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. 2013 Nov 13;10(1):57.
doi: 10.1186/1743-8977-10-57.

Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology

Raymond F Hamilton Jr, Zheqiong Wu, Somenath Mitra, Pamela K Shaw, Andrij Holian  1
Affiliations

Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology

Raymond F Hamilton Jr et al. Part Fibre Toxicol. .

Abstract

Background: Several properties of multi-walled carbon nanotubes (MWCNT) have the potential to affect their bioactivity. This study examined the in vitro and in vivo outcomes of the influence of diameter, length, purification and carboxylation (in vitro testing only) of MWCNT.

Methods: Three original 'as received' MWCNT that varied in size (diameter and length) were purified and functionalized by carboxylation. The resulting MWCNT were characterized and examined for cytotoxicity and inflammasome activation in vitro using THP-1 cells and primary alveolar macrophages from C57BL/6 mice. Oropharyngeal aspiration administration was used to deliver original MWCNT and in vivo bioactivity and lung retention was examined at 1 and 7 days.

Results: Studies with THP-1 macrophages demonstrated that increased length or diameter corresponded with increased bioactivity as measured by inflammasome activation. Purification had little effect on the original MWCNT, and functionalization completely eliminated bioactivity. Similar results were obtained using alveolar macrophages isolated from C57BL/6 mice. The in vivo studies demonstrated that all three original MWCNT caused similar neutrophil influx at one day, but increasing length or diameter resulted in the lavaged cells to release more inflammatory cytokines (IL-6, TNF-α, and IL-1β) ex vivo. Seven-day histology revealed that, consistent with the in vitro results, increasing width or length of MWCNT caused more severe pathology with the longest MWCNT causing the most severe inflammation. In addition, the same two larger MWCNT were retained more in the lung at 7 days.

Conclusions: Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT increased with diameter and length. Purification had no significant modifying effect from the original MWCNT. Functionalization by carboxylation completely eliminated the bioactive potential of the MWCNT regardless of size in in vitro testing.

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Figures

Figure 1
Figure 1
SEM images of MWCNT used in this study. A) narrow/short-original (N/S-O) MWCNT, B) narrow/short-functionalized (N/S-F) MWCNT, C) narrow/short-purified (N/S-P) MWCNT. Length comparison shows in D) narrow/short-functionalized (N/S-F) MWCNT, and E) narrow/long-functionalized (N/L-F) MWCNT. Diameter comparison shows in F) wide/short-functionalized (W/S-F) MWCNT, and G) narrow/long-functionalized (N/L-F) MWCNT.
Figure 2
Figure 2
FTIR Data for narrow/short MWCNT variants. A) N/S-O MWCNT, B) N/S-F MWCNT, and C) N/S-P MWCNT.
Figure 3
Figure 3
Cell viability data and IL-1b release for THP-1 cells exposed to various MWCNT for 24 hours. Data expressed as mean ± SEM percent viable cells compared to no particle control condition for A - C. Data expressed as mean ± SEM pg/ml IL-1β for D – F. A) Cell viability for the 3 variants of the narrow/short MWCNT. B) Cell viability for the 3 variants of the wide/short MWCNT. C) Cell viability for the 3 variants of the narrow/long MWCNT. D) IL-1β release for the 3 variants of the narrow/short MWCNT. E) IL-1β release for the 3 variants of the wide/short MWCNT. F) IL-1β release for the 3 variants of the narrow/long MWCNT. Asterisks indicate significance *** at P < 0.001, ** at P < 0.01, * at P < 0.05 compared to functionalized MWCNT variant at the same concentration. Daggers indicate significance ††† at P < 0.001, †† at P < 0.01, † at P < 0.05 compared to the 0 μg/ml no particle control.
Figure 4
Figure 4
Cell viability and IL-1β release data for C57BL/6 AM cells exposed to various MWCNT for 24 hours. A) Mean ± SEM percent viable cells compared to no particle control condition. B) Mean ± SEM IL-1β as pg/ml. Asterisks indicate significance *** at P < 0.001, ** at P < 0.01 compared to both functionalized MWCNT variants at the same concentration. Daggers indicate significance ††† P < 0.001, †† at P < 0.01, † at P < 0.05 compared to the 0 μg/ml no particle control.
Figure 5
Figure 5
High magnification TEM of MWCNT taken up by C57BL/6 alveolar macrophages 1.5 hr in vitro post-exposure (25 μg/ml). A) N/S-O (narrow/short-original) MWCNT-exposed AM at 30 K x. B) N/S-F (narrow/short-functionalized) MWCNT-exposed AM at 20 K x. C) W/S-O (wide/short-original) MWCNT-exposed AM at 15 K x. D) W/S-F (wide/short-functionalized) MWCNT-exposed AM at 15 K x. E) N/L-O (narrow/long-original) MWCNT-exposed AM at 17 K x. F) N/L-F (narrow/long-functionalized) MWCNT-exposed AM at 20 K x. Black spotted/speckled areas indicate areas of organized phagosomal particle retention in the cytoplasm of the macrophage cell.
Figure 6
Figure 6
Cell count and differential from lung lavage 24 hours post MWCNT instillation. A) Mean ± SEM cell numbers for four instillation conditions. B) Cell differentials expressed as mean ± SEM cell numbers for retrieved cell types. AM – macrophages, PMN – neutrophils, EO – eosinophils, Lymph – lymphocytes. Asterisks indicate significance *** at P < 0.001, * at P < 0.05 compared to same cell type for Vehicle. n = 6 mice per condition.
Figure 7
Figure 7
Ex vivo cytokine release from cells recovered in lung lavage at 24 hours post MWCNT instillation. A) IL-6 release from lavaged cells without LPS co-culture. B) TNF-α release from lavaged cells without LPS co-culture. C) IL-1β release from lavaged cells with LPS co-culture (20 ng/ml). Asterisks indicate significance *** at P < 0.001, ** at P < 0.01, * at P < 0.05 compared to Vehicle. Dagger indicates significance † at P < 0.05 compared to the original narrow/short MWCNT variant. n = 3 mice per condition.
Figure 8
Figure 8
Representative photomicrographs in bright-field microscopy of the four in vivo MWCNT instillation conditions at day 7. A) vehicle-exposed. B) narrow/short-original MWCNT-exposed. C) wide/short-original MWCNT-exposed. D) narrow/long-original MWCNT-exposed. Black arrows indicate areas of particle retention. All images at 200x.
Figure 9
Figure 9
Pathology scoring on a 5-point scale for lung sections at day 7. A score of 0 indicates no deviation from normal. A score of 4 indicates the maximum deviation possible from normal. Data expressed as median pathology score with the dots representing individual scores. There was no statistical significance by non-parametric testing vs. a 0 score. n = 3 mice per condition.
Figure 10
Figure 10
Example of how MWCNT retention is determined in an unstained lung section. A) Low resolution digitized image, composed of auto-fluorescence (tissue) and light loss (MWCNT), of lung section from N/S-O MWCNT-exposed mouse. Blue box indicates the scanning area. B) High resolution digitized image, composed of auto-fluorescence (tissue) and light loss (MWCNT), of scanning area from Figure 10A. C) Gated scatter-gram with total scan areas collected where the purple area indicates positive phantom contours for particle deposition. D) Low resolution digitized image, composed of auto-fluorescence (tissue) and light loss (MWCNT), of lung section from N/L-O MWCNT-exposed mouse. Blue box indicates the scanning area. E) High resolution digitized image, composed of auto-fluorescence (tissue) and light loss (MWCNT), of scanning area from Figure 10C. F) Gated scatter-gram with total scan areas collected where the purple area indicates positive phantom contours for particle deposition.
Figure 11
Figure 11
Particle burden data at day 7 and the relationship to pathology. A) Particle burden data for the four MWCNT instillation conditions at day 7. Data expressed as mean ± SEM percent tissue with particle. Asterisks indicate significance *** at P < 0.001, ** at P < 0.01 compared to Vehicle. Daggers indicate significance †† at P < 0.01, † at P < 0.05 compared to the original narrow/short MWCNT variant. B) Regression of day-7 particle retention verses day-7 pathology score for the MWCNT instillation conditions.
Figure 12
Figure 12
Regression of in vitro THP-1 IL-1β release at 25 μg/ml verses day-7 in vivo pathology scores for the MWCNT instillation conditions.
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