Cytotoxicity of Uncoated and Polyvinyl Alcohol Coated

May 8, 2009 - Institute for Nanoscience and Nanotechnology, Sharif UniVersity of ... Materials Science and Engineering, Sharif UniVersity of Technolog...
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J. Phys. Chem. C 2009, 113, 9573–9580

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Cytotoxicity of Uncoated and Polyvinyl Alcohol Coated Superparamagnetic Iron Oxide Nanoparticles Morteza Mahmoudi,*,† Abdolreza Simchi,*,†,‡ and Mohammad Imani§ Institute for Nanoscience and Nanotechnology, Sharif UniVersity of Technology, Tehran, Iran, Department of Materials Science and Engineering, Sharif UniVersity of Technology, Tehran, Iran, and NoVel Drug DeliVery Systems Department, Iran Polymer and Petrochemical Institute, Tehran, Iran ReceiVed: January 7, 2009; ReVised Manuscript ReceiVed: March 31, 2009

Superparamagnetic iron oxide nanoparticles (SPION) are being increasingly used in various biomedical applications such as hyperthermia, cell and protein separation, enhancing resolution of magnetic resonance imaging, and drug delivery. However, the toxicity data for SPION are limited. In this study, uncoated and single polyvinyl alcohol coated SPION with high chemical reactivity (due to the bigger surface area) were synthesized using a coprecipitation method. Cytotoxicity of these magnetic nanoparticles and their ability to cause arrest in cell life-cycles was investigated. Interaction of these nanoparticles with adhesive mouse fibroblast cell line (L929) was probed using MTT assay. High concentrations of coated SPION (i.e., 100, 200, and 400 mM) demonstrated high cell viability following an exposure to the cells. Treated cells, via coated magnetic nanoparticles, did not showed evident necrosis, apoptosis (via propidium iodide staining), or cell cycle arrest in moderate concentration, i.e., 200 mM. However, the coated nanoparticles at the highest concentration (400 mM) caused both apoptosis and cell cycle arrest in G1 phase, possibly due to the irreversible DNA damage and repair of oxidative DNA lesions. Uncoated nanoparticles showed significant apoptosis amount at the highest concentration. The mentioned damaged occurred because of proteins attachments to the surface of nanoparticles, leading to the formation of protein “corona” on the shell of magnetic particles. The associations of proteins on the surface of nanoparticles were confirmed by UV/Vis spectroscopy. Finally, the effect of particle surface (i.e., uncoated and coated) on the cell cycle was studied. 1. Introduction Due to their highly promising potential in medicine, nanoparticles faced extraordinarily rapid progress and early acceptance in various biomedical applications. However, studies to characterize their effects after exposure and to address their potential toxicity are few in comparison. Cytotoxicity of different types of nanoparticles has been investigated during interaction with a widespread range of cell types by many research groups with a special focus on metals and metal oxides.1-9 Superparamagnetic iron oxide nanoparticles (SPION) have been considered as one of the most promising materials with a wide array of feasible biomedical applications such as drug delivery, cell and protein separation, hyperthermia, and cancer diagnosis and treatment.10-14 In spite of such fortunate attention to the biological applications of the SPIONs, rare reports are available to soundly evaluate their toxicity and/or biocompatibility profile. Karlsson et al.15 have compared different metal oxide nanoparticles (including CuO, TiO2, ZnO, CuZnFe2O4, Fe3O4, and Fe2O3) regarding their cytotoxicity, capability to cause DNA damage, and oxidative lesions (using the comet assay). Intracellular production of reactive oxygen species (ROS) was also determined using an oxidation sensitive fluoroprobe i.e. 2′,7′-dichlorofluorescin diacetate. According to the obtained results, no or low toxicity of magnetite nanopar* Corresponding author. E-mail: [email protected]; [email protected]; [email protected]. † Institute for Nanoscience and Nanotechnology, Sharif University of Technology. ‡ Department of Materials Science and Engineering, Sharif University of Technology. § Iran Polymer and Petrochemical Institute.

ticles have been reported. At lower concentration regimes of SPIONs, i.e., 20 and 40 µg/mL, neither DNA damage nor intracellular ROS toxic effects were observed upon interaction of magnetite nanoparticles to the human lung cancer cell lines, although a little amount of oxidative DNA lesions were observed. Recently, the present authors16 have shown that no cell necrosis and apoptosis occur during interaction of magnetite nanoparticles with L929 mouse fibroblast cell line. Additionally, the biocompatibility of different shapes and sizes of polyvinyl alcohol coated magnetite nanoparticles has been shown via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This observation is in agreement with the other reports showing very low toxicity for iron oxide nanoparticles.3,5 It is worthy to point out that the presence of oxidative stress upon exposure of cells to iron oxide nanoparticle implies the possibility of DNA damage. Here, the early effect would be evidenced in cell cycle progression. DNA damaged cells will accumulate in gap1 (G1), DNA synthesis (S), or in gap2/mitosis (G2/M) phase. In contrast, cells carrying irreversible damages to their genetic content will endure apoptosis, giving rise to the formation of fragmented DNA which would be defined in subG1 phase.17 There is presently significant debate considering biological responses of nanoparticle characteristics containing size, shape, composition, hydrophilicity and hydrophobicity, and surface area.18-20 New and interesting approaches to understanding the impact of interaction with nanoparticles on protein behavior are emerging.21,22 Lynch et al.23 proved that the effective unit of interest in the cell-nanomaterials interaction is not the nanoparticle in itself but also the particle and its “corona” of more or less strongly associated proteins from plasma or other bodily

10.1021/jp9001516 CCC: $40.75  2009 American Chemical Society Published on Web 05/08/2009

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fluids. Ultimately, this corona of native-like or unfolded proteins “expressed” at the surface of the particle is “read” by living cells and is the key phenomenon that scientists need to understand. Given this, it is surprising that the particle-protein complex is so poorly understood. The first reliable analysis of the proteins that associate to a nanoparticle in a complex biological fluid has recently been presented by Cedervall et al.24 The aim of this study is to investigate the interaction of uncoated and coated magnetite nanoparticles with protein corona on the cell cycle. Extension of the cytotoxicity studies to cell cycle assay in order to detect the effective parameters such as apoptosis, cell cycle arrest, and evidence to DNA oxidative damage is presented. In addition, the morphology of cells and the composition of cell medium before and after treatment with the SPION are reported. 2. Materials and Methods 2.1. Materials. Reagent grade iron chloride salts and PVA of 30 000-40 000 g mol-1 nominal molecular weight and 86-89% degree of hydrolysis were supplied by Merck Inc. (Darmstadt, Germany) and Fluka (Ronkonkoma, USA), respectively. The materials were used without further purification. Mouse fibroblast cells (L929) were maintained in Dulbecco’s modified eagles medium (DMEM, Sigma, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, Germany) and 1% penicillin streptomycin (Gibco, Germany). 2.2. Preparation of SPION. Solutions were prepared using deionized (DI) water after 30 min bubbling with argon for deaeration. The iron salts were dissolved in DI water containing 0.5 M HCl where the mole fraction of Fe2+ to Fe3+ was adjusted to 2:1 (1 g of FeCl3:0.368 g of FeCl2) for all samples. The precipitation was performed by dropwise addition of iron salt solutions to NaOH solutions under an argon atmosphere. In order to control mass transfer, which may allow particles to combine and build larger polycrystalline particles, turbulent flow was created by placing the reaction flask in an ultrasonic bath and changing the homogenization rates between 3600 and 9000 rpm in the first 2 min of the reaction. The molarities of the NaOH solution and the stirring rate were fixed at 1.6 and 9000 rpm, respectively. The details of experiments applied for choosing levels of these parameters have been reported elsewhere.14 After 30 min, PVA solution (with a polymer to iron mass ratio of 2) was added by syringe as a stabilizer, and the reaction mixture was stirred at a constant temperature of 35 °C for an additional 30 min. Obviously, this stage was not afforded for the uncoated nanoparticles. The particles were collected by centrifugation at 6000 rpm for 10 min and redispersed in DI water (several times). Finally, the ferrofluid was kept at 4 °C for future use. 2.3. Characterization of SPION and Cell Medium. The synthesized nanoparticles were characterized by various analytical techniques. TEM (ZEISS, EM-10C, Germany) operating at 100 kV was used for size and morphology characterization. XRD (Siemens, D5000, Germany) with Cu KR radiation was used for the phase characterization. The average size of the nanoparticles was determined by using the Scherrer method. Magnetization of the samples was measured in a variable magnetic field using a vibrating sample magnetometer (VSM) with a sensitivity of 10-3 emu and magnetic field up to 8 kOe. The magnetic field was changed uniformly with a time rate of 66 Oe/s. In order to investigate the compositional changes of the cell medium due to the interaction with both uncoated and coated SPION, UV-Vis spectroscopy of the samples was measured

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Figure 1. TEM images of nanosized (a) uncoated and (b) PVA coated SPION.

Figure 2. XRD pattern of magnetite nanoparticles.

Figure 3. Magnetization curve for SPION.

with a Lambda 950 spectrophotometer (Perkin-Elmer, USA) from 200 to 850 nm wavelengths. 2.4. In Vitro Biocompatibility Assessment. Primary mouse fibroblast adhesive cells (i.e., L929) from the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran were seeded on the glass coverslips in 96 well plates at 10 000 cells per well in 150 µL of medium and incubated for 24 h. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. After 24 h incubation period, 40 micro-liter of mediums containing high concentrations of uncoated and coated SPION (100, 200, and 400 mM iron, measured by atomic absorption) were added to the wells and cells were incubated for additional periods ranging from 24 to 72 h. Negative control was provided by culture medium without particles. All particle concentrations and controls were each seeded in ten separate wells.

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Figure 4. (a) Cell viability of MTT assay results for all concentrations of uncoated SPION samples on L929 cells for 24, 48, and 72 h. (b) Optical microscopy of control L929 cells and (c) optical microscopy of L929 cells containing 400 mM of uncoated SPION before MTT treatment, respectively (brown colonies are precipitated SPION).

Cytotoxicity was assessed using the MTT assay, which is a nonradioactive, colorimetric assay. After 24, 48, and 72 h from the interaction of cells with the samples, 100 µL of 1× MTT were added to each well. Following incubation, the medium was removed, and formazan crystals were solublized by incubation for 20 min in 150 µL of acidified isopropanol using HCl. The absorbance of each well, which assesses viable cells, was read at 545 nm on a microplate reader (Stat Fax-2100, AWARENESS, Palm City, USA). The effect of incubation with the nanoparticles on the morphology of cells was examined by optical microscopy (Olympus CK 40).

J. Phys. Chem. C, Vol. 113, No. 22, 2009 9575 2.5. Cell Cycle Assay. In order to obtain maximum exposure of iron oxide nanoparticles on the cell cycle, highly dispersed (i.e., without aggregations) SPIONs were synthesized as previously reported elsewhere.14 Cell cycle assay was carried out by staining of the DNA with Propidium iodide (PI) followed by flow cytometric measurement of the fluorescence. Approximately 106 L929 cells were cultured. Following the treatment with the uncoated and PVA coated SPION (100, 200, and 400 mM) for 72 h, the medium was removed by Pasteur pipet and stored. It is noteworthy that the damaged cells may leave their attached places and be suspended in medium which obligates the medium storage. Cells were detached from the flask via trypsin treatment and harvested using the stored medium. The obtained suspension was centrifuged at 280 g. The collected cells were washed with PBS several times at 200g. The cells have been thoroughly resuspended in PBS by Pasteur pipet in order to have a monodisperse cell suspension at the time of mixing cells with ethanol. Cells were fixed in ethanol via transferring into the tubes containing 70% ethanol and stored in -20 °C. This solution could be stored for several months. Prior to the flow cytometric analysis, the ethanol-suspended cells were centrifuged at 200g for 5 min and the supernatants were decanted thoroughly. The collected cells were washed with PBS and then suspended in 1 mL PI/Triton X-100 staining solution with RNase A followed by keeping either at 37 °C for 15 min or at ambient temperature for 30 min. The stained cells were then analyzed by flow cytometry (FACScan Becton Dickinson, Mountain View, CA) at an excitation wavelength of 488 nm and emission wavelength of 610 nm.

Figure 5. (a) Cell viability of MTT assay results for all concentrations of SPION coated samples on L929 cells for 24, 48, and 72 h. (b) Optical microscopy of control L929 cells; (c and d) optical microscopy of L929 cells containing 200 and 400 mM SPION before MTT treatment, respectively.

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Figure 6. Cell cycle assay results for (a) control and (b) coated SPION (200 mM) treated cells.

2.6. Statistical Analysis. In analyzing data, it is common to find that one value is far from the others. Such a value is often called an outlier. The majority of outliers in MTT may be due to a mistake: poor pipetting, removing useful solution with supernatant, etc. As a result, an outlier value may come from a different population than the others and can be misleading for the interpretation of results. No mathematical method is fully able to ascertain whether or not an outlier comes from the same or different population, given that their distributions are normally unknown. However, assuming a Gaussian distribution, it is possible to determine the probability by which a value is as far from the others as was observed. If this probability is minimal,

then one can conclude that the value is likely to be erroneous (an outlier), and may be excluded from the analysis.25 In the present MTT assay studies, all experiments were carried out at least in triplicate and the results expressed as (mean ( standard deviation). The standard deviation values are indicated as error bars in the subsequent MTT graphs. The results were statistically processed for outlier detection, using a so-called “T procedure”26 in the MINITAB software (Minitab Inc., State College, PA). Statistical judgments were made by a one-way analysis of variances (ANOVA), during which a probability of p < 0.05 was considered as statistically insignificant for a value being outlier.

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Figure 7. Cell cycle assay results for (a) control and (b) coated SPION (400 mM) treated cells.

3. Results and Discussion 3.1. Uncoated and PVA Coated SPION. Transmission electron microscopy indicated that the uncoated magnetic nanoparticles were highly agglomerated due to the absence of surfactant (Figure 1a). In contrast, spherically shaped individual SPION with a narrow size distribution and without aggregation were obtained when the coating process was applied (Figure 1b). Here, no precipitation was observed in the suspension after storage for 6 months. This high stability may be due to the intrinsic properties of PVA as a hydrogel forming polymer which makes the PVA shells to absorb the DI water. Figure 2 shows the X-ray diffraction pattern of the coated nanoparticles. The diffraction peaks reveal the formation of magnetite nanoparticles. The full width at half-maximum (fwhm) of the (311)

reflection was used to determine the average crystallite size of the nanoparticles according to the Scherrer method. The size was found to be 4 nm in agreement with the TEM image (Figure 1b). VSM analysis indicated that the nanoparticles are superparamagnetic (Figure 3); that is, the remanence and coercivity in the hysteresis loop are negligible. Comprehensive data considering chemisorptions of PVA on the surface of SPION can be found elsewhere.14 3.2. Biocompatibility. The treated cells with the uncoated magnetic nanoparticles showed low viability (i.e., toxic effect), Figure 4a. As can be seen in Figure 5a, the coated iron oxide nanoparticles demonstrated acceptable levels of cell viability following exposure, with none demonstrating toxic effects at the concentrations tested. Variation of the shape and morphology

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Figure 8. UV/vis spectrum of fresh cell medium, the extract medium of uncoated and coated SPIONs (DI water defined as reference in UV/ vis spectrum). Upper graph: first (solid line) and second (dash-line) order derivatives of coated SPION with concentration of 400 mM.

of the cells would be the best ocular proof upon their exposure to a toxic material.9 Therefore, control and treatment groups were examined by optical microscopy to ascertain the toxicity or biocompatibility of the synthesized particles. The results are shown in Figure 4, panels b and c, for the uncoated SPION and in Figure 5b-d for the coated particles. 3.3. Influence of SPION on Cell Cycle. The presence of SPION treated cells in each phase of the cell cycle was fully compared to the control ones. High concentrations of coated SPION (i.e., 200 and 400 mM) were selected to facilitate the identification of phase changes of cell cycle due to the interaction with SPION. For comparison with other nanoparticles, it is worthy to mention that silver nanoparticles at concentrations of 25 and 400 µg/mL (around 0.2-3.2 mM/ml) exhibit G2/M arrest.9 We have found that at concentrations of 100 and 200 mM the population of cells in each phase were the same with the control, i.e. no arrest on the cell cycle even at the high concentration of SPION (Figures 6). The same amount of cell population in subG1 phase in control and SPION treated cells clearly proved the absence of apoptosis. The absence of apoptosis for PVA coated SPION treated cells (at a concentration of 200 mM) has also been reported via TUNEL assay.16 As the concentration of SPION increased to 400 mM, a trace apoptosis was observed in the assay. In the control groups, the main percentages of cell population were observed in G1 phase, whereas in SPION treated cells, a decrease in G1 population was detected (∆G1/G1(control) ) -0.12). The same results have previously been reported for Ag nanoparticles at very low concentrations in addition to arrest in G2/M population.9 The results presented in Figure 7 also showed an increasing apoptotic population (∆subG1/subG1(control) ) 0.62) due to the irreversible DNA damage. Since magnetite nano-

Mahmoudi et al. particles showed oxidative DNA lesions via comet assay,15 the arrest in G1 phase may be related to the repair of damaged DNA. In addition, the interaction between the nanoparticles and the proteins in the medium caused the malfunction of associated proteins to particles which certainly had a considerable effect on the cell cycle. The biological outcome may also differ depending on the relative protein exchange rates between the nanoparticles and cellular receptors.22 In order to investigate the adsorption of protein on the surface of SPION, the fresh cell medium and extracted medium (interacted with the uncoated and coated magnetic nanoparticles) were characterized with UV/Vis. Since the color of DMEM medium is significantly affected by minor pH changes, UV/Vis is a suitable method to detect medium color changes during interactions with SPION at wavelength of 560 nm. Figure 8 shows the effect of uncoated and coated magnetic nanoparticles on the cell medium. Iron oxides are often negatively charged in water due to absorption of OH- groups on the surface of SPION.27 Surface charges give rise to an electric field and this will attract counterions. The layer of surface charges and counterions make up the electric double layer.27 The attraction of the electric field is decreased for the coated nanoparticles due to a reduction in amount of the surface charge.27 During the interaction of SPION to Dulbecco’s modified Eagle’s medium (DMEM) which contains many sources of proteins in fetal bovine serum (FBS), the dynamic layer of protein (and other biomolecules) adsorbs to nanoparticles surfaces immediately. Lynch et al.28 have recently reported that protein fibrillation could occur due to the protein-nanoparticle interactions. As a result, the composition of the DMEM medium can be changed, owing to the attraction of proteins in the diffuse layer of magnetic nanoparticles. Mahmoudi et al.29 showed that a more reliable way of identifying cytotoxicity for in vitro assessments is to use particles with saturated surfaces via interactions with DMEM before usage. By using nanoparticles whose surface had been passivated, the changes in the composition of cell medium would be minimal. The rate of association and dissociation of proteins is likely to vary quite considerably with protein and characteristic of the particle such as shape and purity.28 Multiprotein absorbance is detected via the second derivative curves at the wavelength range of 250-300 nm for the uncoated and coated nanoparticles (see Figure 8). The affinity of proteins to the surface of SPION is related to the surface properties of SPION, i.e., the extend of hydrophilicity, which would be the basis of cell-nanoparticle interactions.22 Figure 9 shows the effect of uncoated SPION with a low hydrophilicity on the cell cycle assay. As seen, the granulity of the cells increased. A significant amount of apoptosis was also observed, this can be attributed to the high amount of absorbed protein on the surface of uncoated SPION. 4. Conclusions The nanoparticles considered in this work were uncoated magnetic nanoparticles and SPION composed of a magnetite core and a PVA coating. MTT assay was used to investigate the biocompatibility of the synthesized SPION at relatively high concentrations using L929 cells. The PVA coated SPION demonstrated acceptable levels of cell viability following exposures to 400 mM iron concentration for 72 h. In contrast, the uncoated SPION exhibited toxic effect at the mentioned concentration. Neither apoptosis nor cell cycle arrest were observed for the coated-nanoparticle treated-cells at the concentration of 200 mM. As the coated SPION concentration increased, apoptosis and cell cycle were noticed. DNA damage

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Figure 9. Cell cycle assay results for treated cells to uncoated SPION with concentration of (a) 200 and (b) 400 mM.

is believed to be the prime factor which in fact results in apoptosis and cell cycle arrest following exposure to SPION at a concentration of 400 mM. Since no trace of cell death was observed, it seems that DNA repair pathway was activated. The interaction of magnetic nanoparticles with the living matter was shown to be significantly affected by an adsorbed protein layer, which the proteins may have differing degrees of residual tertiary structure, depending on the details of the system. The uncoated nanoparticles exhibited significant differences in cell cycle results in comparison with the coated particles, possibly due to the different protein affinity to their surfaces.

The results presented in this work demonstrated that the PVA coated nanoparticles have a high biocompatibility with considering the usual concentrations applicable in biological applications. Acknowledgment. Morteza Mahmoudi thanks Prof. Azam Iraji-Zad, the Chair of INST at Sharif University of Technology (SUT), for the invaluable advice that helped the author to construct some synthesis and analyzing instruments in nanolaboratory at SUT. This advice has given the author the unique opportunity to conduct research considering SPION in the past 3 years.

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Mahmoudi et al. (14) Mahmoudi, M.; Simchi, A.; Imani, M.; Milani, A. S.; Stroeve, P. J. Phys. Chem. B 2008, 112 (46), 14470–14481. (15) Karlsson, H. L.; Cronholm, P.; Gustafsson, J.; Moller, L. Chem. Res. Toxicol. 2008, 21, 1726–1732. (16) Mahmoudi, M.; Shokrgozar, M. A.; Simchi, A.; Imani, M.; Milani, A. S.; Stroeve, P.; Vali, H.; Ha¨feli, U. O.; Sasanpour, P.; Bonakdar, S. J. Phys. Chem. C 2009, 113 (6), 2322–2331. (17) Ishikawa, K.; Ishii, H.; Saito, T. DNA Cell Biol. 2006, 25, 406–411. (18) Nel, A.; Xia, T.; Mkdler, L.; Li, N. Science 2006, 311, 622–627. (19) Warheit, D. B.; Webb, T. R.; Sayes, C. M.; Colvin, V. L.; Reed, K. L. Toxicol. Sci. 2006, 91, 227–236. (20) Colvin, V. L. Nat. Biotechnol. 2003, 21, 1166–1170. (21) Lundqvist, M.; Nygren, P.; Jonsson, B. H.; Broo, K. Angew. Chem. 2006, 118, 8349–8353. (22) Linse, S.; Cabaleiro-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S.; Dawson, K. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8691–8696. (23) Lynch, I.; Dawson, K. A.; Linse, S. Science 2006, 327, 14. (24) Cedervall, T.; Lynch, I.; Foy, M.; Berggard, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Angew. Chem. Int. Ed. 2007, 46, 5754–5756. (25) Barnett, V.; Lewis, T. Outliers in Statistical Data; John Wiley & Sons: New York, 1994. (26) Bolton, S. Pharmaceutical statistics: Practical and clinical applications, 2nd ed.; M. Dekker: New York, 1990. (27) Butt, H. J.; Graf, K.; Kappl, M. Physics and Chemistry of Interfaces; Wiley-VCH: Weinheim, Germany, 2003. (28) Lynch, I.; Dawson, K. A. Nanotoday 2008, 3 (1-2), 40–47. (29) Mahmoudi, M.; Simchi, A.; Imani, M.; Milani, A. S.; Stroeve, P. Nanotechnology, 2009, in press.

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