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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.


Raman spectra acquired from live HeLa cells incubated at 4°C in a CoMoCAT DM-SWNT dispersion; both spectra were normalized to the same intensity scale as that in Figure 5.
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Figure 6: Raman spectra acquired from live HeLa cells incubated at 4°C in a CoMoCAT DM-SWNT dispersion; both spectra were normalized to the same intensity scale as that in Figure 5.

Mentions: If the intense G-band signals emanated from DM-SWNTs inside cells, most likely the result of an active uptake process such as endocytosis, then the signals should be absent in cells exposed to DM-SWNTs at 4°C where energy-dependent uptake practically ceases. Figure 6 shows representative Raman spectra acquired from HeLa cells incubated in a DM-SWNT dispersion at 4°C. The peaks detected at ~1608 and 1651 cm-1 in the spectrum acquired from the cytoplasm are presumed to emanate from proteinaceous material, as denoted by the amide-I band at 1650–1659 cm-1 [115-117]. More importantly, the G-band intensities at ~1590 cm-1 recorded from cytoplasmic and nuclear regions were 99.9% less than those recorded from cells incubated at 37°C (Figure 5). In summary, the lack of detectable G-band signals from HeLa cells incubated in DM-SWNT dispersion at 4°C indicates that HeLa cells do not uptake detectable levels of DM-SWNTs when their metabolic activity is low. In addition, the lack of G-band signals from cells incubated at 4°C indicates that there was negligible nonspecific adherence of SWNTs to HeLa cells (i.e., the rinsing procedures were sufficient to remove DM-SWNTs that were on the exterior surface of the plasma membrane).


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)

Raman spectra acquired from live HeLa cells incubated at 4°C in a CoMoCAT DM-SWNT dispersion; both spectra were normalized to the same intensity scale as that in Figure 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Raman spectra acquired from live HeLa cells incubated at 4°C in a CoMoCAT DM-SWNT dispersion; both spectra were normalized to the same intensity scale as that in Figure 5.
Mentions: If the intense G-band signals emanated from DM-SWNTs inside cells, most likely the result of an active uptake process such as endocytosis, then the signals should be absent in cells exposed to DM-SWNTs at 4°C where energy-dependent uptake practically ceases. Figure 6 shows representative Raman spectra acquired from HeLa cells incubated in a DM-SWNT dispersion at 4°C. The peaks detected at ~1608 and 1651 cm-1 in the spectrum acquired from the cytoplasm are presumed to emanate from proteinaceous material, as denoted by the amide-I band at 1650–1659 cm-1 [115-117]. More importantly, the G-band intensities at ~1590 cm-1 recorded from cytoplasmic and nuclear regions were 99.9% less than those recorded from cells incubated at 37°C (Figure 5). In summary, the lack of detectable G-band signals from HeLa cells incubated in DM-SWNT dispersion at 4°C indicates that HeLa cells do not uptake detectable levels of DM-SWNTs when their metabolic activity is low. In addition, the lack of G-band signals from cells incubated at 4°C indicates that there was negligible nonspecific adherence of SWNTs to HeLa cells (i.e., the rinsing procedures were sufficient to remove DM-SWNTs that were on the exterior surface of the plasma membrane).

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.