Single-walled carbon nanotube interactions with HeLa cells

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 MitoSOX™ 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

Retrograde transport of protein toxins under conditions of COPI dysfunction

AbstractRetrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the ϵ-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport

Scavenger Receptor A1 Mediates the Uptake of Carboxylated and Pristine Multi-Walled Carbon Nanotubes Coated with Bovine Serum Albumin

Previously, we noted that carboxylated multi-walled carbon nanotubes (cMWNTs) coated with Pluronic® F-108 (PF108) bound to and were accumulated by macrophages, but that pristine multi-walled carbon nanotubes (pMWNTs) coated with PF108 were not (Wang et al., Nanotoxicology2018, 12, 677). Subsequent studies with Chinese hamster ovary (CHO) cells that overexpressed scavenger receptor A1 (SR-A1) and with macrophages derived from mice knocked out for SR-A1 provided evidence that SR-A1 was a receptor of PF108-cMWNTs (Wang et al., Nanomaterials (Basel) 2020, 10, 2417). Herein, we replaced the PF108 coat with bovine serum albumin (BSA) to investigate how a BSA corona affected the interaction of multi-walled carbon nanotubes (MWNTs) with cells. Both BSA-coated cMWNTs and pMWNTs bound to and were accumulated by RAW 264.7 macrophages, although the cells bound two times more BSA-coated cMWNT than pMWNTs. RAW 264.7 cells that were deleted for SR-A1 using CRISPR-Cas9 technology had markedly reduced binding and accumulation of both BSA-coated cMWNTs and pMWNTs, suggesting that SR-A1 was responsible for the uptake of both MWNT types. Moreover, CHO cells that ectopically expressed SR-A1 accumulated both MWNT types, whereas wild-type CHO cells did not. One model to explain these results is that SR-A1 can interact with two structural features of BSA-coated cMWNTs, one inherent to the oxidized nanotubes (such as COOH and other oxidized groups) and the other provided by the BSA corona; whereas SR-A1 only interacts with the BSA corona of BSA-pMWNTs

Flow cytometry analysis of intracellular MitoSOX™ Red fluorescence from live HeLa cells incubated at 37°C for 60 h in: DMEM/FBS, CoMoCAT DM-SWNTs, DMEM/FBS + MitoSOX™ Red, DM-SWNTs + MitoSOX™ Red, and DMEM/FBS + MitoSOX™ Red + HO

<p><b>Copyright information:</b></p><p>Taken from "Single-walled carbon nanotube interactions with HeLa cells"</p><p>http://www.jnanobiotechnology.com/content/5/1/8</p><p>Journal of Nanobiotechnology 2007;5():8-8.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2131758.</p><p></p> The -axis denotes the MitoSOX™ Red fluorescence detected in the 564–606 nm spectral region and the -axis denotes the number of events recorded for each analysis

Growth curves for living HeLa cells incubated at 37°C for 4 d in DMEM/FBS or DM-SWNTs

<p><b>Copyright information:</b></p><p>Taken from "Single-walled carbon nanotube interactions with HeLa cells"</p><p>http://www.jnanobiotechnology.com/content/5/1/8</p><p>Journal of Nanobiotechnology 2007;5():8-8.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2131758.</p><p></p> The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File ) and SWNT lengths were estimated to be 100–400 nm (Additional File )

Representative differential image contrast (DIC) images of live HeLa cells incubated for 60 h at 37°C in DMEM/FBS or CoMoCAT DM-SWNTs

<p><b>Copyright information:</b></p><p>Taken from "Single-walled carbon nanotube interactions with HeLa cells"</p><p>http://www.jnanobiotechnology.com/content/5/1/8</p><p>Journal of Nanobiotechnology 2007;5():8-8.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2131758.</p><p></p