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April 05, 2013

C 2013 American Chemical Society

Carbon-Based Nanomaterials:Multifunctional Materials forBiomedical EngineeringChaenyung Cha,†,‡ Su Ryon Shin,†,‡,§ Nasim Annabi,†,‡,§ Mehmet R. Dokmeci,†,‡ and

Ali Khademhosseini†,‡,§,*

†Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts 02139,United States, ‡Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States,and §Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States

Graphite is one of the oldest andmostwidely used natural materials. Moretraditionally known as the main in-

gredient of pencil lead, from which thename “graphite” originated, it is now morewidely used in several large-scale industrialapplications, such as carbon raising in steel-making, battery electrodes, and industrial-grade lubricants.1 Due to its high demand,the consumption of synthetic graphitehas significantly increased in recent years.Extensive scientific investigation into gra-phite has revealed that its unique combina-tion of physical properties stems from itsmacromolecular structure, which consists ofstacked layers of hexagonal arrays of sp2

carbon.1

With the deeper appreciation and devel-opment of nanofabrication techniques andnanomaterials that have progressed withinthe last two decades, graphite is now beingactively used as a starting material to en-gineer various types of carbon-based nano-materials (CBNs), including single or multi-walled nanotubes, fullerenes, nanodiamonds,and graphene (Figure 1).2 These CBNs pos-sess excellent mechanical strength, electri-cal and thermal conductivity, and optical

properties; much of the research effortshave been focused on utilizing these ad-vantageous properties for various applica-tions, such as high-strength compositematerials and electronics.1,2

The field of biomedical engineering hasalso embraced the growing popularity andinfluence of CBNs in recent years becausemany of its applications rely heavily on theperformance of biomaterials. Carbon-basednanomaterials have been widely regardedas highly attractive biomaterials due to theirmultifunctional nature. In addition, incor-porating CBNs into existing biomaterialscould further augment their functions.Therefore, CBNs have found their way intomany areas of biomedical research, includ-ing drug delivery systems, tissue scaffoldreinforcements, and cellular sensors.3

Carbon Nanotubes. Since their discovery,carbon nanotubes (CNTs) have becomethemost widely used CBNs.4,5 Carbon nano-tubes are commonly synthesized by arcdischarge or chemical vapor deposition ofgraphite. They have a cylindrical carbonstructure and possess a wide range of elec-trical and optical properties stemming notonly from their extended sp2 carbon but

* Address correspondence toalik@rics.bwh.harvard.edu.

Published online10.1021/nn401196a

ABSTRACT Functional carbon-based nanomaterials (CBNs) have become im-

portant due to their unique combinations of chemical and physical properties (i.e.,

thermal and electrical conductivity, high mechanical strength, and optical

properties), and extensive research efforts are being made to utilize these materials

for various industrial applications, such as high-strength materials and electronics.

These advantageous properties of CBNs are also actively investigated in several

areas of biomedical engineering. This Perspective highlights different types of

carbon-based nanomaterials currently used in biomedical applications.

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also from their tunable physicalproperties (e.g., diameter, length,single-walled vs multiwalled, sur-face functionalization, and chirality).5

Due to their diverse array of use-ful properties, CNTs have been ex-plored for use in many industrialapplications.6 For example, CNTsare well-known for their superb me-chanical strength: their measuredrigidity and flexibility are greaterthan that of some commerciallyavailable high-strength materials(e.g., high tensile steel, carbonfibers, and Kevlar). Thus, theyhave been utilized as reinforc-ing elements for composite mate-rials such as plastics and metalalloys, which have already led toseveral commercialized products.7

However, the possibility of CNT-incorporated composites as superhigh-strength load-bearing mate-rials has not been met with satis-factory results, mostly due to theirpoor interaction with the sur-rounding matrices, which leads toinefficient load transfer from thematrices to the CNTs.7

Many recent research efforts havebeen geared toward incorporatingCNTs into various materials to utilizetheir multifunctional nature (i.e., elec-trical and thermal conductivity andoptical properties) rather than focus-ing purely on composite mechanicalstrength. For example, the excellentelectrical properties of CNTs coupledwith their nanoscale dimensions areof great interest in electronics for theconstruction of nanoscale electroniccircuitry.8,9 In addition, CNTs areknown to have low threshold electricfields for field emission, as com-pared with other common fieldemitters.10,11 Thus, CNTs are activelyexplored in high-efficiency electronemission devices such as electronmicroscopes, flat display panels, andgas-discharge tubes. Carbon nano-tubes also display strong lumines-cence from field emission, whichcould be used in lighting elements.9

Carbon Nanotubes in Biomedical Engi-neering. There is considerable inter-est in using CNTs for various bio-medical applications. The physicalproperties of CNTs, such asmechan-ical strength, electrical conductivity,and optical properties, could be ofgreat value for creating advancedbiomaterials.3 Carbon nanotubescan also be chemically modified topresent specific moieties (e.g., func-tional groups, molecules, and poly-mers) to impart properties suited forbiological applications, such as in-creased solubility and biocompat-ibility, enhanced material compati-bility, and cellular responsiveness.12

Their applications include cell andtissue labeling agents, injectabledrug delivery systems, and bioma-terial reinforcements.

Cell and Tissue Labeling and Imag-

ing. The possibility of using CNTsas labeling and imaging agents hasbeen discussed since their discov-ery due to their unique optical prop-erties. Carbon nanotubes have opti-cal transitions in the near-infrared(NIR) region, which has been shownto be useful in biological tissue be-cause NIR has greater penetrationdepth and lower excitation scatter-ing.3 In addition, fluorescence in theNIR region displays much lowerautofluorescence than do the ultra-violet or visible ranges. These prop-erties make CNTs potent imagingagents with higher resolution andgreater tissue depth for NIR fluores-cence microscopy and optical co-herence tomography. For example,Cherukuri et al. successfully moni-tored CNTs in phagocytic cells andthose intravenously administeredinto mice using NIR fluorescence.13

Raman spectroscopy has beenused extensively to characterizethe structural features of CNTs, asthey are highly sensitive to Ramanscattering because of their exten-sive symmetric carbon bonds.14 Thecharacteristic Raman signatures ofCNTs have also been utilized ascellular probes. For example, Liuet al. detected CNTs in various tis-sues after intravenous delivery intomice using Raman spectroscopy.15

Drug Delivery Systems. Drug de-livery has benefited greatly from theadvances in nanotechnology byusing a variety of nanomaterials(i.e., liposomes, polymersomes,

Figure 1. Various types of carbon-based nanomaterials.

The physical properties

of carbon nanotubes,

such as mechanical

strength, electrical

conductivity, and

optical properties,

could be of great value

for creating advanced

biomaterials.Carbon-based

nanomaterials have

found their way into

many areas of

biomedical research,

including drug delivery

systems, tissue scaffold

reinforcements, and

cellular sensors.

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microspheres, and polymer conju-gates) as vehicles to deliver thera-peutic agents.16 Carbon nanotubeshave also been investigated exten-sively as drug delivery systems sinceCNTs have been shown to interactwith various biomacromolecules(i.e., proteins and DNA) by physicaladsorption.17 In addition, severalchemical modification schemeshave been developed to conjugatetherapeutic molecules or targetingmoieties covalently to CNTs.12

In one interesting study, Zhenget al. provided important insightinto the interactions betweenCNTs and DNA molecules.18 Carbonnanotubes were shown to be effec-tively dispersed in aqueous mediain the presence of single-strandedDNA (ssDNA). Spectroscopic andmicroscopic analyses provided evi-dence of strong interactions be-tween DNA molecules and CNTs,resulting in individual dispersion.Molecular dynamics modelingshowed that the base of ssDNAinteracted with the surface of CNTviaπ�π stacking, resulting in helicalwrapping of ssDNA chains aroundthe CNT (Figure 2). This researchhighlights the potential use of CNTsfor gene delivery, as well as DNA-specific separation techniques formolecular electronics.

Reinforcing Tissue Engineering

Scaffolds. The use of CNTs as com-posite reinforcements for tissue en-gineering scaffolds to date hasprimarily been focused on enhan-cing their mechanical properties.19

Commonly used scaffold materials,such as hydrogels and fibrous scaf-folds, are inherently soft in order tomimic the stiffness of natural tissuesand thus often lack structuralstrength and support. IncorporatingCNTs into these materials has beenshown to enhance their mechanicalproperties. For example, Shin et al.

demonstrated that incorporatingCNTs into photo-cross-linkable ge-latin hydrogels resulted in signifi-cant increases in their tensilestrengths (Figure 3).20 Sen et al. alsodemonstrated improvement in thetensile strengths of CNT-reinforced

polystyrene and polyurethane fi-brous membranes.21

More recently, researchers haveturned their attention to utilizingthe multifunctional nature of CNTs

in engineering tissue scaffolds. Mostnotably, CNTs have been incorpo-rated to fabricate electrically con-ductive scaffolds. Most of the bio-materials used for tissue engineering

Figure 2. Bindingmodel of a carbonnanotube (CNT)with a poly(T) DNA sequence.The DNAwraps around the CNT in a right-handed helical structure. The bases (red)orient to stack with the surface of the nanotube and extend away from thesugar�phosphate backbone (yellow). The DNA wraps to provide a tube withinwhich the CNT can reside, hence converting it into a water-soluble object.Reprinted with permission from ref 18. Copyright 2003 Nature Publishing Group.

Figure 3. (a) Schematic description of gelatinmethacrylate (GelMA) coated onto acarbon nanotube (CNT). (b) High-resolution TEM image of CNT-GelMA. (c) Stress�strain curves of CNT-GelMA hydrogels with various concentrations of CNTs. (d)Tensile moduli of CNT-GelMA hydrogels. (e) 3T3 fibroblasts cultured on GelMAhydrogel (A) and CNT-GelMA hydrogel (B) (scale bar: 100 μm). Reprinted from ref20. Copyright 2011 American Chemical Society.

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applications are electrically insulat-ing, as they are made from noncon-ductive polymers.22�24 However, cer-tain applications, such as neural andcardiac tissues, would benefit greatlyfrom conductive scaffolds that caneffectively propagate electrical sig-nals across the tissue constructs forproper electrophysiological func-tions. For example, Kam et al. ap-plied electrical stimulation to neuralstem cells (NSCs) grown on CNT�la-minin composite films and demon-strated improved action potentialsof NSCs anddifferentiation into func-tional neural networks.23 Shin et al.

cultured cardiomyocytes on CNT-reinforced gelatin hydrogels wherethe cells showed enhanced electro-physiological activities and ulti-mately developed functional cardiactissue.25 These works highlight theability of CNTs to impart electricalconductivity successfully to otherwisenonconductive biomaterials.

Cytotoxicity of CNTs. Despitemany successful applications in bio-medical engineering, there is agrowing concern for safety withCNTs. Some recent in vitro studieshave reported increased cytotoxi-city of CNTs due to their cellularuptake, agglomeration, and in-duced oxidative stress.26�28 Theseconflicting results regarding thebiocompatibility of CNTs largelystem from the variability of CNTs(i.e., size, surface properties, andfunctionalization) and testing pro-tocols (i.e., in vitro vs in vivo, types ofcells, tissues, and animals tested). Inaddition, increased cytotoxicity hasoften been attributed to incompleteremoval of metal catalysts used toprepare CNTs.27 Most in vivo studiesusing CNTs have shown insignifi-cant toxicity and reported renalclearance from the body, althoughsmall portions of CNTs have beenshown to accumulate in certain or-gans, such as lungs, liver, and spleenand may cause inflammation.26�28

However, the cytotoxicity seems tobe more highly variable and morepronounced at the cellular level,based on several in vitro cell culturestudies.29,30

With the continued growth ofCNTs in various biomedical fields,more systematic biological evalua-tions of CNTs having various chemi-cal and physical properties arewarranted in order to determinetheir precise pharmacokinetics, cyto-toxicity, and optimal dosages. Never-theless, many studies have shownthat toxicity can effectively be mini-mized by functionalizing the CNTswith biocompatible polymers or sur-factants to prevent aggregation.31,32

Graphene. Graphene is the latestnanomaterial to burst onto thescene. The ground-breaking workby Geim and Novoselov provided asimple method for extracting gra-phene from graphite via exfoliation

and explored its unique electricalproperties.33,34 Graphene and CNTspossess similar electrical, optical,and thermal properties, but thetwo-dimensional atomic sheet struc-ture of graphene enables more di-verse electronic characteristics; theexistence of quantum Hall effectand massless Dirac fermions helpexplain the low-energy charge ex-citation at room temperature andthe optical transparency in infraredand visible range of the spectrum.34

In addition, graphene is structurallyrobust yet highly flexible, whichmakes it attractive for engineeringthin, flexible materials.35,36

Graphene in Biomedical Engineering.Research into utilizing graphene for

Figure 4. (a) Grapheneoxide (GO)was funtionalizedwith folic acid (FA) as a targetingmolecule (FA-NGO), then loaded with anticancer drug, doxorubicin (DOX) or camp-tothecin (CPT). (b) FA-NGO was localized into MCF-7 (human breast cancer) cells,identified with fluorescent labeling with rhodamine. (c) Greater anticancer activity ofDOX was observed when FA-NGO was used as a drug carrier. Reprinted withpermission from ref 39. Copyright 2010 Wiley-VCH Verlag GmbH & Co.

Figure 5. (a) Class of C60 derivatives that display anti-HIV activity.44 (b) Molecularstructureof a5nmdiameter nanodiamond (ND). TheND ismostlymadeupof sp3 car-bon, but theouter layer is functionalizedwith sp2 carbonandother functional groups.Reprinted with permission from ref 45. Copyright 2012 Nature Publishing Group.

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biomedical applications has beenlimited to date, as graphene re-search itself is still in its infancy.Graphene oxide (GO), produced byoxidation of graphite under acidicconditions, is more commonly used,as it offers several advantages overusing pure graphene.37 First, GO isdispersible in aqueous media, whichis essential for biological applica-tions. Second, GO presents hydro-philic functional groups that enablechemical functionalization. Third,GO has broader ranges of physicalproperties than pure graphene dueto its structural heterogeneity. Sev-eral biomedical applications includ-ing injectable cellular labeling agents,drug delivery systems, and scaffoldreinforcements have been exploredusing GO, as has similarly beendone with CNTs.38 For example,Zhang et al. functionalized GO withfolic acid (FA) as a cancer-targetingmolecule and loaded doxorubicinand camptothecin, known cancerdrugs, onto the large surface areaof GO via π�π stacking (Figure 4).39

The drug-loaded FA-GO showed im-proved cancer-targeting capabilityand anticancer activity as comparedwith drugs delivered alone or drugscarried with unmodified GO. Sunet al. also demonstrated that poly-(ethylene glycol)-conjugated GOwith targeting molecules could beused as a cellular sensor by utilizingthe intrinsic photoluminescenceproperty of GO at the NIR region.40

In another study, Zhang et al. incor-porated GO into poly(vinyl alcohol)hydrogels to improve theirmechan-ical strength.41

Other Carbon-Based Nanomaterials.Buckminsterfullerene (C60), alsocommonly known as the buckyball,is a spherical closed-cage structure(truncated icosahedron) made of 60sp2 carbon. Its discovery in 1985 andsubsequent investigation led to theuncovering of electronic properties,stemming from its highly symme-trical structure, and potential appli-cations, culminating in aNobel Prizein 1996.42 It can be argued that thescientific pursuit of CBNs and theirpotential applications began withthe discovery of C60. The popularityof C60 has somewhat diminished inrecent years with the rise of morescalable and practical CBNs such asCNTs and graphene. However, itsuniform size and shape as well asavailability for chemical modifica-tion led many scientists to developC60 derivatives for therapeutic pur-poses.43 Perhaps the most fascinat-ing and highly promising aspect ofC60 is its anti-human immunodefi-ciency virus (HIV) activity. Schinaziet al. first discovered a group ofwater-soluble C60 derivatives cap-able of inhibiting HIV protease ac-tivity by binding to its active site,due to their uniquemolecular struc-ture andhydrophobicity (Figure 5a).44

Various C60 derivatives have sincebeen developed that display anti-HIV activity by targeting other im-portant HIV enzymes, such as re-verse transcriptase.43 These resultsdemonstrate that C60 derivativesmay become apotent group of AIDStherapeutics in the future.

Nanodiamond (ND) has also gen-erated interest in the field of bio-medical engineering in recent years(Figure 5b).45 Nanodiamonds aresynthesized by high-energy treat-ment of graphite, most commonlyvia detonation, and are smaller than10 nm. They have similar physicalproperties as bulk diamond, such asfluorescence and photolumines-cence, as well as biocompatibility.Unlike other CBNs, NDs aremade upmostly of tetrahedral clusters of sp3

carbon. The surface of nanodia-monds, however, is functionalizedwith various functional groups or

sp2 carbon for colloidal stability,which enables chemical modificationfor targeted drug and gene deliveryand tissue labeling. For example, Lienet al. recently used fluorescent andmagnetic NDs for cell labeling.46

Zhang et al. also demonstrated thatpolyethyleneimine (PEI)-conjugatedNDs were highly effective as genecarriers, without the cytotoxicity as-sociated with PEI alone.47

CONCLUSIONS AND FUTUREOUTLOOK

Extensive research efforts overthe last two decades have elevatedthe status of CBNs as one of themost widely used classes of nano-materials. Owing to their uniquecombinations of mechanical, opti-cal, and electrical properties, CBNshave been explored in various in-dustrial applications. Several areasof biomedical engineering havealso benefited greatly from CBNsin recent years because incorpor-ating CBNs is effective not only asinjectable nanoscale devices butalso as components to enhancethe function of existing biomater-ials significantly. Despite safetyconcerns over CBNs, many studieshave reported the successful useof CBNs in biological applications.In addition, several chemical mod-ification strategies have been de-veloped to circumvent toxicity issuesand to increase the biocompatibilityand functionality of CBNs. Never-theless, it should be noted thatmore systematic toxicology studiesare needed to determine the toxi-city and pharmacokinetics of CBNs.This paper has introduced several

successful applications of CBNs indrug delivery, tissue imaging, andscaffold reinforcement.With the po-pularity of CBNs as highly versatileand useful nanomaterials, we ex-pect to see continued use of CBNsin many facets of biomedical engi-neering. In particular, there is greatpromise in applying the biocompa-tible and multifunctional nature ofCBNs to the areas that interconnectmechatronics and biology, suchas microelectromechanical systems

Several biomedical

applications including

injectable cellular

labeling agents, drug

delivery systems, and

scaffold reinforcements

have been explored

using graphene oxide.

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(“MEMS”) for biological sensors andactuators.48 Furthermore, more re-cent studies suggest that CBNs mayalso be used to regulate cellularbehavior.49,50 Although research ef-forts have largely been focused onutilizing CNTs, other types of CBNs;especiallygraphene,whichhasgainedwide recognition in recent years;areexpected to be investigated exten-sively in the near future.

Conflict of Interest: The authors de-clare no competing financial interest.

Acknowledgment. The authors wouldlike to acknowledge funding from theNational Science Foundation CAREERAward (DMR 0847287), the Office of NavalResearch Young Investigator Award, theNational Institutes of Health (HL092836,DE019024, EB012597, AR057837, DE021468,HL099073, EB008392), and the Presiden-tial Early Career Award for Scientists andEngineers (PECASE). N.A. acknowledgesthe support from the National Health andMedical Research Council of Australia.

Note Added after ASAP Publication.Due to a production error, this paperpublished to the Web on April 5, 2013with several missed corrections. Therevised version was reposted on April 10,2013.

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