Pluronic-coated carbon nanotubes do not induce degeneration of cortical neurons in vivo and in vitro

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Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 96 – 104 www.nanomedjournal.com

Original Article: Toxicology

Pluronic-coated carbon nanotubes do not induce degeneration of cortical neurons in vivo and in vitro Giuseppe Bardi, PhD, a,⁎ Paola Tognini, MSc, a Gianni Ciofani, MSc, b Vittoria Raffa, PhD, b Mario Costa, PhD, a Tommaso Pizzorusso, PhD c a

b

Abstract

Key words:

Institute of Neurosciences, National Research Council, Pisa, Italy Center for Research in Microengineering, Scuola Superiore Sant'Anna, Pisa, Italy c Department of Psychology, University of Florence, Florence, Italy

Carbon nanotubes (CNTs) are nanodevices with important potential applications in biomedicine such as drug and gene delivery. Brain diseases with no current therapy could be candidates for CNT-based therapies. Little is known about toxicity of CNTs and of their dispersion factors in the brain. Here we show that multiwall CNTs (MWCNTs) coated with Pluronic F127 (PF127) surfactant can be injected in the mouse cerebral cortex without causing degeneration of the neurons surrounding the site of injection. We also show that, contrary to previous reports on lack of PF127 toxicity on cultured cell lines, concentrations of PF127 as low as 0.01% can induce apoptosis of mouse primary cortical neurons in vitro within 24 hours. However, the presence of MWCNTs can avoid PF127-induced apoptosis. These results suggest that PF127-coated MWCNTs do not induce apoptosis of cortical neurons. Moreover, the presence of MWCNTs can reduce PF127 toxicity. © 2009 Elsevier Inc. All rights reserved. Carbon nanotubes; Neurons; Surfactant; Biocompatibility; Toxicity

Among the huge set of the “nanomaterials” and “nanodevices” interacting with biological systems, carbon nanotubes (CNTs) are intensively studied for their properties.1 Among CNTs, single-wall CNTs consist of a single layer of graphite lattice rolled into a perfect cylinder, whereas sets of concentric cylindrical graphite shells form multiwall CNTs (MWCNTs). Neurobiology is one of the fields where the potential applications seem to be very promising.2 Neurons and neuronal cell lines can grow and differentiate on CNT substrates.3-5 Furthermore, the electrical interactions between nanomaterials and neurons has been recently shown developing an in vitro integrated CNT-neuron system to study electrical stimulation delivered by CNTs to the neurons and its effects on neuronal signaling.6 These observations have Received 14 March 2008; accepted 27 June 2008. The activity presented in this work was supported by the NINIVE (NonInvasive Nanotransducer for in Vivo Gene Therapy, STRP 033378) Project, cofinanced by the 6FP of the European Commission. ⁎Corresponding author. Institute of Neurosciences, National Research Council, Pisa Italy. E-mail address: [email protected] (G. Bardi).

raised the possibility of using CNTs as therapeutic agents for brain diseases in which targeted electrical stimulation, gene and drug delivery, and cell ablation might be required.7 In many brain diseases no cure is available, and there is a strong need of new therapeutic approaches. However, to use CNTs as therapeutic devices the issue of their toxicity must be solved. There is a debate in the literature concerning CNT toxicity with data in favor of or against a damaging interaction of CNTs with biological tissues.8 Unfortunately, these studies have used different types of CNTs, different coatings, and different bioassays. All these factors have been proven to influence toxicity. Therefore, it seems crucial that validation of CNTs as therapeutic nanodevices for biomedicine should use defined CNTs with standardized coating and should adopt biological assays as close as possible to the in vivo situation. To release CNTs in the brain or in any biological system we first need to disperse them in solution using special surfactants.9 This operation is already a big challenge, because CNTs tend to form bundles and clusters.10 Precoating CNTs with the nonionic surfactant Pluronic F127 (PF127; Sigma, St. Louis, Missouri) has been successful in promoting CNTs

1549-9634/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2008.06.008 Please cite this article as: G. Bardi, P. Tognini, G. Ciofani, V. Raffa, M. Costa, T. Pizzorusso, Pluronic-coated carbon nanotubes do not induce degeneration of cortical neurons in vivo and in vitro. Nanomedicine: NBM 2009;5:96-104, doi:10.1016/j.nano.2008.06.008

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Figure 1. Dispersion of MWCNTs in PF127 and primary neurons cultured in the presence of pristine CNTs. FIB picture of a bundle of pristine MWCNTs before, (A), at 6500× magnification, and after, (B) dispersed, fragmented PF127 MWCNTs at magnification of 10,000×; ion beam current of 5 pA. (C) Confocal microscopy image of mixed primary neuronal culture in the presence of pristine MWCNTs (yellow arrows).

dispersion in water solutions. PF127 has been shown to have low toxicity when tested in macrophage or keratinocyte cell lines,11-13 being substantially nontoxic at the concentrations required for CNT dispersion. However, to use PF127 for CNT delivery to differentiated tissues, experiments on the relatively undifferentiated cell lines are of limited usefulness. In particular, no information is available about toxicity of PF127-based solutions of CNTs in primary neuronal cultures or after stereotaxic injection into the intact brain.

Here we show a parallel in vivo–in vitro investigation of the biocompatible features of MWCNTs. We released PF127-coated MWCNTs in the mouse brain cortex by microinjection and observed the histology of the surrounding tissue. We did not find CNT-induced damages of the cellular brain structures. A cellular detailed investigation has been conducted on primary cortical neurons in cultures. We proved that PF127 alone can induce apoptosis of the neurons already at a concentration of 0.01% in the cell medium.

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Table 1 Multiwall carbon nanotubes (MWCNTs) description and properties Properties

MWNTs, 97%

Available form

Black powder

Diameter

10-30 nm

Average length ⁎

2 μm

Carbon content (purity)

97.06%

Metal particles

2.94%

Amorphous carbon and other carbon impurities (in the predetermined carbon content)

b1%

Raman peaks

D = 1359 cm-1 G = 1584 cm-1

Bulk density

0.15 g/cm3

Odor

Odorless

⁎After dispersion treatment.

Intriguingly, PF127-coated MWCNTs do not show any cell death, so the presence of CNTs is not toxic. Methods Cell cultures Primary mouse cortical cultures were prepared as follows: Brains were removed from mice at postnatal days 0-2 and placed in dissection medium (16 mM glucose, 22 mM sucrose, 10 mM HEPES, 160 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.22 mM KH2PO4; pH 7.4; 320-330 mOsm). Cortices were dissected and meninges removed. Chopped small pieces of cortex were triturated with a Pasteur pipette in the presence of 0.25% trypsin and DNase to reach a single-cell suspension. After 5 to 10 minutes a small volume of suspension at the top was placed in an equal volume of Dulbecco's minimal essential medium (DMEM) supplemented with 10% horse serum. Cells were then counted and directly cultured on coverslips or Petri dishes previously coated with polylysine. After 24 hours DMEM–horse serum was replaced with Neurobasal-A medium (Invitrogen, Carlsbad, California) supplemented with B27, 2 mM glutamine, and antibiotics. After 10 days fully differentiated neurons lay on glia cells growing in a layer underneath the neurons. CNTs MWCNTs were supplied by Nanothinx (Rio Patras, Greece). Purity of nanotubes was estimated with a postdeposition thermo gravimetric analysis treatment by the supplier, which revealed a carbon content of about 97.2% and a minimal amount of carbon soot (b1%). Apoptosis assays and microscopy Hoechst 33342 (1 μg/mL) staining and Annexin VApoAlert Kit (Clontech Laboratories, Mountain View, California) were

used to evaluate differences between normal and apoptotic cells after treatments. Anti-mouse NeuN antibodies to stain neurons were used at a concentration of 20 μg/mL, and Alexa 488 secondary antibody were used at 2.5 μg/mL. Phase contrast and fluorescence cell images were acquired by a camera-mounted Zeiss Axioskop microscope (Carl Zeiss MicroImaging Inc., Thornwood, New York) and by a Leica TCS NT confocal microscope (Leica Microsystem, Wetzlar, Germany). Intracerebral injections Animals were used in accordance with protocols approved by the Italian Minister for Scientific Research. Mice were anesthetized with avertin (0.5 mL/100 g) and mounted on a stereotaxic apparatus. Injections are made at specific stereotaxic locations in the visual cortex by means of a glass pipette (30-μm tip diameter) mounted on a motorized (0.1-μm step) three-axis micromanipulator connected to an injector (Sutter Instruments, Novato, California). A total of 350 nL were released at 700 μm and another 350 nL were released 400 μm below the cortical surface to allow homogeneous dispersion of CNTs along the cortical depth. During injections, animals were oxygenated and heated by means of a blanket with a thermostat to ensure a 37°C rectal temperature. After surgery the antibiotic gentamicin was topically administered to prevent infections. In these conditions the whole procedure requires about 20 minutes, and recovery from anesthesia occurs after 60 to 90 minutes. After recovery animals were returned to their home cages. Injected mice were transcardially perfused with 4% paraformaldehyde in phosphatebuffered saline (PBS) solution (0.1 M). Brains were sectioned on a sliding microtome in 40-μm sections, and cresyl violet staining was performed. Images were acquired on a cameramounted Zeiss Axioskop microscope, and lesion area was measured using the Metamorph software (Molecular Devices, Downingtown, Pennsylvania). Focused ion beam imaging Samples were observed after drying with a focused ion beam (FIB) system that allows imaging, localized milling, and deposition of conductors and insulators with high precision. The FIB system used for the imaging in the present work is a FEI 200 (focused ion beam localized milling and deposition, from FEI Company, Hillsboro, Oregon) delivering a 30-keV beam of gallium (Ga+) ions, with beam currents varying between 1 pA and 11 nA. MTT assay MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, M2128 from Sigma, St. Louis, Missouri) assays were carried out after 24 hours of incubation with or without CNT-modified medium. Cells were incubated with MTT 0.5 mg/mL for 2 hours. After cell treatment with 100 μL of dimethyl sulfoxide (D8418 from Sigma) absorbance at 550 nm was measured with a VERSAMax microplate reader (Molecular Devices, Union City, California).

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Figure 2. Sections of mouse brain at the site of CNT injection. (A) The injection site is labeled with a star. cc, cerebral cortex; wm, white matter. Calibration bar is 1.2 mm. (B) The injection site in mice treated for 3 days at higher magnification. Note the black precipitate at the center of the injection site and the area with reduced density of large cells corresponding to the lesioned area. Normal neuronal density and tissue layering is present outside the lesioned site. Lesioned area is outlined with a dashed line. Calibration bar is 400 μm. (C) Higher magnification of the transition between the lesioned and the unlesioned area (on the right). Note the clear difference in the size and the density of the cells between the unlesioned and the lesioned area. The transition between the lesioned and the unlesioned area is outlined with a dashed line. Control mice (D, E) and MWCNT-injected mice (F, G) brain cortices present a scar of glial cells surrounding the injection site. Higher magnifications of (E) the control and (G) the MWCNT-injected mice are shown.

Statistics To evaluate the statistical significance of all the described in vitro experiments, ten microscopic fields per coverslip were counted and three coverslips/treatment were used for each experiment. Three independent experiments were performed in triplicate. One-way statistical analysis of variance followed by analysis with the Student-NewmanKeuls method were performed. Results Multiwalled carbon nanotubes are very efficiently dispersed in the presence of PF127 Pristine CNTs tend to aggregate in large particles in the micron size range when released in saline solution (Figure 1, A). However, the presence of PF127 at concentration below 0.1% is sufficient to solubilize MWCNTs up to a concentration in the range of micrograms

per milliliter (Figure 1, B). The MWNTs production method is based on the synthesis of carbon nanostructures by catalytic chemical vapor deposition of hydrocarbon sources on substrates of alumina impregnated with metal catalysts (iron).14 Noncovalent wrapping of the tubular surface was performed with PF127 surfactant (steric dispersion). 15 Aqueous solutions of MWNTs were obtained with PF127 (polyoxyethylene-polyoxypropylene block copolymer, purchased from Sigma), a water-soluble surfactant with MW = 12,600. A water-PF127 solution (0.1%) containing 0.5 mg/ mL of MWNTs was placed over a hot plate (at 70°C) under magnetic stirring for 4 hours; the resulting mixture was sonicated with a Branson sonicator 2510 (Bransonic; Branson, Danbury, Connecticut) at 20 W for 12 hours. The mixture was then centrifuged at 900 g for 10 minutes to remove nondispersed nanotubes. The concentration of the CNT solution measured via spectrophotometric analysis16 at 270 nm was in the range 30-150 μg/mL. Nanotubes treated according to this procedure retain their pristine electronic properties.17 MWCNT properties are given in Table 1.

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Figure 3. PF127 induces apoptosis of mouse primary neurons in vitro. (A) Pictures of primary mixed cortical cultures are taken in phase contrast and fluorescence after 24 hours at 37°C without or with increasing concentration of PF127 (from 0.001% to 0.05%). Cell nuclei have been stained using Hoechst 33342 (1 μg/mL). (B), (C), The percentage of (B) neurons and (C) glia after treatments. The percentage of cell numbers as compared to control cells is shown (mean ± SEM) (treatments versus control, *P b .05; **P b .01; ***P b .001). (D), Fluorescence (nuclei) and phase contrast (whole cell body) pictures of mouse cortical neurons and glia treated for 24 hours at 37°C with PF127 (0.05% treatment as an example) are shown. In the middle panel is shown the difference of pycnotic cell nuclei and nuclei of healthy cells. The Hoechst staining of the neuron nuclei overlay with the NeuN (green labeling in the bottom panel). (E), Quantification and statistical analysis of apoptotic nuclei without or with increasing concentration of PF127 (from 0.001% to 0.05%). The percentage of apoptotic nuclei as compared to control cells is shown (mean ± SEM) (treatments versus control, *P b .05; **P b .01; ***P b .001).

If the powder of pristine MWCNTs is directly released in cell culture medium they are visible as large aggregates (Figure 1, C, yellow arrows). However, the pristine MWCNTs particles do not affect cell viability in culture, as shown by Figure 1, C and the MTT measurement in Figure 5 (dark stripes filled white bar).

PF127-MWCNTs solution does not induce damage in mouse brain To understand whether MWCNTs could be really used as nanodevices for brain applications we investigated the ability of our PF127-MWCNTs solution to induce damage in vivo.

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Figure 4. Dispersed MWCNTs avoid cell death induced by PF127. Treatments for 24 hours or 48 hours at 37°C without (white bars) or with 0.01% of PF127 (yellow bars) were performed in the presence of 3.5 μg/mL MWCNTs (dark stripes filled yellow bars). Percentage of (A) neurons and (B) glia under the different treatments. (C) Apoptotic neurons after 24-hour treatments in the presence of 0.01% of PF127 alone (yellow bars) or with 3.5 μg/mL MWCNTs (dark stripes filled yellow bars); 0.05% of PF127 alone (orange bars) or with 17.5 μg/mL MWCNTs (dark stripes filled orange bars); untreated cells (white bars). Percentages of cell numbers are shown compared to control as mean ± SEM (treatments versus control, *P b .05; **P b .01; ***P b .001) (PF127 + CNTs versus PF127 treatment alone, #P b .01). (C) The percentage of apoptotic nuclei as compared to control cells is shown (mean ± SEM). (D), Confocal microscopy images of primary neurons stained with Annexin V–FITC (green) and propidium iodide (red) after treatment with 0.05% PF127 in the presence or absence of 17.5 μg/mL MWCNTs. Yellow arrows indicate visible aggregates of MWCNTs.

We found that injected PF127-MWCNTs (Figure 2) provoked no damage to the overall organization of the mouse brain and that the organization in layers of the tissue surrounding the lesion site was unaffected after 3 days (Figure 2, A, B). Close to the lesion site a area of small injury area was present (Figure 2, C). By computer-assisted morphometry we reconstructed the volume of the injury to quantify the damage induced by the MWCNT solution or its

vehicle as a control. Injected MWCNTs (1 μL of 35 μg/mL in 0.1% PF127-PBS solution) did not increase the volume of the injury created by the injection itself (average lesion volume 0.155 ± 0.03 mm3 in mice treated with CNTs and 0.208 ± 0.144 mm3; no difference was present between the two groups; t-test, P = .88; n = 3 per group). Eighteen days after the injection, control and CNTtreated mice present the formation of a glial scar in the

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PF127 induces apoptosis of neurons

Figure 5. Cell metabolism is decreased in the presence of PF127 but not with PF127-MWCNTs. The bar graph shows MTT results after 24-h treatments without (white bars) or with 0.01% of PF127 (yellow bars); 0.01% of PF127 in the presence of 3.5 μg/mL MWCNTs (dark stripes filled yellow bars); 0.05% of PF127 alone (orange bars) or with 17.5 μg/mL MWCNTs (dark stripes filled orange bars). Pristine MWCNTs added to the cell medium (dark stripes filled white bar). Absorbances of MTT of samples are shown compared to control as mean ± SEM (treatments versus control, ***P b .001) (PF127 + CNTs versus PF127 treatment alone, ^P b.001).

cortex (Figure 2, D-G) localized at the injection site without major alterations of the cortical structure. CNT-treated mice present a local (diameter b200 μm) gliosis engulfing the nanotubes (Figure 2, F, G) without disruption of the surrounding tissue structure. These data demonstrate that injection of PF127-MWCNTs does not increase the damage induced by the mechanical insult of the microsyringe. Moreover, after a long period of time this damage is reduced even in the presence of CNTs. PF127 decreases the number of cells in mouse primary mixed neuronal cultures Among several applications, for example to facilitate the loading of polar molecules across the cell membrane, PF127 has been used as surfactant for dispersion of MWCNTs. In cell lines PF127 is toxic only at very high concentrations ranging from 1% to 5% (ref. 13), but no data are available on its effects on primary neuronal cultures at the lower concentrations effective in dispersing MWCNTs. Thus, we tested PF127 toxicity on mouse primary cortical neurons (Figure 3). When these adherent cells die they usually detach from the plate and cannot be seen and counted. Neurons, whose cell bodies are visible and recognizable on the top of a flat layer of glia cells, were found to be slightly more sensitive to PF127 (Figure 3, A, B). At a concentration of only 0.01%, PF127 was able to reduce significantly the number of neurons (Figure 3, C) and glia (Figure 3, C). The number of cells in culture decreased by more than 50% when PF127 was added at a final concentration of 0.05%.

Figure 3, D and E show that in the presence of PF127 the number of apoptotic cells was increased. Cell death was evaluated 24 hours after treatment. Hoechst 33342 (1 μg/mL) was used to evaluate differences between normal and apoptotic nuclei.18 The difference between Hoechst-stained nuclei of neurons (red arrow), glia (blue arrow), and apoptotic cells (yellow arrow) is emphasized in the lower panel of Figure 2, D). By phase contrast pictures and anti-NeuN staining (Figure 3, D, bottom panel) of the cultures we found that each apoptotic nucleus belongs to neurons. We cannot exclude that some glia cells could be apoptotic, because we cannot exclude an involvement of glia in the induction of apoptosis. However, in every experiment that we have performed an apoptotic nucleus corresponded to a neuronal cell (Figure 3, D, yellow arrow tips). As we can see by Figure 3, E, the trend of apoptotic death in the presence of increasing concentration of PF127 is inversely proportional to the results of Figure 1. Apoptotic cell death is already significant in the presence of 0.01% PF127 and reaches almost 50% at a concentration of 0.05% of PF127. To further establish the apoptotic cell death we decided to detect changes in the position of phospatidylserine in the cell membrane using Annexin V–fluorescein isothiocyanate (FITC). Phospatidylserine molecules are localized in the inner layer of a nonapoptotic cell plasma membrane. Soon after induction of apoptosis phospatidylserine molecules redistribute to the outer layer of the membrane and become exposed to the Annexin V, which shows strong avidity for phospatidylserine. As shown by confocal microscopy images in Figure 4, D, PF127-treated cells present high affinity for FITC (green)–labeled Annexin V. These data indicate that PF127 induces an apoptotic cell death of primary neurons, albeit the molecular mechanisms triggering PF127-induced apoptosis remain unknown. Dispersed MWCNTs prevent PF127-induced apoptotic neuronal cell death Either the reduction of total cell number or the increased apoptotic cell nuclei present in primary cultures treated with PF127 were not present in primary cultures treated with PF127 dispersed MWCNTs at microgramsper-milliliter concentrations. As seen before (Figure 3), 0.01% of PF127 in the medium is able to reduce by almost 25% the number of neurons and 15% of glia. Whereas a concentration of 350 ng/mL of MWCNTs does not avoid PF127-induced cell death after 24 hours (data not shown), ten times more concentrated MWCNTs are able to save cells (Figure 4, A, B). Thus, 3.5 μg/mL of MWCNTs dispersed in 0.01% PF127 in vitro did not affect cell number. After 48 hours of treatment we observed a further reduction of cells in the presence of PF127. Nevertheless, the presence of dispersed MWCNTs in the cell medium was able to significantly reduce cell death (Figure 4, A,

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B). The count of apoptotic neurons following the same treatment with MWCNTs and PF127 was also significantly reduced (Figure 4, C). Moreover, a higher concentration of MWCNTs (17.5 μg/mL) dispersed in 0.05% PF127 was also able to significantly reduce apoptotic cell death (Figure 4, C, D) after 24 hours, albeit not comparable with the untreated control. The dose-response curve of increasing concentration of PF127 indicates that cell metabolism is strongly reduced (Figure 5). However, the presence of MWCNTs in the PF127 solution significantly avoids this reduction, as demonstrated by the dark stripes filled bars in Figure 5. Pristine CNTs do not induce any difference in the basal metabolism of neurons (Figure 5, dark stripes filled white bar). It is worth mentioning that MWCNTs at a concentration of 17.5 μg/mL begin to aggregate in the cell medium (yellow arrows, Figure 4, D), possibly reducing their effectual concentration. These data show that PF127 dispersed MWCNTs are well tolerated by primary neurons.

Discussion In vivo brain injection of MWCNTs has been performed by our group to test a real biocompatibility in prospective of future nanomaterials application to brain diseases. In parallel, a more detailed study related to molecular mechanisms of MWCNT-induced toxicity was performed in vitro. Our results emphasize the compatibility of CNTs with cells or organized cellular environments such as the cerebral cortex. In the attempt to introduce nanotubes in vivo it is necessary to solubilize them. Great efforts have been made, and are today in progress, by many laboratories to mix nanomaterials in solutions able to reach biological targets not easily accessible. The brain is undoubtedly one of the most complex biological structures to deal with, if not the most complex. Other studies19 have already shown in vitro biocompatibility with neurons or neuron-like cells. However, no one had shown before this whether the biocompatibility could be possible in vivo and whether the presence of CNTs released in the brain could damage a biologically highly organized structure. The in vitro observation that PF127-induced apoptosis of primary neurons, at lower concentration than in previous studies on more resistant but less physiological cell lines, could raise some concern about the choice of this surfactant for biomedical use. Probably the negative effects of PF127 on cell survival are due to PF127 action on the cell membrane affecting the regulation of intracellular calcium signaling.20 We must not forget that PF127 has been widely used to aid in cell membrane penetration and solubilization of several substances, for example intracellular Ca 2+ indicators.21 On the contrary, the presence of MWCNTs significantly avoids Pluronic-induced apoptosis of the

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neurons (Figure 4), probably by a simple mechanism of surfactant sequestration from the solution. Many authors propose that CNTs will have many different bioapplications in the future. Therefore, the significance of in vivo administration is crucial to progress in this field of exploration. Our data suggest that Pluroniccoated CNTs are biocompatible devices. However, the formation of a glial scar surrounding the injected nanotubes led us to believe that a more intensive investigation of longterm toxicity, carcinogenicity, and inflammatory properties of CNTs is needed before any application could be transferred to human research. Acknowledgments The authors thank Dr. Lisa Gherardini, Orazio Vittorio, and Riccardo Parra for technical assistance, and Prof. Alfred Cuschieri for his supervision. References 1. Iijima S. Helical microtubules of graphitic carbon. Nature 1991;354: 56-8. 2. Malarkey EB, Parpura V. Applications of carbon nanotubes in neurobiology. Neurodegener Dis 2007;4:292-9. 3. Galvan-Garcia P, Keefer EW, Yang F, Zhang M, Fang S, Zakhidov AA, et al. Robust cell migration and neuronal growth on pristine carbon nanotube sheets and yarns. J Biomater Sci Polym Ed 2007;18:1245-61. 4. Jan E, Kotov NA. Successful differentiation of mouse neural stem cells on layer-by-layer assembled single-walled carbon nanotube composite. Nano Lett 2007;7:1123-8. 5. Mattson MP, Haddon RC, Rao AM. Molecular functionalization of carbon nanotubes and use as substrates for neuronal growth. J Mol Neurosci 2000;14:175-82. 6. Mazzatenta A, Giugliano M, Campidelli S, Gambazzi L, Businaro L, Markram H, et al. Interfacing neurons with carbon nanotubes: electrical signal transfer and synaptic stimulation in cultured brain circuits. J Neurosci 2007;27:6931-6. 7. Lacerda L, Raffa V, Prato M, Bianco A, Kostarelos K. Cell-penetrating carbon nanotubes in the delivery of therapeutics. Nano Today 2007;2: 38-43. 8. Lacerda L, Bianco A, Prato M, Kostarelos K. Carbon nanotubes as nanomedicines: from toxicology to pharmacology. Adv Drug Deliv Rev 2006;58:1460-70. 9. Vaisman L, Wagner H, Marom G. The role of surfactants in dispersion of carbon nanotubes. Adv Colloid Interfac 2006;128-130:37-46. 10. Ajayan PM, Tour JM. Nanotubes composites. Nature 2007;447:1066-8. 11. Dutta D, Sundaram SK, Teeguarden JG, Riley BJ, Fifield LS, Jacobs JM, et al. Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials. Toxicol Sci 2007;100:303-15. 12. Zhang LW, Zeng L, Barron AR, Monteiro-Riviere NA. Biological interactions of functionalized single-wall carbon nanotubes in human epidermal keratinocytes. Int J Toxicol 2007;26:103-13. 13. Monteiro-Riviere NA, Inman AO, Wang YY, Nemanich RJ. Surfactant effects on carbon nanotube interactions with human keratinocytes. Nanomedicine 2005;1:293-9. 14. Kouravelou K, Sotirchos S, Verykios X. Catalytic effects of production of carbon nanotubes in a thermogravimetric CVD reactor. Surf Coat Technol 2007;201:9226-31. 15. Tasis D, Tagmatarchis N, Bianco A, Prato M. Chemistry of carbon nanotubes. Chem Rev 2006;106:1105-36. 16. Li Z, Luo G, Zhou W, Wei F, Xiang R, Liu Y. The quantitative characterization of the concentration and dispersion of multi-walled

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