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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.


Related in: MedlinePlus

Raman spectra acquired from CoMoCAT SWNT dispersions (10-min probe sonication and two 2-min centrifugations) prepared in various solutions (water or DMEM ± FBS); all spectra were normalized to the same intensity scale.
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Figure 4: Raman spectra acquired from CoMoCAT SWNT dispersions (10-min probe sonication and two 2-min centrifugations) prepared in various solutions (water or DMEM ± FBS); all spectra were normalized to the same intensity scale.

Mentions: Confocal microRaman spectrometer acquisition methods and the interpretation of the Raman spectra of various SWNT dispersions prepared using our sonication and centrifugation protocol have been detailed previously [19,45,47,49]. A representative Raman spectrum for a DM-SWNT dispersion is shown in Figure 4 (blue spectrum; DMEM + 5% FBS). The spectrum clearly shows a number of well characterized SWNT resonances [100,107,108], in particular, two predominant radial breathing modes at ~281 and ~301 cm-1, the D-band at ~1303 cm-1, and the G-band in the 1550–1610 cm-1 region. Control spectra of DMEM/FBS without SWNTs did not display detectable resonances under these operating conditions (data not shown). Spectrometer stability was assessed by monitoring the reproducibility of the G-band peak intensity at ~1590 cm-1 since it is the most prominent Raman peak indicative of intrinsic SWNT features [109]. In brief, the relative standard deviation (RSD) of G-band peak intensities acquired from the same region of a SWNT dispersion was <1%, the RSD of G-band peak intensities acquired from four different regions of a SWNT dispersion was <10%, and the correlation coefficient for the linear relationship between the G-band peak intensity and relative SWNT concentration was 0.982 (Figures S3-S5 in Additional File 3). In summary, the data indicates that the FBS components coating the SWNTs did not significantly affect the G-band profile of SWNTs dispersed in this fashion, which is in agreement with previous reports using non-covalently modified SWNTs dispersed in aqueous solutions of peptides [19,41,45] and proteins [53,62].


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 CoMoCAT SWNT dispersions (10-min probe sonication and two 2-min centrifugations) prepared in various solutions (water or DMEM ± FBS); 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 4: Raman spectra acquired from CoMoCAT SWNT dispersions (10-min probe sonication and two 2-min centrifugations) prepared in various solutions (water or DMEM ± FBS); all spectra were normalized to the same intensity scale.
Mentions: Confocal microRaman spectrometer acquisition methods and the interpretation of the Raman spectra of various SWNT dispersions prepared using our sonication and centrifugation protocol have been detailed previously [19,45,47,49]. A representative Raman spectrum for a DM-SWNT dispersion is shown in Figure 4 (blue spectrum; DMEM + 5% FBS). The spectrum clearly shows a number of well characterized SWNT resonances [100,107,108], in particular, two predominant radial breathing modes at ~281 and ~301 cm-1, the D-band at ~1303 cm-1, and the G-band in the 1550–1610 cm-1 region. Control spectra of DMEM/FBS without SWNTs did not display detectable resonances under these operating conditions (data not shown). Spectrometer stability was assessed by monitoring the reproducibility of the G-band peak intensity at ~1590 cm-1 since it is the most prominent Raman peak indicative of intrinsic SWNT features [109]. In brief, the relative standard deviation (RSD) of G-band peak intensities acquired from the same region of a SWNT dispersion was <1%, the RSD of G-band peak intensities acquired from four different regions of a SWNT dispersion was <10%, and the correlation coefficient for the linear relationship between the G-band peak intensity and relative SWNT concentration was 0.982 (Figures S3-S5 in Additional File 3). In summary, the data indicates that the FBS components coating the SWNTs did not significantly affect the G-band profile of SWNTs dispersed in this fashion, which is in agreement with previous reports using non-covalently modified SWNTs dispersed in aqueous solutions of peptides [19,41,45] and proteins [53,62].

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.


Related in: MedlinePlus