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Diameters of single-walled carbon nanotubes (SWCNTs) andrelated nanochemistry and nanobiology

Jie MA1, Jian-Nong WANG2, Chung-Jung TSAI3, Ruth NUSSINOV3,4, Buyong MA (✉)3

1 Shanghai Key Laboratory for Laser Processing and Materials Modification, School of Materials Science and Engineering,Shanghai Jiao Tong University, Shanghai 200240, China

2 Shanghai Key Laboratory for Metallic Functional Materials, Key Laboratory for Advanced Civil Engineering Materials (Ministry of Education),School of Materials Science and Engineering, Tongji University, Shanghai 200092, China

3 Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, NCI-Frederick, NIH, Frederick, MD 21702, USA4 Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine,

Tel Aviv University, Tel Aviv 69978, Israel

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Abstract We reviewed and examined recent progressesrelated to the nanochemistry and nanobiology of signal-walled carbon nanotubes (SWCNTs), focusing on thediameters of SWCNTs and how the diameters affect theinteractions of SWCNT with protein and DNA, whichunderlay more complex biological responses. The dia-meters of SWCNTs are closely related to the electronicstructure and surface chemistry of SWCNTs, and subse-quently affect the interaction of SWCNTs with membrane,protein, and DNA. The surfaces of SWCNT with smallerdiameters are more polar, and these with large diametersare more hydrophobic. The preference of SWCNT tointeract with Trp/Phe/Met residues indicates it is possiblethat SWCNT may interfere with normal protein-proteininteractions. SWCNT-DNA interactions often changeDNA conformation. Besides the promising future ofusing SWCNTs as delivering nanomaterial, thermaltherapy, and other biological applications, we shouldthoroughly examine the possible effects of carbonnanotube on interrupting normal protein-protein interac-tion network and other genetic effects at the cellular level.

Keywords carbon nanotube (CNT), nanobiology, pro-tein, DNA, toxicity, cancer

1 Introduction

Cancer nanotechnology presents both challenge andpromise for controlling cancer cell [1,2]. Nanoparticleswith the size range of 10–100 nm can be used for drug

delivery, imaging, and sensing in cancer research [3]. Forexample, iron oxide nanoparticles can be used todistinguish normal cells from cancer breast cells [4], andmagnetic nanocrystal hybrided with polymer and anti-cancer drug has been used for targeted detection and hassynergistic therapeutic effects on breast cancer cell [5].Functionalized single-walled carbon nanotube (SWCNT)can carry small interfering RNA (siRNA) into cells,illustrating a potential useful therapeutic strategy forchronic myelogenous leukemia cells [6]. On the otherhand, the nanocarrier systems may induce cytotoxicity [7].Clearly, the nanotechnology not only provided aninnovative tool but also calls for deeper understanding ofnanobiology and nanosystem introduced, which willenable us to fight diseases with greater power.Carbon nanotube (CNT) is one group of carbon-based

nanosystems. CNTs comprise a carbon fibrous formconsisting of one (single-walled carbon nanotubes –SWCNTs) to tens of coaxial tubes (multiwalled carbonnanotubes – MWCNTs) of carbon elements with adjacentgraphene sheets separated by 0.334 nm, with diametersranging from 0.4 to 50 nm. Different synthesis methodsoften produce different types of CNT (either multiplewalled or single walled) with different diameters. SWCNTrepresents a unique nanosystem not only with potentialapplications for cancer therapy [8], but also an excellentnanosystem to test biological reaction. Structurally simple,SWCNTs with different diameters provided a deliverysystem with variable surface area and internal volume. Thesurface chemistries (with and without modification) allowvarious interactions with other biomolecules. The thermaleffect with near infrared frequencies can be used to targetspecific cancer cell for photothermal therapy. Electricproperties of SWCNTs can be used as biosensor [9,10].

Received September 9, 2009; accepted September 30, 2009

E-mail: [email protected]

Front. Mater. Sci. China 2010, 4(1): 17–28DOI 10.1007/s11706-010-0001-8

In the last decades, an enormous number of works havebeen published about biological properties of the CNT,and many reviews have been devoted to describing theprogress and potential biological applications [1–3,8,11–13]. It is increasingly clear that dispersed property and thediameters of the CNT are the two important factors relatedto biological responses of CNT. The surface propertiesand electronic structures of the SWCNTs are all related totheir diameters. However, even though there are variousreports linking the biological effects with the diameter/size of CNTs, there is no comprehensive study tosummarize the overall effects. The understanding ofCNT interactions with protein/DNA is fundamental tounderstand complex biological responses to the nanoma-terial. Here we review some recent studies related to thenanochemistry and nanobiology of SWCNTs, focusing onthe controlling of diameters of SWCNTs and how thediameters of SWCNTs change their interactions withprotein and DNA, which underlie more complex biolo-gical responses. In order to allow the readers with differentbackgrounds to appreciate the progresses and challenges inthe area, we put equal weights in nanotube preparationsand the interactions of nanotube with biomolecules. In thelast section, we will discuss the outlook in the researchfield.

2 Synthesis methods that control walls anddiameters of CNT

CNTs are formed either through vaporizations of carbonsource (graphite) at extremely high temperature generatedthrough electric arc or laser, or by pyrolytic reaction (i.e.,decomposition at high temperature) of organic chemicals.Unlike modern chemical synthesis methods to obtain targetmolecules, the formations of carbon nanotubes at theseextreme conditions are hard to control. Up to now, thecontrollable synthesis of this nanostructured carbonmaterial is still a great challenge, and exploration ofsimple synthetic methods suitable for economical andlarge-scale production is still an important task.Three basic chemical events lead to the formation of

CNT: (i) generation of atomic carbon or carbon clusters;(ii) formation of CNT seeds; (iii) growth of CNTs. Usually,the “synthesis” methods are classified by the way togenerate atomic carbon or carbon clusters, namely, (a)electric arc method (arc discharge [14–16]), (b) laservaporization method (laser ablation [17,18]), and (c)chemical vapor deposition (CVD) methods [19–22]. Allthe three technologies need metals such as iron, nickel, andcobalt as the catalysts to catalyze the growth of CNTs, tocontrol the nature of nanotube (MWCNTs or SWCNTs),and to adjust the diameters of the CNT. The diameters ofthe SWCNT are normally within the range of 1–2 nm. Ifthe diameters are more than 3 nm, it can be called as large-diameter SWCNTs (LD-SWCNTs).

2.1 Electric arc and laser vaporization methods

The electric arc method, initially used for producing C60

fullerenes, is the most common and perhaps the easiestway to produce CNTs. MWCNTs were discovered in 1991by Iijima [23] by the arc-discharge evaporation technique.SWCNTs were produced subsequently in 1993 by thesame group [24,25]. In this method, electric arc createdbetween two graphite electrodes leads to extremely hightemperature which is sufficient to sublimate carbon. EitherMWCNTs or SWCNTs can be formed when the carbonvapor cools and condenses. Generally, MWCNT will beformed when there is no catalyst particles between twographite electrodes; and the SWCNT can be generatedwhen adding Fe, Ni, or Co as catalysts. The catalysts canbe introduced by packing metal powder into a hole in theanode. The metal was consumed along with the graphiteand created catalyst particles favoring small-diameterSWCNTs (SD-SWCNTs) [25].In 1995, Smalley’s group reported the synthesis of

carbon nanotubes by laser ablation [26]. The laser ablationmethod uses a pulsed or continuous laser to vaporize agraphite target in an oven, which is filled with helium orargon gas to keep pressure. The laser ablation is similar tothe arc discharge, both taking advantage of the very hightemperature generated, with the similar optimum back-ground gas and catalyst mix observed. The very similarreaction conditions needed indicated that the reactionsprobably occur with the same mechanism for both the laserablation and electric arc methods.Three kinds of catalysts can be used in the laser ablation

and electric arc methods to control the diameters ofSWCNT: (i) primary catalysts (Co, Fe, Ni, or their mixture)mainly contribute to the formation of SWCNTs; (ii)promoters (S, Y, Bi, or Pb) can change the size ofSWCNT; and (iii) gaseous reaction environments may alsohave effects. The mechanism by adding S, Y, Bi, Pb, or FeSto control the growth of nanotubes is not yet clear.However, it is clear that no SWCNTs can be formedwithout primary catalyst, and LD-SWCNTs were producedonly in the presence of a promoter.Among the primary catalyst, it seems that Fe has large

effects on the diameters of SWCNTs. Zhang et al. [27]reported that adding Fe into a conventional NiCo catalystenables the production of LD-SWCNTs by the laserablation. The three promoter elements (Bi, Pb, and S) notonly improve overall catalytic activities, but also cocata-lyze the formation of LD-SWCNTs [28] with the electricarc method. SWCNTs of diameter as large as 7 nm wereobserved, and electron diffractions indicated that thesenanotubes have a variety of helical structures. Lebedkin etal. [29] studied the effect of hydrogen gas (as an H2

admixture to Ar) and sulfur (as a FeS additive to carbontargets) on SWCNTs using the pulsed laser vaporizationmethod with Ni and Co as metal catalysts. Their resultsshow that the addition of hydrogen and sulfur leads to the

18 Front. Mater. Sci. China 2010, 4(1): 17–28

efficient formation of SWCNTs with large diametersapproaching 6 nm. However, Raman spectra showed thatthe diameter of SWCNTs had a wide distribution [29].The promotion of the SWCNT production by sulfur and

hydrogen has been observed by many other researchers.The possible mechanism could be that the hydrogen andsulfur modified the catalytic activity of metal nanoparticlesand suppress the formation of carbides and graphiticoverlayers on catalysts’ surface. At the extremely hightemperature of more than 1200°C generated by either laserablation or electric arc, graphite can be transformed intomolten carbon first. The droplets of the molten Ccontaining metal catalysts (C-metal droplets) cool downand will form metal clusters with diameters of 1–2 nmwhich catalyze the formation of SWCNTs with the samediameters. The introduction of Fe or other promotersprobably increases the size of metal clusters leading to LD-SWCNTs. The metal clusters could also have a large sizewith the presence of sulfur and hydrogen.For the procedures described above, the LD-SWCNT

was only produced occasionally as by-products, and mostCNTs still have diameters around 1–2 nm. Even the yieldsof SWCNTs could be relatively low, and SWCNTs withlarge diameter were even lower. Overall, these techniquesproduce a mixture of components requiring separatingnanotubes from the soot; and the catalytic and promotermetals presented in the crude products remain a potentialproblem in purification for biological application.

2.2 Catalytic CVD methods

Catalytic CVD synthesis is achieved by putting a carbonsource in the gas phase and using plasma or a resistivelyheated coil to heat the gaseous carbon containingmolecules. Commonly used gaseous carbon sourcesinclude methane, carbon monoxide, and acetylene. Theheat is used to “crack” the molecule into reactive atomiccarbon. When the carbon diffuses towards a catalyst(usually a first-row transition metal such as Ni, Fe, or Co),CNTs will be formed if the proper conditions aremaintained. The currently used CVD methods includeplasma-enhanced CVD, thermal chemical CVD, hotfilament CVD, microwave-enhanced CVD, aero gel-supported CVD, and laser-assisted CVD.Similar to the electric arc or laser methods, the diameters

of CNT formed by the CVD are still controlled by the sizeof the catalytic particles. Unlike the electric arc or lasermethods which vaporize solid carbon, the CVD methodsuse gaseous carbon sources. Therefore, the size anduniformity of CNT can be conceivably controlled easier.The nature of completely gaseous reaction environment inCVD also suggests another growth mechanism such as thepolyyne nuclear growth model (SWCNTs grow in the gasphase, where planner carbon rings are the nuclei for tubeformation). Here we only use the catalytic particle size

mechanism to illustrate the principle to control the CNTdiameters.Hydrocarbon molecules and enhanol are often used as

carbon sources, and ferrocene (FeCp2) as catalyst. Forexample, Yang et al. [30] obtained SWCNTs with a meandiameter of 3.23 nm through the catalytic decomposition ofa hydrocarbon with hydrogen/helium as the carrier gases.Lupo et al. [31] prepared SWCNTs with diameters of 2–3.5 nm through the catalytic decomposition of ethanol withargon as carrier gas. The nanotube diameter can only reachup to 3.5 nm with high pyrolytic temperature and highFeCp2 concentration. By the catalytic particle sizemechanism, it is possible that larger Fe catalytic particlesare generated with higher FeCp2 concentration, assuggested by Huang et al. [32] for the fast-heating CVD.Overall, these CVD methods produce film-like CNTsdeposited only on the inner surface of the silica tubelocated outside the reaction zone. It is interesting to notethat both MWCNTs and SWCNTs can be formed throughthe reported processes [31].The strategies to synthesize LD-SWCNTs materials

continuously and with large-scale production ability havebeen developed [33,34]. The method uses thiophene aspromoter to enhance the yield of SWCNTs and control thediameter of SWCNTs. The SWCNTs with averagediameter of 5.8 nm can be continuously and reliablyobtained with a floating catalyst. Generally, a higherconcentration of thiophene leads to a larger tube diameter.The schematic illustration of grown mechanism of LD-SWCNTs with thiophene is shown in Fig. 1. The additionof thiophene could increase the size of catalytic Fe-Sregions on the metal surface, which favors the rupture ofthe bond of carbon source and increases the formation ofcarbon filament. Thus, the more the thiophene added, thelarger the diameter of the SWCNT (Fig. 1). Usingthiophene as a controlling factor, the diameters of theSWCNT can be controlled with the range from SD- to LD-SWCNTs.The SWCNTs generated from the thiophene procedures

can be easily separated. Other methods often have aproblem of producing bundles entangled by SWCNTs. Inthe thiophene procedure, the main product is the separatedLD-SWCNTs without entangled bundles (Fig. 2(a)).Subsequently, the LD-SWCNTs can be easily dispersedin water by ultrasonic vibration (Fig. 2(b)). The diametersof the SWCNTs using the thiophene method mostly lie inthe range of 4–10 nm, with Gaussian mean diameter being5.8 nm. The produced SWCNTs can be brought out of thereaction zone by argon gas continuously, rather thandeposited in the reaction zone as in other methods.

2.3 Growth mechanism of SWCNT

Unlike the progress in the synthesis of CNTs, theknowledge about the details of kinetics and microscopic

Jie MA et al. Diameters of SWCNTs and related nanochemistry and nanobiology 19

growth mechanisms is still seriously missing [35].Computational modelling [36] and experimental in situobservations [35,37] are adding more clues to thenucleation and growth of the formation of CNTs.In a study of the beginning of the process to generate

atomic carbons by exposing Ni tip to acetylene in hightemperature, mass spectrum revealed that the mostabundant species are C1, C2, and C3, with occasionallyC4 as well [35]. The atomic C1 forms first, and thenfollowed by C2 and C3. Combined with Monte Carlosimulation, it was found that adsorption of carbon ispreferentially at the step edge leading to linear andconnected atomic structure, finally to defective graphene-like sheet on the relatively large Ni surface (Fig. 3(a),according to Ref. [35]). When using Ni50 cluster forsimulation, the similar process leads to the formation ofcarbon cap (Fig. 3(b), according to Ref. [38]), which can beviewed as formation of CNT. In these models catalyzed byNi, there is no chemical bond formed between Ni and C.But in a work using density-functional tight-bindingmolecular dynamics (DFTB/MD) approach [36], directFe-C bond can form on a Fe38 cluster. Similar to Niclusters, the formation of carbon sheet also was foundwrapping the iron cluster. Thus, CNT seed can be directlyformed on the iron cluster surface or containing all iron

cluster. Three types of rings (five-ring, six-ring, and seven-ring) can coexist from the DFTB/MD simulation.In the simulations of the Ni and Fe clusters, CNTs grow

through adding carbons on the open edge of the nanotubeseeds [36,38]. The simulation of the iron cluster demon-strated that long polyyne chains with more than 4 carbonsare not necessarily required for nanotube growth. Instead,short C1 and C2 extensions are more frequent. In afascinating in situ monitoring of CNT growth, it wasobserved that closed cap is able to grow or shrink [37].Since the round-shaped cap has no contact with catalyst,one may infer that the catalyst is only needed for the CNTseed formation or for generating atomic carbons. Jin et al.proposed two schemes of the possible pathways toincorporate a C2 into a hexagon and a heptagon. Followingthe Jin-Suenaga-Iijima schemes, we tested to add C2 into aclosed cap to simulate a detailed procedure. As shown inFig. 4, the repeated adding of C2 gradually extends theCNT wall. If adding of C2 is evenly distributed, a roundcap may be preferred. However, if C2 breaks existingsymmetry of the nanotube wall, a nanohorn-like cap maybe formed.SWCNTs can be “cloned” via open-end growth

mechanism [39]. Active open-end SWCNT can beprepared by cutting long SWCNT using electron beam

Fig. 2 (a) HRTEM images of the samples synthesized at 1150°C containing LD-SWCNTs without entanglement; (b) Dispersion of LD-SWCNTs is much easier than that of SD-SWCNTs in water with a concentration of 0.7 mg/mL (Reprinted from Ref. [34])

Fig. 1 The schematic illustration of the mechanism to control diameters of SWCNTs. At low thiophene concentration, the Fe cluster hasonly scattered small Fe-S regions, which leads to SWCNTwith small diameters. With increasing thiophene concentration, the Fe-S regionenlarges to catalyze the formation of LD-SWCNTs.


lithography or other methods, and the duplicate SWCNTcan be made by putting the open-end seeds in CVDfurnace. There are two key characteristics of the “clone”

experiment: (i) there is no other catalyst involved; and (ii)the “cloned” SCWNTs have the same (n, m) chirality as theinitial seeded short SWCNTs. Thus, Yao’s experiments

Fig. 3 Growth mechanism of SWCNT (a) on flat Ni surface and (b) around Ni cluster.

Fig. 4 Growth mechanism of SWCNT through addition of C2 into existing nanotube. The red and blue carbons are walls of SWCNT,and the ray carbons are caps. The newly inserted carbons are shown in green.


[39] again support the idea that the catalyst is onlyimportant for the initial seed formation stage. After seed isformed with the original (n,m) chirality parameter fixed foran SWCNT, the after-growth mostly will follow the patternin the seed.

3 The size of CNT and related biologicalissues

3.1 Diameter-dependent electronic structure and surfaceproperties of CNT

The structure of CNTs can be visualized as the cylindricalroll-up of one or more flat graphene sheets containingcarbon atoms in a honeycomb arrangement (Fig. 5). ForSWCNTs and MWCNTs, the diameter ranges from 0.4 to6 nm and 2 to 50 nm, respectively. The diameter andhelicity are the key parameters that control the unusualphysical, mechanical, and chemical properties of CNTs.The structure of an ideal straight, infinitely long SWCNTscan be uniquely described by two integers (n, m), whichrefer to the number of unit vectors in the carbon walllattice. The tube diameter d is related to the (n, m) by d = a(n2+m2+ nm)1/2/π, where a = 0.249 nm. In Fig. 5, weillustrated four types of SWCNT, with both atomicstructures (left panels) and distributions of electron density(right panels). Figure 5(a) is an SWCNTwith lattice of (6,6) and diameter of 0.8 nm, and Fig. 5(b) with lattice of (8,4) and also diameter of 0.8 nm. Figures 5(c) and (d) are theSWCNTs with diameters of 4 nm, one with lattice of (30,30) (Fig. 5(c)) and the other of (40, 20) (Fig. 5(d)).The electronic structure of SWCNTs is related to the

tube diameter d and depends on the pair (n, m) values.SWCNTs exhibit either zero bandgap (i.e., metallic for n =m, like Fig. 5(a) and (c)), small bandgap (ca. 10 meV, i.e.,semimetallic for n –m = 3k, where k is an integer), or largebandgap (0.6 eV and above, i.e., semiconducting forn –m≠3k, as shown in Fig. 5(b) and (d)) [40]. Typically,semimetallic tubes are treated as metallic owing to theirsmall bandgap at room temperature. The electronic bandstructure also relates to the optical absorptions ofSWCNTs. These electronic transitions produce prominentfeatures in the NIR (near infra-red) spectral range, whichcan be used to analyze the type, purity, and diameterdistribution of SWCNTs. The unique feature of opticalexcitation of SWCNT at NIR range makes it possible to beused as thermal therapy to target cancer cell with NIRirradiation to heat CNT (reviewed in Ref. [8]). Forexample, NIR light can kill targeted cell without damagingreceptor-free cells [41], and the SWCNT functionalizedwith specific monoclonal antibodies can target breastcancer cells and kill the cancer cell upon the near-infraredphototherapy (reviewed in Ref. [8]).The CNT surface contains sp2-hybridized carbon atoms.

For an infinite, flat grapheme sheet, these p-orbital

electrons organize in broad valence (π) and conduction(π*) bands, providing a semimetallic character owing totheir theoretically zero bandgap [42]. On the other hand,when the graphene sheet is rolled to the cylindricalstructure of SWCNT, the π and π* electron cloudsexperience significant curvature, which causes partial σ-πhybridization [43]. As a result, the electron density aroundSWCNTwith smaller diameter is more diffuse, and that forLD-SWCNT is more confined to the atomic core region. Interms of biologically relevant surface properties, we cansee that the CNTs with smaller diameters (Fig. 5(a) and (b))are more polar, and the surfaces of the CNT with largediameters are more hydrophobic (Fig. 5(c) and (d)).SWCNTs are usually synthesized as a mixture of

metallic and semiconducting CNTs. Several methodswere reported to separate metallic and semiconductingCNTs. For example, the semiconducting CNTs can beetched away by a gas reaction, while metallic ones can beretained [44]. On the other hand, the metallic SWCNT canbe suppressed and converted to semiconducting SWCNTsby cycloaddition reactions of fluorinated olefins [45]. Themetallic or semiconducting properties of the SWCNTs canalso be modulated by adsorption of anionic surfactants onnanotube surface in ionic liquids. It was demonstrated thatimidazolium-based ionic liquid can be used to effectivelydisperse of SWCNTs and modify their band structures[46]. The band structure changes depend on SWCNT typeand diameter, and also depend on the anionic surfactantconcentration. Further studies have shown that onlysurfactant with small cations (H+, Li+, and Na+) canselectively modify the band structure, indicating a possiblemechanism of pi-cation interactions. The significance ofthis study also illustrates that upon interacting with proteinor DNA (reviewed in next sections), the electronicstructure of SWCNTs can be changed as well. Thephenomena not only underlie that mechanism to useSWCNTas biosensors, but also imply possible interplay ofelectronic properties of SWCNTs and the biomoleculesadsorbed on SWCNT surface.

3.2 Solubility and biodistributions related to the size ofSWCNTs

The two types of nanotube (MWCNTs and SWCNTs)differed significantly with respect to their size, shape,length, and chemical surface state, and therefore, they havedifferent biocompatibility. Fraczek et al. [47] implantedtwo types of CNTs (SWCNTs and MWCNTs) into theskeletal rat muscle and found that the reactions of tissue-carbon materials are dependent on the tendency of CNTs togroup together. SWCNTs created smaller particles, whichwere easily dispersed in the tissue environment and weresubsequently transported to the local lymph nodes bymacrophages. In contrast, MWCNTs created aggregates ofrelatively large size in the vicinity of the implantation site,and such aggregates increased with time. Undesirable


cytotoxicity may be caused by the presence of abundantmultinucleated cells attached to the MWCNTs agglomera-tions and the transport of SWCNTs from the implant sitesto the lymph nodes [47].The large-diameter nanotubes may form less aggrega-

tion, however, with less mobility. After effectivelydispersed, CNTwith smaller size may have more mobility,especially helped by lipid molecules. In a study to testdiameter-selective solubility of SWCNTs by lipid micelles,

it was found that solubilization occurs through wrapping oflipid molecules around the CNTs, yielding lipid mono-layers on the graphitic sidewalls. Raman spectroscopyshowed that the dispersion and centrifugation process leadsto an effective enrichment of the stable aqueous suspensionin carbon nanostructures with smaller diameters [48].The high solubility and mobility of smaller SWCNTs

may imply higher membrane penetration. It is good fornanodelivering system. Furthermore, the membrane

Fig. 5 CNTs (left panels) and their electron density distribution on surface (right panels): (a) SWCNTwith lattice of (6, 6) and diameterof 0.8 nm; (b) SWCNTwith lattice of (8, 4) and also diameter of 0.8 nm; (c)(d) SWCNTs with diameters of 4 nm, one with lattice of (30,30) (c) and another of (40, 20) (d).


damage ability could be a double-sided knife. It may implyhigher toxicity for SWCNT with smaller diameters;however, it can also be used to kill cells in purpose. Byusing pristine SWCNTs with a narrow diameter distribu-tion, it was demonstrated that cell membrane damageresulting from direct contact with SWCNTs aggregatescould damage bacterial cell membrane and kill thebacterial cell. Direct comparison of the size effectsconfirmed that the diameter of CNTs is a key factorgoverning their antibacterial effects. This finding may beuseful in the application of SWCNTs as building blocks forantimicrobial materials [49]. Experiments with well-characterized SWCNTs and MWCNTs demonstrate thatSWCNTs are much more toxic to bacteria than MWCNTs.Gene expression data show that in the presence of bothMWCNTs and SWCNTs, Escherichia coli expresses highlevels of stress-related gene products, with the quantity andmagnitude of expression being much higher in thepresence of SWCNTs [50].The large attraction area of MWCNTs and resulted

aggregates may be carcinogenic. If the MWCNTs formfibrous or rod-shaped particles of length around 10 to 20 μmwith an aspect ratio of more than 3, it could have similarcarcinogenic effects as asbestos. This has been demon-strated in a study of p53 heterozygous mice which aresensitive to asbestos, indicating the possibility that carbon-made fibrous or rod-shaped micrometer particles mayshare the carcinogenic mechanisms postulated for asbestos[51]. Similar effects were also found for SWCNTs. In a cellbehavior study of SWCNT raw material and purifiedSWCNT (SWCNT bundles), it was found that the cellproliferation, cell activity, cytoskeleton organization, apopto-sis, and cell adhesionwere dependent on cell type, SWCNTquality (purified or not), andSWCNTconcentration [52].

3.3 CNT-protein interactions: aromatic preference orhydrophobic preference?

Both functionalized CNT and nonfunctionalized CNT caninteract with protein, for example, hemoglobin [53]. CNTsare often functionalized to increase either dispersion inwater or biocompatibility. Introduction of polar or chargedfunctional group increases the interaction of CNT withcharged polypeptide like polylysine [54,55]. In the case ofnonfunctionalized CNT, the protein-CNT interactions canbe dominated by hydrophobic and aromatic π-π interac-tions for peptides with aromatic side chains. Such effectcannot be overestimated. It seems that the aromaticinteractions of peptides with CNTsurface are even strongerthan charged interactions of polylysine with functionalizedCNT, as evidenced by stronger interaction of polytrypto-phan with both MWCNT and SWCNT [54].The general preference of tryptophan to interact with the

CNT can be illustrated by selection of SWCNT-bindingpeptides. Phage-displayed libraries have been used toselected peptides with high-affinity binding to CNTs,

including SWCNTs and MWCNTs. One unique feature ofthese peptides is that their amino acid sequences are rich intryptophan and histidine residues. Analysis of experimen-tal and computational data suggests that the peptide-SWCNT interaction can be defined by the electronic natureof tryptophan and the peptide conformation. It was foundthat the highest occupied molecular orbital of thetryptophan residue in the peptide interacts with the lowestunoccupied molecular orbital (LUMO) from the SWCNT[56]. It was also revealed [57] that the high affinity bindingof the motif to SWCNTs required constrained peptideconformations, which can be achieved through either theextension of the amino acid sequence or the addition of aconstrained disulfide bond.The involvements of molecular orbital of aromatic

residues with the LUMO of SWCNT [56] suggest thatSWCNTwith smaller diameters may have stronger LUMOinteractions and thus stronger aromatic preference. By thisargument, MWCNTs may have less orbital interaction dueto overall larger outer wall diameter.The SWCNT with large diameter may also have less

orbital interaction and have more hydrophobic interactionsin nature. If this is the case, the protein-CNT interactionmay not be necessarily dominated by aromatic interaction.Indeed, in a genetic analysis of CNT-binding proteins [58]enrichment by cell-surface display, a different trend wasfound. Trp residue was found not to be absolutely requiredfor a peptide to bind CNT, and no histidine was presentedin the optimized sequence. Meantime, we noticed that inthe surface-selected CNT-binding peptides, Met, Phe, andTrp often exist [58]. As found in our study, Trp, Phe, andMet are hotspot residues enriched in normal protein-protein interactions [59]. If the hotspot residues can beselectively targeted by CNT, CNT might interfere withnormal protein-protein interaction network. For example,the selective interaction of SWCNT with the hotspotresidues may also change protein aggregation pattern toeither promote [60] protein-protein aggregation or theother way around. Generally, nanoparticles have largesurface areas and could enhance the rate of proteinfibrillation by decreasing the lag time for nucleation [60].On the other hand, the CNT hotspot residue interactionmay decrease protein aggregation by shielding thehydrophobic residues, which are often critical to proteinaggregation [61,62].The interactions of CNT and protein have been shown to

be selective to protein types. Nonwoven SWCNTs clearlydisplayed greater adsorption preference for fibrinogen thanfor albumin. As a result, the function of adsorbedfibrinogen to mediate platelet recognition, adhesion,activation, and aggregation was significantly suppressed,which induced extremely low levels of platelet adhesionand activation [63]. Therefore, the binding behavior ofCNT with proteins needs to be carefully evaluated beforethe full potential of CNT in biological studies can berealized.


3.4 Easy disturbance of DNAs by CNT interactions

The selective binding of hydrophobic and aromaticsgroups by CNTmay also interfere with DNA conformationand function. Since DNA molecules are genetic in nature,the effect of the CNT-DNA interaction goes beyond themolecular interaction and could trigger large-scale biolo-gical response related to the gene network.At the molecular level, it was found that SWCNTs bind

to human telomeric i-motif DNA under molecular-crowding conditions. The results indicate that SWCNTsmay have the potential to modulate the structure of humantelomeric DNA in vivo, like DNA B-A transitions and B-Zchanges on SWCNTs in live cells [64]. Indeed, moleculardynamics simulations have shown that the hydrophobicend groups of DNA are attracted to the hydrophobicSWCNT surface, while the hydrophilic backbone of DNAdoes not bind to the uncharged SWCNT. The adsorptionprocess appears to have negligible effect on the internalstacking structure of the DNA molecule but significantlyaffects the A to B form conversion of A-DNA. Theadsorption of A-DNA onto an uncharged SWCNT inhibitsthe complete relaxation of A-DNA to B-DNA within thetime scale of the simulations. In contrast, binding of the A-DNA onto a positively charged SWCNT may promoteslightly the A to B conversion [65]. In another simulation,it was found that SWCNT induces ssDNA (single-strandedDNA) to undergo a spontaneous conformational changethat enables the hybrid to self-assemble via the π-πstacking interaction between ssDNA bases and SWCNTsidewall. ssDNA is observed to spontaneously wrap aboutSWCNT into compact right- or left-handed helices within afew nanoseconds [66]. DNA destabilization and conforma-tional transition induced by SWCNTs are sequence-dependent [54,67]. It was found that for GC homopolymer,DNA melting temperature was decreased by 40°C bySWCNTs; but no change for AT-DNA was observed.Conformational transition caused the melting temperaturechange. SWCNTs can induce B-A transition for GC-DNA,but AT-DNA resisted the transition, possibly becauseSWCNTs bind to the DNA major groove with GCpreference [54,67].Such DNA interaction can change the cell cycle,

indicating that the interaction of the SWCNT with itshost biological system goes beyond DNA molecule level.In the case of the SWCNT binding to human telomeric i-motif DNA, such interaction leads to significant accelera-tion of S1 nuclease cleavage rate [68]. Even though it washoped that SWCNTs might have the intriguing potential tomodulate human telomeric DNA structures in vivo, itsbiological outcome must be carefully balanced. In a studyto evaluate the genotoxic potential of SWCNT, cytotoxi-city tests showed loss of viability in a concentration- andtime-dependent manner after exposure of cells to SWCNT.The induction of DNA damage was found after only 3 h ofincubation with 96 μg/cm2 of SWCNT [69]. Other types of

stresses also have been observed after applying SWCNT ina cell, for example, inducing oxidative stress in rat lungepithelial cells and also activating specific signaling path-way [70]. DNA damage induced by MWCNTs was alsofound in mouse embryonic stem cells. These resultssuggest that careful scrutiny of the genotoxicity ofnanomaterials is needed even for those materials thathave been previously demonstrated to have limited or notoxicity at the cellular level [71].

4 Summary and outlook

In the conventional electric arc and laser vaporizationmethods, the SWCNTwith large diameter can be obtainedby adding Fe as primary catalyst and several elements (S,Y, Bi, or Pb) as promoters. Recent advances in the catalyticCVD methods make it possible to systematically controlthe diameter and the size of CNT, mainly by controlling thethiophene added. These progresses promote us to examinethe relationship of the diameter of CNT, the electronicstructure and surface chemistry of SWCNT, and theinteractions of SWCNT with biomolecules.SWCNTs have been observed to interact directly with

membrane, protein, and DNA, the three important classesof biomolecules in cell. Such interactions depend on thesize and diameter of SWCNT. The SWCNT with smallersize and diameter is easier to interact with lipids and haslarger mobility in cell. The interactions of SWCNT withprotein are mostly hydrophobic, with Trp, Phe, and Metbeing preferred to bind on the SWCNT surface. Since theTrp/Phe/Met are hotspot residues controlling protein-protein interaction interface, we suspect that SWCNTmay disrupt cell protein-protein interaction network.Another significant consequence of SWCNT interactionis selectively interfering GC-rich DNA conformation,resulting in cell responses similar to DNA damage (forexample, by irradiations). Such interactions may underliethe toxic mechanism of SWCNT, but also provide themethods to kill cancer cell when SWCNT can be deliveredto cancer cell. The possible ways include NIR thermaltherapy or taking advantage of DNA damage effect ofSWCNT.Clearly, more systematic studies are needed to evaluate

the CNT at both molecular and cellular levels. Theapplication of CNT in biological system must be carefullyplanned to minimize the possible long-term and geneticdamages. The selection of CNT in biological applicationshould consider purity, size distribution, and surfaceproperties. This may need close collaborations amongmaterial scientists, biochemists, and biologists.

Acknowledgements This project has been funded in whole or in part withFederal Funds from the NCI, National Institutes of Health, under contractnumber HHSN261200800001E. The content of this publication does notnecessarily reflect the views or policies of the Department of Health and


Human Services, nor does the mention of trade names, commercial products,or organizations implies endorsement by the U.S. Government. This researchwas supported (in part) by the Intramural Research Program of the NIH, NCI,Center for Cancer Research. Prof. Jian-Nong Wang is thankful to the supportfrom the National Natural Science Foundation of China (Grant No.50871067) and the fund for the National 863 Project of 2007AA05Z128from the Ministry of Science and Technology of China.

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