Advanced Materials Research Online: 2013-03-11 ISSN: 1662-8985, Vol. 667, pp 155-159 doi:10.4028/www.scientific.net/amr.667.155 2013 Trans Tech Publications, Switzerland Carbon Nanotubes in Cancer Treatment: A Preliminary In Vitro Study Using Neuroblastoma Cells I. Nurulhuda 1,3,a, M.Z. Mazatulikhma 3,b, R. Poh 4,c, and M. Rusop 1,2,d 1 NANO-SciTech Centre, Institute of Science, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia 2 NANO-ElecTronic Centre, Faculty of Electrical Engineering, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia 3 Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia 4 Department of Molecular Medicine, Faculty of Medicine, 50603 University of Malaya, Malaysia a nurulnye@gmail.com, b mazatul70@gmail.com, c rozaiday@yahoo.com, d nanouitm@gmail.com Keywords: Carbon nanotubes; neuroblastoma cells; cells viability; cytotoxicity; EMEM medium Abstract. Carbon nanotubes (CNTs) were reported to cause severe toxicity in in vivo and in vitro studies. On the other hand, CNTs has the potential as a mechanism for drug delivery across the blood brain barrier due to its nano-sized dimensions. The present preliminary study examined the cytotoxic effect of CNTs in serum-free and serum-containing EMEM medium on neuroblastoma cells growth. The MTS assay showed that CNTs did not cause any cytotoxicity at concentrations below 0.1 mg/ml at 24 and 48 h incubation in serum-containing EMEM medium. This finding suggested that below 0.1 mg/ml, CNTs can be used as a mechanism for drug delivery for the treatment of neuronal cancer. However, CNTs showed slighty toxic towards neuroblastoma cells at concentration 0.1 mg/ml at 48h incubation in serum-free EMEM medium. This finding indicates that the present of serum play a role for toxicity effect of CNTs in cells. Introduction Carbon nanotubes (CNTs) are classified as nanoparticles which are particles having diameters of less than 100 nm. The CNTs are rod shape of carbon with a cylindrical nanostructure. They can be single-walled or multi-walled. They are found to be potentially useful in many applications of nanotechnology especially in biomedical engineering, tissue engineering, drug delivery, nanoinjectors, gene therapy and biosensor technology [1]. The outer part of CNTs is usually coated with a hydrophilic polymer such as polyethylene glycol (PEG) or Tween 80 to disperse CNTs homogenously in cells [2,3]. In biomedical applications, many researchers have attempted to develop a good drug transporter based on the small size and high surface area of CNTs. Carbon nanotubes may be used to carry a minimal amount of drug to specific target cells for treatment, thus causing fewer side effects of the drugs in patients [4]. However, it has been reported that CNTs may cause toxicity in vitro [5-7]. Most of the toxicity that has been associated with using CNTs may be caused by agglomeration or accumulation of CNTs in the preparation [8]. In this study, the cytotoxicity of intact CNTs on neuroblastoma cells was measured by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay which involved the conversion of tetrazolium salt according to mitochondrial activity of viable cells at 37 C. Thus, the amount of formazan produced by dehydrogenase enzymes is directly proportional to the number of living cells in culture and can be measured at 490 nm [9]. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, www.ttp.net. (#69805558, Pennsylvania State University, University Park, USA-17/09/16,23:42:40)
156 Nanosynthesis and Nanodevice Materials and Methods CNTs with a physical dimension of 30-50 nm diameters and >95 % purity was purchased from (Chengdu Organic Chemicals, China). The neuroblastoma cells SH-SY5Y which had been discussed by Dila et al. [10] was chosen as cells model in this experiment. The cells culture is presently maintained in the Tissue Culture Laboratory of Synthesis and Chemical Biology, Institute of Science, Universiti Teknologi Mara. complete culture medium of EMEM-F12 supplemented with 2 mm glutamine (Sigma, USA), 1 % non-essential amino acid (PAA Laboratories GmbH, Austria), 1 % gentamicin (PAA Laboratories GmbH, Austria) and 10 % fetal bovine serum (PAA Laboratories GmbH, Austria) and incubated in a humidified incubator (Contherm) at atmosphere containing 5 % CO 2 at 37 C. The CNTs were first dissolved in DMSO to a final concentration of 10 mg/ml. Stock suspensions of CNTs were prepared both in serum-containing and serum-free EMEM media. Serial dilutions of CNTs in serum and serum-free media were carried out in a 96-well microplate at 0.0001, 0.001, 0.01 and 0.1 mg/ml for test concentrations [11]. The neuroblastoma cells were harvested by spreading 1 ml of 0.25 % trypsin over the stationary cultures and incubating at 37 C for 5 min. This was followed by the addition of 3 ml of EMEM medium to stop the trypsin reaction on cells. The cells were collected directly. The neuroblastoma cells were collected by centrifugation at 25 000 rpm for 2 min at room temperature. The pellet was washed twice with PBS. The viability of cells processed in this way was determined by using the Cellometer (nexcelom). One hundred ml aliquotes of neuroblastoma cells (2 x 10 4 cells) were seeded into the wells of 96-well plates. The cells were incubated for 24 h. After that, the cells were then replaced with 100 ml of serial dilutions of CNTs with serum-containing and serum-free EMEM media for 24 and 48 h treatment. Each test concentration was done in triplicates. After 24 and 48 h post-treatment with CNTs, the medium was replaced with MTS reagent. The MTS reagent was added to each well for each test concentration. The 96-well plates were wrapped with aluminium foil as the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2h-tetrazolium (MTS) reagent is sensitive to light. The level of fluorescence produced by the cells in the MTS assay was measured by using a microplate reader (Glomax Integrated System, Promega, USA) at the absorbance 490 nm. Statistical analyses were carried out using one-way analysis of variance (ANOVA) because of differences among treatment and control groups, followed by pair-wise comparisons between groups using Tukey's test. A p value < 0.05 was considered statistically significant. Results and Discussion In vitro cytotoxicity test of CNTs with neuroblastoma cell. The effect of CNTs on cellular viability was assessed by using the MTS assay [12]. In serum-containing EMEM medium, the data obtained for 24 h treatment (Fig. 1 and Fig. 2 (a)) indicated that CNTs showed less toxicity towards the cells compared to those tested using serum-free EMEM medium (Fig. 1 and Fig. 2 (b)).
Advanced Materials Research Vol. 667 157 Percentage of cell viability versus CNTs concentration % cell viability 100 80 60 40 20 Control 0.0001 0.001 0.01 0.1 CNTs concentrations (mg/ml) % cells viability (no serum) % cells viability (serum) Fig. 1, Histogram of cell viability of SH-SY5Y cells post-exposure to CNTs in serum-containing and free-serum EMEM medium at 24 h. Less cellular viability was observed in serum-containing and free-serum cells treated at 0.0001 mg/ml (1%,4%), 0.001 mg/ml (4%,2%), 0.01 mg/ml (3%) compared to control, whereas at 0.1 mg/ml the viability of cells was higher than control by 7% in serum-containing and less 8% in free-serum medium. Fig. 2, (a): Photo of SH-SY5Y cells post-exposure to CNTs in serum-containing EMEM medium at 24 h for CNTs at 0.0001 mg/ml and (b): photo of cells in free-serum EMEM medium at 0.0001 mg/ml. At 48 h incubation, for serum-containing EMEM medium, the percentage of cellular viability at 0.1 mg/ml was decreased by 11% compared to control (Fig. 3 and Fig. 4 (a)). Similarly, in the absence of serum in the medium, cytotoxicity at 0.1 mg/ml showed approximately 43% less cell viability compared to control (Fig. 3 and Fig. 4 (b)). Percentage of cell viability versus CNTs concentration % Cell viability 120 100 80 60 40 20 0 Control 0.0001 0.001 0.01 0.1 % cells viability (serum) % cells viability (no serum) CNTs concentrations (mg/ml) Fig. 3, Histogram of cell proliferation (MTS) of SH-SY5Y cells post-exposure to CNTs in serumcontaining and free-serum EMEM medium at 48 h. Decrease in cell viability was observed in cells treated at 0.0001 mg/ml (19%,5%), 0.001 mg/ml (11%, 19%), 0.01 mg/ml (21%) and 0.1 mg/ml (11%,43%) in serum-containing and free-serum medium respectively.
158 Nanosynthesis and Nanodevice Fig. 4, Photo of SH-SY5Y cells post-exposure to CNTs in serum-containing EMEM medium at 48 h. a: photo for CNTs at 0.1 mg/ml in serum-containing medium. b: photo for CNTs at 0.1 mg/ml in free-serum medium. Overall for cells incubated for both 24 h and 48 h in the presence of serum-containing EMEM medium, CNTs showed less toxicity compared to control as the percentage of cell viability was more than 80%. Thus there was no significant cytotoxicity following 24 h and 48 h exposure of neuroblastoma cells in serum-containing media. Similarly, in serum-free EMEM medium, the viable cells decreased at 48 h compared to 24 h post-exposure to CNTs. However, the statistical analysis confirmed that there were no significant differences for CNTs in serum-containing EMEM medium compared to CNTs in serum-free EMEM medium at 48 h even though the cell viability was above the EC 50 value (ANOVA; p>0.05). Prolonged incubation of CNTs in a serum-containing medium maintained the high viability of cells with no apoptosis or oxidative stress [13]. It appeared that serum-containing EMEM medium may enhance cellular growth, thus reducing the toxicity of CNTs on cells. The next stage of the present study will be use a similar range of concentration of CNTs to conjugate with the selected drug to observe the effect of the drug on neuroblastoma cells in the presence of serum-containing media. Some challenges were encountered during the preparation of the CNT solution. The CNTs on their own would hardly dissolve in water, therefore dimethyl sulfoxide (DMSO) solvent was used to enhance the dispersion of CNTs. However, CNTs in DMSO remained homogenous for only one minute, after which the CNTs usually started to settle at the bottom of the microcentrifuge tube. Thus immediately prior to introduction into the cell culture medium, the CNT preparation was mixed well by pipetting several times to avoid agglomeration which can be toxic to cells. Conclusion Carbon nanotubes were less toxic in the presence of serum-containing EMEM medium, compared to serum-free EMEM medium, although statistically the difference was not significant. Further studies on the effect of CNTs on neuroblastoma cells will be conducted using the complete EMEM medium at both 24 and 48 h incubation. Acknowledgements We thank the Tissue Culture Laboratory of Institute of Science, UiTM Shah Alam for providing the facilities to conduct this study, and also the financial support from Higher Education, Malaysia and Research Management Institute, UiTM.
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