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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.


Growth curves for living HeLa cells incubated at 37°C for 4 d in DMEM/FBS or DM-SWNTs. The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File 1) and SWNT lengths were estimated to be 100–400 nm (Additional File 2).
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Figure 11: Growth curves for living HeLa cells incubated at 37°C for 4 d in DMEM/FBS or DM-SWNTs. The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File 1) and SWNT lengths were estimated to be 100–400 nm (Additional File 2).

Mentions: A crucial question amongst reports concerning the adherence and/or uptake of CNTs by cultured cells [1,7,9-18,37,43,65-73,75,77,78,81-84,86,88,91-94,113,114] is whether CNTs are toxic. Previously, we observed that the growth rates of HeLa cells incubated for 4 d in ~100 μg/mL HiPco SWNTs dispersed in a peptide solution or in media supplemented with serum were statistically similar to controls [19]. The evaluation of CoMoCAT DM-SWNTs also involved monitoring growth rates over the course of 4 d. First, there were no discernable differences in the morphologies of HeLa cells incubated in DM-SWNTs for 60 h (Figures 5 and 10B) relative to controls (Figure 10A; cells incubated in DMEM/FBS). Next, the growth rates of HeLa cells continuously exposed to DM-SWNTs were quantitated by calculating population double times (PDTs). A PDT is a measure of cell numbers at the early log growth phase and is used for comparisons of normal cell growth. PDTs were obtained from the slopes of the lines of a plot of the natural log of cell numbers versus time [120]. Figure 11 shows such a plot over a time period of 4 d for cells cultured in DM-SWNTs and control cells (DMEM/FBS only). For both samples, the respective number of HeLa cells counted on days 1, 2, 3, and 4 were statistically similar at a 95% confidence level. The control HeLa cell PDT was 27 h and was statistically similar to the PDT of 29 h observed with HeLa cells cultured in DM-SWNTs. In summary, the data from this sensitive test argue that our preparations and concentrations of purified CoMoCAT DM-SWNT dispersions do not affect HeLa cell growth rates.


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)

Growth curves for living HeLa cells incubated at 37°C for 4 d in DMEM/FBS or DM-SWNTs. The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File 1) and SWNT lengths were estimated to be 100–400 nm (Additional File 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Growth curves for living HeLa cells incubated at 37°C for 4 d in DMEM/FBS or DM-SWNTs. The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File 1) and SWNT lengths were estimated to be 100–400 nm (Additional File 2).
Mentions: A crucial question amongst reports concerning the adherence and/or uptake of CNTs by cultured cells [1,7,9-18,37,43,65-73,75,77,78,81-84,86,88,91-94,113,114] is whether CNTs are toxic. Previously, we observed that the growth rates of HeLa cells incubated for 4 d in ~100 μg/mL HiPco SWNTs dispersed in a peptide solution or in media supplemented with serum were statistically similar to controls [19]. The evaluation of CoMoCAT DM-SWNTs also involved monitoring growth rates over the course of 4 d. First, there were no discernable differences in the morphologies of HeLa cells incubated in DM-SWNTs for 60 h (Figures 5 and 10B) relative to controls (Figure 10A; cells incubated in DMEM/FBS). Next, the growth rates of HeLa cells continuously exposed to DM-SWNTs were quantitated by calculating population double times (PDTs). A PDT is a measure of cell numbers at the early log growth phase and is used for comparisons of normal cell growth. PDTs were obtained from the slopes of the lines of a plot of the natural log of cell numbers versus time [120]. Figure 11 shows such a plot over a time period of 4 d for cells cultured in DM-SWNTs and control cells (DMEM/FBS only). For both samples, the respective number of HeLa cells counted on days 1, 2, 3, and 4 were statistically similar at a 95% confidence level. The control HeLa cell PDT was 27 h and was statistically similar to the PDT of 29 h observed with HeLa cells cultured in DM-SWNTs. In summary, the data from this sensitive test argue that our preparations and concentrations of purified CoMoCAT DM-SWNT dispersions do not affect HeLa cell growth rates.

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.