Photo-Induced Cytotoxicity of Malonic Acid [C60]Fullerene Derivatives and Its Mechanism

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Toxicology in Vitro 16 (2002) 41–46 www.elsevier.com/locate/toxinvit

Photo-induced cytotoxicity of malonic acid [C60]fullerene derivatives and its mechanism

X.L. Yang*,C.H. Fan,H.S. Zhu Research Center of Materials Science, Beijing Institute of Technology, PO Box 327, Beijing 100081, PR China

Accepted 10 August 2001

Abstract The biological activities of fullerenes have attracted extensive attention in recent years. The aim of this paper is to study the relation of the photo-induced cytotoxicity of fullerene derivatives to their chemical structures as well as the possible cellular mechanism involved in the photocytotoxicity. Three C60 derivatives with two to four malonic acid groups (DMA C60,TMA C 60 and QMA C60) were prepared and the cytotoxicity of these compounds against HeLa cells was determined by MTT. Cell cycle was measured by ﬂow cytometry. The results showed that the cytotoxicity of the malonic acid C60 derivatives was irradiation- and dose- dependent. The sequence of their photo-induced growth inhibition was DMA C60>TMA C60 >QMA C60. Hydroxyl radical quencher mannitol (10 mm) was not able to prevent cells from the damage induced by irradiated DMA C60. DMA C60,together with irradiation,was found to have an ability of inducing a decrease in the number of G 1 cells from 63 to 42% and a rise in that of G2+M cells from 6 to 26%. These data indicated that the number of malonic acid molecules added to C60 played an important role in the phototoxicity,and the blockage of cell cycle might be a mechanism of this activity. # 2002 Published by Elsevier Science Ltd.

Keywords: Cell cycle; Cell growth; Malonic acid C60 derivatives; Photo-induced; Reactive oxygen species

1. Introduction 1996; Schuster et al.,1996; Tsuchiya et al.,1996). Among various kinds of C60 derivatives,the malonic In recent years,aqueous solutions of C 60 and its deri- acid adducts of C60,first synthesised by Lamparth and vatives have been prepared by various methods to study co-workers (Hirsch et al.,1994; Lamparth and Hirsch, their biological effects (Yang et al.,1998). Some pro- 1994),may have very potent applications in the biome- gress has been made in their activities against enzymes dical field,due not only to their unique physical and (Ando et al.,1993; Fridman et al.,1993; Sijbesma et al., chemical properties,but also to their simple massive 1993; Schinazi et al.,1994; Nacsa et al.,1997; Iwata et preparation. These properties included: (i) minor mod- al.,1998),DNA molecules (Tokuyama et al.,1993; ification in the structure of parent C60 cage; (ii) good Boutorine et al.,1994; An et al.,1996) and free radicals solubility and poor aggregation formation in aqueous (Nagano et al.,1994; Chiang et al.,1995; Sun et al., solution when the number of addends is bigger than 1997; Cheng et al.,2000a). There have been a few one,and this aggregation will lead to the significant papers concerning the influence of C60 and its deriva- reduction of the lifetime of the excited triplet state tives on cell growth as well (Tokuyama et al.,1993; Li et (Guldi and Asmus,1999); (iii) reliable availability of a al.,1994; Schinazi et al.,1994; Scrivens et al.,1994; variety of regioisomers with defined three-dimensional Tsuchiya et al.,1995; Irie et al.,1996; Nakajima et al., structures (Lamparth and Hirsch,1994). Therefore, many recent papers have dealt with the biological con-

Abbreviations: DMA C60,dimalonic acid C 60; MTT,methylthiazo- cerns of the malonic acid adducts of C60 (Okuda et al., lyldiphenyl-tetrazolium bromide; OD,optical density; QMA C 60, 1996,2000; Dugan et al.,1997; Satoh et al.,1997a,b; quadrimalonic acid C60; ROS,reactive oxygen species; TMA C 60,tri- Huang et al.,1998; Guldi and Asmus,1999; Mashino et malonic acid C60 * Corresponding author. Tel.: +8610-6891-1949; fax: +8610-6891- al.,1999). For example,two regioisomers with C 3 or D3 5023. symmetry,containing three malonic acid groups per E-mail address: [email protected] (X.L. Yang). molecule,were found to be neuroprotective in mice,

0887-2333/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0887-2333(01)00102-3 中国科技论文在线 http://www.paper.edu.cn 42 X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46

both in vitro and in vivo and able to prevent apoptosis 2.3. Preparation of aqueous solutions of C60 derivatives in human hepatoma cells (Dugan et al.,1997; Huang et al.,1998). However,the photo-induced cytotoxicity of The stock solutions of DMA C60,TMA C 60 and malonic acid derivatives of C60 and the related QMA C60 were prepared in pH8 buffer (0.2 m mechanism have not yet been examined. In this pre- NaH2PO4/Na2HPO4). Sterile solutions for cell experi- liminary study,three malonic acid derivatives of C 60 ments were obtained by filtering these solutions with 0.2 were chosen to investigate their effects on the growth mm pore membranes. and cell cycle of human cervix uteri tumor-derived HeLa cells. They were dimalonic acid C60 (DMA C60), 2.4. Cytotoxicity determination trimalonic acid C60 (TMA C60) and quadrimalonic acid C60 (QMA C60),the structures of which are shown in Human cervix uteri tumor-derived HeLa Cells were Fig. 1. Our results showed their cytotoxicity was irra- cultured in an atmosphere with 5 CO2 and at 37 C pro- diation dependent and decreased with the number of vided by a NAPCO CO2 incubator in RMPI 1640 med- malonic acid molecules added to C60. It was also indi- ium containing 10% heat-inactivated fetal calf serum and cated cell-cycle blockage is possibly related to the 100 U/ml penicillin and 100 mg/ml streptomycin. photo-induced cytotoxic activity of the fullerene com- Cytotoxicity determination of C60 derivatives was pound. carried out with 24-well plates. Four repeats were undertaken for each sample. When cells were attached to the bottom of culture 2. Materials and methods plates,the culture medium was replaced by the fresh medium containing 2% fetal calf serum and C60 deriva- 2.1. Chemicals and technical equipment tives or/and mannitol. Then the cultures were irradiated with a 300 W halogen lamp set at 30 cm over the cul- C60 (99% purity) was prepared as previously reported tures. To avoid heat damage to the cells,the heat pro- (Cheng et al.,2000a). RMPI 1640 medium,fetal calf duced by the lamp was removed by a piece of heat- serum,MTT reagent and propidium iodide were pur- shield glass. Irradiation was carried out three times, chased from Sigma Chemical Co. The NAPCO CO2 each time for 0.5 h with a 24-h interval between each incubator and the MODEL 550 microplate reader were irradiation. After the third irradiation,the viable cell products of Precision Scientific and Bio-Rad,Inc., number was determined immediately by MTT assay respectively. Cell-cycle analysis was carried out with a (Mosmann,1983; Zhu et al.,2000). MTT reagent can be FACS 420 flow cytometer (Becton Dickinson Co.). converted into blue formazan only by viable cells. Lysis of cells produced a blue solution,the optical density 2.2. Synthesis of malonic acid derivatives of C60 value (OD value) of which was measured with a MODEL 550 microplate reader in a dual wavelength, Malonic acid derivatives of C60 were synthesised as with the measurement wavelength at 450 nm and the described in our previous paper (Cheng et al.,2000a). reference wavelength at 655 nm. The data obtained were Briefly,C 60 and diethyl bromomalonate was mixed at a statistically analyzed using Student’s t-test. ratio of 1:6 at room temperature under an Ar atmo- Growth inhibition was represented as (1Àmean OD sphere,so three malonate derivatives of C 60 with two to value of sample/mean OD value of control)Â100%. four additional groups could be produced simulta- neously and then separated macroscopically by silica-gel 2.5. Cell-cycle analysis chromatography. Hydrolysis with NaH and CH3OH led to the formation of the corresponding acids. Flow cytometry was used to analyze the cell cycle according to the method described previously (Zhu et al.,2000). Briefly,cells with or without treatment of DMA C60 or/and irradiation were collected by cen- trifuge (1000 rpm/10 min) after trypsinization and then fixed in 75% ethanol overnight at 4 C. Fixed cells were stained with propidium iodide to show the DNA content of each cell. The strength of the fluo- rescence within a single cell was detected under a FACS 420 flow cytometer. 100,000 cells were counted in total for each sample. The percent of cells in differ- Fig. 1. The malonic acid derivatives of C60 used for cytotoxic investi- ent phases (G1,S and G 2+M) were obtained by the gation in the present experiment. They included dimalonic acid C60 (DMA C60),trimalonic acid C 60 (TMA C60) and quadrimalonic acid utilization of the ‘Multicycle’ software attached to the C60 (QMA C60). flow cytometer. 中国科技论文在线 http://www.paper.edu.cn X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46 43

3. Results (63%)>TMA C60 (55%)>QMA C60 (31%),suggesting that the increase of side group number in C60 cage may 3.1. Photo-induced cytotoxicity of malonic acid C60 reduce its photo-induced activity.

The photo-induced cytotoxicity of dimalonic acid C60 3.2. Influence of hydroxyl radical quencher mannitol on (DMA C60) is shown in Fig. 2. Without irradiation, photo-induced cytotoxicity of DMA C60 DMA C60 in the range of 0–64 mm did not produce any toxicity against HeLa cells. When irradiation was Several studies in cell-free systems indicated that C60 applied,however,DMA C 60 significantly inhibited cell and C60 derivatives could produce various reactive oxy- growth at the concentration of 32 mm (P<0.05),and its gen species (ROS) under light irradiation (Arbogast et inhibitory efficiency became higher when its concentra- al.,1991; Orfanopoulos and Kambourakis,1995; tion doubled (P<0.01). Therefore,the photo-induced Hamano et al.,1997; Kamat et al.,1998; Yang et al., cytotoxic effect of DMA C60 was dose dependent. 1999; Cheng et al.,2000b). The previous studies by Moreover,when observed under an inverted micro- Kamat et al. and our studies suggested hydroxyl radical scope,the shape of many cells became round,indicating might be the major effective ROS (Kamat et al.,1998; severe damage to cells might take place (not shown). Cheng et al.,2000b),so we tested whether hydroxyl To clarify the relationship between the photo-induced radical quencher mannitol could influence the photocytotoxicity and the side group number of C60 com- induced cytotoxic activity. The growth inhibition by 32 pounds,three oligoadducts of malonic acid C 60 (DMA mm DMA C60 alone was 32%,and it remained almost C60,TMA C 60 and QMA C60) were considered in the the same when 10 mm mannitol was added (Table 2). present experiment. These compounds were selected Therefore,it can be concluded that mannitol was not because they had not only good solubility,but also poor effective on the photo-induced activity of DMA C60. cluster formation in aqueous solution (Guldi and Asmus,1999; Cheng et al.,2000a). The final concentra- 3.3. Cell-cycle arrest by DMA C60 in the presence of tion of 64 mm DMA C60,TMA C 60 and QMA C60, irradiation could be prepared easily in the present experiments. Table 1 shows the photo-induced cytotoxicity of these As an approach to explore the mechanism involved in compounds against HeLa cells. They were all remark- the photocytotoxicity of fullerene derivatives,we exam- ably cytotoxic at the concentration of 40 mm and under ined further the influence of DMA C60 on HeLa cell the irradiation conditions used (P<0.01),while no cycle by flow cytometry. The result was shown in obvious effects were examined in the absence of irra- Table 3. The percent of each phase was 63% of G1,29% diation. Furthermore,the ability of their photo-induced inhibition appeared to be sequenced as DMA C 60 Table 1 Photo-induced growth inhibition of three malonic acid derivatives of

C60 (DMA C60,TMA C60 and QMA C60) against HeLa cells. The concentration of each compound was 40 mm. Cells were kept in the dark or irradiated. The cytotoxicity of these compounds was found irra-

diation-dependent and sequenced DMA C60>TMA C60 >QMA C60

Group Growth inhibition (%)

Dark Irradiated

Control 0 0

DMA C60 À463 TMA C60 À655 QMA C60 531

Table 2 Inﬂuence of hydroxyl radical inhibitor mannitol on the photo-induced

cytotoxicity of DMA C60

Group Growth inhibition (%) m Fig. 2. Dose-dependent eﬀects of DMA C60 (064 m ) on the growth Control 0 of HeLa cells. Cells were kept in the dark (Dark) or irradiated (Irra- DMA C60 (32 mm) alone 32 diation). The cytotoxicity of DMA C60 was found to be irradiation- Mannitol (10 mm) alone 3 and dose-dependent. Data in each column were obtained from four DMA C60 (32 mm)+mannitol (10 mm)34 repeats and statistically analyzed by Student’s t-test. 中国科技论文在线 http://www.paper.edu.cn 44 X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46

of S and 8% of G2+M in the absence of both DMA-C60 This will be helpful in finding new a C60 derivative and irradiation. Either the irradiation by a 300 W lamp or effective enough to apply in the photodynamic therapy the addition of 10 mm DMA C60 alone caused little variety for tumor treatment (Irie et al.,1996). In addition,the in the percent of each phase during the cell cycle. How- sequence of their cytotoxicity was ultimately the same as ever,treatment of DMA C 60 combined with the irradia- that of their photosensitive activity in the production of tion showed an obvious effect. It induced a decrease of G1 singlet oxygen and hydroxyl radical in the cell-free system from 63 to 42%,accompanied by a rise of G 2+M from 8 (Hamano et al.,1997; Cheng et al.,2000b). These results to 26%. These data indicated that DMA C60 under light implied that the photosensitive efficiency was probably a irradiation was able to prevent cells from entering G1. critical factor for the photo-induced cytotoxicity of C60 compounds. However,further investigation is needed into the cellular uptake of these compounds in order to 4. Discussion assess to what extent it might influence their activities. Some evidence from the cell-free system showed that Some researchers found C60 derivatives had photo- various ROS were generated by C60 and its derivatives induced cytotoxicity (Tokuyama et al.,1993; Nakajima under irradiation (Arbogast et al.,1991; Orfanopoulos et al.,1996; Tsuchiya et al.,1996); however,others pre- and Kambourakis,1995; Hamano et al.,1997; Kamat et sented different results (Li et al.,1994; Scrivens et al., al.,1998; Yang et al.,1999; Cheng et al.,2000b) and led 1994; Irie et al.,1996; Schuster et al.,1996). We consider to the oxidative damage of lipids and proteins on the that a principal reason for this discrepancy is that the cell membranes (Kamat et al.,1998). Thus most authors structures of the C60 derivatives used were very differ- thought the ROS was the critical mediator for the ent. For example,C 60 and carboxylic acid C60 keep photo-induced cytotoxicity of C60 and its derivatives. almost all the original conjugated double bonds of C60 However,addition of superoxide dismutase,a specific cage,but fullerols destroy them very severely,and their inhibitor of superoxide radical,did not reverse the side-chains are quite different too. Thus the relationship cytotoxicity of C60–ethylene diamine–PEG-COOH con- between the structures and biological activities of C60 jugates against mouse fibroblast cell line L929 cells derivatives needs to be systemically examined. In this (Nakajima et al.,1996). In this study,hydroxyl radical paper,efforts were made to investigate,for the first time, quencher mannitol was also found ineffective on the the photo-induced cytotoxicity of three manolic acid photo-induced activity of DMA C60 against HeLa cells derivatives of C60. Their structures are given in Fig. 1. (Table 2). Therefore,in the case of the cellular system, Three malonic acid derivatives of C60 (DMA C60, whether C60 compound is able to act as the intracellular TMA C60 and QMA C60) were all toxic towards HeLa ROS producer that contributes to its photo-induced cells under light irradiation (Fig. 2,Table 1). These data cytotoxicity is still unclear. were in accordance with the photo-induced cytotoxicity Several C60 compounds have been reported to be of other C60 derivatives previously reported (Tokuyama photo-induced cytotoxic (Tokuyama et al.,1993; et al.,1993; Nakajima et al.,1996; Tsuchiya et al., Nakajima et al.,1996; Tsuchiya et al.,1996),and it is 1996). It was also found that the photosensitive cyto- interesting to question whether the cell cycle can be toxicity of C60 adducts decreased with the rise in the varied by these compounds. In the present experiment, additive number (Table 1). In other words,the integrity DMA C60 was found to induce a decrease of G1 and a of conjugated double bonds in C60 cage and the rise of G2+M during HeLa cell cycle,possibly indicat- decrease of the adduct groups appeared helpful for the ing prevention of cells from entering G1 (Table 3). Fur- photosensitive effect. Therefore,the maintenance of thermore,light irradiation was necessary both to its integrity of parent C60 cage should be carefully con- blocking effect on the cell cycle and its inhibitory effect sidered for the drug design related to photo-dynamics. on cell growth (Fig. 2,Table 3). This implied that C 60 compounds might inhibit the cell growth possibly by Table 3 blocking the cell cycle at G2+M. Flow cytometric analysis of cell-cycle distribution (percent of G1,S In conclusion,the present study showed that the and G2+M) of HeLa cells treated with DMA C60 (10 mm). 100,000 photo-induced cytotoxicity of C60 malonic acid adducts cells were counted in total for each sample. The data were mean values decreased with the increase of the additive number,and of those obtained from two experiments. The results showed a cell-cycle blockage might play a role in the mechanism decrease in the number of G1 cells and a rise in that of G2+M cells by DMA C60 in the presence of irradiation of the photocytotoxicity of C60 derivatives.

Group Percent of G1,S and G 2+M

Control G1: 63%; S: 29%; G2+M: 8% Acknowledgements DMA C60 alone G1: 65%; S: 29%; G2+M: 6% Irradiation alone G1: 63%; S: 31%; G2+M: 6% This work supported by the National Natural Science DMA C +irradiation G : 42%; S: 32%; G +M: 26% 60 1 2 foundation of China (No. 59702009). 中国科技论文在线 http://www.paper.edu.cn

X.L. Yang et al. / Toxicology in Vitro 16 (2002) 41–46 45

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