Dispersion of single-walled carbon nanotubes by a natural lung surfactant for pulmonary in vitro and in vivo toxicity studies

Abstract

Background: Accumulating evidence indicate that the degree of dispersion of nanoparticles has a strong influence on their biological activities. The aims of this study were to develop a simple and rapid method of nanoparticle dispersion using a natural lung surfactant and to evaluate the effect of dispersion status of SWCNT on cytotoxicity and fibrogenicity in vitro and in vivo.

Results: The natural lung surfactant Survanta® was used to disperse single-walled carbon nanotubes (SWCNT) in a biological medium. At physiologically relevant concentrations, Survanta® produced well dispersed SWCNT without causing a cytotoxic or fibrogenic effect. In vitro studies show that Survanta®-dispersed SWCNT (SD-SWCNT) stimulated proliferation of lung epithelial cells at low doses (0.04-0.12 μg/ml or 0.02-0.06 μg/cm² exposed surface area) but had a suppressive effect at high doses. Non-dispersed SWCNT (ND-SWCNT) did not exhibit these effects, suggesting the importance of dispersion status of SWCNT on bioactivities. Studies using cultured human lung fibroblasts show that SD-SWCNT stimulated collagen production of the cells. This result is supported by a similar observation using Acetone/sonication dispersed SWCNT (AD-SWCNT), suggesting that Survanta® did not mask the bioactivity of SWCNT. Likewise, in vivo studies show that both SD-SWCNT and AD-SWCNT induced lung fibrosis in mice, whereas the dispersing agent Survanta® alone or Survanta®-dispersed control ultrafine carbon black had no effect.

Conclusions: The results indicate that Survanta® was effective in dispersing SWCNT in biological media without causing cytotoxic effects at the test concentrations used in this study. SD-SWCNT stimulated collagen production of lung fibroblasts in vitro and induced lung fibrosis in vivo. Similar results were observed with AD-SWCNT, supporting the conclusion that Survanta® did not mask the bioactivities of SWCNT and thus can be used as an effective dispersing agent. Since excessive collagen production is a hallmark of lung fibrosis, the results of this study suggest that the in vitro model using lung fibroblasts may be an effective and rapid screening tool for prediction of the fibrogenic potential of SWCNT in vivo.

Figure 1

Comparison of Survanta^®-dispersed and non-dispersed SWCNT. (A) The non-dispersed SWCNT suspension (0.1 mg/ml in PBS) shows visible clumping of the SWCNT (left panel) with corresponding light microscopy (middle panel, 100x) and hyperspectral imaging of an individual clump (right panel, 400x). (B) The Survanta^®-dispersed SWCNT suspension at the same concentration shows much improved dispersion with no visible large clumps (left panel). Corresponding light microscopy (middle panel) and hyperspectral imaging (right panel) show a uniform dispersion of the particles. (C-E) Field emission scanning electron microscopy of non-dispersed, Survanta^®-dispersed, and aerosolized SWCNT at low magnification (400x, left panel) and high magnification (30,000x, right panel). Aerosolization of SWCNT was performed according to the method previously described by our group [14].

Figure 2

Effect of Survanta^® on SWCNT particle size and distribution. SWCNT were dispersed in PBS in the presence or absence of Survanta^® as described in Figure 1. (A) Width distribution of non-dispersed and Survanta^®-dispersed SWCNT. (B) Length distribution of non-dispersed and Survanta^®-dispersed SWCNT. CMW = count median width; CML = count median length; GSD = geometric standard deviation. The size and distribution values were determined from triplicate experiments with each experiment measuring a minimum of 300 particles.

Figure 3

Effect of Survanta^® on cell toxicity and cell number. Subconfluent (80%) cultures of human lung epithelial BEAS-2B cells were exposed to various concentrations of Survanta^® and, at one and two days after the treatment, cells were analyzed for cytotoxicity and cell number by LDH assay (A) and cell counting (B). Plots are mean ± S.D. (n = 4). No significant changes over non-treated controls were observed in all measurements at p < 0.05.

Figure 4

Effect of SWCNT on cell proliferation and cell viability. Subconfluent (80%) cultures of lung epithelial BEAS-2B cells were either left untreated or treated with the indicated concentrations of non-dispersed (ND-), Survanta^®-dispersed (SD-), or acetone/sonication-dispersed (AD-) SWCNT, or Survanta^® alone, or no treatment (No Tx) for 24 h. (A) Cell proliferation was determined by hemocytometry. (B) Cell toxicity was determined by LDH assay. Plots are mean ± S.D. (n = 4). *p < 0.05 versus non-treated control.

Figure 5

Effect of SWCNT on fibroblast collagen production. Subconfluent cultures of human lung fibroblast CRL-1490 cells were either left untreated (No Tx) or treated with Survanta^® alone, Survanta^®-dispersed SWCNT (SD-SWCNT), Survanta^®-dispersed ultrafine carbon black (SD-UFCB), or acetone/sonication-dispersed SWCNT (AD-SWCNT) at a particle concentration of 0.02 μg/cm² or 0.04 μg/ml. At 2 days after the treatment, cells were washed, lysed, and analyzed for total collagen content by the Sircol^® assay (A) or collagen I expression by Western blotting (B), as described in the Methods section. Equal amounts of total protein per sample were used in all measurements. β-actin was used as a loading control for Western blotting. The blot signals were quantified by densitometry and mean data from independent experiments (one of which is shown here) were normalized to the result obtained in cells without treatment. Plots are mean ± S.D. (n = 4). *p < 0.05 versus non-treated control.

Figure 6

Effect of SWCNT on lung fibrosis in mice. Mice were pharyngeal aspirated with Survanta^® alone, Survanta^®-dispersed SWCNT (SD-SWCNT) or acetone/sonication-dispersed SWCNT (AD-SWCNT), or Survanta^®-dispersed ultrafine carbon black SD-UFCB) at the particle concentration of 10 μg/mouse. At 2 weeks after the treatment, mice were sacrificed and the lungs were isolated, lysed, and analyzed for collagen content by the Sircol^® assay (A) and Western blotting (B). Equal amounts of total protein per sample were used. β-actin was used as a loading control for Western blotting. The blot signals were quantified by densitometry and mean data from three independent experiments were normalized to the result obtained in cells without treatment. Plots are mean ± S.D. (n = 3). *p < 0.05 versus non-treated control (No Tx).