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Single-walled carbon nanotube interactions with HeLa cells.

Yehia HN, Draper RK, Mikoryak C, Walker EK, Bajaj P, Musselman IH, Daigrepont MC, Dieckmann GR, Pantano P - J Nanobiotechnology (2007)

Bottom Line: Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACT
This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

No MeSH data available.


Representative Raman spectra acquired from five different live HeLa cells that were incubated at 37°C in CoMoCAT DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. All spectra were normalized to the same intensity scale. The G-band intensities increased in a linear fashion (R2 = 0.932) over the course of 12–60 h (n = 8 cells analyzed at each time point).
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Figure 7: Representative Raman spectra acquired from five different live HeLa cells that were incubated at 37°C in CoMoCAT DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. All spectra were normalized to the same intensity scale. The G-band intensities increased in a linear fashion (R2 = 0.932) over the course of 12–60 h (n = 8 cells analyzed at each time point).

Mentions: In another series of experiments, the time-dependence of DM-SWNT uptake was evaluated. First, the heterogeneous distribution of DM-SWNTs was taken into consideration. As shown in Figure 5, the G-band intensities detected from the cytoplasm ranged from 20 to 500 a.u. and those for the nuclear region ranged from 10 to 350 a.u. It was therefore decided to perform all time-dependent studies with the Raman laser focused on the center of a cell's nuclear region. This selection was influenced by our previous observations of SWNT accumulation around the nuclear region as revealed through confocal fluorescence imaging of HeLa cells exposed to SWNTs dispersed with a fluorescent-labeled peptide [118], and by Strano and co-workers through Raman spectral mapping of 3T3 cells exposed to SWNTs dispersed with DNA [9]. Figure 7 shows Raman spectra from HeLa cells that were incubated at 37°C in DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. In all cases, the number of cells displaying detectable G-band signals increased as the DM-SWNT incubation time increased. Typically, the G-band intensities acquired from HeLa cells incubated in DM-SWNTs for 60 h was 90% greater than those detected at 12 h. Specifically, <10% of the cells analyzed after 12 h incubation displayed detectable G-band signals, while >90% of cells analyzed after 60 h displayed significant G-band signals (n = 40 cells analyzed). In summary, the combined Raman evidence indicated that the observed G-band intensities emanate from DM-SWNTs inside HeLa cells, and that the uptake of DM-SWNTs by HeLa cells is a time- and temperature-dependent process. While complete elucidation of the mechanism(s) of SWNT uptake by cells still requires further investigation, our results are consistent with the work of Dai and co-workers [12] and Cherukuri et al. [81] who have demonstrated that CNTs are transported inside cells via a temperature-dependent mechanism, and contrast the work of Bianco and co-workers who provide evidence that CNT uptake follows a temperature- and endocytosis-independent mechanism [14,37,43].


Single-walled carbon nanotube interactions with HeLa cells.

Yehia HN, Draper RK, Mikoryak C, Walker EK, Bajaj P, Musselman IH, Daigrepont MC, Dieckmann GR, Pantano P - J Nanobiotechnology (2007)

Representative Raman spectra acquired from five different live HeLa cells that were incubated at 37°C in CoMoCAT DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. All spectra were normalized to the same intensity scale. The G-band intensities increased in a linear fashion (R2 = 0.932) over the course of 12–60 h (n = 8 cells analyzed at each time point).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131758&req=5

Figure 7: Representative Raman spectra acquired from five different live HeLa cells that were incubated at 37°C in CoMoCAT DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. All spectra were normalized to the same intensity scale. The G-band intensities increased in a linear fashion (R2 = 0.932) over the course of 12–60 h (n = 8 cells analyzed at each time point).
Mentions: In another series of experiments, the time-dependence of DM-SWNT uptake was evaluated. First, the heterogeneous distribution of DM-SWNTs was taken into consideration. As shown in Figure 5, the G-band intensities detected from the cytoplasm ranged from 20 to 500 a.u. and those for the nuclear region ranged from 10 to 350 a.u. It was therefore decided to perform all time-dependent studies with the Raman laser focused on the center of a cell's nuclear region. This selection was influenced by our previous observations of SWNT accumulation around the nuclear region as revealed through confocal fluorescence imaging of HeLa cells exposed to SWNTs dispersed with a fluorescent-labeled peptide [118], and by Strano and co-workers through Raman spectral mapping of 3T3 cells exposed to SWNTs dispersed with DNA [9]. Figure 7 shows Raman spectra from HeLa cells that were incubated at 37°C in DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. In all cases, the number of cells displaying detectable G-band signals increased as the DM-SWNT incubation time increased. Typically, the G-band intensities acquired from HeLa cells incubated in DM-SWNTs for 60 h was 90% greater than those detected at 12 h. Specifically, <10% of the cells analyzed after 12 h incubation displayed detectable G-band signals, while >90% of cells analyzed after 60 h displayed significant G-band signals (n = 40 cells analyzed). In summary, the combined Raman evidence indicated that the observed G-band intensities emanate from DM-SWNTs inside HeLa cells, and that the uptake of DM-SWNTs by HeLa cells is a time- and temperature-dependent process. While complete elucidation of the mechanism(s) of SWNT uptake by cells still requires further investigation, our results are consistent with the work of Dai and co-workers [12] and Cherukuri et al. [81] who have demonstrated that CNTs are transported inside cells via a temperature-dependent mechanism, and contrast the work of Bianco and co-workers who provide evidence that CNT uptake follows a temperature- and endocytosis-independent mechanism [14,37,43].

Bottom Line: Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACT
This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

No MeSH data available.