High-COOH (Lot #1) (c). Representative XPS plots of the carbon 1 s regions for both the pristine MWCNT (d), MWCNT Low-COOH (e) and MWCNT High-COOH (f) display the relative increase in oxidized carbon bonds

Fig. 2

Effects of MWCNT on the DNFB induced CHS ear swelling responses after 24 h. Mice were sensitized with 30 μl of 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the lower dorsum on day 0. On day 5, the mice were challenged with either vehicle or 0.2% DNFB. The right ears (black bars) are controls that were not treated with MWCNT. The left ears (gray bars) were treated with either pristine MWCNT, MWCNT Low-COOH, MWCNT High-COOH, or MWCNT High-COOH 0.45 μm filtrate. The DNFB increased ear swelling significantly, compared to all vehicle treated mice. Only ears treated with DNFB and MWCNT High-COOH displayed significantly increased swelling responses, compared to DNFB only. Graphs represent changes in mean ear swelling after 24 h (+/− SEM), N = 5. A Student’s T-test was used to analyze all data presented here, since comparisons were between corresponding right and left ears. Significance is defined at a p-value < 0.05, and # indicates significance between groups

Fig. 3

MWCNT High-COOH nanoparticles induce increases in epidermal thickness and PMN recruitment in skin. Mice were sensitized with 30 μl 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the abdomen on day 0. On day 5 the mice were collared to prevent grooming and challenged with 50 μl of either vehicle (a), 5 μg MWCNT (b), 0.2% DNFB (c), or 0.2% DNFB with 5 μg MWCNT (d) on a 5 cm² area of dorsal skin for 24 h. Skin was fixed in 10% formalin, paraffin embedded, and stained with hematoxylin and eosin to assess the skin histology. Representative images of H&E stained samples are included, and the black arrows indicate the epidermis in each image. Higher magnification images of representative polymorphonuclear cells (PMN) are included, and white arrows indicate the PMN cells. The epidermal skin thickness (e) and total number of PMN cells (f) are quantified. The graphs represent the mean (+/− SEM), N = 6. Significance is defined at a p-value < 0.05. * indicates significance compared to control, # indicate significance within groups

Fig. 4

MWCNT High-COOH nanoparticles induce mast cell/ basophil degranulation in skin. Mice were sensitized with 30ul 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the abdomen on day 0. On day 5 the mice were collared to prevent grooming and challenged with 50 μl of either vehicle (a), 5 μg MWCNT (b), 0.2% DNFB (c), or 0.2% DNFB with 5 μg MWCNT (d) on a 5 cm² area of dorsal skin for 24 h. Skin was fixed in 10% formalin, paraffin embedded, and stained with toluidine blue to assess the number of degranulating mast cells/basophils. Representative images of toluidine blue stained samples are included, and black arrows indicate degranulating mast cells. The total number of mast cells/ basophils (e) and the percentage of degranulating mast cells/ basophils (f) are quantified. The graphs represent the mean (+/− SEM), N = 6. Significance is defined at a p-value < 0.05. * indicates significance compared to control, # indicate significance within groups

Fig. 5

MWCNT High-COOH nanoparticles exacerbate DNFB induced MHCII skin cell expression and granulocyte infiltration. Mice were sensitized with 30 μl 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the abdomen on day 0. On day 5 the mice were collared to prevent grooming and challenged with 50 μl of either vehicle, 5 μg MWCNT, 0.2% DNFB, or 0.2% DNFB with 5 μg MWCNT on a 5 cm² area of dorsal skin for 24 h. A 2 cm² section of skin was processed into a single cell suspension and stained for flow cytometry. The cell populations were gated into total APCs (MHCII+) (a), granulocytes (CD11b+, F4/80-, SSC high) (b), macrophages (MHCII+, F4/80+, CD11c-) (c), and Langerhans cells (MHCII+, F4/80-, CD11c+) (d). The graphs represent the mean percentage of positive cells in the total live cell population (+/− SEM), N = 6. Significance is defined at a p-value < 0.05. * indicates significance compared to control, # indicate significance within groups

Fig. 6

MWCNT High-COOH nanoparticles augment skin expression of inflammatory cytokines. Mice were sensitized with 30 μl 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the abdomen on day 0. On day 5 the mice were collared to prevent grooming and challenged with 50 μl of either vehicle, 5 μg MWCNT, 0.2% DNFB, or 0.2% DNFB with 5 μg MWCNT on a 5 cm² area of dorsal skin for 24 h. A 1 cm² piece of skin was homogenized and the cytokine protein expression levels were analyzed by Luminex assay. The graphs represent the mean (+/− SEM), N = 6. All data was analyzed by two-way ANOVA except for MCP-1, which was analyzed by one-way ANOVA, due to some data being below the limit of detection. Significance is defined at a p-value < 0.05. * indicates significance compared to control, # indicate significance within groups

Fig. 7

MWCNTs oxidized for 6 or more hours exacerbate the CHS ear swelling response. Mice were sensitized with 30 μl 0.05% DNFB in a 4:1 acetone/olive oil vehicle on the lower dorsum on day 0. On day 5 the mice were challenged with 20 μl per ear of 0.2% DNFB on the right ear (black bars) and 0.2% DNFB with 1 μg/ear of the different oxidized MWCNTs on the left ear (gray bars). Graphs represent the change in mean ear swelling, after 24 hours, (+/- SEM), N=5. Data were analyzed by a Student’s T-test, comparing the DNFB and DNFB + MWCNT groups. Significance is defined at a p-value < 0.05. # indicate significance compared to DNFB control