Chemically Functionalized Single-Walled Carbon Nanotube Films

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Chemically Functionalized Single-Walled Carbon Nanotube Films Modulate the Morpho-Functional and Proliferative Characteristics of Astrocytes Manoj K. Gottipati,† Josheua J. Samuelson,† Irina Kalinina,‡ Elena Bekyarova,‡,§ Robert C. Haddon,*,‡,∥ and Vladimir Parpura*,†,⊥ †

Departments of Neurobiology and Biomedical Engineering, University of Alabama, Birmingham, Alabama 35294, United States Departments of Chemistry and Chemical Engineering and Center for Nanoscale Science and Engineering, University of California, Riverside, California 92521, United States § Carbon Solutions, Inc., Riverside, California 92507, United States ∥ Department of Physics, King Abdulaziz University, Jeddah 21589, Saudi Arabia ⊥ Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia ‡

S Supporting Information *

ABSTRACT: We used single-walled carbon nanotube (CNT) films to modulate the morpho-functional and proliferative characteristics of astrocytes. When plated on the CNT films of various thicknesses, astrocytes grow bigger and rounder in shape with a decrease in the immunoreactivity of glial fibrillary acidic protein along with an increase in their proliferation, changes associated with the dedifferentiation of astrocytes in culture. Thus, CNT films, as a coating material for electrodes used in brain machine interface, could reduce astrogliosis around the site of implantation. KEYWORDS: Carbon nanotube films, astrocytes, glial fibrillary acidic protein, morphology, proliferation

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outgrowth of neurites, number of growth cones, and neuronal body area.8 However, the effect that the CNT films of varying thicknesses have on astrocytes is not understood. Such knowledge will aid in a recommendation on the amount of coating well-suited for a brain implant. In this study, we show the extent to which the various thicknesses of CNT strata, in the range previously used with neurons, affect astrocyte area and cell form/roundness, along with changes in the immunoreactivity of glial fibrillary acidic protein (GFAP), and the adhesion and proliferation of astrocytes in culture. The SWCNT-PEG graft copolymers were prepared using a procedure described elsewhere.3,12 These graft copolymers were dispersed in double-deionized water by ultrasonication using a bath sonicator to create a homogeneous dispersion. This dispersion was then sprayed onto precleaned13 round glass coverslips (12 mm in diameter), placed on a hot plate (T ∼ 100 °C), to create uniform 10, 30, and 60 nm thick CNT films7,8 (for details on the synthesis and characterization of the CNT films see Supporting Information, Scheme S1 and Figures S1− S5). In all the experiments, as a reference, we coated the glass coverslips with polyethyleneimine (PEI), a standard stratum, commonly used to promote cell adhesion and growth. We

arbon nanotubes (CNTs) have shown much promise in neural applications.1,2 Single-walled carbon nanotubes covalently linked to polyethylene glycol (SWCNT-PEG), applied as colloidal solutes, are biocompatible and can modulate the morpho-functional properties of neurons and astrocytes in vitro,3,4 and neurons in vivo;5 inhibition of stimulated endocytosis was implicated as a mechanism underlying neuronal morphological changes.6 SWCNT-PEGs were also sprayed onto hot glass coverslips to make retainable films/planar strata of various thicknesses with well understood electronic properties.7 CNT planar strata were shown to modulate neuronal growth and neurite outgrowth in culture8 and can affect the electrical properties of neurons,9 effects that appear to sprout from electrical shortcuts, that is, direct physical interactions between the CNTs embedded within the film and the neurons grown on it.10 CNTs found use in brain machine interface (BMI) applications as a coating layer for tungsten and stainless steel wire electrodes; such surface coating has shown to improve electrical stimulation of neurons and recordings from these cells.11 While one can simply use electrical performance of the electrodes to guide optimal amount/ thickness of the CNT material applied, it is obvious that additional guidelines should come from the effect that the amount of a CNT coating may exert on the two major neural cells, neurons, and astrocytes. Indeed, in a narrow range of thicknesses (10, 30, and 60 nm) the CNT layers can affect the © 2013 American Chemical Society

Received: June 18, 2013 Revised: August 6, 2013 Published: August 12, 2013 4387

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Figure 1. Single-walled carbon nanotube films induce morphological changes in mouse cortical astrocytes. (a) Images of astrocytes in culture plated onto PEI- and CNT- (10, 30, and 60 nm thick) coated coverslips and loaded with the vital fluorescent dye calcein. Scale bar, 20 μm. (b) Summary graphs showing the effects of CNT films on the morphology of astrocytes. Number of astrocytes studied in each condition is given in parentheses. The dashes represent medians with interquartile range and the open bars represent mean ± standard error of mean. Asterisks indicate a statistical difference when compared to PEI. The other difference is marked by the bracket. KWA followed by Dunn’s test except for occupancy tested by oneway ANOVA followed by Fisher’s LSD test; *p < 0.05, **p < 0.01.

the CNT films of different thicknesses have differential effects on the astrocytes (Figure 1b; KWA followed by Dunn’s test or one-way ANOVA followed by Fisher’s least significant difference (LSD)). Changes in astrocytic morphology have been linked to changes in the level of GFAP with functional consequences on astrocytic physiology,18,19 which further affects neuronal excitability.18−20 To assess whether CNT films affect this functionality of astrocytes, we labeled the astrocytes plated onto PEI- and CNT-coated coverslips for GFAP using indirect immunocytochemistry4,14 (Figure 2a). The cells were also preloaded with a dipeptide β-Ala-Lys conjugated to 7-amino-4methylcoumarin-3-acetic acid (AMCA) (20 μM),21 which is specifically taken up by the astrocytes into their cytoplasm, to get the total area of the cells (Supporting Information and Figure S7 and S8). Astrocytic GFAP immunoreactivity (GFAPir) and β-Ala-Lys-Nε-AMCA were visualized using a fluorescence microscope and tetramethylrhodamine isothiocyanate (TRITC) and 4′,6-diamidino-2-phenylindole (DAPI) filter sets, respectively. We quantitatively assessed the GFAP-ir parameters, that is, density (average fluorescence intensity per pixel of the total cell area), content (density × total cell area) and occupancy (positive/total cell area). Though there was a decreasing trend in the density of GFAP-ir with an increase in the thickness of the films, a significant decrease was only seen in the astrocytes grown on the 60 nm films, in comparison to the cells grown on PEI (Figure 2b, left). The content of GFAP-ir was not significantly different across the astrocytes grown on the different CNT substrates when compared to the cells grown on PEI (Figure 2b, middle). The occupancy of GFAP-ir was also not significantly different in the astrocytes plated on the CNT films in comparison to that on PEI. However, the astrocytes plated on the 10 nm CNT films showed an increased GFAP-ir occupancy in comparison to that on the 60 nm CNT films (Figure 2b, right; KWA followed by Dunn’s test). This result further emphasizes that the CNT strata of different thicknesses can have differential effects on the astrocytes. It

initially assessed the effect that these CNT films have on the morphology of astrocytes. After 3 days in culture, we loaded the astrocytes with the acetoxymethyl ester of calcein (calcein AM, 1 μg/mL),4,14,15 a nonfluorescent compound, hydrolysis of which, caused by the intracellular esterases, results in the intracellular accumulation of calcein anion, a vital fluorescent dye.16 We imaged calcein-loaded solitary astrocytes, devoid of contact with other cells (to avoid cell−cell interactions) on the coverslip, using a fluorescence microscope and a standard fluorescein isothiocyanate (FITC) filter set. We found that all the cells plated onto PEI- (n = 34) and CNT-coated coverslips (n = 109) accumulated calcein, indicating the viability of the cells and the biocompatibility of the CNT films (Figure 1a). Upon the analysis of calcein fluorescence densities, we found no statistical difference in the signal obtained from the astrocytes grown on PEI- and CNT-coated coverslips (Kruskal−Wallis one-way ANOVA (KWA), p = 0.73; Supporting Information, Figure S6b), indicating that the enzymatic activity of esterases in astrocytes is unaffected by the growth of these glial cells on the CNT films. We analyzed the calcein images using a self-designed algorithm4 and quantitatively assessed the morphological parameters of the astrocytes by measuring the area and perimeter values of the cells, which in turn were used to calculate the form factor17 (FF) defined by the equation FF = 4π [area(μm2)]/ [perimeter(μm)]2. Form factor is a measure of the circularity or the roundness of a cell/object; FF = 1 describes a perfectly round/circular object while a FF ≈ 0 describes a line. We found that the 10 nm films did not cause any significant change in the area and perimeter values of the astrocytes compared to PEI, but caused a significant increase in the FF. However, the thicker films (30 and 60 nm CNT films) caused a significant increase in the area of the astrocytes with no apparent change in the perimeter, and an increase in the FF (Figure 1b). Also, the FF of the astrocytes plated onto the 10 nm films was significantly higher compared to the FF of the astrocytes plated onto the 60 nm films (Figure 1b). Taken together, these results show that 4388

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Figure 2. Single-walled carbon nanotube films induce functional/biochemical alterations in astrocytes, as seen by the changes in cellular GFAP immunoreactivity parameters. (a) Images of astrocytes plated onto PEI- and CNT- (10, 30, and 60 nm thick) coated coverslips, and labeled for GFAP using indirect immunocytochemistry. Scale bar, 20 μm. Gray scale is a linear representation of the fluorescence intensities, expressed in fluorescence intensity units (iu), of the pixels in the images. (b) Summary graphs showing the median effect of CNT films on the quantitative GFAP immunoreactivity parameters. Density is shown in fluorescence intensity units (iu) per area (pixel). Asterisks indicate a statistical difference when compared to PEI. The other difference is marked with the bracket. KWA followed by Dunn’s test; *p < 0.05, **p < 0.01.

on the CNT surfaces (10, 30, and 60 nm thick films) show a statistically significant increase in their relative density of live cells, 4 h postplating, implying that there is a preferential adhesion of the astrocytes onto the CNT films in comparison to PEI. We also found that there is a significant increase in the relative density of live cells on the 60 nm film in comparison to that on the 10 nm film implying that the adhesion of the astrocytes increases with increasing thickness of the strata. The relative density of live cells at the 4 day time point was significantly higher compared to the relative density of live cells at the 4 h time point across all the groups implying that the cells do undergo proliferation on the CNT films as well. The effects that CNT films exert on cellular proliferation were disproportionately higher than expected from the enhanced adhesion of astrocytes onto the CNT films, when compared to PEI. Though the cells grown on the 10 nm films did not show a significant increase in their relative density of live cells (when compared to the cells grown on PEI), 4 days postplating, the cells grown on the thicker films (30 and 60 nm films) showed a statistically significant increase, implying that there is an increased proliferation of the astrocytes on the CNT films in comparison to PEI. The astrocytes grown on the thicker CNT films also showed a statistically significant increase in their relative density of live cells in comparison to that on the 10 nm films, 4 days postplating (Figure 3c; KWA followed by Newman-Keuls’ test). The percentage of dead cells, however, remained insignificant across the groups, both at the 4 h and 4 day time points (Figure 3d; KWA, p = 0.75 and 0.24, respectively). Taken together, the results of the proliferation study indicate that the CNT films show an enhanced adhesion and proliferation of astrocytes in culture compared to PEI. The effect of the CNT films on the adhesion and proliferation of astrocytes appears to be graded with respect to the thickness of the strata. In this study, we investigated the role of CNT films of varying thicknesses, on which the cells are grown in culture, in affecting the morpho-functional/biochemical and proliferative

should be noted that these measurements were not substantially affected by the optical properties, that is, absorption of light, of the CNT films themselves (Supporting Information and Figures S6, S9). Taken together, our results implicate that the CNT films at a high thickness cause a decrease in the density of GFAP-ir in astrocytes while maintaining a similar content and occupancy values of this astrocyte-specific intermediate filament in comparison to the values obtained from the astrocytes grown on the standard substrate, PEI. It should, however, be noted that any change in GFAP-ir may be due to a change in the synthesis of GFAP and/ or post-translational modifications of GFAP by phosphorylation or proteolysis.22,23 The changes in GFAP expression are often associated with changes in the proliferation of astroglia.24−27 To assess this possibility, astrocytes were plated onto PEI- and CNT-coated coverslips (n = 6 coverslips in each group), loaded with calcein (Figure 3a, middle panel), labeled with Hoechst 33342 (5 μg/ mL) (Figure 3a, left panel), and imaged using a fluorescence microscope, 4 h and 4 days postplating. The 4 h time point gives an estimate about the initial plating density of the astrocytes as well as the adhesion of astrocytes onto the CNT films in comparison to PEI, while the 4 day time point provides an estimate about the proliferation of astrocytes. Since Hoechst 33342 is a cell permeant nuclear stain, calcein was used to differentiate the live cells (calcein positive) from the dead (Hoechst stain not associated with calcein) (Figure 3a, merge). We observed that the median initial plating of the astrocytes onto PEI, 4 h postplating, was about 22 800 cells per cm2, which increased to about 28 000 cells per cm2, 4 days postplating, confirming that the cells do undergo some proliferation (∼23% increase in cell number) in culture (Figure 3b; Wilcoxon Signed-Rank test). We then calculated the relative density of live cells across the different CNT strata by normalizing the number of live cells in each group and time point to the median number of live cells on the PEI-coated coverslips, 4 h postplating. We found that the astrocytes grown 4389

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Figure 3. Single-walled carbon nanotube films enhance the proliferative characteristics of astrocytes in culture. (a) Images of astrocytic nuclei labeled with the cell permeable nuclear dye Hoechst 33342 (left column) and astrocytes loaded with the vital fluorescent dye calcein (middle column). Right column shows the merge of the images. First and third rows show images of astrocytes plated onto PEI-coated coverslips, 4 h and 4 days postplating. Second and fourth rows show images of astrocytes plated onto 60 nm thick CNT-coated coverslips, 4 h and 4 days postplating. Scale bar, 50 μm. (b) Summary graph showing the median number of live cells per cm2 on the PEI-coated coverslips, 4 h and 4 days (4d) postplating. (c) Summary graph showing the relative density of live cells plated onto PEI- and CNT- (10, 30, and 60 nm thick) coated coverslips normalized against the median number of live cells, 4 h postplating, on the PEI-coated coverslips. (d) Summary graph showing the percentage of dead cells plated onto PEI- and CNT-coated coverslips. We used six coverslips per time point per group for the proliferation assay. Asterisks indicate a statistical difference when compared to PEI. Other differences are marked with brackets. Wilcoxon Signed-Rank test in b and c, between 4 h and 4 day time points KWA followed by Newman-Keuls’ test in c; *p < 0.05, **p < 0.01.

characteristics of astrocytes. Our results suggest that CNT films within a range of thicknesses modulate the various characteristics of astrocytes by making the cells larger and rounder while decreasing the immunoreactivity of GFAP and increasing the adhesion and proliferation in culture. These results, together with our previous study on neurons,8 suggest that the thickness of the CNT strata with associated properties, such as surface roughness and/or stratum conductivity, may play a critical role in influencing the properties of both electrically excitable cells, that is, neurons, and electrically nonexcitable cells, that is, astrocytes, that are present in the brain. Namely, the CNT strata used here showed an increase in the surface roughness (Ra) and conductivity (σRT) with their thickness (Supporting Information, Figures S4 and S5, respectively). Indeed, it has been shown previously that the nanoscale roughness of the surface can affect the cellular growth, adhesion, and proliferation characteristics of various cell types.28−31 Similarly, the conductivity of the CNT films can affect neuronal growth and neurite outgrowth.8 Hence, it is plausible that the differential effects we observed could be a result of the roughness and/or passive conductivity of the CNT films.

The decrease in GFAP-ir, rounder morphology, and increased proliferation of astrocytes caused by the CNT films could point to a dedifferentiation of these glial cells. Indeed, the astrocytes can dedifferentiate into neural stem cells like type B cells that are GFAP positive proliferating cells and precursors giving rise to neuroblasts,32 which is the phenotype seen when astrocytes were grown on the CNT films with increased thickness. This is a desirable effect that could be extended to the astrocytes surrounding the CNT-coated metal electrodes implanted in the brain. However, there is a potential danger that the dedifferentiation of astrocytes into neural stem cells could lead to malignant transformation resulting in the formation of gliomas and perhaps even teratomas.33 Malignant astrocytes do express GFAP, which is perhaps the most clinically useful and specific marker for the subclassification of astrocytomas within the gliomas.34 In such astrocytic neoplasms, the extent of anaplasia is inversely proportional to the number of cells expressing GFAP.35 We have not observed GFAP negative astrocytes in this study, however. It is possible that a further increase in the thickness of the CNT films could lead to further suppression of GFAP expression and eventually 4390

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Information); area and perimeter of the cells were obtained using a self-designed algorithm and were further used to calculate FF as we described elsewhere.4 This morphometric analysis included the image stitching/autoleveling step when the cell size exceeded an individual image frame. For the measurement of calcein density (Supporting Information and Figure S6b), however, only astrocytes that could be imaged within a single frame were analyzed (compare sample size in Figure 1b and Supporting Information Figure S6b) in order to avoid errors in the quantification of fluorescence intensity that would otherwise occur during the stitching/autoleveling step. Indirect immunocytochemistry was performed to assess the immunoreactivity of GFAP4 (also see Supporting Information). Astrocytes were loaded with β-Ala-Lys-Nε-AMCA21 (Supporting Information), labeled for GFAP, and analyzed for the cell area by manual tracing (based on the β-Ala-Lys-Nε-AMCA images acquired using a standard DAPI filter set), and for the density, content and occupancy of GFAP-ir (based on the images acquired using a standard TRITC filter set). Here, as in the analysis of calcein fluorescence intensity, only the astrocytes that could be imaged within a single frame were analyzed. To assess the proliferative characteristics, astrocytic nuclei were labeled with Hoechst 33342, imaged using a DAPI filter set 4 h and 4 days postplating, and the number of nuclei per view field (0.15 mm2) were counted (Supporting Information). All the data are reported as medians with the interquartile range or means ± standard errors of means. Statistical analysis was done using the GB-Stat v6.5 software (Dynamic Microsystems Inc., Silver Spring, MD) and SAS Software, version 9.2 of the SAS software for Windows (SAS Institute Inc., Cary, NC). The number of subjects required for individual set of experiments was estimated using power analysis (set at 80% and α = 0.05). For the subgroups that deviated from normality based on Shapiro-Wilk test for normality, nonparametric statistics were used. To test the difference between the 4 h and 4 day time points in the proliferation study, the two correlated groups were compared using Wilcoxon Signed-Rank test (Figure 3). For all the other experiments with multiple groups, the multiple independent groups were analyzed using KWA followed by Dunn’s or Newman-Keuls’ test, or one-way ANOVA followed by Fisher’s LSD (significance established at p < 0.05) to find a statistical significance of the effect induced by the CNT films when compared to PEI.

its disappearance in astrocytes. It should be noted, however, that the loss of GFAP seen in astrocytomas does not represent a step in cancer development but rather a manifestation of the undifferentiated state of astrocytes.35 Nonetheless, a great care should be taken when using CNT films to enhance the BMI electrode performance. At present, we recommend that only here established thicknesses of the CNT films should be utilized to avoid potential tumorigenesis. Decrease in the density of GFAP-ir could also mean that the CNT films can cause a decrease in reactive astrogliosis, the response of astrocytes to injuries like the ones induced by a “stab” wound due to electrode implantation, leading to a scar formation that prevents the regrowth of damaged neurons.36 A coating of platinum implants with polypyrrole/SWCNT composite films has been shown to decrease the intensity of GFAP-ir within a distance of 100 μm from the implant interface.37 Studies with multiwalled carbon nanotube-coated electrodes have also shown a decrease in the intensity of GFAP immunostaining around the implant site when compared to the noncoated implants.38 Previous work on cultured neurons has shown that the CNT strata in a narrow range of thicknesses from 10 to 30 nm, caused an increase in neurite outgrowth and cell area, respectively.8 These observations, together with our findings, could implicate the potential role of CNT strata in neuronal regeneration by reducing reactive astrogliosis and hence the scar formation along with enhancing the neurite outgrowth. CNT-coated electrodes have been shown to outperform the traditional brain implants by enhancing the neuronal recordings and stimulation, both in vitro and in vivo.11,39 Interaction of the CNT strata with neurons has been shown to increase neuronal excitability by rearranging the charge along the surface of the membrane.40 This is particularly advantageous for neural implants as the charge required for the stimulation of neurons would be greatly reduced. The increased cell adhesive property of the CNT strata, as we observe in this study and also the study conducted on neurons,41 is important as it increases the tissue-electrode interaction, reducing the electrical charge transfer, and removing the need for coating the implants with adhesion promoting layers like laminin or polylysine. The low semiconductive range of CNT strata, as used in our study, would be amenable with electrochemical detection of various neurotransmitters and therapeutic neuromodulation approaches.42 Therefore, retainable and biocompatible CNT films could serve as a promising stratum for applications in neural prostheses. Methods Summary. Here, we summarize the methods used in this study, while additional details of our procedures are available in the Supporting Information. Astrocytes isolated from the visual cortices of 0−2 day old C57BL/6 mice were purified and maintained in cell culture as we previously described in detail elsewhere.4 In the present work, the sole modification presents itself in growing the astrocytes on CNTcoated glass coverslips in addition to the standard plating on PEI-coated coverslips. CNT films were synthesized and characterized as we previously described7,8 (also see Supporting Information). All imaging experiments were done at room temperature (22−25 °C) using a light microscope equipped with differential interference contrast (DIC) and epifluorescence illumination. To quantify the morphological changes, astrocytes were loaded with calcein and imaged using a standard FITC filter set, as we described in detail elsewhere4,14,15 (also see Supporting

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ASSOCIATED CONTENT

S Supporting Information *

Additional information and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (V.P.) [email protected]; (R.C.H.) [email protected]. Author Contributions

V.P. and R.C.H. were responsible for the overall project conception, design and supervision. M.K.G., J.J.S., and V.P. designed the experiments. I.K. and E.B. synthesized and characterized the carbon nanotube films. M.K.G. performed all the experiments on astrocytes and analyzed the data; J.J.S. performed a pilot study. M.K.G. and V.P. wrote the paper. All authors discussed the results and commented on the manuscript. 4391

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Notes

(28) Zamani, F.; Amani-Tehran, M.; Latifi, M.; Shokrgozar, M. A. J. Mater. Sci. Mater. Med. 2013, 24, 1551−60. (29) Chung, T. W.; Liu, D. Z.; Wang, S. Y.; Wang, S. S. Biomaterials 2003, 24, 4655−61. (30) Fan, Y. W.; Cui, F. Z.; Hou, S. P.; Xu, Q. Y.; Chen, L. N.; Lee, I. S. J. Neurosci. Methods 2002, 120, 17−23. (31) Sorkin, R.; Greenbaum, A.; David-Pur, M.; Anava, S.; Ayali, A.; Ben-Jacob, E.; Hanein, Y. Nanotechnology 2009, 20, 015101. (32) Doetsch, F.; Caille, I.; Lim, D. A.; Garcia-Verdugo, J. M.; Alvarez-Buylla, A. Cell 1999, 97, 703−16. (33) Amariglio, N.; Hirshberg, A.; Scheithauer, B. W.; Cohen, Y.; Loewenthal, R.; Trakhtenbrot, L.; Paz, N.; Koren-Michowitz, M.; Waldman, D.; Leider-Trejo, L.; Toren, A.; Constantini, S.; Rechavi, G. PLoS Med. 2009, 6, e1000029. (34) Furnari, F. B.; Fenton, T.; Bachoo, R. M.; Mukasa, A.; Stommel, J. M.; Stegh, A.; Hahn, W. C.; Ligon, K. L.; Louis, D. N.; Brennan, C.; Chin, L.; DePinho, R. A.; Cavenee, W. K. Genes Dev. 2007, 21, 2683− 710. (35) Wilhelmsson, U.; Eliasson, C.; Bjerkvig, R.; Pekny, M. Oncogene 2003, 22, 3407−11. (36) Ridet, J. L.; Malhotra, S. K.; Privat, A.; Gage, F. H. Trends Neurosci. 1997, 20, 570−7. (37) Lu, Y.; Li, T.; Zhao, X.; Li, M.; Cao, Y.; Yang, H.; Duan, Y. Y. Biomaterials 2010, 31, 5169−81. (38) Zhou, H.; Cheng, X.; Rao, L.; Li, T.; Duan, Y. Y. Acta Biomater. 2013, 9, 6439−49. (39) Ansaldo, A.; Castagnola, E.; Maggiolini, E.; Fadiga, L.; Ricci, D. ACS Nano 2011, 5, 2206−14. (40) Lovat, V.; Pantarotto, D.; Lagostena, L.; Cacciari, B.; Grandolfo, M.; Righi, M.; Spalluto, G.; Prato, M.; Ballerini, L. Nano Lett. 2005, 5, 1107−10. (41) Galvan-Garcia, P.; Keefer, E. W.; Yang, F.; Zhang, M.; Fang, S.; Zakhidov, A. A.; Baughman, R. H.; Romero, M. I. J. Biomater. Sci., Polym. Ed. 2007, 18, 1245−61. (42) Parpura, V.; Silva, G. A.; Tass, P. A.; Bennet, K. E.; Meyyappan, M.; Koehne, J.; Lee, K. H.; Andrews, R. J. J. Neurochem. 2013, 124, 436−53.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V.P. acknowledges the support of this work by a National Science Foundation Award CBET 0943343. We thank William Lee for help with a pilot study.

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