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Physica E 40 (2008) 2565–2569

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Estimation of multi-walled carbon nanotubes toxicity in vitro

S.V. Prylutskaa, I.I. Grynyuka, O.P. Matyshevskaa, V.M. Yashchukb,Yu.I. Prylutskyyc,�, U. Ritterd, P. Scharffd

aDepartment of Biochemistry, Kyiv National Shevchenko University, Volodymyrska Street 64, 01033 Kyiv, UkrainebDepartment of Physics, Kyiv National Shevchenko University, Volodymyrska Street 64, 01033 Kyiv, Ukraine

cDepartment of Biophysics, Kyiv National Shevchenko University, Volodymyrska Street 64, 01033 Kyiv, UkrainedInstitute of Physics, Technical University of Ilmenau, D-98684 Ilmenau, Germany

Available online 10 August 2007

Abstract

It was shown that the multi-walled carbon nanotubes at concentrations up to 25 mg/ml do not show toxic effects on rat erythrocytes

and thymocytes in cell suspension, while at concentration 50mg/ml cause the acceleration of erythrocyte hemolysis, decrease of the

number of viable thymocytes in suspension and inhibition of mitochondrial electron-transporting chain.

r 2007 Elsevier B.V. All rights reserved.

PACS: 61.46.+w

Keywords: Multi-walled carbon nanotubes; Optical absorption spectra; Cytotoxicity

1. Introduction

The establishment of the ways of the biomedical use ofcarbon nanotubes (CNT) is one of the priority directions innanobiotechnology. CNT are supposed to be the promisingpharmacological compounds of a new class with the widespectrum of therapeutic action and unique physico-chemical properties [1,2]. According to the study [3],CNT are able to penetrate into the cell, for example, viaendocytosis and can be used as the materials for developingbiocatalysts and biosensors. At the same time the problemof CNT toxicity in vitro [4] and their bioactivity is to beelucidated more completely.

The aim of this work is to study the ways of formation ofthe high-stability multi-walled CNT (MWCNT) watersolutions for the transport of MWCNT into the biologicalmedium, to study the structural and spectral characteristicsof the MWCNT water solutions and to estimate MWCNTcytotoxicity in vitro.

e front matter r 2007 Elsevier B.V. All rights reserved.

yse.2007.07.017

ing author.

ess: prylut@biocc.univ.kiev.ua (Y.I. Prylutskyy).

2. Samples and methods

Chemical vapour deposition was used to produceMWCNT with high purity. The key parameters innanotube growth are the hydrocarbons, the catalysts andthe growth temperature. They are all together responsiblefor the properties of the obtained tubes. Our method isbased on the catalytic decomposition of benzene (as thecarbon source) and ferrocene (catalyst) in a tube furnace atdifferent temperatures. To fill the reactor volume simulta-neously with the components, the experimental set-upconsists of an aerosol generator and a tube furnace. Thequartz glass tube (length: 70 cm, diameter: 3 cm) ispreheated to a temperature between 800 and 900 1C. Theaerosol generator is made from quartz glass and thefunction is based on a spray-aerosol principle. Argon as acarrier gas is feeding the reactor with an aerosol of benzeneand ferrocene (0.01%). The aerosol flow rate is variedbetween 4 and 5L/min argon. The aliened nanotubes growon the quartz glass reactor wall. The reaction time is mostlyresponsible for the length of the tubes. A short duration ofthe reaction in the range of 2–5min gives nanotubesbetween 1 and 5 mm length, whereas a longer reaction time(up to 1 h) leads to nanotubes with up to 100 mm length.

ARTICLE IN PRESSS.V. Prylutska et al. / Physica E 40 (2008) 2565–25692566

The nanotubes, which are used for further reactions, arein the range of 1–4 mm (reaction time 1–4min). At the endof the reaction the aerosol generator is shut down and thereactor is cooled in an argon flow (0.5 L/min; cooling timeis 5 h). After the nanotube collection they are analyzed bySEM/TEM (Fig. 1) and thermogravimetric analysis. TheMWCNT samples were first washed with de-ionized water.The air-dried samples were then heated at 100 1C for 0.5 hunder vacuum to degas absorbed carbon dioxide andwater.

Fig. 1. SEM (FEI, type XL-30 with LaB6 cathode) and HRTEM (Tecnai

20 S-Twin) micrographs of the MWCNT. The picture shows a typical

CNT sample. The estimated diameter of the MWCNT in the sample is

between 20 and 40 nm. The calculated length of the MWCNT is 1–4mm.

In a typical experiment, 100mg of MWCNT was stirred in50.00ml of water under argon for 48h. Prolonged stirringwas required to allow the solid MWCNT to go partly in thewater solution. The mixture was then filtered through amembrane (pore size of 1.2mm). The MWCNT werecollected on the membrane. After drying the amount ofCNT on the membrane was weighted. The weight lossof CNT is less than 0.01%. The filtrate has a brown colorand contains small amounts of short MWCNT in aconcentration of less than 100mg/mL. The content of theCNT in the solution could only be determined indirectly byspectroscopic methods. The partly solubility of MWCNT inwater is due to some surface carboxyl groups on theMWCNT. The surface carbonyl groups result from thepartly oxidation of dangling bonds on the CNT surface in air.The amount of these surface carbonyl groups is much lowerthen that what we receive by acid treatment of the CNT. Thecarbonyl group content could be determined by XPSmeasurements and in some cases by acid-base titration [5].Erythrocytes, isolated from the heparinized rat blood,

were incubated at 24 1C with or without MWCNT atdifferent concentrations. Erythrocytes hemolysis was in-duced by addition of hydrochloric acid to the finalconcentration of 2mM. Measurements of the hemolysisdynamics were carried out for 2min with 10 s intervalsusing the ‘‘Scinco’’ spectrophotometer (Germany) atl ¼ 630 nm.Thymocytes were isolated by the passaging rat thymus

(Wistar line) through neulone mesh sieve and incubated for4 or 24 h at 37 1C into RPMI 1640 medium completed by8mM NaHCO3, 20mM HEPES, 5% bovine serum, 10mgstreptomycin and 10U/ml penicillin with or without theaddition of MWCNT. The number of viable cells wascalculated in the Goriaev’s chamber after cell staining with0.4% trypan blue solution.Cell metabolic state was estimated by the activity of

mitochondrial electron-transport chain in MTT test.Reaction with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolium bromide, ‘‘Sigma’’) was carried out in96-well plate at 37 1C in thermostate [6]. The cells wereincubated for 4 and 24 h in RPMI 1640 medium in thepresence or absence of MWCNT, then MTT was added,and incubation was continued for 2 h. The content ofgenerated formazane was evaluated by spectrophotometryat l ¼ 570 nm using the digital spectrophotometer IFCO-2(ABOTEK, Russia).The optical absorption spectra of MWCNT in water

solution were registered in the range of the wavenumbern ¼ 15,000–50,000 cm�1 at room temperature using thespectrophotometer ‘‘Scinco’’ (Germany).Statistical analysis of the results was performed using

‘‘Microsoft Excel 98’’ program.

3. Results and discussion

Fig. 2 shows the optical absorption spectra of MWCNTin water solution at different concentrations. The arising of

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Fig. 2. The optical absorption spectra of the MWCNT in water at T ¼ 293K. Concentrations of MWCNT: 1—50mg/ml, 1a—25mg/ml, 1b—16.7mg/ml,

1c—12.5mg/ml.

0 20 40 60 80 1000

5

10

15

20

25

30

% e

ryth

rocyte

s

32

14

t, s

Fig. 3. Dependence of erythrocytes stability to hemolysis on the time of

incubation without (1) or in the presence of 12.5mg/ml (2), 25 mg/ml (3) or

50mg/ml (4) MWCNT.

S.V. Prylutska et al. / Physica E 40 (2008) 2565–2569 2567

absorption takes place starting from n�1000 cm�1. Thisfact demonstrates that the optical electrons that areresponsible for absorption do not delocalize along allMWCNT length (1–4 mm). The spectra shapes prove thatthe absorption in the region n�10,000–41,000 cm�1 possiblybelongs to the first electronic transition; nX41,000 cm�1 isthe next electronic transitions. The examination of theoptical density dependencies on MWCNT concentration inwater solution proves that in this case the Buher law is notvalid. It may be connected with aggregation of MWCNT inwater solution.

For estimation of the cytotoxicity of different com-pounds at the cellular level such parameters of theirbiological effects as hemolytical activity, influence on thecontent of viable cells in suspension and on the activity ofmitochondrial electron-transport chain estimated by MTTtest are widely used.

The influence of chemical compounds on the structuralintegrity of erythrocytes, which is evaluated by erythro-cytes stability to hemolysis, is the important parameter oftheir action at the membrane level. Therefore, weconducted the study of the stability of erythrocytespopulation to the action of acidic hemolytic.

The time keeping of hemolysis was monitored from themoment of hemolytic introduction into the probe, whichcontained erythrocytes, preliminarily incubated during 1 hwithout or in the presence of MWCNT in erythrocytessuspension. The destruction of erythrocytes was accom-panied by the gradual drop in extinction, which reflects twoprocesses: firstly, the destruction of erythrocytes and theefflux of hemoglobin into the medium; secondly, thealternate of the hemolytic destruction of erythrocytesdepending on their stability. The corresponding erythro-grams (the dependence of the percentage of erythrocytes inthe suspension on the time of hemolysis) were built taking

into account the data on the kinetics of erythrocyteshemolysis (Fig. 3).Fig. 3 (curve 1) shows the dependence of the hemolysis of

erythrocytes, preliminarily incubated in the medium, whichdid not contain MWCNT, at the time of incubation. Theprocess of hemolysis was accelerated through 30 s after theintroduction of hemolytic into the test, the maximum ofhemolysis (22%) was observed at 50 s, the duration of thehemolysis process was 90 s.In the case of the preliminary incubation of erythrocytes

in the presence of MWCNT, the dynamics of hemolysiswas dependant on the MWCNT concentration in themedium of preincubation. The erythrogram parameters forthe erythrocytes, preincubated in the presence of 12.5 and25 mg/ml MWCNT, were not differed from control (Fig. 3,

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0

20

40

60

80

100

120

% fro

m c

ontr

ol

4 h 24 h

1 3

∗

42 1 3 42

Fig. 4. Content of viable cells in suspension incubated in the presence of

5mg/ml (1), 12.5mg/ml (2), 25 mg/ml (2) or 50mg/ml (3) MWCNT.�Pp0.05 compared to the control.

0

0.1

0.2

0.3

0.4

0.5

A540

1 2 3

4 h 24 h

∗

4 1 2 3 4

Fig. 5. The level of MTT reduction in thymocytes after 4 or 24 h

incubation without (1) or in the presence of 12.5mg/ml (2), 25mg/ml (3) or

50mg/ml (4) MWCNT. �Pp0.05 compared to the control.

S.V. Prylutska et al. / Physica E 40 (2008) 2565–25692568

curves 2 and 3), whereas after the preincubation oferythrocytes in the presence of 50 mg/ml MWCNT, theprocess of hemolysis was accelerated and finished alreadyin 70 s. The content of the hemolysized cells in 10 s afterthe introduction of hemolytic was increased by 80%in comparison with the control, the part of the hemoly-sized erythrocytes in 50 s was increased to 30% (Fig. 3,curve 4).

It is possible to assume that at concentration 50 mg/mlMWCNT the hydrated clusters form. This assumption isapproved by optical absorption spectra of MWCNT inwater solution. If MWCNT are localized on the surface oferythrocytes, the structural organization of the polar partof the integral proteins is changed and the membranestability to the hemolytic is reduced.

The following task was to study the content of viablethymocytes in suspension after incubation in the presenceof MWCNT, assuming the content of the viable cells in 4and 24 h of incubation in control for 100%. After 4 h ofthymocytes incubation in the presence of MWCNT theviability of cells was not changed, whereas in 24 h ofthe incubation in the presence of 50 mg/ml MWCNT thecontent of viable cells was decreased by 32% (Fig. 4).

For the evaluation of MWCNT influence on the generalmetabolic state of thymocytes, we have used MTT test,which is based on the MTT reduction by the reductasesystem, which contains the mitochondrial succinatedehy-drogenase. The MTT test is the indicator of the ratiobetween reduced and oxidized forms of nicotineamidecoenzymes and the activity of electron-transport chain ofmitochondria [7].

In thymocytes, incubated in the presence of 12.5 and25 mg/ml MWCNT, the MTT reduction rate was notchanged independently of the preincubation time (Fig. 5).The negative influence of MWCNT on the MTT reductionrate in the cells was revealed after their addition to thethymocytes suspension in the concentration of 50 mg/ml:after 4 h of preincubation the activity of electron-transportchain was reduced by 17%, and after 24 h—by 24% incomparison with control.

The results obtained agree well with available literaturedata. In particular, the decrease of keratinocytes viabilityafter 48 h of incubation in the presence of MWCNT(0.1–0.4mg/ml) was observed [8]. Muller et al. [9] revealedthe cytotoxic effect of MWCNT, which was evaluated bylactate dehydrogenase efflux after 24 h of the incubation ofperitoneal and alveolar macrophages in the presence ofMWCNT (0.05–0.1mg/ml).

4. Conclusions

The obtained optical absorption spectra of MWCNT inwater solution testify the possibility of MWCNT aggrega-tion in water solution.According to the obtained biological data, the influence

of MWCNT on cells depends on MWCNT concentrationand duration of incubation. At low concentrations,p25 mg/ml MWCNT do not affect the stability oferythrocytes to hemolysis, a number of viable thymocytesin suspension and the rate of MTT reduction in thymo-cytes. At concentration 50 mg/ml the negative effect ofMWCNT was observed: the acceleration of the hemolysisprocess, the decrease of a number of viable thymocytes andthe activity of the electron-transporting chain of themitochondria.

Acknowledgements

This work was partly supported by the BMBF Grant(Ukr 04-008). S.V.P. is grateful to the INTAS (N 05-109-4328) for the support too.

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