mattson paper

IntroductionCarbon nanotubes are strong, flexible, conduct

electrical current (Rao et al., 1997; Wong et al., 1997;Tans et al., 1998), and can be functionalized withdifferent molecules (Chen et al., 1998; Wong et al.,1998), properties that may be useful in basic andapplied neuroscience research. The mammalian

nervous system is the most complex cellular com-munication network known, containing over 1011nerve cells (neurons), each of which has an elabo-rate morphology extending neurites (axons anddendrites) over long distances. The growth of neu-rites and the formation of synapses during devel-opment and regeneration is controlled by a highlymotile structural specialization at the tip of the neu-

Journal of Molecular Neuroscience 175 Volume 14, 2000

Molecular Functionalization of Carbon Nanotubes and Use as Substrates for Neuronal Growth

Mark P. Mattson,*,1,2 Robert C. Haddon,3 and Apparao M. Rao4

1Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY 40536; *,2Laboratory of Neurosciences, National Institute

on Aging, 5600 Nathan Shock Boulevard, Baltimore, MD 21224; 3Department of Chemistry and Physics, University of Kentucky, Lexington, KY 40506; 4Center for Applied Energy Research

and Department of Physics and Astronomy, University of Kentucky, Lexington, KY 40511

Received December 17, 1999; Accepted March 8, 2000

Abstract

Carbon nanotubes are strong, flexible, conduct electrical current, and can be functionalizedwith different molecules, properties that may be useful in basic and applied neuroscienceresearch. We report the first application of carbon nanotube technology to neuroscience research.Methods were developed for growing embryonic rat-brain neurons on multiwalled carbon nan-otubes. On unmodified nanotubes, neurons extend only one or two neurites, which exhibit veryfew branches. In contrast, neurons grown on nanotubes coated with the bioactive molecule 4-hydroxynonenal elaborate multiple neurites, which exhibit extensive branching. These find-ings establish the feasability of using nanotubes as substrates for nerve cell growth and as probesof neuronal function at the nanometer scale.

Index Entries: Brain; growth cones, hippocampus; hydroxynonenal; nanotechnology.

Journal of Molecular NeuroscienceCopyright 2000 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0895-8696/00/14:175182/$12.00

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

176 Mattson, Haddon, and Rao

Journal of Molecular Neuroscience Volume 14, 2000

rite called the growth cone (Goodman, 1996; Kateret al., 1998). One commonly used approach to study-ing mechanisms that regulate neurite outgrowthemploys cultures of dissociated neurons frombrains or spinal cords of embryonic rodents. In suchcultures the neurons are seeded into dishes in whichthe culture surface has been coated with uniformlayer of adhesive molecules that promotes neuriteoutgrowth. Although studies of such cultures haveled to the identification of a number of moleculesthat promote or inhibit neurite outgrowth (e.g.,neurotrophic factors and neurotransmitters) (Matt-son, 1988; Song and Poo, 1999), a large gap in ourunderstanding of the regulation of neurite out-growth exists because of the lack of methods formanipulating the growth environment at thenanometer scale.

Methods

Production, Dispersion, and Modificationof Multi-Walled Carbon NanotubesMultiwalled nanotubes were synthesized via the

catalytic decomposition of a ferrocene-xylene mix-ture as described previously (Andrews et al., 1999).Nanotubes were dispersed by bath sonication for5 min in 100% ethanol. The dispersed nanotubeswere applied to 22 mm2 glass coverslips that hadbeen coated with a thin layer of polyethyleneimine.The ethanol was allowed to evaporate under ambi-ent conditions, resulting in firm adherance of thenanotubes to the coverslips. The coverslips werethen placed in 35-mm diameter plastic culturedishes, sterilized by a 5-min exposure to UV light,and culture medium was added to the dishes. Thenext day dissociated embryonic rat hippocampalneurons were seeded into the cultures. Adhesionof 4-hydroxynonenal (4-HNE) to nanotubes wasaccomplished by first dispersing the nanotubes in100% ethanol, and then incubating the dispersednanotubes in an acidic solution, pH 5.0, of 50%ethanol (4 mg nano-tubes/mL) containing 200 M4-HNE (Cayman Chemical Co.). The mixture wasthen sonicated for 20 min in a bath sonicator, andnanotubes were applied to polyethyleneimine-coated glass coverslips. The coverslips were thenwashed extensively with phosphate-buffered saline(PBS), sterilized by exposure to UV light for 5 min,and placed in culture dishes containing medium.

Methods for Primary Neuronal Culture and MicroscopyPrimary hippocampal cell cultures were estab-

lished from E18 rat embryos using methodsdescribed previously (Mattson et al., 1988). Disso-ciated cells were seeded onto polyethyleneimine-coated 22 mm2 glass coverslips, and incubated inNeurobasal medium containing B-27 supplements(Gibco-BRL) plus 2 mM L-glutamine, 1 mM HEPES,and 0.001% gentamicin sulfate (Sigma). Methodsfor confocal laser-scanning microscope analysis ofimmunostained nanotubes were similar to thosedescribed previously (Mattson and Partin, 1999).Briefly, cells were visualized under visible light andthen scanned with the laser (488 nm excitation and510 nm emission). Transmitted light images wereacquired by scanning the sample with the laserturned off. For SEM analysis cells were fixed in asolution of 2% glutaraldehyde in PBS, dehydratedin solutions of increasing ethanol concentration,and critical-point dried. Samples were then coatedwith a thin layer of gold particles and examinedand photographed using a S900FE Hitachi SEM.

ImmunochemistryNanotubes on coverslips were incubated for 10

min in PBS containing 4% normal horse serum, fol-lowed by a 3-h incubation in PBS containing 4%horse serum plus a 1!100 dilution of mouse mon-oclonal antibody (MAb) against 4-HNE (Mattsonand Kater, 1988; Mattson et al., 1997). For confocalmicroscopy, samples were then incubated for 1 hin PBS containing a 1!200 dilution of biotinylatedhorse antimouse IgG, followed by a 30-min incu-bation in PBS containing 4 g/mL fluorescein-avidin conjugate (Vector Laboratories). For SEManalysis samples were incubated for 2 h in a sec-ondary (sheepantimouse) antibody conjugated to5 nm gold particles.

Results and DiscussionTechnologies have recently been developed that

allow large-scale production of highly purifiedcarbon nanotubes under conditions in which tubediameters and lengths, and tube composition (e.g.,single-walled and multiwalled) can be reliably con-trolled (Thess et al., 1996; Journet et al., 1997; Renet al., 1998; Fan et al., 1999). Our studies employed

Nanotubes and Neurite Growth 177


multi-walled nanotubes with diameters of approx20 nm and lengths of 20100 m, which are pro-duced as sheets in which the nanotubes arearranged in parallel arrays using a method basedon a controlled decomposition of a ferrocenexylene mixture using a two-stage quartz reactor(Andrews et al., 1999). We found that nanotubes insuch sheets can be dispersed to varying extents bysuspension in ethanol followed by sonication. Weapplied the dispersed nanotubes to glass coverslipsthat had been coated with polyethyleneimine, asubstrate for neuronal growth, and then seeded dis-sociated embryonic rat hippocampal neurons ontothe coverslips in culture dishes. Cultures wereexamined and photographed using a light micro-

scope with phase-contrast optics, and some of thecultures were prepared for examination with a scan-ning electron microscope (SEM). Examples of lightand SEM micrographs of neurons growth for 3 don nanotubes are shown in Fig. 1.

Neurons attached to the nanotubes and extendedone or two neurites, which grew to distances ofapproximately 7090 m within 3 d (Fig. 1). Neu-rons on nanotubes survived and continued to growthrough at least 8 d in culture indicating that thenanotubes support long-term cell survival. SEMexamination of neurons growing on nanotubesrevealed that, although the nanotubes permittedneurite outgrowth, they did not appear to influ-ence the direction of growth (Fig. 1). Thus, neurites

Fig. 1. Multiwalled carbon nanotubes support neuronal survival and provide a permissive substrate for neuriteoutgrowth. Phase-contrast (A) and (B) and scanning electron (C) and (D) micrographs showing embryonic hip-pocampal neurons growing on dispersed nanotubes and adjacent culture substrate. Axons extend from neuronalcell bodies across nanotubes and onto the polyethyleneimine-coated glass surface. Arrows point to a growth cone(panel A) and a branchpoint of an axon (panel B). Asterisks mark neuronal cell bodies (panels C and D). Scalebars: (A) and (B), 10 m; (C) and (D), 5 m.



grew straight across the surfaces of nanotubesarranged in various orientations with respect to thedirection of neurite outgrowth. In cases where neu-rites grew across nanotubes and then onto the

polyethyleneimine-coated glass coverslips, theneurites typically did not form branches on the nan-otubes, but did form branches on the coverslips(Fig. 1B,D), suggesting that unmodified nanotubes

Fig. 2. Multiwalled nanotubes can be coated with 4-hydroxynonenal (4-HNE). (A) and (B) Confocal laser scan-ning microscope images of nanotubes that had been reacted with 4-HNE and then processed for immunofluores-cence-based detection of 4-HNE. Panel (A) is a transmitted light micrograph showing the distribution of thenanotubes on the glass culture substrate. Panel (B) is a fluorescence image of the same microscope field showinglocalization of 4-HNE (green). (C) SEM micrograph showing nanotubes reacted with 4-HNE and then processedfor immuno-gold based detection of 4-HNE. The arrows point to gold particles that decorate sites of binding ofthe 4-HNE antibody. (D) Nanotubes reacted with a solution containing 4-HNE that had been preadsorbed with a 100-fold molar excess of the amino acid histidine and then processed for immuno-gold based detection of 4-HNE.Note that essentially no gold particles are associated with the nanotubes. Scale bars: (A) and (B), 10 m; (C) and(D) 20 nm.



do not provide a substrate that is conducive forbranch formation.

Recent studies have demonstrated that nan-otubes can be modified with a variety of moleculesincluding carboxyl and amine groups (Chen et al.,1998; Wong et al., 1998; Hamon et al., 1999). In orderto determine whether molecules that effect neuritegrowth can be coupled to nanotubes, and to studythe influence of such modified nanotubes on neu-

rite outgrowth, we established methods for cou-pling the aldehyde 4-hydroxynonenal (4-HNE) tonanotubes. 4-HNE is a lipid peroxidation productthat covalently modifies proteins on cystine, histi-dine, and lysine residues (Esterbauer et al., 1991;Uchida and Stadtman, 1992). Previous studies hadshown that 4-HNE can induce increases of intra-cellular Ca2+ levels (Mark et al., 1997) and modifycytoskeletal proteins (Mattson et al., 1997), signal-

Fig. 3A. Neurite outgrowth and branching is enhanced in neurons growing on nanotubes coated with 4-HNE.(A) SEM micrographs showing neurons that had grown for 3 d on unmodified control nanotubes (upper) and nan-otubes coated with 4-HNE (lower). The right panel is a high magnification view of the region of the neurite des-ignated by the arrow in the left panel. Scale bars: left panels, 5 m; right panels, 100 nm.



ing mechanisms that regulate neurite outgrowth inmany types of neurons including cultured embry-onic hippocampal neurons (Mattson and Kater,1987; Waeg et al., 1996). Coupling of 4-HNE to nan-otubes was accomplished by sonicating the nan-otubes in an acidic solution containing 200 M4-HNE. The nanotubes were then applied to glasscoverslips and were either prepared for immuno-chemical analyses or cell culture. In order to deter-mine whether 4-HNE was bound to the nanotubes,an antibody against 4-HNE (Waeg et al., 1996; Mattson et al., 1997) was employed. Examinationof the nanotubes using confocal laser-scanningmicroscopy and SEM showed that 4-HNE antibodybound strongly to the nanotubes that had beenreacted with 4-HNE, but not to untreated nanotubesor to nanotubes reacted with 4-HNE that had beenpreincubated with a 100-fold molar excess of his-tidine (Fig. 2). 4-HNE was present along the lengthof the nanotubes. These findings demonstrate a

physical association of 4-HNE with the nanotubesunder the conditions employed.

We next cultured embryonic hippocampal neu-rons on nanotubes coated with 4-HNE, and on con-trol nanotubes that were either untreated or reactedwith 4-HNE that had been preadsorbed with excesshistidine. SEM examination suggested that neuronsgrown on nanotubes modified with 4-HNE hadmore elaborate neuritic arbors than did neuronsgrown on unmodified nanotubes (Fig. 3A). We there-fore quantified numbers of neurites/neuron, totalneurite length/neuron, and numbers of branches/neurite in neurons grown on nanotubes coated with4-HNE, and neurons grown on control nanotubes(Fig. 3B). Whereas neurons grown on control nan-otubes possessed only one or two neurites, thosegrown on 4-HNE-modified nanotubes elaborated46 neurites. Total neurite length was increasedmore than twofold, and number of branches/neu-ritewere increased approximately threefold, in neu-rons grown on 4-HNE-modified nanotubes. Thesefindings demonstrate a striking effect of nanotube-bound 4-HNE on neurite outgrowth, and establishthe feasibility of using chemically modified carbonnanotubes as a tool for studying and manipulatingneurite outgrowth.

Our findings show, for the first time, that neu-rons can attach to and grow across the surfaces ofcarbon nanotubes. Previous studies have demon-strated that substrate adhesiveness plays animportant role in the process of growth cone motil-ity and neurite branching (Lustgarten et al., 1991).We found that surfaces of unmodified nanotubeswere permissive for neurite outgrowth, but did notpromote neurite branching, suggesting that adhe-sion of growth cones to the carbon surface was rel-atively weak. However, when nanotubes werecoated with 4-HNE neurite branching and totalneurite outgrowth were greatly enhanced. Thesefindings suggest the possibility that 4-HNE enhancesadhesion of growth cones to the nanotubes. Previ-ous studies have shown that Ca2+ influx can regu-late growth cone motility and neurite elongation(Kater et al., 1988), and that 4-HNE can modulateintracellular Ca2+ levels in cultured hippocampalneurons (Mark et al., 1997), as well as in nonneu-ronal cells (Carini et al., 1996). It is therefore pos-sible that that the neurite outgrowth-enhancingeffect of 4-HNE involves changes in intracellularCa2+ levels, although this remains to be established.

Fig. 3B. Neurons were grown for 3 d on unmodifiednanotubes, nanotubes reacted with 4-HNE, or nanotubesthat had been preadsorbed with a 100-fold molar excessof the amino acid histidine. The indicated parameters ofneurite outgrowth were then quantified. Values are themean and SD of measurements made in eight separatecultures (1015 neurons assessed/culture). For each out-growth parameter the value for neurons grown on nan-otubes coated with 4-HNE was significantly greater thanthe value for each of the other two conditions (p < 0.001in each case; ANOVA with Scheffes post-hoc tests).



The convergence of two previously distinctfields, nanotube technology and neurobiology,paves the way for future studies in which nanotubesare used to study the formation and function ofnerve cell networks, and possibly to restore func-tion of damaged neuronal circuits. Nanotubes ofdefined diameters ranging from 1 nm (single-walled nanotubes) to 10100 nm (multiwalled nan-otubes) can now be routinely produced. Thesediameters are similar to those of small nerve fibers,growth cone filopodia, and synaptic contacts. Func-tionalization of such nanotubes with one or morespecific bioactive molecules will thus allow a dis-section of the molecular control of neuronal archi-tecture at focal microdomains similar to those thatoccur in vivo. This contrasts with current culturemethods in which neurons are grown on a uniformflat substrate that bears little resemblance to thecell surfaces and extracellular matrix structures thatneurons encounter in vivo. Because nanotubes areexcellent conductors, they will also prove valuablefor electrophysiological analyses of neuronal micro-circuitry. Moreover, the ability to functionalizenanotubes with molecules that promote neurite out-growth suggests clinical applications of this tech-nology, in light of the limited success of attempts topromote the regeneration of nerves in the spinalcord and brain by the use of crude syntheticbridges (Lustgarten et al., 1991). In contrast to theartificial growth substrates employed thus far, nan-otubes have diameters similar to those of neuritesand may therefore allow for the kinds of localizedmolecular interactions that may be required for for-mation of functional neuronal circuits.

AcknowledgmentsWe thank D. Jacques and R. Gonzalez for pro-

viding the multiwalled nanotube samples and forhelp with electron microscopy. Supported by theNational Institute on Aging and the National Sci-ence Foundation.

ReferencesAndrews R., Jacques D., Rao A. M., Derbyshire F.,

Qian D., Fan X., et al. (1999) Continuous produc-tion of aligned carbon nanotubes: a step closer tocommercial realization. Chem. Phys. Lett. 303, 467.

Carini R., Bellomo G., Paradisi L., Dianzani M. U.,and Albano E. (1996) 4-Hydroxynonenal triggers

Ca2+ influx in isolated rat hepatocytes. Biochem. Bio-phys. Res. Commun. 18, 772776.

Chen J., Hamon M. A., Hu H., Chen Y., Rao A. M.,Eklund P. C., and Haddon R. C. (1998) Solutionproperties of single-walled carbon nanotubes. Science 282, 9598.

Esterbauer H., Schaur R. J., and Zollner H. (1991)Chemistry and biochemistry of 4-hydroxynonenal,malonaldehyde and related aldehydes. Free Rad.Biol. Med. 11, 81128.

Fan S., Chapline M. G., Franklin N. R., Tombler T. W.,Cassell A. M., and Dai H. (1999) Self-oriented reg-ular arrays of carbon nanotubes and their fieldemission properties. Science 283, 512514.

Goodman C. S. (1996) Mechanisms and molecules thatcontrol growth cone guidance. Annu. Rev. Neurosci.19, 341377.

Hamon M. A., Chen J., Hu H., Chen Y., Rao A. M.,Eklund P. C., and Haddon R. C. (1999) Dissolutionof single-walled carbon nanotubes. Adv. Mater. 11,834840.

Journet C., Maser W. K., Bernier P., Loiseau A., Lamyde la Chapelle M., Lefrant S., et al. (1997) Largescale production of single wall carbon nanotubesby the electric arc technique. Nature 388, 756758.

Kater S. B., Mattson M. P., Cohan C., and Connor J.(1988) Calcium regulation of the neuronal growthcone. Trends Neurosci. 11, 315321.

Lustgarten J. H., Proctor M., Haroun R. I., Avellino A. M., Pindzola A. A., and Kliot M. (1991) Semi-permeable polymer tubes provide a microenvi-ronment for in vivo analysis of dorsal rootregeneration. J. Biomech. Eng. 113, 184188.

Mark R. J., Lovell M. A., Markesbery W. R., UchidaK., and Mattson M. P. (1997) A role for 4-hydroxy-nonenal in disruption of ion homeostasis and neu-ronal death induced by amyloid `-peptide.J. Neurochem. 68, 255264.

Mattson M. P. (1988) Neurotransmitters in the regu-lation of neuronal cytoarchitecture. Brain Res. Rev.13, 179212.

Mattson M. P. and Kater S. B. (1987) Calcium regula-tion of neurite elongation and growth cone motil-ity. J. Neurosci. 7, 40344043.

Mattson M. P. and Kater S. B. (1988) Intracellular mes-sengers in the generation and degeneration of hippo-campal neuroarchitecture. J. Neurosci. Res. 21,447464.

Mattson M. P., Dou P., and Kater S. B. (1988) Out-growth-regulating actions of glutamate in isolatedhippo-campal pyramidal neurons. J. Neurosci. 8,20872100.



Mattson M. P., Fu W., Waeg G., and Uchida K. (1997)4-hydroxynonenal, a product of lipid peroxidation,inhibits dephosphorylation of the microtubule-associated protein tau. NeuroReport 8, 22752281.

Mattson M. P. and Partin J. (1999) Evidence for mito-chondrial control of neuronal polarity. J. Neurosci.Res. 56, 820.

Rao A. M., Richter E., Bandow S., Chase B., Eklund P. C., Williams K. A., et al. (1997) Diameter-selec-tive Raman scattering from vibrational modes incarbon nanotubes. Science 275, 187191.

Ren Z. F., Huang Z. P., Xu J. W., Wang J. H., Bush P.,Siegal M. P., and Provencio P. N. (1998) Synthesisof large arrays of well-aligned carbon nanotubeson glass. Science 282, 11051107.

Song H. J. and Poo M. M. (1999) Signal transductionunderlying growth cone guidance by diffusible fac-tors. Curr. Opin. Neurobiol. 9, 355363.

Suter D. M. and Forscher P. (1998) An emerging linkbetween cytoskeletal dynamics and cell adhesionmolecules in growth cone guidance. Curr. Opin.Neurobiol. 8, 106116.

Tans S. J., Vershueren A. R. M., and Dekker C. (1998)Room temperature transistor based on a singlecarbon nanotube. Nature 393, 4952.

Thess A., Lee R., Nikolaev P., Dai H., Petit P., RobertJ., et al. (1996) Crystalline ropes of metallic carbonnanotubes. Science 273, 483487.

Uchida K. and Stadtman E. R. (1992) Modification of histidine residues in proteins by reaction with4-hydroxynonenal. Proc. Natl. Acad. Sci. USA 89,45444548.

Waeg G., Dimsity G., and Esterbauer H. (1996) Mon-oclonal antibodies for detection of 4-hydroxy-nonenal modified proteins. Free Rad. Res. 25,149159.

Wong E. W., Sheehan P. E., and Lieber C. M. (1997)Nanobeam mechanics: elasticity, strength, andtoughness of nanorods and nanotubes. Science 277,19711975.

Wong S. S., Joselevich E., Woolley A. T., Caung C. L.,and Lieber C. M. (1998) Covalently functionalizednanotubes as nanometre-sized probes in chemistryand biology. Nature 394, 5255.

mattson paper

Documents

abstractcarbon nanotubes

introductioncarbon nanotubes

unmodified nanotubes

university of kentucky

growth of neu

nerve cells neurons

embryonic ratbrain neurons

department of physics