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Cytotoxic effect of poly-dispersed single walled carbon nanotubes on erythrocytes in vitro and in vivo.

Sachar S, Saxena RK - PLoS ONE (2011)

Bottom Line: Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
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.

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Effect of treatment with control and acid functionalized SWCNTs on hydrophobicity of erythrocyte membrane.Erythrocytes (5×106 /ml in PBS+1%FBS at 37°C) from Swiss (panel A) and C57Bl6 (panel B) mice were incubated with 25 µg/ml of SWCNTs and AF-SWCNTs for 1 h. Cells were washed and incubated in PBS with 4 mM ANS for 20 min in dark at 37°C, after which fluorescence spectra was determined on a fluorimeter.
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pone-0022032-g004: Effect of treatment with control and acid functionalized SWCNTs on hydrophobicity of erythrocyte membrane.Erythrocytes (5×106 /ml in PBS+1%FBS at 37°C) from Swiss (panel A) and C57Bl6 (panel B) mice were incubated with 25 µg/ml of SWCNTs and AF-SWCNTs for 1 h. Cells were washed and incubated in PBS with 4 mM ANS for 20 min in dark at 37°C, after which fluorescence spectra was determined on a fluorimeter.

Mentions: Exposure of hydrophobic regions in erythrocyte membrane is associated with cell damage [8]. In order to see if membrane hydrophobicity is induced in erythrocyte membrane exposed to AF-SWCNTs, freshly isolated mature erythrocytes were incubated with AF-SWCNTs at 37°C for 1 h and examined for membrane changes by using ANS (8-anilino naphthalene sulfonic acid), a dye that binds to hydrophobic patches on the cell membrane [13], [14]. Results in Figure 4 show that there was a significant increase in the binding of ANS to erythrocytes treated with AF-SWCNTs. A significant shift of emission maxima (blue shift) from 520 to 480 nm was also noted in AF-SWCNT treated erythrocytes. These results indicate a significant damage of erythrocyte membrane upon exposure to AF-SWCNTs.


Cytotoxic effect of poly-dispersed single walled carbon nanotubes on erythrocytes in vitro and in vivo.

Sachar S, Saxena RK - PLoS ONE (2011)

Effect of treatment with control and acid functionalized SWCNTs on hydrophobicity of erythrocyte membrane.Erythrocytes (5×106 /ml in PBS+1%FBS at 37°C) from Swiss (panel A) and C57Bl6 (panel B) mice were incubated with 25 µg/ml of SWCNTs and AF-SWCNTs for 1 h. Cells were washed and incubated in PBS with 4 mM ANS for 20 min in dark at 37°C, after which fluorescence spectra was determined on a fluorimeter.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139600&req=5

pone-0022032-g004: Effect of treatment with control and acid functionalized SWCNTs on hydrophobicity of erythrocyte membrane.Erythrocytes (5×106 /ml in PBS+1%FBS at 37°C) from Swiss (panel A) and C57Bl6 (panel B) mice were incubated with 25 µg/ml of SWCNTs and AF-SWCNTs for 1 h. Cells were washed and incubated in PBS with 4 mM ANS for 20 min in dark at 37°C, after which fluorescence spectra was determined on a fluorimeter.
Mentions: Exposure of hydrophobic regions in erythrocyte membrane is associated with cell damage [8]. In order to see if membrane hydrophobicity is induced in erythrocyte membrane exposed to AF-SWCNTs, freshly isolated mature erythrocytes were incubated with AF-SWCNTs at 37°C for 1 h and examined for membrane changes by using ANS (8-anilino naphthalene sulfonic acid), a dye that binds to hydrophobic patches on the cell membrane [13], [14]. Results in Figure 4 show that there was a significant increase in the binding of ANS to erythrocytes treated with AF-SWCNTs. A significant shift of emission maxima (blue shift) from 520 to 480 nm was also noted in AF-SWCNT treated erythrocytes. These results indicate a significant damage of erythrocyte membrane upon exposure to AF-SWCNTs.

Bottom Line: Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus