Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells

Cytotoxic Effect of Poly-Dispersed Single Walled Carbon Nanotubes on Erythrocytes In Vitro and In Vivo

Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age <10 days) and old (age>40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed

Effect of cytochalasin D on the uptake of AF-SWCNTs by PMs and PLE cells.

<p>PMs and PLE cells were cultured in 48 well culture plates in the presence or absence of 2.5 µg/ml cytochalasin D for 1 h. Fluorescence probe tagged AF-SWCNTs 5 µg/ml) were added and incubation continued for an additional 24 h. At the end of the incubation period, cells were washed with PBS, harvested by trypsinization and analyzed on FACS. Representative flow histograms for AF-SWCNTs uptake in control and cytolchalasin D treated PMs and PLE cells are shown in panel A where cells in M2 window represent AF-SWCNT⁺cells. Bar histograms in the lower panel represents Mean ± SEM of results obtained from 3 independent experiments.</p

Effect of AF-SWCNTs on the recovery of PMs and PLE cells in culture.

<p>PMs and PLE cells were cultured in 48 well plate with or without AF-SWCNTs (10 or 50 µg/ml). After 24, 48 and 72 h, cells were washed, detached by trypsinization and suspended in 0.2% trypan blue solution in PBS. Recoveries of viable cell numbers of PMs (panel A) and PLE cells (panel B) were assessed by using a hemocytometer. Each point represents Mean ±SEM values obtained from 3 replicate assay wells. *p<0.05 for difference between control and AF-SWCNTs treated cell cultures.</p

Examination by confocal microscopy of uptake of fluorescence tagged AF-SWCNTs by PMs and PLE cells.

<p>PMs and PLE cells were cultured on glass cover slip. After one day cells were washed with complete media and continued in culture for 2 more days. Fluorescence labeled AF-SWCNTs (5 µg/ml) were added to cell cultures and after 24 h cells were washed with PBS, fixed with paraformaldehyde, washed twice with quencher (Ammonium chloride) and examined by Confocal Laser Scanning Microscope. Two z-sections each of PLE cells (top two panels) and PMs (bottom two panels) show the presence of fluorescenated AF-SWCNTs in cytoplasm. (Magnification 60×).</p

Effect of AF-SWCNTs on antigen presentation activity of PMs and PLE cells.

<p>Peritoneal macrophages (panel A) and PLE cells (panel B) (2×10⁵) were cultured with BCG sonicate (sBCG, equivalent to a MOI of 100∶1) with or without AF-SWCNTs (50 µg/ml) for 24 h. Excess antigen and particles were removed by washing and fixation was performed with glutaraldehyde and quenched with L-lysine. CD4⁺ T cells (3×10⁵ cells, purity >98%) isolated from spleens of BCG infected mice were added to the wells containing BCG pulsed PMs or PLE cells. The culture supernatants were collected after 24 h and the amount of IL-2 determined by ELISA. Each value represents Mean ± SEM of IL2 levels in 3 replicate assay wells. *(p<0.05).</p

Effect of AF-SWCNTs on PLE cell cycle.

<p>Freshly isolated PLE cells were cultured in 6-well plate with or without of AF-SWCNTs (50 µg/ml). After 24 h, cells were washed and isolated by trypsinization. Cell were fixed, treated with RNase and stained with propidium iodide for flow cytometric analysis.. Data was analyzed by using Modfit software that enumerated proportion of cells in G1/Go phase (left peaks in all histograms), S phase (cross hatched peaks) and G2/M phase (right dark peaks in all histograms).</p

In vivo Uptake of fluorescence tagged AF-SWCNTs by lung epithelial cells and bronchoalveolar lavage (BAL) cells.

<p>Fluorescenated AF-SWCNTs (20 µg) were intratracheally administered in C57Bl6 mice. After 2, 24 and 48 h of the instillation of AF-SWCNTs, BAL cells were harvested and lung tissue processed for isolation of PLE cells. Uptake of AF-SWCNTs as percentage of cells positive for AF-SWCNT fluorescence was assessed flow cytometrically in PLE cells (left panel) and BAL cells (right panel). Each value represents Mean ± SEM of observations from 4 mice.</p

Uptake of fluorescence tagged AF-SWCNTs by PMs and PLE cells.

<p>Peritoneal macrophages and PLE cells were cultured in 48 well plates in presence of fluorescent tagged AF-SWCNTs (5 µg/ml). At different time periods, cells were washed with PBS and harvested by trypsinization. Percentage of cells positive for AF-SWCNTs was determined by using a flow cytometer. Each point represents Mean ±SEM of values obtained from 3 replicate assays.</p