ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

�CorrespondE-mail addr

Biomaterials 28 (2007) 5437–5448

www.elsevier.com/locate/biomaterials

The mechanism of cell-damaging reactive oxygen generation bycolloidal fullerenes

Zoran Markovica, Biljana Todorovic-Markovica, Duska Kleuta,Nadezda Nikolica, Sanja Vranjes-Djurica, Maja Misirkicb,c, Ljubica Vucicevicb,

Kristina Janjetovicb,c, Aleksandra Isakovicd, Ljubica Harhajib, Branka Babic-Stojica,Miroslav Dramicanina, Vladimir Trajkovicc,�

aVinca Institute of Nuclear Sciences, Belgrade, SerbiabInstitute for Biological Research, Belgrade, Serbia

cInstitute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, SerbiadInstitute of Biochemistry, School of Medicine, University of Belgrade, Belgrade, Serbia

Received 12 July 2007; accepted 1 September 2007

Available online 19 September 2007

Abstract

Because of the ability to induce cell death in certain conditions, the fullerenes (C60) are potential anticancer and toxic agents. The

colloidal suspension of crystalline C60 (nano-C60, nC60) is extremely toxic, but the mechanisms of its cytotoxicity are not completely

understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen

species (ROS)-mediated cytotoxicity of different nC60 suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC60)

and ethanol (EtOH/nC60), or by extended mixing in water (aqu/nC60). With regard to their capacity to generate ROS and cause

mitochondrial depolarization followed by necrotic cell death, the nC60 suspensions are ranked in the following order: THF/nC604EtOH/

nC604aqu/nC60. Mathematical modelling of singlet oxygen (1O2) generation indicates that the 1O2-quenching power (THF/

nC60oEtOH/nC60oaqu/nC60) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause

cell damage. These data could have important implications for toxicology and biomedical application of colloidal fullerenes.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Carbon; Nanoparticle; Cytotoxicity; Free radical; Modelling

1. Introduction

Owing to its unique chemical and physical propertiesthat enable interaction with living cells, the C60 fullerenehas recently gained considerable attention both as apromising candidate for many biomedical applications[1,2] and a potentially toxic agent [3,4]. One of thebiologically most relevant features of C60 is the ability toform a long-lived triplet excited state upon photosensitiza-tion, acquiring the potential to generate singlet oxygen(1O2) [5], a highly reactive form of molecular oxygen.Singlet oxygen reacts with a wide range of biological

e front matter r 2007 Elsevier Ltd. All rights reserved.

omaterials.2007.09.002

ing author. Tel./fax: +381 11 265 7258.

ess: vtrajkovic@eunet.yu (V. Trajkovic).

targets including lipids, proteins, nucleic acids andcarbohydrates, and is known to be involved in bothcellular signaling and cell damage [6]. However, theexploitation of this property of C60 has been greatlyhampered by its extremely low solubility in water, as wellas by the fact that covalent attachment of variousfunctional groups (–OH, –COOH, –NH2 and others) tothe fullerene core, while providing water solubility, changesthe photophysical properties of C60 and decreases itscapacity for 1O2 generation [7,8]. Moreover, some watersoluble C60 derivatives, such as polyhydroxylated C60

(fullerol) or malonic acid derivatives (carboxyfullerenes),are very efficient quenchers of cell-damaging reactiveoxygen species (ROS) [9], displaying a cytoprotectiveeffect in various ROS-dependent in vitro and in vivo

ARTICLE IN PRESSZ. Markovic et al. / Biomaterials 28 (2007) 5437–54485438

experimental models of cell death [10–13]. In contrast,Sayes et al. [14] have recently shown that pure C60 broughtinto water by means of solvent extraction forms water-stable crystalline aggregates (nano-C60 or nC60) able togenerate high amount of ROS and kill both normal andtumor cells at extremely low (ppb) concentrations. Thesame group subsequently demonstrated that cytotoxicactivity of nC60 was mediated through ROS-mediated cellmembrane lipid peroxidation [15], which was also observedin the fish brains upon exposure to nC60 [16]. By directlycomparing the ROS-generating properties/cytotoxicityof nC60 and fullerol in different experimental systems, wehave confirmed their mainly pro-oxidant/cytotoxic andantioxidant/cytoprotective properties, respectively [17].

Having in mind the envisaged widespread use of C60 inconsumer products, as well as possible unintentionalgeneration of C60 aggregates in aqueous environments,the cytotoxicity of nC60 could have important toxicologicalimplications. On the other hand, as tumor-selectivedelivery of the large drug molecules through abnormalendothelial pores in tumor vasculature represents a validtherapeutic strategy [18], the size of nanocrystalline C60

(up to 400 nm) [19] makes it a potential candidate for ananticancer agent and/or drug carrier. However, there is amajor controversy regarding the mechanisms underlyingthe extremely potent cytotoxicity of nC60. While Sayes etal. [14] argue that pro-oxidant/cytotoxic properties areinherent to pure, underivatized C60, an alternative hypoth-esis suggests that ROS-mediated cytotoxicity of nC60 couldactually stem from the residual presence of tetrahydrofuran(THF), the organic solvent used for nC60 preparation andremaining intercalated into its lattice [20]. In support of thelatter assumption, C60 suspension prepared by long-termstirring in water was significantly less toxic to variousaquatic organisms [21,22], and detergent-solubilized pureC60 was even protective in ROS-mediated liver injury inrats [23]. Accordingly, our recent results show thatg-irradiation-mediated decomposition of THF within C60

nanocrystals results in a complete loss of their capacity forROS production and cell killing [24]. It is, however,important to note that THF alone was completely unableto cause cell death even at concentrations4100-fold higherthan its estimated residual presence (10%) in the nC60 [25].Therefore, one must envisage some kind of interactionbetween C60 and the organic solvent, leading to generationof ROS and subsequent cytotoxicity. While the knowledgeof the exact mechanisms underlying nC60 cytotoxicity isrequired for the assessment of its toxic and anticancerpotential, the studies to that aim have not been performedthus far.

In an attempt to resolve the controversy regarding themechanisms responsible for nC60 pro-oxidant/cytotoxicproperties, we examined the cytotoxicity/ROS productionof nC60 colloids prepared using different solvents. Our dataindicate that the ROS-quenching ability of the solvententrapped within nC60 crystals could determine their pro-oxidant/cytotoxic capacity. These predictions are verified

by mathematical modelling of singlet oxygen generation bydifferent nC60 suspensions.

2. Materials and methods

2.1. Preparation and characterization of different fullerene colloids

The fullerenes were produced by carbon arc discharge and extracted in

Soxlet extractor using toluene as solvent. The extract was mixture of

fullerenes, composed of approximately 80% of C60 and 20% of C70. By

employing the solvent exchange method according to the previously

reported procedure [19], we prepared THF/nC60 and EtOH/nC60 using

THF or ethanol, respectively. Briefly, powdered fullerenes was molecu-

larly dispersed in the fresh THF of HPLC purity (Carlo Erba, Milan,

Italy) or distilled ethanol at a concentration of 25 or 11mg/l, respectively.

After the mixture was purged with argon to remove any dissolved oxygen,

an equal amount of MiliQ water was then added to the THF/C60 or

EtOH/C60 filtrate at a rate of 2 l/min, while being continuously stirred.

The more volatile THF or ethanol was subsequently removed from

the solution using a rotary evaporator at 45 or 68 1C, respectively.

The aqu/nC60 was prepared through extended mixing of 200mg of

powdered fullerenes in 500ml of water, using magnetic stirrer at 500 rpm

for 4 weeks [26]. The fullerene suspensions were filtered through a

0.45mm nylon filter and concentration was estimated by absorption

procedure (the concentrations were 12, 6 and 2.1 mg/ml for THF/nC60,

EtOH/nC60 and aqu/nC60, respectively). Stored at 4 1C, the obtained

solutions were stable for at least 3 months, showing no visible

precipitation. The UV–vis spectra of the nano-C60 suspensions were

scanned within the wavelength range of 200–550nm using Contron

UV–vis spectrophotometer. All UV–vis measurements were carried

out at 20 1C and were automatically corrected for the suspending

medium (water). The particle size distribution of the C60 suspensions

was obtained using Brookhaven Instruments light-scattering system

equipped with a BI-200SM goniometer, a BI-9000AT correlator, a

temperature controller and a Coherent INNOVA 70C argon-ion laser.

Dynamic light-scattering (DLS) measurements were performed using

135mW laser excitation on 514.5 nm at a 901 detection angle, and

normalized particle size distribution was calculated using Brookhaven

Instruments particle-sizing software.

2.2. Cell cultures

The human keratinocyte cell line NTCC 2544, normal human dermal

fibroblasts NHDF, mouse fibrosarcoma cell line L929 and mouse

melanoma cell line B16 were maintained at 37 1C in a humidified

atmosphere with 5% CO2, in an HEPES (20mM)-buffered RPMI 1640

cell culture medium (Sigma, St. Louis, MO) supplemented with 5% fetal

calf serum, 2mM L-glutamine, 10mM sodium pyruvate, and 100 IU/ml

penicillin and streptomycin (all from Sigma). The cells were prepared for

experiments using trypsin/EDTA and incubated in flat-bottom 96-well

(1� 104 cells/well) or 6-well (5� 105 cells/well) cell culture plates (Sarstedt,

Newton, NC) for the cell viability assessment or flow cytometry analysis,

respectively. For the fluorescent microscopy assessment of ROS produc-

tion, cells (3� 104) were incubated in appropriate chamber slides. After

being rested for 24 h, cell cultures were washed and incubated alone

(control) or with different nC60 suspensions. Working solutions of nC60

were prepared by addition of the appropriate amounts of 10-fold

concentrated culture medium and deionized water. Cells were treated

with nC60 under the ambient light and incubated in the dark at 37 1C in a

humidified atmosphere with 5% CO2.

2.3. Cytotoxicity and apoptosis/necrosis assessment

Cytotoxicity of nC60 was assessed by measuring cell number, mito-

chondrial dehydrogenase activity and cell membrane damage by crystal

violet staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ARTICLE IN PRESSZ. Markovic et al. / Biomaterials 28 (2007) 5437–5448 5439

(MTT) reduction and lactate dehydrogenase (LDH) release assay. The

tests were performed exactly as previously described [27] and the results

were presented as fold increase in cell number or % of control value

(untreated cells). The type of cell death (apoptotic or necrotic) was

analyzed by double staining with annexin V-FITC and propidium iodide

(PI), in which annexin V bound to the apoptotic cells with exposed

phosphatidylserine, while PI labeled the necrotic cells with a membrane

damage. Staining was performed according to the instructions by the

manufacturer (BD Pharmingen, San Diego, CA), and flow cytometric

analysis was conducted on a FACSCalibur flow cytometer (BD). The

percentage of apoptotic (annexin+/PI�) and necrotic (annexin+/PI+) cells

was determined using CellQuest Pro software.

2.4. Mitochondrial depolarization

Mitochondrial depolarization was assessed using DePsipher (R&D

Systems, Minneapolis, MN), a lipophilic cation susceptible to the changes

in mitochondrial membrane potential. It has the property of aggregating

upon membrane polarization forming an orange-red fluorescent com-

pound. If the potential is disturbed, the dye cannot access the

transmembrane space and remains or reverts to its green monomeric

form. The cells were stained with DePsipher as described by the

manufacturer, and the green monomer and the red aggregates were

detected by flow cytometry. The results were presented as a green/red

fluorescence ratio (geomean FL1/FL2), the increase of which reflects

mitochondrial depolarization.

2.5. Fluorescence-based measurement of ROS generation

The production of ROS was determined by measuring the intensity of

green fluorescence emitted by redox-sensitive dyes dihydrorhodamine 123

(DHR; Sigma) or 20,70-dichlorofluorescein diacetate (DCFDA; Invitrogen,

Carlsbad, CA). ROS generation in nC60 water mixture with DHR (5mM)or DCFDA (10mM) was assessed using fluorescence microplate reader

(Chameleon; Hidex, Finland) equipped with a 488 nm excitation filter and

a 535 nm emission filter. The results of ROS production by nC60 in water

were presented as fold increase in DHR or DCFDA fluorescence in

comparison with the control (water alone). For intracellular ROS

production, DHR was added to cell cultures 10min prior to nC60

treatment at a concentration of 1mM. At the end of incubation, cells were

detached by trypsinization, washed in PBS, and the green fluorescence

intensity (FL1) in treated cells was analyzed using a FACSCalibur flow

cytometer. Alternatively, DHR fluorescence in nC60-treated cell cultures

was examined with a fluorescent microscope (Carl Zeiss, Berlin,

Germany).

2.6. Detection of singlet oxygen by electron paramagnetic

resonance (EPR)

The EPR spectroscopy was used to monitor the generation of

singlet oxygen in aqueous solutions. The method is based on scavenging

of 1O2 by a diamagnetic and water-soluble substrate molecule tetra-

methylpiperidine (TMP), yielding a paramagnetic product, the stable

nitroxide radical TEMPOL [28,29]. The unpaired electron is located

on the NO group of TEMPOL, which leads to the hyperfine splitting of

the EPR signal into three narrow lines due to interaction between the

unpaired electronic spin and the nitrogen 14N nucleus. The EPR

experiments were performed at room temperature on a Varian E-line

spectrometer operating at a nominal frequency of 9.5GHz. The mixture

containing 0.18mM TMP (Sigma) and different nC60 colloids (2mg/ml)

was thoroughly ultrasonicated and incubated at room temperature for

24 h. The 7 ml aliquots of TMP-nC60 mixture were then transferred

into 3mm i.d. quartz tube and the TEMPOL signal was analyzed

by EPR. Quantification of the signals was carried out by calculating the

mean value of EPR signal amplitudes, and the data are expressed in

arbitrary units.

2.7. Mathematical modelling of singlet oxygen production by nC60

A simple kinetic model was developed to describe singlet oxygen

generation of fullerene nanocrystals based on the model for fullerene

solutions [30]. It is assumed that upon addition of water, fullerenes

dissolved in organic solvent form nanocrystals and that certain amount of

molecules of organic solvent remains intercalated in the fullerene

crystalline lattice [19]. Therefore, nanocrystal is composed of fullerenes

with the property to produce singlet oxygen and organic solvent molecules

that quench singlet oxygen. We have assumed in the model a diffusion of

triplet oxygen from water (or cell culture medium) into the interior

of nanocrystal. The following set of reactions inside nanocrystal is

considered.

The absorption of light by ground state fullerene and singlet–triplet

transition

C60ðS0Þ þ hn�!tT

C60ðT1Þ. (1)

The decay of triplet fullerene

C60ðT1Þ �!t2

C60ðS0Þ. (2)

The generation of S oxygen

C60ðT1Þ þO2ð3S�!

k3C60ðS0Þ þO2ð

1SÞ. (3)

The quenching of singlet S oxygen by ground state fullerenes

C60ðS0Þ þO2ð1S�!

k2C60ðT1Þ þO2ð

3SÞ. (4)

The transition between the state 1S and 1D in an oxygen molecule

O2ð1SÞ�!

t4O2ð

1DÞ. (5)

The quenching of singlet oxygen by molecules M (water, ethanol and

THF) intercalated in the fullerene nanocrystal

O2ð1SÞ þM�!

tqO2ð

3SÞ þM. (6)

We have considered steady-state irradiation regime with a radiation

intensity I ¼ 20mW/cm2 that corresponded to ambient light in the

laboratory. The effective time of excitation of triplet state tt ¼ 4.17 s.

The lifetime t2 of triplet state T1 of fullerene C60 is 143ms [31]. The lifetime

of 1S state of oxygen in solutions is t4 �10�10 s [32]. The constansts

k2 and k3 have values of 1� 10�15 cm3/s and 3.3� 10�12 cm3/s, respectively

[33]. The characteristic lifetimes tq of singlet oxygen in water, ethanol

and THF are 4.1, 13 and 480ms, respectively [34–36]. The following systemof kinetic equations that describe reactions (1)–(6) is the basis of the

model:

dN1

dt¼ �

N1

tT

þN2

t2þ k3N2N3 � k2N1N4,

dN2

dt¼

N1

tT

�N2

t2� k3N2N3 þ k2N1N4,

dN3

dt¼ �k3N2N3 þ

N5

tq

þ k2N1N4, (7)

dN4

dt¼ k3N2N3 �

N4

t4� k2N1N4,

dN5

dt¼

N4

t4�

N5

tq

.

Quantities N1–N5 are the concentrations (in cm�3) of C60(S0), C60(T1),

O2(3S), O2(

1S) and O2(1D), respectively. The initial conditions for Eq. (7)

are given by

N1ð0Þ ¼ NF ¼ 1021cm�3; N3ð0Þ ¼ NO2¼ 1013cm�3;

N2ð0Þ ¼ N4ð0Þ ¼ N5ð0Þ ¼ 0: ð8Þ

ARTICLE IN PRESS

Fig. 1. Characterization of fullerene colloids: (A) UV–vis absorbance

spectra of different nC60 colloids. (B) Normalized particle size distribution

of different nC60 colloids.

Z. Markovic et al. / Biomaterials 28 (2007) 5437–54485440

Oxygen concentration dissolved in the water at atmospheric

pressure and room temperature has order of magnitude of 1017 cm�3

[37]. We assumed in this calculation that steady-state concentration of

oxygen was NO2¼ 1013 cm�3 in the interior of the crystal due to a

hampered diffusion of oxygen from water (or cell culture medium) into the

vacancies of the crystalline lattice.

2.8. Statistical analysis

The statistical significance of the observed differences was analyzed by

ANOVA followed by the Student–Newman–Keuls test for multiple

comparisons. The value of po0.05 was considered significant.

3. Results

3.1. Characterization of nC60 colloids

The UV absorption spectra of THF/nC60 and EtOH/nC60 were typical for C60, although there was a slight

displacement of peaks (Fig. 1A). On the other hand, aqu/nC60 did not show typical features of C60 absorptionspectrum—while it shared with the other two colloids thehigh value of absorption in the deep UV region, thecharacteristic C60 peaks were barely perceptible (Fig. 1A).This could be due to a significantly lower C60 concentrationin our aqu/nC60 preparation (2.1 mg/ml) in comparisonwith THF/nC60 (12 mg/ml) or EtOH/nC60 (6 mg/ml).The average size of particles in THF/nC60, aqu/nC60 andEtOH/nC60 was determined by DLS to be 20.5, 29.2 and35.1 nm, respectively (Fig. 1B). Small percentage (1%) ofparticles in aqu/nC60 had an average diameter of 92 nm,while particle size distribution of EtOH/nC60 displayedsomewhat larger full width at half maximum than the othertwo colloids.

3.2. The cytotoxicity of nC60 depends on the solvent used for

its preparation

We next compared the influence of different C60 colloidson the viability of NTCC human keratinocytes. Morpho-logical analysis performed by inverted microscopy revealedthat only the THF-prepared nC60 caused significantmorphological changes in NTCC cells—they becamesmaller, round and detached from cell culture plastic,which is consistent with the induction of cell death(Fig. 2A). Conversely, the majority of cells treated withEtOH/nC60 or aqu/nC60 retained the polygonal or spindle-like shape observed in untreated control cells (Fig. 2A). Inaccordance with the miscroscopy data, crystal violetstaining has shown that the THF/nC60 markedly reducedcell numbers in a dose- and time-dependent manner, whileEtOH/nC60 and aqu/nC60 exerted minimal cytotoxicity(Fig. 2B and C). The results were not assay-, cell type, orspecies-specific, as the similar results were obtained usingMTT test for mitochondrial activity and LDH releaseassay for cell membrane damage (Fig. 2D), as well as withother normal (HDFF human fibroblasts) and tumor celllines (L929 mouse fibrosarcoma, B16 mouse melanoma)(Fig. 2E and F). The flow cytometric analysis hasconfirmed our previous findings that THF/nC60 inducednecrosis, a type of cell death characterized by cellmembrane damage, detected by intracellular presence ofred-fluorescent PI (Fig. 3A, upper right quadrant).Accordingly, no increase was observed in the number ofcells undergoing apoptosis, another type of cell death inwhich cell membrane remains intact and aberrant exposureof phosphatydilserine on the outer side of cell membrane isdetected by green-fluorescent annexin V-FITC (Fig. 3A,lower right quadrant). In comparison with THF/nC60, theother two colloids caused rather small, but evident increasein the number of necrotic cells (Fig. 3A). We have alsoobserved that treatment of NTCC cells with THF/nC60 ledto a significant loss of mitochondrial membrane potential,as demonstrated by an increase in green-to-red (FL1/FL2)fluorescence ratio of mitochondria-binding dye DePsipher(Fig. 3B and C). Therefore, mitochondrial depolarization

ARTICLE IN PRESS

Fig. 2. The effect of different nC60 colloids on cell morphology and viability: (A) The inverted microscopy photographs of NTCC cells treated with

different nC60 suspensions (1mg/ml, 48 h). (B) Time-dependent influence of different nC60 suspensions (1 mg/ml) on NTCC cell number (crystal violet-CV).

(C) Concentration-dependent influence of different nC60 colloids on NTCC cell number (crystal violet, 48 h). (D) Mitochondrial activity (MTT) and cell

membrane damage (LDH release) in NTCC cells treated with different nC60 suspensions (1mg/ml, 48 h). (E) Cell number (crystal violet) and (F)

mitochondrial activity (MTT) in HDF, L929 and B16 cell cultures treated with different nC60 solutions (1mg/ml, 48 h). (B–F) Results from representative

of at least three experiments are presented as mean values7SD of triplicate observations (* #po0.05 refers to all other treatments* or control#).

Z. Markovic et al. / Biomaterials 28 (2007) 5437–5448 5441

ARTICLE IN PRESS

Fig. 3. The ability of different nC60 colloids to induce necrosis/apoptosis and mitochondrial depolarization: (A) Flow cytometry analysis of apoptosis/

necrosis in NTCC cultures treated with different nC60 solutions (1mg/ml, 36 h). (B) Flow cytometry analysis of mitochondrial membrane potential in

NTCC keratinocytes treated with THF/nC60 (1mg/ml, 24 h). The increase in green fluorescence (FL1) associated with the decrease in red fluorescence

(FL2) upon THF/nC60 treatment indicates depolarization of mitochondrial membrane. (C) Mitochondrial depolarization in NTCC cells treated with

different nC60 (1mg/ml, 24 h), expressed as fold increase in DePsipher FL1/FL2 ratio. (A, C) Results are mean values7SD from three independent

experiments (* #po0.05 refers to all other treatments* or control#).

Z. Markovic et al. / Biomaterials 28 (2007) 5437–54485442

could contribute to THF/nC60-triggered necrotic celldeath. On the other hand, EtOH/nC60 and aqu/nC60

were markedly less potent than THF/nC60 in causingmitochondrial depolarization in human keratinocytes(Fig. 3C), thus further confirming that the cytotoxicpotential of nC60 depends on the solvent used for itspreparation.

3.3. The cytotoxicity of different nC60 colloids correlates

with their ability for ROS production

As it has previously been demonstrated that thecytotoxicity of THF/nC60 is ROS mediated [15,17,24], wecompared the ability of different nC60 colloids to produceROS. To that effect, we used redox-sensitive fluorescent

ARTICLE IN PRESS

Fig. 4. ROS production by different nC60 colloids: (A) Time-dependent DHR fluorescence-based measurement of ROS production in water solution of

nC60 (0.5 mg/ml). (B, C) Concentration-dependent ROS production by different nC60 in water (120min), measured as increase in DHR (B) or DCFDA

fluorescence (C). (D) Influence of 1O2 quenchers (NaN3, tryptophan-Trp) on THF/nC60-mediated ROS production (control), measured as DHR

fluorescence after 120min. (E, F) EPR measurement of 1O2 generation by different nC60 in comparison with control sample containing H2O. (A–F) The

results from representative of at least three experiments are presented. The data in (A–D) are mean values of triplicate observations (7SD in D; the SD

values in A–C were within 10% of the mean; *po0.05).

Z. Markovic et al. / Biomaterials 28 (2007) 5437–5448 5443

ARTICLE IN PRESS

Fig. 5. ROS generation in nC60-treated NTCC cells: (A) Flow cytometry

or (B) fluorescent microscopy detection of intracellular ROS production

(DHR fluorescence, 180min) in NTCC cells treated with different nC60

(1mg/ml).

Z. Markovic et al. / Biomaterials 28 (2007) 5437–54485444

reporter dyes DHR and DCFDA, the former beingrecently shown to be a highly sensitive probe for detectionof singlet oxygen [38]. The data of DHR and DCFDAfluorescence-based measurement of dose- and time-dependent ROS production in a cell free system(Fig. 4A–C) revealed a positive correlation between thecytotoxic capacity of the investigated nC60 colloidsand their capacity to produce reactive oxygen(THF/nC604EtOH/nC604aqu/nC60). The fairly selective1O2 quenchers tryptophan and sodium azide [39,40]prevented THF/nC60-induced DHR fluorescence in adose-dependent manner, suggesting that synglet oxygenwas one of the ROS produced by THF/nC60 (Fig. 4D).Accordingly, the EPR spectra of different nC60 prepara-tions showed that very strong TEMPOL signal wasgenerated by THF/nC60, while marginal or no signalincrease was observed with EtOH/nC60 and aqu/nC60,respectively (Fig. 4E and F). The ability of the three C60

colloids to cause oxidative stress in human keratinocyte cellline NTCC was tested by flow cytometric analysis orfluorescent microscopy of DHR-stained cells (Fig. 5). Inaccordance with the ROS measurements in a cell-freesystem, a significant amount of ROS was observed in thecells treated with THF/nC60, while only a slight increase inDHR fluorescence could be detected in cells treated withEtOH/nC60 or aqu/nC60 (Fig. 5A and B). Therefore, thenature of the solvent used for preparation of nC60 crystalscould determine their ability to produce ROS and to induceoxidative stress and subsequent cytotoxicity.

3.4. Theoretical analysis of ROS production by different

fullerene colloids

In order to get additional insight into the mechanismsunderlying the observed differences in ROS-dependentcytotoxicity of nC60, a kinetic model was developed tocalculate the rate of ROS production by different nC60

colloids. Since our experimental data suggested that nC60 inwater suspension retained the ability of C60 molecularlydispersed in organic solvents to produce singlet oxygen [5],we have chosen to model 1O2 production by nC60. Based onthe data that a significant amount of THF (approximately10%) remains intercalated in the fullerene crystalline latticeduring nC60 preparation [19], we assumed for the purposeof mathematical modelling that the interior of the nC60

crystal is impregnated by the solvent (THF, ethanol orwater). Therefore, the nanocrystal is apparently composedof fullerenes with the property to produce singlet oxygenand solvent molecules that quench singlet oxygen. Mathe-matical modelling of time-dependent 1O2 generation fornC60 crystals with different intercalated molecules ispresented in Fig. 6A. Results of calculation show thatsteady-state concentrations of 1O2 are 1.1, 2.9 and9.4� 1012 cm�3 for fullerene nanocrystals impregnatedwith water, ethanol and THF, respectively. While weassumed in the calculation the oxygen concentration of1013 cm�3 within the nC60 crystals, the similar patterns

of 1O2 generation efficiency were obtained for the oxygenconcentration ranging from 1012 to 1015 cm�3 (data notshown). These data indicate that THF/nC60 should bemore effective singlet oxygen-generator than EtOH/nC60

or aqu/nC60, transforming the ground state oxygeninto a singlet reactive state with the efficiency 4 90%.

ARTICLE IN PRESS

Fig. 6. Theoretical modelling of singlet oxygen generation by nC60 impregnated with different solvents: (A) Mathematical modelling of singlet oxygen

generation by nC60. The parameters were P ¼ 20mW/cm2, NF ¼ 1021 cm�3 and NO2¼ 1013 cm�3. (B) Schematic representation of the role of 1O2

quenching power of the solvent in the cytotoxicity of nC60.

Z. Markovic et al. / Biomaterials 28 (2007) 5437–5448 5445

The mathematical prediction of 1O2 efficiency generation(THF/nC60 4 EtOH/nC60 4 aqu/nC60) correlated extre-mely well with the experimental measurement of ROSproduction and cytotoxicity, thus laying grounds for theconclusion that prooxidant/cytotoxic capacity of nC60

could be determined by the capacity of intercalated solventfor capturing singlet oxygen (Fig. 6B).

4. Discussion

The present study clearly demonstrates that the ability ofdifferent colloidal C60 suspensions to produce ROS and killcells depends on the solvent used for their preparation.With regard to the capacity to generate ROS and exertROS-dependent mitochondrial dysfunction and necrotic

cell death, different nC60 preparations are ranked in thefollowing order: THF/nC60 4 EtOH/nC60 4 aqu/nC60.Mathematical modelling of singlet oxygen productionindicates that the 1O2-quenching power (THF/nC60oEtOH/nC60oaqu/nC60) of the solvent intercalated in thenC60 crystals could be crucial for determining the ROS-generating ability and subsequent cytotoxicity of nC60

(summarized in Fig. 6B).To ensure that the observed differences in cytotoxicity of

nC60 were not related to species, cell type or methodologyfor assessing cell death, we have used different tumor celllines and primary cells of both human and mouse origin, aswell as an array of different tests for cell viability. WhileMTT assay is routinely used to assess cell death, it hasrecently been reported that carbon nanotubes can directly

ARTICLE IN PRESSZ. Markovic et al. / Biomaterials 28 (2007) 5437–54485446

affect the reduction of MTT in the absence of cells [41],thus raising doubts regarding the validity of the MTT assayfor testing the viability of cells treated with carbonnanomaterials. However, since we obtained similar resultsusing microscopy for morphological examination, thecrystal violet test for cell number, flow cytometry-basedanalysis of cell membrane integrity and MTT-basedanalysis of mitochondrial respiration, it appears that thelatter is fairly applicable for testing the cytotoxicity ofnC60. We have also confirmed our previous finding thatTHF/nC60 mainly induced necrosis [17], a type of cell deathcharacterized by cell membrane damage, and not apopto-sis, a programmed cell death in which cell membraneremains intact, but DNA is fragmented [42]. It has beenproposed that the opening of the mitochondrial perme-ability transition pore associated with mitochondrialdepolarization could play a central role in some forms ofoxidative stress-mediated necrosis [43]. Using the flowcytometric analysis, we show for the first time that celltreatment with THF/nC60 leads to a significant loss ofmitochondrial membrane potential. Therefore, mitochon-drial depolarization and the subsequent opening of themitochondrial permeability transition pore, in addition topreviously described cell membrane damage [15,17], couldcontribute to THF/nC60-triggered necrotic cell death.Having in mind the established fact that the cytotoxicityof THF/nC60 is ROS-mediated [15,17,24], the results of thetheoretical and experimental analysis of ROS productionby the three different nC60 preparations correlated wellwith their cytotoxicity, i.e. THF/nC60 was markedly moreefficient in generating ROS than EtOH/nC60 or aqu/nC60.Interestingly, although aqu/nC60 was completely unable togenerate ROS in water, both EtOH/nC60 and aqu/nC60

induced low level of necrotic cell death, which has alsobeen confirmed by crystal violet and MTT assay. This isconsistent with the recent data describing the genotoxicityof both aqu/nC60 and EtOH/nC60 [44], and indicates thatnC60-induced cell death could be partly ROS independent.In accordance with such an assumption, we havepreviously demonstrated that treatment with antioxidantsprovides an incomplete recovery of THF/nC60-treatedcells [24].

The EPR data, together with the experiments employingsinglet oxygen quenchers, for the first time demonstratea singlet oxygen production in water suspension ofTHF/nC60. Moreover, mathematical modelling of singletoxygen generation indicates that the ROS-generatingcapacity of nC60 might primarily depend on 1O2-quenchingpower of the solvent used for nC60 preparation. Therefore,an extremely toxic effect of THF-impregnated C60 nano-crystal could result from nearly total transformation of theground-state oxygen into a singlet reactive state. Accord-ingly, a poor capacity for ROS production, resulting in analmost negligible cytotoxicity of EtOH/nC60 and aqu/nC60,could be ascribed to a very high 1O2-quenching power ofethanol and water. While the model-predicted singletoxygen production is consistent with the data obtained

using EPR and 1O2 quenchers, it should be noted that thereactivity of non-specific ROS reporter dyes DHR andDCFDA indicates that reduced active oxygen species suchas superoxide anion (O2

�), hydroxyl radical (OH) andhydrogen peroxide (H2O2), might also be generated inTHF/nC60 water suspension. Accordingly, Sayes et al. [14]have previously demonstrated the production of super-oxide by nC60, using superoxide-sensitive fluorescentreporter dye iodophenol and xanthine as a superoxidequencher. While this is consistent with the ability ofdetergent-solubilized C60 to generate both superoxide andhydroxyl radical in aqueous solutions [45], the chemistryunderlying the production of various ROS by nC60 requiresfurther investigation.Interestingly, as we did not intentionally expose the nC60

water suspension or nC60-treated cells to the light, itappears that a minimal exposure to ambient light duringsolution preparation, cell treatments and inspection of thecells by an inverted microscope, could have initiated ROSformation in our experiments. For that reason, we haveused in a mathematical model a radiation intensity value(20mW/cm2) that roughly corresponds to ambient light inthe laboratory. In accordance with our observation, it hasbeen previously reported that an additional exposure tovisible light could not further increase the ability of nC60 toproduce ROS [14]. While this implies that photophysicalproperties of nanocrystalline C60 could differ from thoseof molecular C60, the reasons underlying this discrepancyare still to be revealed. An apparent limitation of ourmathematical model is its failure to incorporate thepossible differences in the size of nC60 crystals. The resultsof the DLS measurements, however, show that THF/nC60

crystals, possessing the highest ROS-generating power,have smaller size in comparison with those in EtOH/nC60

or aqu/nC60 suspension. Nevertheless, since the differencesin particle size were not very pronounced, it does not seemvery likely that distinct surface-to-volume ratio could becrucial in determining ROS-producing capacity of nC60.This is also indicated by a disagreement of the ROS-producing order (THF/nC60 4 EtOH/nC60 4 aqu/nC60)with that of the surface-to-volume ratio (THF/nC60 4aqu/nC60 4 EtOH/nC60). To further support this assump-tion, we have compared the ROS-generating capacitiesof the two THF/nC60 preparations with differentparticle size (30 and 50 nm, prepared by varying the rateof water addition to THF/water mixture), and did notfind a significant difference (Isakovic et al., unpublishedobservation).

5. Conclusion

The present study challenges the opposing and some-what simplistic views that nC60-induced ROS generationand cell death are either completely mediated by, or totalyindependent of the residual solvent (THF) [14,20]. Basedboth on experimental evidence and mathematical model-ling, we propose that solvent intercalated into nC60

ARTICLE IN PRESSZ. Markovic et al. / Biomaterials 28 (2007) 5437–5448 5447

crystalline lattice does not directly contribute to, butactually plays a permissive role for ROS generation andresultant mitochondria-dependent necrotic cell damage.While questioning the relevance of using THF-preparednC60 for ecotoxicological testing, our data neverthelesswarrant awareness concerning the potential low-leveltoxicity of pure C60 water suspension. More importantly,by revealing the basic requirements for the cytotoxic actionof different colloidal C60 preparations, these findingsprovide important clues for their further development aspotential tools for anticancer therapy and other biomedicalapplications.

Acknowledgments

This work was supported by the Ministry of Scienceand Environmental Protection of the Republic of Serbia(Grant no. 145073).

References

[1] Bosi S, Da Ros T, Spalluto G, Prato M. Fullerene derivatives: an

attractive tool for biological applications. Eur J Med Chem 2003;

38:913–23.

[2] Tagmatarchis N, Shinohara H. Fullerenes in medicinal chemistry

and their biological applications. Min Rev Med Chem 2001;1:

339–48.

[3] Fiorito S, Serafino A, Andreola F, Togna A, Togna G. Toxicity and

biocompatibility of carbon nanoparticles. J Nanosci Nanotechnol

2006;6:591–9.

[4] Oberdorster G, Oberdorster E, Oberdorster J. Nanotoxicology: an

emerging discipline evolving from studies of ultrafine particles.

Environ Health Perspect 2005;113:823–39.

[5] Guldi DM, Prato M. Excited-state properties of C60 fullerene

derivatives. Acc Chem Res 2000;33:695–703.

[6] Briviba K, Klotz LO, Sies H. Toxic and signaling effects of

photochemically or chemically generated singlet oxygen in biological

systems. Biol Chem 1997;378:1259–65.

[7] Prat F, Stackow R, Bernstein R, Qian W, Rubin Y, Foote CS.

Triplet-state properties and singlet oxygen generation in a homo-

logous series of functionalized fullerene derivatives. J Phys Chem A

1999;103:7230–5.

[8] Prat F, Marti C, Nonell S, Zhang X, Foote CS, Moreno RG, et al.

Fullerene-based materials as singlet oxygen C60 O2(1Dg) photosensi-

tizers: a time-resolved near-IR luminescence and optoacoustic study.

Phys Chem Chem Phys 2001;3:1638–43.

[9] Bensasson RV, Brettreich M, Frederiksen J, Gottinger H, Hirsch A,

Land EJ, et al. Reactions of e�aq, CO2� �, HO � , O2

� � and O2(1Dg) with a

dendro[60]fullerene and C60[C(COOH)2)]n (n ¼ 2–6). Free Radic Biol

Med 2000;29:26–33.

[10] Dugan LL, Gabrielsen JK, Yu SP, Lin TS, Choi DW. Buckmin-

sterfullerenol free radical scavengers reduce excitotoxic and

apoptotic death of cultured cortical neurons. Neurobiol Dis 1996;

3:129–35.

[11] Dugan LL, Turetsky DM, Du C, Lobner D, Wheeler M, Almli CR,

et al. Carboxyfullerenes as neuroprotective agents. Proc Natl Acad

Sci USA 1997;94:9434–9.

[12] Lin AM, Chyi BY, Wang SD, Yu HH, Kanakamma PP, Luh TY,

et al. Carboxyfullerene prevents iron-induced oxidative stress in rat

brain. J Neurochem 1999;72:1634–40.

[13] Monti D, Moretti L, Salvioli S, Straface E, Malorni W, Pellicciari R,

et al. C60 carboxyfullerene exerts a protective activity against

oxidative stress-induced apoptosis in human peripheral blood

mononuclear cells. Biochem Biophys Res Commun 2000;277:711–7.

[14] Sayes C, Fortner J, Lyon D, Boyd A, Ausman K, Tao Y, et al. The

differential cytotoxicity of water-soluble fullerenes. Nano Lett

2004;4:1881–7.

[15] Sayes CM, Gobin AM, Ausman KD, Mendez J, West JL, Colvin VL.

Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials

2005;26:7587–95.

[16] Oberdorster E. Manufactured nanomaterials (fullerenes, C60) induce

oxidative stress in the brain of juvenile largemouth bass. Environ

Health Perspect 2004;112:1058–62.

[17] Isakovic A, Markovic Z, Todorovic-Markovic B, Nikolic N,

Vranjes-Djuric S, Mirkovic M, et al. Distinct cytotoxic mecha-

nisms of pristine versus hydroxylated fullerene. Toxicol Sci 2006;

91:173–83.

[18] Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G,

Roberge S, et al. Openings between defective endothelial cells

explain tumor vessel leakiness. Am J Pathol 2000;156:1363–80.

[19] Fortner JD, Lyon DY, Sayes CM, Boyd AM, Falkner JC, Hotze EM,

et al. C60 in water: nanocrystal formation and microbial response.

Environ Sci Technol 2005;39:4307–16.

[20] Andrievsky G, Klochkov V, Derevyanchenko L. Is the C60 fullerene

molecule toxic?!. Fuller Nanotub Carbon Nanostruct 2005;13:363–76.

[21] Oberdorster E, Zhu S, Blickley TM, McClellan-Green P, Haasch

ML. Ecotoxicology of carbon-based engineered nanoparticles:

effects of fullerene (C60) on aquatic organisms. Carbon 2006;44:

1112–20.

[22] Zhu S, Oberdorster E, Haasch ML. Toxicity of an engineered

nanoparticle (fullerene, C60) in two aquatic species, Daphnia and

fathead minnow. Mar Environ Res 2006;62(Suppl):S5–9.

[23] Gharbi N, Pressac M, Hadchouel M, Szwarc H, Wilson SR, Moussa

F. [60]fullerene is a powerful antioxidant in vivo with no acute or

subacute toxicity. Nano Lett 2005;5:2578–85.

[24] Isakovic A, Markovic Z, Nikolic N, Todorovic-Markovic B,

Vranjes-Djuric S, Harhaji L, et al. Inactivation of nanocrystalline

C60 cytotoxicity by gamma-irradiation. Biomaterials 2006;27:

5049–58.

[25] Harhaji L, Isakovic A, Raicevic N, Markovic Z, Todorovic-

Markovic B, Nikolic N, et al. Multiple mechanisms underlying the

anticancer action of nanocrystalline fullerene. Eur J Pharmacol 2007;

568:89–98.

[26] Cheng X, Kan AT, Tomson MB. Naphthalene adsorption and

desorption from aqueous C60 fullerene. J Chem Eng Data 2004;49:

675–83.

[27] Kaludjerovic GN, Miljkovic D, Momcilovic M, Djinovic VM,

Mostarica Stojkovic M, Sabo TJ, et al. Novel platinum(IV)

complexes induce rapid tumor cell death in vitro. Int J Cancer 2005;

116:479–86.

[28] Lion Y, Delmelle M, van de Vorst A. New method of detecting

singlet oxygen production. Nature 1976;263:442–3.

[29] Vileno B, Lekka M, Sienkiewicz A, Marcoux P, Kulik AJ, Kasas S,

et al. Singlet oxygen (1Dg)-mediated oxidation of cellular and

subcellular components: ESR and AFM assays. J Phys Condens

Matter 2005;17:S1471–82.

[30] Belousova IM, Mironova NG, Yurev MS. A mathematical model of

the photodynamic fullerene–oxygen action on biological tissues. Opt

Spectrosc 2005;98:349–56.

[31] Ausman KD, Weisman RB. Kinetics of fullerene triplet states. Res

Chem Intermed 1997;23:431–51.

[32] Snelling DR. Production of singlet oxygen in the benzene oxygen

photochemical system. Chem Phys Lett 1968;2:346–8.

[33] Arbogast JW, Darmanyan AO, Foote CS, Rubin Y, Diederich FN,

Alvarez MM, et al. Photophysical properties of C60. J Phys Chem

1991;95:11–2.

[34] Studer SL, Brewer WE, Martinez ML, Chou PT. Time-resolved study

of the photooxygenation of 3-hydroxyflavone. J Am Chem Soc

1989;111:7643–4.

[35] Naether DU, Gilchrist JR, Gensch T, Roeder B. Temporal and

spectral separation of singlet oxygen luminescence from near infrared

emitting photosensitizers. Photochem Photobiol 1993;57:1056–9.

ARTICLE IN PRESSZ. Markovic et al. / Biomaterials 28 (2007) 5437–54485448

[36] Scurlock RD, Ogilby PR. Effect of solvent on the rate constant for

the radiative deactivation of singlet molecular oxygen (1DgO2). J Phys

Chem 1987;91:4599–602.

[37] Battino R, Rettich TR, Tominaga T. The solubility of

oxygen and ozone in liquids. J Phys Chem Ref Data 1983;12:

163–78.

[38] Costa D, Fernandes E, Santos JL, Pinto DC, Silva AM, Lima JL.

New noncellular fluorescence microplate screening assay for scaven-

ging activity against singlet oxygen. Anal Bioanal Chem 2007;387:

2071–81.

[39] Matheson IB, Etheridge RD, Kratowich NR, Lee J. The quenching

of singlet oxygen by amino acids and proteins. Photochem Photobiol

1975;21:165–71.

[40] Harbour JR, Issler SL. Involvement of the azide radical in the

quenching of singlet oxygen by azide anion in water. J Am Chem Soc

1982;104:903–5.

[41] Worle-Knirsch JM, Pulskamp K, Krug HF. Oops they did it again!

Carbon nanotubes hoax scientists in viability assays. Nano Lett

2006;6:1261–8.

[42] Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and

autophagy. Curr Opin Cell Biol 2004;16:663–9.

[43] Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP,

Nishimura Y, et al. The mitochondrial permeability transition in cell

death: a common mechanism in necrosis, apoptosis and autophagy.

Biochim Biophys Acta 1998;1366:177–96.

[44] Dhawan A, Taurozzi JS, Pandey AK, Shan W, Miller SM, Hashsham

SA, et al. Stable colloidal dispersions of C60 fullerenes in water:

evidence for genotoxicity. Environ Sci Technol 2006;40:7394–401.

[45] Yamakoshi Y, Umezawa N, Ryu A, Arakane K, Miyata N, Goda Y,

et al. Active oxygen species generated from photoexcited fullerene

(C60) as potential medicines: O2� versus 1O2. J Am Chem Soc

2003;125:12803–9.