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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 morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d.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.


Related in: MedlinePlus

Raman spectra acquired from cytoplasmic (A) and nuclear (B) regions of the same live HeLa cell that was incubated at 37°C for 60 h in a CoMoCAT DM-SWNT dispersion. The colored arrows in the optical micrographs denote the specific regions of the HeLa cell where spectra were acquired; spectra were also acquired from cell-free regions of the culture dish ~5 μm away from the nearest cell (dark-blue arrows). All spectra were normalized to the same intensity scale.
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Figure 5: Raman spectra acquired from cytoplasmic (A) and nuclear (B) regions of the same live HeLa cell that was incubated at 37°C for 60 h in a CoMoCAT DM-SWNT dispersion. The colored arrows in the optical micrographs denote the specific regions of the HeLa cell where spectra were acquired; spectra were also acquired from cell-free regions of the culture dish ~5 μm away from the nearest cell (dark-blue arrows). All spectra were normalized to the same intensity scale.

Mentions: Herein, the presence of CoMoCAT G-band intensities emanating from inside living cells incubated in DM-SWNT dispersions was evaluated using confocal microRaman spectroscopy. In the first series of experiments, cells were incubated in DM-SWNT dispersions for 60 h at 37°C. A representative transmitted white-light image of a single HeLa cell acquired through the Raman microscope is shown in Figure 5. Typical HeLa cells were observed to possess 10–30-μm widths and 40–70-μm lengths. The relatively large dimensions of HeLa cells, coupled with the 4-μm lateral resolution of the confocal microscope system, permitted Raman spectra to be acquired from distinct cellular regions [19]. For example, Figure 5 shows Raman spectra acquired from a cell that was incubated in a DM-SWNT dispersion. Intense G-band signals were observed from both cytoplasmic (Figure 5A) and nuclear (Figure 5B) regions. In the latter case, it should not be implied that SWNTs are in the nucleus because the detected G-band resonances could emanate from SWNTs located in the perinuclear region and/or in the cytoplasm immediately above or below the nucleus. Finally, control cells incubated in DMEM/FBS (without DM-SWNTs) had no detectable SWNT Raman signatures under these conditions (data not shown), and no SWNT resonances were detected from cell-free regions of the dish adjacent (≤5 μm) to cells (Figures 5A and 5B, dark blue spectra).


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 cytoplasmic (A) and nuclear (B) regions of the same live HeLa cell that was incubated at 37°C for 60 h in a CoMoCAT DM-SWNT dispersion. The colored arrows in the optical micrographs denote the specific regions of the HeLa cell where spectra were acquired; spectra were also acquired from cell-free regions of the culture dish ~5 μm away from the nearest cell (dark-blue arrows). All spectra were normalized to the same intensity scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Raman spectra acquired from cytoplasmic (A) and nuclear (B) regions of the same live HeLa cell that was incubated at 37°C for 60 h in a CoMoCAT DM-SWNT dispersion. The colored arrows in the optical micrographs denote the specific regions of the HeLa cell where spectra were acquired; spectra were also acquired from cell-free regions of the culture dish ~5 μm away from the nearest cell (dark-blue arrows). All spectra were normalized to the same intensity scale.
Mentions: Herein, the presence of CoMoCAT G-band intensities emanating from inside living cells incubated in DM-SWNT dispersions was evaluated using confocal microRaman spectroscopy. In the first series of experiments, cells were incubated in DM-SWNT dispersions for 60 h at 37°C. A representative transmitted white-light image of a single HeLa cell acquired through the Raman microscope is shown in Figure 5. Typical HeLa cells were observed to possess 10–30-μm widths and 40–70-μm lengths. The relatively large dimensions of HeLa cells, coupled with the 4-μm lateral resolution of the confocal microscope system, permitted Raman spectra to be acquired from distinct cellular regions [19]. For example, Figure 5 shows Raman spectra acquired from a cell that was incubated in a DM-SWNT dispersion. Intense G-band signals were observed from both cytoplasmic (Figure 5A) and nuclear (Figure 5B) regions. In the latter case, it should not be implied that SWNTs are in the nucleus because the detected G-band resonances could emanate from SWNTs located in the perinuclear region and/or in the cytoplasm immediately above or below the nucleus. Finally, control cells incubated in DMEM/FBS (without DM-SWNTs) had no detectable SWNT Raman signatures under these conditions (data not shown), and no SWNT resonances were detected from cell-free regions of the dish adjacent (≤5 μm) to cells (Figures 5A and 5B, dark blue spectra).

Bottom Line: 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.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.


Related in: MedlinePlus