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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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Uptake of fluorescenated AF-SWCNTs by erythrocytes in vitro.Erythrocytes were incubated with fluorescenated AF-SWCNTs (50 µg/ml) for 1 h and examined flow cytometrically either before (panel B) or after (panel C) three washings with fresh medium. Panel A shows the flow histogram for control unstained erythrocytes. Panel D shows the dose dependent uptake of AF-SWCNTs by erythrocytes with or without three washings. Results of a representative experiment have been shown. In panel D all data points represent mean ± SEM of three observations.
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pone-0022032-g002: Uptake of fluorescenated AF-SWCNTs by erythrocytes in vitro.Erythrocytes were incubated with fluorescenated AF-SWCNTs (50 µg/ml) for 1 h and examined flow cytometrically either before (panel B) or after (panel C) three washings with fresh medium. Panel A shows the flow histogram for control unstained erythrocytes. Panel D shows the dose dependent uptake of AF-SWCNTs by erythrocytes with or without three washings. Results of a representative experiment have been shown. In panel D all data points represent mean ± SEM of three observations.

Mentions: For examining the interactions between AF-SWCNTs and erythrocytes, AF-SWCNT preparations tagged with fluorescence probe were used. Erythrocytes freshly derived from female C57BL/6 mice were incubated with fluorescence tagged AF-SWCNT particles at 50 µg/ml in vitro for 1 h and analysed on flow cytometer before and after washing the erythrocyte preparations. Results in Figure 2 (panel B) show that 69% of erythrocytes incubated with fluorescence tagged AF-SWCNTs were positive for fluorescence indicating an uptake or association with AF-SWCNTs. After washings 18.40% of the erythrocytes (panel C) were still positive for uptake of tagged AF-SWCNTs. These results suggest that AF-SWCNTs could associate with erythrocytes in a loose as well as a relatively stronger manner. Erythrocytes incubated with fluorescence tagged AF-SWCNTs were also examined by confocal microscopy. Results in Figure 3 show the localization of fluorescence in z-sections of erythrocytes and strongly suggest that some fluorescence tagged AF-SWCNTs could enter erythrocytes.


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

Sachar S, Saxena RK - PLoS ONE (2011)

Uptake of fluorescenated AF-SWCNTs by erythrocytes in vitro.Erythrocytes were incubated with fluorescenated AF-SWCNTs (50 µg/ml) for 1 h and examined flow cytometrically either before (panel B) or after (panel C) three washings with fresh medium. Panel A shows the flow histogram for control unstained erythrocytes. Panel D shows the dose dependent uptake of AF-SWCNTs by erythrocytes with or without three washings. Results of a representative experiment have been shown. In panel D all data points represent mean ± SEM of three observations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022032-g002: Uptake of fluorescenated AF-SWCNTs by erythrocytes in vitro.Erythrocytes were incubated with fluorescenated AF-SWCNTs (50 µg/ml) for 1 h and examined flow cytometrically either before (panel B) or after (panel C) three washings with fresh medium. Panel A shows the flow histogram for control unstained erythrocytes. Panel D shows the dose dependent uptake of AF-SWCNTs by erythrocytes with or without three washings. Results of a representative experiment have been shown. In panel D all data points represent mean ± SEM of three observations.
Mentions: For examining the interactions between AF-SWCNTs and erythrocytes, AF-SWCNT preparations tagged with fluorescence probe were used. Erythrocytes freshly derived from female C57BL/6 mice were incubated with fluorescence tagged AF-SWCNT particles at 50 µg/ml in vitro for 1 h and analysed on flow cytometer before and after washing the erythrocyte preparations. Results in Figure 2 (panel B) show that 69% of erythrocytes incubated with fluorescence tagged AF-SWCNTs were positive for fluorescence indicating an uptake or association with AF-SWCNTs. After washings 18.40% of the erythrocytes (panel C) were still positive for uptake of tagged AF-SWCNTs. These results suggest that AF-SWCNTs could associate with erythrocytes in a loose as well as a relatively stronger manner. Erythrocytes incubated with fluorescence tagged AF-SWCNTs were also examined by confocal microscopy. Results in Figure 3 show the localization of fluorescence in z-sections of erythrocytes and strongly suggest that some fluorescence tagged AF-SWCNTs could enter erythrocytes.

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