Carbon Nanomaterials as Antibacterial Colloids

19 Pages·2016·2.84 MB·English

by  Michael Maas

materials
Review
Carbon Nanomaterials as Antibacterial Colloids
MichaelMaas
FacultyofProductionEngineering,AdvancedCeramics,MAPEX—CentreforMaterialsandProcesses,
UniversityofBremen,Bremen28359,Germany;[email protected];Tel.:[phone]
AcademicEditor:ShahinHomaeigohar
Received:16June2016;Accepted:15July2016;Published:25July2016
Abstract: Carbon nanomaterials like graphene, carbon nanotubes, fullerenes and the various
forms of diamond have attracted great attention for their vast potential regarding applications
in electrical engineering and as biomaterials. The study of the antibacterial properties of carbon
nanomaterialsprovidesfundamentalinformationonthepossibletoxicityandenvironmentalimpact
of these materials. Furthermore, as a result of the increasing prevalence of resistant bacteria
strains, the development of novel antibacterial materials is of great importance. This article
reviewscurrentresearcheffortsoncharacterizingtheantibacterialactivityofcarbonnanomaterials
from the perspective of colloid and interface science. Building on these fundamental findings,
recentfunctionalizationstrategiesforenhancingtheantibacterialeffectofcarbonnanomaterialsare
described. Thereviewconcludeswithacomprehensiveoutlookthatsummarizesthemostimportant
discoveriesandtrendsregardingantibacterialcarbonnanomaterials.
Keywords: carbon;nanotubes;graphene;fullerene;diamond;nanomaterial;environment;bacteria;
toxicity;antibacterial
1. Introduction
Carbon is able to form different allotropes based on the formation of either sp2 or sp3 bonds
betweentheindividualcarbonatoms. Theseconfigurationsareespeciallysignificantatthenanoscale
whereeachallotropeexhibitsuniqueproperties. Applicationsofcarbonnanomaterialsincludetheir
utilizationincompositematerials,asbiomaterials,asdrugcarriersorasmaterialsforenergystorage.
Mostnotably,carbonnanotubes(CNT)andgraphenearestudiedfortheirvastpotentialinelectrical
engineering.Concurrently,albeittoalesserextent,investigationsontheotherallotropesofnanocarbon
likefullerenesandnanodiamondaresteadilygainingmomentum. Withtheincreasingprevalence
ofcarbonnanomaterials(CNMs)innanotechnology,theinteractionwithlivingtissueandtheirfate
in organisms as well as consequences for the environment have come into focus. Most allotropic
formsofcarbon,grapheneoxides,carbonnanotubesandfullerenesweresoonidentifiedasmoderately
toxictomostlivingcells,bothforeukaryoticandespeciallyforprokaryoticcells[1,2]. Onereasonfor
theirtoxicitymightbetheirhydrophobiccharacter,whichenablespenetrationofcellmembranes[3].
Severalstudiesreportedthegenerationofreactiveoxygenspecies(ROS)causingcellulartoxicity[4–6].
Becausecarbonnanomaterialsareoftensynthesizedinthepresenceofmetalliccatalysts,heavymetal
residuescanadditionallyaffectcellularprocessesashasbeenshownforcarbonnanotubes[7,8].
Studiesregardingthetoxicologyandsafetyofnanomaterialsareoftencomplicatedbyalackof
comparabilitypartlyduetoanunnecessaryneglectofstandardizationaswellasshortcomingsinthe
physico-chemical characterization of the investigated materials [9]. Additionally, well-established
biologicaltestingmethodsaresometimescompromisedbyinteractionswithnanomaterials[10].Thisis
especiallytrueformoreinvolvedtestsetupsbasedoneukaryoticcells,tissuesandanimalmodels
wheregreatcontroversyexistsregardingtheinherenttoxicityofnanomaterials. Hence,investigating
toxicityforbacteriaoffersawelcomesimplificationofexistingmodelsystems,whichallowsabetter
Materials2016,9,617;doi:10.3390/ma9080617 www.mdpi.com/journal/materials
Materials2016,9,617 2of19
Materials 2016, 9, 617 2 of 18
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Figure 1. The different allotropes of nanocarbon and their antibacterial action (adapted from [13], with
Figure1.Thedifferentallotropesofnanocarbonandtheirantibacterialaction(adaptedfrom[13],with
permission from the American Chemical Society 2008).
permissionfromtheAmericanChemicalSociety2008).
For the described studies, bacterial viability was assessed via the expression of stress-related
For the described studies, bacterial viability was assessed via the expression of stress-related
genes [20] or via the minimum inhibitory concentration based on colony forming units [21–24]. A
genes[20]orviatheminimuminhibitoryconcentrationbasedoncolonyformingunits[21–24].Adirect
direct inter-study comparison of the effect of the nanomaterial on bacteria viability is usually not
inter-studycomparisonoftheeffectofthenanomaterialonbacteriaviabilityisusuallynotpossible,
possible, since biological test systems differed significantly, including nanomaterial dosages and
since biological test systems differed significantly, including nanomaterial dosages and exposure
exposure times [10].
times[10].
2.1. Graphene
2.1. Graphene
Graphene is a monomolecular layer of carbon that is linked via sp2 bonds. Despite its many
Graphene is a monomolecular layer of carbon that is linked via sp2 bonds. Despite its many
interesting properties, individual graphene sheets were first prepared as late as 2003 [25], triggering
interestingproperties,individualgraphenesheetswerefirstpreparedaslateas2003[25],triggering
a vibrant and rapidly developing research field. However, dispersing the extremely hydrophobic
a vibrant and rapidly developing research field. However, dispersing the extremely hydrophobic
material proved to be a challenge. In the last years, several dispersing methods for graphene were
materialprovedtobeachallenge. Inthelastyears,severaldispersingmethodsforgraphenewere
developed, e.g., the preparation of aqueous dispersions of graphene oxide sheets from graphite using
oxidizing acids [26,27]. The resulting graphene oxide nanosheets exhibit lateral dimensions of up to
Materials2016,9,617 3of19
developed, e.g., the preparation of aqueous dispersions of graphene oxide sheets from graphite
usingoxidizingacids[26,27]. Theresultinggrapheneoxidenanosheetsexhibitlateraldimensions
ofuptoseveralmicrometerswhilejustbeingoneatomiclayerthick. Thesheetscarryamixtureof
oxygencontainingsurfacegroups,mainlycarboxylicacid. Dependingonpretreatmentandprocessing
route,includingthechoiceofacidsandotheroxidizingagents,grapheneoxidecanshowdiverging
toxicological behavior as was shown by Chng and Pumera for adherent lung epithelial cells [28].
Grapheneoxidecanbeconvertedbacktographenebyreducingagents,forexampleusinghydrazine,
orevenmetal-reducingbacteriafromthegenusShewanella[29]. However,unlessdispersionconditions
arenotcarefullycontrolled,thechemicallyconvertedgraphene(alsocalledreducedgrapheneoxide)
is usually no longer stable in aqueous dispersion leading to aggregation and sedimentation [26].
Properdispersionisofcourseacriticalrequirementforsystematicbacteriastudies.
Theantibacterialactivityofgrapheneandgrapheneoxideiswidelydocumentedandhasalready
been utilized to create antibacterial materials for specific applications [15,16,19,30–33]. Liu et al.
comparedtheantibacterialactivityofgraphite, graphiteoxide, grapheneoxide(GO)andreduced
grapheneoxide(rGO),showingthatGOandrGOarebothstronglyantibacterial(Figure2)[34].Inthese
experiments,GOshowedstrongerantibacterialactivitythanrGO.Conversely,rGOshowedthehighest
oxidationcapacitytowardsglutathione,whichisanindicatorforoxidativestress. Thisdiscrepancyhas
beenexplainedbythemainantibacterialmechanismofdispersedgrapheneorgrapheneoxidewhich
isgovernedbymembranedamagecausedbystrongdispersioninteractionsbetweenphospholipids
andgraphenesheets,whichdirectlyrelatestothesuperiordispersionstabilityofthemorehydrophilic
GO.Intheirdetailedstudy,Tuetal.,combiningbothbacteriaexperimentsandsimulations,identified
twodifferentmechanismsformembranedamagecausedbygrapheneoxide[35]: Thefirstmechanism
is based on severe insertion and cutting away of large areas of the cell membrane. The second
mechanism is described as a destructive extraction of lipid molecules. Here, phospholipids from
the cell membrane spread on partially inserted graphene sheets. An additional mechanical effect
wasreportedbyLiuetal.,whosuggestedasize-dependentwrappingofbacteriabygrapheneoxide
sheetsthatresultedinhigherantibacterialactivityforlargersheets[36,37]. Usingdissipativeparticle
dynamicssimulations,Dallavalleandco-authorsstudiedthevariouspotentialinteractionsofgraphene
withlipidbilayersandemphasizedtheresultingadverseeffectsforcellmembranes[38].
However,reactiveoxygenspecieswerealsostronglyincreasedinbacteriaaffectedbygraphene
andgrapheneoxide[34,39–41]. IncreaseinROSwasattributedtosuper-oxideanion-independent
oxidativestressonbacterialcells[34]. Oxidativestressisparticularlyincreasedwhenbacterialcells
areincontactwithconductivereducedgrapheneoxideandgraphite(thelattershowingonlymarginal
antibacterialactivity)ascomparedtoinsulatinggrapheneoxideorgraphiteoxide. However,since
grapheneoxideexhibitshigherdispersionstability,cellcontactismorelikelycomparedtotheless
stablegraphene. Consequently,bacterialdeathviathemembranedamagemechanism,whichmore
stronglydependsonwell-dispersednanosheets,dominatestheoverallantibacterialactivity.
ThefindingsfordispersedgraphenesheetsareconfirmedbyastudybyAkhavanandGhaderi,
who investigated the antibacterial activity of deposited graphene nanowalls, i.e., graphene sheets
thatwereperpendicularlydepositedonasubstrate[42]. Inthisrespect,Phametal. foundthatthe
antibacterialactivityofsurface-depositedgraphenestronglydependsonthedensityofgrapheneedges
onthesurface[43]. InAkhavan’sandGhaderi’swork,contrarytodispersedgraphenenanosheets,
rGOshowshigherantibacterialactivitythanGO,sincethedifferencesindispersabilityarenolonger
anissueforthesurface-depositednanosheets. Thus,sincerGOcauseshigheroxidativestressthan
GO,andthemechanicalinteractionsarecomparable,rGOshowsthestrongereffect. Thesamestudy
demonstratesthatgram-positivebacteriaarelesssusceptibletomembranedamagethangram-negative
bacterialackingathickprotectivepeptidoglycan-layer[42].
Materials2016,9,617 4of19
Materials 2016, 9, 617 4 of 18

Figure 2. Left: (a) Viability of E.coli after incubation with Gt (graphite), GtO, GO, and rGO dispersions;
Figure2.Left:(a)ViabilityofE.coliafterincubationwithGt(graphite),GtO,GO,andrGOdispersions;
(b) time-dependent antibacterial activities of GO and rGO with E. coli; and (c) concentration-dependent
(b)time-dependentantibacterialactivitiesofGOandrGOwithE.coli;and(c)concentration-dependent
antibacterial activities of GO and rGO, E. coli was incubated for 2 h. Right: Oxidation of glutathione
antibacterialactivitiesofGOandrGO,E.coliwasincubatedfor2h.Right:Oxidationofglutathione
by graphene-based materials: (a) loss of GSH (0.4 mM) after in vitro incubation with Gt, GtO, GO,
bygraphene-basedmaterials: (a)lossofGSH(0.4mM)afterinvitroincubationwithGt,GtO,GO,
and rGO dispersions for 2 h; (b) time dependent GSH (0.4 mM) oxidation by GO and rGO dispersions
andrGOdispersionsfor2h;(b)timedependentGSH(0.4mM)oxidationbyGOandrGOdispersions
(40 μg/mL) after incubation from 0 to 4 h; and (c) concentration dependent GSH (0.4 mM) oxidation
(40µg/mL)afterincubationfrom0to4h;and(c)concentrationdependentGSH(0.4mM)oxidation
by GO and rGO after incubation for 2 h. Obtained from [34], with permission from the American
byGOandrGOafterincubationfor2h. Obtainedfrom[34],withpermissionfromtheAmerican
Chemical Society 2011.
ChemicalSociety2011.
The findings for dispersed graphene sheets are confirmed by a study by Akhavan and Ghaderi,
who iHnvoewsteivgeart,edno thtea lalnsttiubdacietesroianl aincttievriatyct oiof ndsepoofsgitreadp hgreanpehweniteh nbaancotweraiallss,h i.oew., garnapihnehnibei stohreyetes ftfheactt .
wFoerree xpaemrppelen,dinicaunlaortlhye rdsetupdosyi,tAedk hoanv aan saunbdsGtrhaated e[r4i2[]4. 4I]nr etphoisrt rthesapteEc.tc, oPlihaarema belte atol. refoduuncde gtrhaapth tehnee
aonxtiidbeacstheereiatls ,afcotrivmitiyn gorf GsOur,fwachei-ldeespigosniitfiedca ngrtaapnhtiebnaec tsetrrioanlgalcyt ivdietpyeoncdcsu rorned thoen ldyeonnsictey thofe ggrraapphheennee
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ninacnroesahseeedtsc, ormGOpa srhedowtos hthigehbear raenStiibOa2ctseuribaslt raactteiv.itTyh tihsarne sGuOlt,s siinncae sthelef -dliimffeitrienngcegsr oinw dthispoefrbsaacbtielirtiya
aorne gnroa lpohnegneer aonx iidsesuceo faoter dthseu sbusrtfraactee-sd.eNpootseittehda nt,ainnotshhiesesttsu. Tdhy,uGs,O sinwcaes rGdeOp ocasiutseeds rhaingdhoerm olxyidoantitvhee
ssturebssst rtahtaenw GitOho, uantdth tehfeo mrmeachtiaonnicoafls ihnaterpranctainoonws aarlels caosminpathraebalfeo, rreGmOe nshtioownesd thsetu sdtryo.nAgesirm efilfaerctr.e Tsuhelt
siasmreep sotruteddy bdyemDoenllsietruaetetsa tlh.awt hgorasmtu-dpioesditCivVe Dbagcrtoerwian agrrea lpehsse nsuesficlemptsibolne gtoo lmdeamndbrcaonpep dearmsuabgset rtahtaens .
gHraerme,-ntheegafltaivtley bdaecpteorsiiat eldacgkrianpgh ae ntheicskh opwrostencotiavnet ipbeapcttiedroiagllaycctainv-itlayyoefr i[t4s2o]w. nandevendecreasesthe
antibHacotwereiavleer,f fneoctt aolfl csotupdpieers boyn binlotcekrainctgiocnosp opfe grrioapnhfleonwe wtoiwtha brdacttheeriba aschtoerwia a[n4 5in].hibitory effect. For
examCpolen, vine rasneloyt,hReur isztuedtyal, .Arekphoarvtatnh aatngdr aGphhaedneer,i b[4o4th] rienpcoorltl othidaat lEf.o cromli aanred adbelep otos irteedduocne sgurbaspthraetnees
oexnihdaen scheesebtas,c tfeorrimalignrgo rwGtOh,[ 4w6]h.ilIen stihgensiefiecxapnet raimnteibnatsc,teprrieacl iapcittaivtiiotyn oocfccuorlrleodid oanlGlyO onwciet hthbea cgtrearpiahwenaes
sheets have been reduced. In this study, growth of E. coli on the GO coated substrate is slightly
increased compared to the bare SiO2 substrate. This results in a self-limiting growth of bacteria on
Materials2016,9,617 5of19
observed,whichseeminglyresultedinagrowthareaforbiofilmswithintheprecipitate. However,
thesefindingsaremerelyjudgedonthebasisofmeasuringtheopticaldensityofthedispersion(which
shouldbestronglydependentontheGOconcentration[47])andmicroscopy. Inthecontextofthis
study,itshouldalsobenotedthattestingforbacterialgrowthinhibitionzonesonagarplatesshould
onlyproducenegatives(noinhibition)forgraphene,sincetheantibacterialeffectisnotbasedonthe
leachingofsolublespeciesliketoxicions.
In accordance with the previously described study by Akhavan and Ghaderi [44], a slight
enhancementofbacterialgrowthonrandomlydepositedgrapheneoxidewasreported. Theenhanced
growthofbacteriaonrough(butnon-perpendicular)GOfilmsisutilizedbyWangetal. toenhance
theactivityofanaerobicammoniumoxidationbacteria[48].
Theapparentdiscrepancybetweenbiocompatibilityanddifferingantibacterialactivitieshasbeen
explainedbyHuietal.[47]Intheirwork,theauthorsshowedthattheadsorptionofproteinsandother
biomoleculesthatarecommonlyfoundinnutrientbrothonbasalplanes(i.e.,aflatsurface)ofgraphene
deactivatesitsantibacterialactivity. However,mostantibacterialtestsareperformedinsimplesaline
solutionsthatdonotcontainingredientsthatarepronetoadsorbongraphene. Consequently,results
oftheantibacterialactivityofgraphene(andothernanomaterials[49])havetobeinterpretedregarding
whethernutrientbrothsorsimilarmediacontainingbiomoleculesthatarestronglysurfaceactiveand
readilyadsorbon(carbon)nanomaterialswereusedinthebiologicalexperiments.Writtenconcurrently
tothisoverview,thesefindingsontheantibacterialactivityhavealsobeenveryrecentlysummarized
inseveralextensiveanddetailedreviews[50–52].
2.2. CarbonNanotubes
Carbonnanotubes(CNT)canbedescribedashollowstructureswithanextremelyhighaspect
ratio,whichareformedbyrolledgraphenesheets. Dependingontherollingangle,carbonnanotubes
canbemetallicorsemi-conductive.Furthermore,nanotubesarecategorizedassingle-wallednanotubes
(SWNTs) and multi-walled nanotubes (MWNTs). The latter consist of several single-walled tubes
thatarenestedinsideeachother. Carbonnanotubes,especiallySWNTshaveasignificantlyhigher
antibacterialeffectthanmostcarbonnanomaterials[12]. TheantibacterialactivityofpurifiedSWNTs
wasfirstdemonstratedbyKangetal.[53]. Intheirdetailedfollow-upstudy,Kangetal. showthat
purified SWNTs and MWNTs seriously impact bacterial membrane integrity upon direct contact.
Accordingly,metabolicactivityandmorphologyarecompromisedaswell[20]. Theyalsoshowthat
SWNTsexhibitastrongerantibacterialactivitythanMWNTs,probablycausedbytheirsmallersizethat
facilitatesmembraneperturbationandprovidesalargersurfacearea(Figure3). Oxidativestresslikely
playsanadditional,albeitminor,roleintheantibacterialmechanism[20].Liuetal.addfurtherdetailto
theinvestigationofmechanicaleffectsthatgoverntheantibacterialactivityofCNTs[54]. Inaccordance
withtheworkbyChenetal.[55],theyaddweighttothehypothesisthatSWNTcanactas“nano-darts”
thatpiercebacterialmembranesbycomparingbacteriawithdifferingmembranerobustnessandruling
outothermechanismsbyperformingextensivecontrolexperiments. Furtherstudiesindicatedthat
MWNTsshowednomutagenicactivityinbacteriaassayswithE.coliandS.typhimurium[56].
Adistinctfeatureofsp2 carbonnanomaterials,includingnanotubes,istheirspecialelectronic
structurecausingsemi-conductivity,or,inthecaseofsomeCNTs,even(pseudo)metallicconductivity.
ThisaspectwasinvestigatedbyVecitisetal.[57]whocouldclearlydemonstratethatmetallicnanotubes
exhibit a much higher antibacterial activity than semi-conducting CNTs. Consequently, electronic
effectscanalsocontributetotheantibacterialactivityofnanotubesandthismightalsoapplyforother
carbonnanomaterials. Similartotheeffectdescribedinmoredetailforfullerenes(seebelow),CNTs
canbeactivatedviaphotosensitizationcausingtheformationofadditionalROS[58].
Materials2016,9,617 6of19
Materials 2016, 9, 617 6 of 18

Figure 3. Left: Scanning electron microscopy (SEM) images of E.coli cells exposed to CNTs: (a) cells
Figure3.Left:Scanningelectronmicroscopy(SEM)imagesofE.colicellsexposedtoCNTs:(a)cells
incubated with MWNTs for 60 min; and (b) cells incubated with SWNTs for 60 min. The bars in both
incubated with MWNTs for 60 min; and (b) cells incubated with SWNTs for 60 min. The bars in
images represent 2 µm. Right: Bacterial cytotoxicity of carbon nanotubes: (a) fluorescence-based
bothimagesrepresent2µm.Right:Bacterialcytotoxicityofcarbonnanotubes:(a)fluorescence-based
toxicity assay of SWNTs and MWNTs for suspended- and deposited-type experiments with E. coli;
toxicityassayofSWNTsandMWNTsforsuspended-anddeposited-typeexperimentswithE.coli;
and (b) metabolic activity test of E. coli cells deposited on a CNT-coated filter and a control PVDF
and(b)metabolicactivitytestofE.colicellsdepositedonaCNT-coatedfilterandacontrolPVDF
membrane. Obtained from [20], with permission from the American Chemical Society 2008.
membrane.Obtainedfrom[20],withpermissionfromtheAmericanChemicalSociety2008.
A distinct feature of sp2 carbon nanomaterials, including nanotubes, is their special electronic
With CNTs, it is especially important to define the dispersion state of the fibrous colloids.
structure causing semi-conductivity, or, in the case of some CNTs, even (pseudo) metallic
UnfunctionalizedCNTsareamphiphobic,whichmeansthattheyarenearlyinsolubleinmostsolvents.
conductivity. This aspect was investigated by Vecitis et al. [57] who could clearly demonstrate that
Accordingly, dispersions of CNTs can show a wide range of aggregation states that define the
metallic nanotubes exhibit a much higher antibacterial activity than semi-conducting CNTs.
accessiblesurfaceareathatmightinteractwithbacteria[59]. SomestudiesdifferentiatebetweenCNTs
Consequently, electronic effects can also contribute to the antibacterial activity of nanotubes and this
thataredepositedonasubstrateanddispersedCNTs, showingwidelydivergingbacteriatoxicity
might also apply for other carbon nanomaterials. Similar to the effect described in more detail for
(Figure 3) [57], while other studies completely fail to address these critical aspects. Furthermore,
fullerenes (see below), CNTs can be activated via photosensitization causing the formation of
toxicologicalassessmentsofcarbonnanotubesexposuresshouldtakeintoconsiderationsignificant
additional ROS [58].
presenceofcatalyticallyactiveironembeddedwithinthenanotubes[60],aswellasotherbyproductsof
With CNTs, it is especially important to define the dispersion state of the fibrous colloids.
productionorprocessing. Possibleinteractionswithtestsystems(e.g.,MTTassay)havebeenreported
Unfunctionalized CNTs are amphiphobic, which means that they are nearly insoluble in most
aswell[11].Finally,CNTdispersionsseldomcontainunfunctionalizednanotubes.Aswillbediscussed
solvents. Accordingly, dispersions of CNTs can show a wide range of aggregation states that define
later,surfaceadsorbedmoleculesorcovalentfunctionalgroupssignificantlyalterbacterialreactions.
the accessible surface area that might interact with bacteria [59]. Some studies differentiate between
CNTs that are deposited on a substrate and dispersed CNTs, showing widely diverging bacteria
2.3. Fullerenes
toxicity (Figure 3) [57], while other studies completely fail to address these critical aspects. Furthermore,
Fullerenesaresphericalcarbonmolecules. ThemoststudiedfullereneistheBuckminsterfullerene
toxicological assessments of carbon nanotubes exposures should take into consideration significant
(abbreviated as C ), which consists of exactly 60 carbon atoms that are assembled in a pattern
presence of catalyti6c0ally active iron embedded within the nanotubes [60], as well as other byproducts
resemblingasoccerball.C isstronglyhydrophobicandisonlymarginallysolubleinwater.However,
of production or processi6n0g. Possible interactions with test systems (e.g., MTT assay) have been
C canbedispersedinwaterascolloidalaggregates(nano-C ornC )undervariousconditions.
re6p0orted as well [11]. Finally, CNT dispersions seldom contain 6u0nfunct6io0nalized nanotubes. As will
Consequently,nC shouldnotbeconfusedwithindividualC moleculeswithdiametersof1nm.
be discussed later6,0 surface adsorbed molecules or covalent f6u0nctional groups significantly alter
Instead,thecolloidalnC suspensionconsistsofcrystallineaggregateswithsizesbetween25and
bacterial reactions. 60
500nmthatareexpectedtohaveadifferentsetofpropertiesascomparedtobulkC orindividual
60
2C.360. Fmuollleercenueles s[61]. Althoughfullerenesarereportedasnotbeingverytoxictoeukaryoticcellsin
comparisontonanotubesandothercarbonmaterials[5], theyarefoundtobepotentantibacterial
Fullerenes are spherical carbon molecules. The most studied fullerene is the
agents. Fullerenewatersuspensions(nC )weretestedforantibacterialactivityusingB.subtilisby
60
BLuycoknmeitnaslt.er[f2u1l]leTrhenise st(uabdbyresvhioawteedd atsh aCt6f0r)a, cwtiohnicsho fconnCsistsc oonft aeinxaincgtlys m60a llcearrfbuolnle raetnoemasg gthraetg aatrees
60
assembled in a pattern resembling a soccer ball. C60 is strongly hydrophobic and is only marginally
soluble in water. However, C60 can be dispersed in water as colloidal aggregates (nano-C60 or nC60)
Materials 2016, 9, 617 7 of 18
under various conditions. Consequently, nC60 should not be confused with individual C60 molecules
with diameters of 1 nm. Instead, the colloidal nC60 suspension consists of crystalline aggregates with
sizes between 25 and 500 nm that are expected to have a different set of properties as compared to
bulk C60 or individual C60 molecules [61]. Although fullerenes are reported as not being very toxic to
eukaryotic cells in comparison to nanotubes and other carbon materials [5], they are found to be
potent antibacterial agents. Fullerene water suspensions (nC60) were tested for antibacterial activity
Materials2016,9,617 7of19
using B. subtilis by Lyon et al. [21] This study showed that fractions of nC60 containing smaller
fullerene aggregates showed greater antibacterial activities. Furthermore, different pretreatment and
sphroowceesdsinggr eaaltseor aafnfetcibtsa cbtaecrtiearliaa cttoixviictiiteys .[2F1u].r Itth herams boeree,n driefpfeorretnedt pthreattr feualtlmereenntesa naddsporrobc oenss binagctearlisaol
amffeemctbsrbaancetse r[i6a1t]o. xHicoiwtye[v2e1r],. iInt hcaosnbtreaesnt rteop gorratepdhethnae tofru lnlearneonteusbaeds,s ofurblleornenbeasc tdeori anlomt esmeebmra tnoe sca[u61se].
Halotewraetvioern,si ntoc obnatcrtaesrtiatol mgreamphbernaneeosr [n2a1n,6o1t,u6b2e].s ,Cfuonllterraernielys dtoo Lnyootnse eetm alt.o [6ca3u],s Feaanltge reatt iaoln. sshtoowbaecdt etrhiaalt
mnCe6m0 bisr aanbelse [t2o1 ,a6l1t,e6r2 ]b.aCctoenritarla rmilyemtobrLaynoen pehtaasle. [b6e3h],avFiaonrg [6et4]a. l.Aslthhoowugedh tfhualltenreCn6e0s iscaanb laectto aasl taenr
boaxcidteirziianlgm aegmenbtr,a Lneyopnh aest eabl.e rheapvoiortr t[h6a4t] .tAhelt hanotuigbhacftuelrliearle ancetsivciatny aisc tnaost acnauosxeiddi zbiyn gROagSe n[6t,2L].y Ionnsteetaadl,.
rfeuplloerrtetnheast tahree aanbtlieb atcot ecraiaulsaec tRivOitSy iinsdneoptecanudseendt boyxiRdOatSiv[6e2 s].trIenssst,e awdh,ifcuhll eirse nthees amreaianb lreetaoscoanu sfoerR tOhSe
ianndteibpaecntedreinalt oaxctidivaittiyv eofs tfruesllse,rwenheics h[6is3]t.h eAmccaoirndrineagslyo,n tfhoer tahnetiabnatcitbearciatel raiacltiaocnti voift yfuolflefruelnleerse nise sm[6o3s]t.
Alikcceolyr dcianugsleyd, tbhye aunntsipbaeccitfeirci arleaaccttiioonnso wffiuthll emreenmebsriasnme opsrtoltiekienlsy acnadu soetdhebry vuitnaslp mecoilfieccureleasc t[i6o2n]s. Nwoitthe
mtheamt tbhreasnee epfrfoetcetisn sonalnyd eomtheerrgve itianl smaolilneec umleesd[i6a2 ]o.rN oottheetrh amtitnhiemseale fbfeucftfsero nsylysteemmesr.g Ief imnosarlei nceommepdleiax
onruotrtihteiornm mineidmiaa labrue fufesredsy, sthteem asn.tIifbmacoterreiacol macptilveixtyn uoftr nitCio6n0 dmiseadpipaeaarresu, osewdi,ntgh etoa nintiabcaticvteartiianlga rcetiavcittiyonosf
noCf meddisiaap bpieoamrso,loewcuilnegs twoiitnha ctthivea ftuinllgerreenacet isounrsfaocfem aenddia ebniohmanocleedcu alegsgwreigthattihoen fudluleer eton ehsiugrhfearc eioannidc
60
esntrheanngctehd [6a5g]g. rDegisactuiosnsidngu ethtoe henigvhireorniomneicntsatrl einmgpthac[t6 5o]f. CDNisTcu osnsi nthget bhaeseisn voifr obnamcteernitaa sltiumdpieasc,t LoyfoCnN eTt
oanl. tahsesebrat stihsaot ffuballcetreerniaess taurde iteosx,iLcy toon beatctael.riaas, sbeurtt tiht aist dfuifllfeicruenlte tsoa friendto ax ircetaolibstaicct teersiat ,sbyustteimti sthdaitf fidcouelst
tnoofit naldtear rdeiaslpisetrisciotens tbseyhsatvemiort hanatdd toheuss nthote atoltxeircd riessppeornsisoen [6b1e]h. aviorandthusthetoxicresponse[61].
AA ssttrriikkiinngg ffeeaattuurree ooff ffuulllleerreenneess iiss tthheeiirr ssttrroonngg pphhoottooccaattaallyyttiicc aaccttiivviittyy,, wwhhiicchh iiss ccaauusseedd bbyy tthheeiirr
aabbiilliittyy ttoo pprroodduuccee ooxxyyggeenn rraaddiiccaallss uuppoonn UUVV raraddiaiatitoionn (F(Figiguurer e4)4 )[5[85,86,66,66,76]7.] P.hPohtootcoactaaltyaltyict iaccaticvtiitvyi tiys
iwsewlle lklnkonwown nfofro rsesmemiciocnodnudcutcintign gnnananopoparatritcilcelse slilkike eTTiOiO2.2 .HHeerere, ,pphhoottoonnss wwiitthh aa ssppeecciiffiicc mmiinniimmuumm
eenneerrggyy ((uussuuaallllyy wwiitthhiinn tthhee UUVV rreeggiimmee)) aarree aabbllee ttoo eexxcciittee eelleeccttrroonnss iinnttoo tthhee ccoonndduuccttiinngg bbaanndd ccrreeaattiinngg
aa ccoonndduuccttiinngg eelleeccttrroonn aanndd aann eelleeccttrroonn hhoollee.. TThhee nneexxtt sstteepp aafftteerr pphhoottoo--iinndduucceedd cchhaarrggee sseeppaarraattiioonn iiss
tthhee ttrraannssffeerr ooff tthhee iinndduucceedd cchhaarrggee ttoo ddoonnoorr oorr aacccceeppttoorr ssuubbssttrraatteess oonn tthhee ppaarrttiiccllee ssuurrffaaccee [[6688]].. IInn tthhee
ccaassee ooff ffuulllleerreenneess, ,sisningglelte otxoyxgyegne n(1O(12O) a2n)da nsudpseuropxeirdoex irdaedircaadlsi c(aOls2−)( Oar2e´ c)reaarteedcr aeta ttheed paatrttihcelep saurrtfiacclee
s(Fuirgfaucree 4(F) [ig69u]r.e Th4)es[e6 9o]x.ygTehne sraedoicxaylgs eanrer iand tiucranls aabrlee tion ctruearnte afubrlethteor RcrOeaSt aenfdu irnthfleicrt RoxOidSaatinvde sintrfleiscst.
oBxriudnaettiv eet satlr.e csos.mBpruarneedt ethtea lp. choomtopaacrtievdittyh eofp fhuoltloeraecntievsi twyiotfhf uthllaetr eonf ewsewlli-tshtuthdaietdo fTwiOe2ll -psaturtdicieleds T[6iO6]2.
pDaerptiecnledsin[6g6 o].nD perep-etnredaitnmgeonnt, pnroet- atrlel afutmlleernetn,en wotaatellr fsuullsepreennseiownas tsehroswusepde andsdiointisonshalo bwaecdteraidad toitxioicnitayl
buapcotner iUaVto xriacditiyatuiopno, nbUutV trhaodsiea ttihoant, bshuotwtheods eththisa tesfhfeocwt eddidt hsios eaftf emctudcihd sloowaetrm puacrhtilcolew (efrupllaerrteinclee)
(cfounllceernentrea)ticoonnsc ethnatrna TtiioOn2s. t hanTiO2.

Figure 4. Mechanism of ROS production by fullerenes. Obtained from [69], with permission of the
Figure4. MechanismofROSproductionbyfullerenes. Obtainedfrom[69],withpermissionofthe
American Chemical Society 2009.
AmericanChemicalSociety2009.
2.4. Nanodiamond
2.4. Nanodiamond
In contrast to the other carbon nanomaterials, nanodiamond is comprised of sp3 hybridized
Incontrasttotheothercarbonnanomaterials,nanodiamondiscomprisedofsp3hybridizedcarbon.
carbon. However, since sp3 hybridized carbon would create dangling bonds at the surface,
However, since sp3 hybridized carbon would create dangling bonds at the surface, nanodiamond
nanodiamond surface chemistry is rich with different oxygen species and residual graphene. The
surfacechemistryisrichwithdifferentoxygenspeciesandresidualgraphene. Thesurfacechemistry
surface chemistry is further enhanced by the faceted nature of the nanocrystals contributing slightly
isfurtherenhancedbythefacetednatureofthenanocrystalscontributingslightlydifferentreactivities

atthedifferentcrystalplanes. Theactualcompositionofthesurfacechemistryandtheamountof
defectsinthecrystallatticearearesultofthesynthesisroutefornanodiamond,whichiseitherbased
ondetonatingTNTandhexagonorsimilarcompoundsinapressurizedcontainer(bottom-up)or
bymillinglargerdiamonds(top-down). Detonationsynthesisyieldsnanodiamondswithadiameter
of about 5 nm, while milled diamonds are usually larger. The final surface chemistry is defined
Materials2016,9,617 8of19
duringpre-treatment,whichistypicallybasedonvariouswashingstepswithoxygenatingacidsthat
removeresidualgraphene. Alsoincontrasttotheotherdiscussedcarbonnanomaterials, andasa
resultofthehighamountofoxygenspecies(mainlycarboxylgroups)atthesurface,nanodiamond
forms very stable aqueous dispersions with high zeta-potentials. However, proper protocols for
dispersingindividualnanodiamondshaveonlybeenestablishedaroundtenyearsago,whichexplains
thecomparativelysparsepublicationrecordonbiologicalinteractionswithnanodiamond.
The current (early) consensus on the biocompatibility of NDs is that they are nontoxic for
eukaryotic cells [70–73]. NDs are assumed to have the highest biocompatibility in comparison to
allothercarbon-basednanomaterialsincludingcarbonblacks,single-andmulti-wallednanotubes,
andfullerenes[74]. StudiesontheimpactofNDsonsmallorganismsorprokaryotesarerare,but
results more recently drifted into a slightly adverse direction. Though Mohan et al. reported the
nontoxicnatureofNDswithnodetectablestressforthewormCaenorhabditiselegans[75],Linetal.
assumedthatNDsmightbemoretoxicformicroorganismsthanforanimal/humancells. Thiswas
tentativelyconfirmedbyinvestigatingthetoxicpropertiesof5nmand100nmNDsontheprotozoa
ParameciumcaudatumandTetrahymenathermophile. WhilesmallerNDsweremoretoxicthanbigger
particles,carboxylatedNDswerelesstoxicthanthenon-carboxylatedNDs. Althoughthemeasured
toxicity was relatively low, it was found to be significant [21]. A study on embryonic stem cells
was the first hint that NDs might be toxic for eukaryotic cells. Here, carboxylated and oxidized
detonationdiamonds(4–5nm)purifiedbyacidtreatmentwerefoundtoinduceDNAdamage[76].
FirststudiesontheimpactofNDsonbacteriaweremicroscopicinvestigations. Forthese,highND
concentrations were applied leading to the coverage of bacterial cells [77,78]. Shortly afterwards,
Beranovaetal. demonstratedtheinhibitingeffectsofdetonationNDonE.coligrowth[79]. Thesame
authorsreportedthatdetonationNDsareantibacterial,whilelargerdiamondnanoparticlesobtained
frommillingarenot[80].Thisstudylackedanexplanationfortheantibacterialpropertiesofdetonation
ND,butitwassuggestedthatuntreatedNDsaremoreeffectiveinkillingbacteriathantheoxidized
form. Inourownstudy[81],whichwaspublishedaroundthesametimeasBeranova’spaper,we
found that the antibacterial activity of ND strongly depends on its surface chemistry. While fully
carboxylated NDs do not show any antibacterial activity, NDs that are merely partially oxidized
showveryhighantibacterialactivitythatissimilarinpotencytosilvernanoparticles. Mostlikely,
carboxylanhydride groupsand otherreactive oxygengroupsonthe NDsurfacearethecause for
thestrongeffect. Thesefindingsaresomewhatcomplicatedbythefactthatthesurfacechemistryof
nanodiamondsisnotverywellcontrolledbymanufacturers[82],whichmightexplainthediffering
findingsregardingthebiocompatibilityofND.Additionally,andanalogoustotheotherdescribed
CNMs,wefoundthattheantibacterialeffectofNDdisappearsaftercontactwithbiomoleculesfrom
nutritionmedia,mostlikelyduetounspecificreactionswithmediabiomolecules[81,83].
Sincenanodiamondscanbesemi-conductiveasaresultofdefectsinthecrystallattice,NDshave
thepotentialforphotocatalyticactivity,ascouldbedemonstratedbyJangetal.[84]. However,atthe
pointofwriting, anenhancementoftheantibacterialeffectofNDthatiscausedbyphotocatalytic
activityhasnotyetbeenreported.
2.5. Diamond-LikeCarbon,DiamondThinFilms
Diamond-likecarbon(DLC)isaclassofcarboncoatings. Althoughtheyarenotcolloidsperse,
thecomparisonofthesecoatingswithdispersedCNMsisvaluableforunderstandingtheantibacterial
action of CNMs in general. DLC coatings have attracted great attention from the biomaterials
communitybecausetheyimpartbiocompatibility,chemicalinertness,alowfrictioncoefficient,high
hardness,wearandcorrosionresistancetomedicaldevicesurfaces[85,86]. DLCconsistsofamorphous
carbon and/or carbon crystallites and possesses a disordered structure with a mixture of sp2 and
sp3 hybridizedcarbon[87](Figure5a). Dependingonthesp2/sp3 ratio,thepropertiesofDLCcan
varysignificantly. Ultrananocrystallinediamond(UNCD)isverysimilartoDLC,buthasahighersp3
contentanddistinctnanocrystallinedomains.
Materials 2016, 9, 617 9 of 18
mixture of sp2 and sp3 hybridized carbon [87] (Figure 5a). Depending on the sp2/sp3 ratio, the
Mpratoepriaelrst2i0e1s6 ,o9f, 6D17LC can vary significantly. Ultrananocrystalline diamond (UNCD) is very simi9laorf 1to9
DLC, but has a higher sp3 content and distinct nanocrystalline domains.

(A) (B)
Figure 5. (A) DLC film; and (B) number of E. coli and B. subtilis colonies on DLC and UNCD films and
Figure5.(A)DLCfilm;and(B)numberofE.coliandB.subtiliscoloniesonDLCandUNCDfilmsand
the respective antibacterial efficiency. Obtained from [87], with permission of John Wiley & Sons 2013.
therespectiveantibacterialefficiency.Obtainedfrom[87],withpermissionofJohnWiley&Sons2013.
Both ultrananocrystalline diamond films and DLC exhibit strong antibacterial properties [85,87–
Both ultrananocrystalline diamond films and DLC exhibit strong antibacterial
89] (Figure 5b). Nanocrystalline diamond surfaces with larger crystallite sizes than DLC or UNCD
properties[85,87–89] (Figure 5b). Nanocrystalline diamond surfaces with larger crystallite
also show bactericidal and anti-adhesive properties [90–92], but bacteria toxicity disappears if the
sizesthanDLCorUNCDalsoshowbactericidalandanti-adhesiveproperties[90–92],butbacteria
crystallites are in the micrometer range [92]. Jelinek et al. compare UNCD and DLC films with varying
toxicitydisappearsifthecrystallitesareinthemicrometerrange[92]. Jelineketal. compareUNCD
sp3 content showing that all those materials exhibit high antibacterial activity (Figure 4) [87]. Since
andDLCfilmswithvaryingsp3 contentshowingthatallthosematerialsexhibithighantibacterial
DLC films are extremely smooth, interfacial roughness or sharp features (as in graphene films) are
activity(Figure4)[87]. SinceDLCfilmsareextremelysmooth,interfacialroughnessorsharpfeatures
not important parameters for the antibacterial properties [88]. Instead, the strong hydrophobicity of
(as in graphene films) are not important parameters for the antibacterial properties [88]. Instead,
the coatings might lead to alterations of the cell membrane, resulting in bacterial death [87].
thestronghydrophobicityofthecoatingsmightleadtoalterationsofthecellmembrane,resultingin
Hydrophobicity and reactivity of the chemically stable coatings also depend on the hydrogen content,
bacterialdeath[87]. Hydrophobicityandreactivityofthechemicallystablecoatingsalsodependon
with increased hydrogen content resulting in lower antibacterial activity [93]. Various other studies
thehydrogencontent,withincreasedhydrogencontentresultinginlowerantibacterialactivity[93].
have investigated the interactions of nanocrystalline diamond surfaces with eukaryotic cells and
Variousotherstudieshaveinvestigatedtheinteractionsofnanocrystallinediamondsurfaceswith
biological molecules [86,91,94,95]. Here, hydrophilic, oxygen-terminated surfaces were described to
eukaryotic cells and biological molecules [86,91,94,95]. Here, hydrophilic, oxygen-terminated
be the anchor point of interaction on the otherwise anti-adhesive surfaces. Furthermore, diamond
surfaces were described to be the anchor point of interaction on the otherwise anti-adhesive
thin films were shown to exhibit semiconducting properties [96]. The electrically active surface
surfaces. Furthermore, diamond thin films were shown to exhibit semiconducting properties [96].
showed the capacity to form chemical bonds with biomolecules from the surrounding media.
Theelectricallyactivesurfaceshowedthecapacitytoformchemicalbondswithbiomoleculesfromthe
Therefore, nanocrystalline diamond surfaces might interact with bacterial cell membranes resulting
surroundingmedia. Therefore,nanocrystallinediamondsurfacesmightinteractwithbacterialcell
in an effective membrane-distortion mechanism. This could hinder bacterial adhesion and the
membranesresultinginaneffectivemembrane-distortionmechanism. Thiscouldhinderbacterial
subsequent bacterial colonization on the surface [92]. It should be noted that none of the cited
adhesionandthesubsequentbacterialcolonizationonthesurface[92]. Itshouldbenotedthatnoneof
antibacterial studies for DLC test for ROS or other oxidative stress that might originate from the redox
thecitedantibacterialstudiesforDLCtestforROSorotheroxidativestressthatmightoriginatefrom
active properties of the sp2 portions of DLC and nanocrystalline diamond.
theredoxactivepropertiesofthesp2portionsofDLCandnanocrystallinediamond.
Furthermore, not all studies for DLC films clearly differentiate between antibacterial and anti-
Furthermore, not all studies for DLC films clearly differentiate between antibacterial and
adhesive properties. In this context, antibacterial would mean inhibition of bacterial growth in the
anti-adhesive properties. In this context, antibacterial would mean inhibition of bacterial growth
surrounding medium, as is known, for example, for copper or silver surfaces through the leaching of
in the surrounding medium, as is known, for example, for copper or silver surfaces through the
toxic ions. Anti-adhesive simply means that bacteria cannot adhere to the surface and thus are not
leachingoftoxicions. Anti-adhesivesimplymeansthatbacteriacannotadheretothesurfaceand
able to form colonies on the material. This can be easily investigated using different methods: for example,
thus are not able to form colonies on the material. This can be easily investigated using different
the anti-adhesive properties can be tested using stamps that are equipped with the investigated
methods: for example, the anti-adhesive properties can be tested using stamps that are equipped
surface. Here, only adherent bacteria are transferred to a nutrition medium and quantified [92].
with the investigated surface. Here, only adherent bacteria are transferred to a nutrition medium
Conversely, simple incubation tests that monitor the bacteria concentration in a medium containing
andquantified[92]. Conversely,simpleincubationteststhatmonitorthebacteriaconcentrationina
the investigated material provide information on the antibacterial properties of a (macroscopic)
mediumcontainingtheinvestigatedmaterialprovideinformationontheantibacterialpropertiesof
material [87]. Finally, a coating could be antibacterial only to bacteria that adsorb at the surface
a(macroscopic)material[87]. Finally,acoatingcouldbeantibacterialonlytobacteriathatadsorbat
without significantly influencing bacteria in the surrounding medium, thus featuring a bactericidal
thesurfacewithoutsignificantlyinfluencingbacteriainthesurroundingmedium,thusfeaturinga
b actericidalsurface. ThisisusuallytestedbySEMofbacteriaonthesubstratesurfaceorbyapplyinga
Materials2016,9,617 10of19
smalldropletofbacteriamediumontothetestedsurfacefollowedbyincubationoveracertainperiod
oftime. Afterwards,survivingbacteriaarequantified[89]. Differentiatingbetweenthesedifferenttest
setupsiscriticalforcomparabilityandgeneralvalidityofantibacterialtestswithmacroscopicsurfaces,
equallyimportantasconsideringdispersionstatesincolloidaldispersionsofcarbonnanomaterials.
Asinthecasefornanodiamond,ithasbeenshownthatDLCcanbephotocatalyticallyactive[97].
Again, at the point of writing, an enhancement of the antibacterial effect of DLC caused by
photocatalyticactivityhasnotyetbeenreported.
3. FunctionalizationofCarbonNanomaterialsforTailoringAntibacterialProperties
The body of literature dealing with functionalization, enhancement and tailoring of carbon
nanomaterials for different applications is overwhelming. However, since the mechanisms of the
antibacterialactivityofCNMsarestillapointofdebate,muchfewerpublicationsexistthatdealwith
CNMsthataretailoredforenhancedantibacterialproperties. Thefollowingisaselectionofengineered
CNMsystemswithimprovedantibacterialactivity. Nexttodirectchemicalfunctionalization,CNMs
have mostly been enhanced by combining them with other materials, for example in the form of
nanocomposites,byloadingthemwithfunctionalbiomoleculesorbydopingdiamond.
3.1. Graphene
Most publications dealing with antibacterial materials based on graphene describe
nanocompositesofgraphenewithantibacterialnanoparticlesormolecules. Thisworkisgoverned
by the principle that the antibacterial activity of the single component is lower than that of the
nanocomposites. Forexample,countlesspapersdescribetheformationofantibacterialcomposites
ofgrapheneandsilvernanoparticles,improvingtheantibacterialactivityofsilver[98–105]. Hybrids
ofTiO andgraphenehavebeenproducedinordertoenhancethephotocatalyticactivityofTiO ,
2 2
whichinturnincreasestheantibacterialactivity. Here,grapheneservesaselectronacceptor,while
TiO isthephotocatalyticallyactivecomponent[106]. Workonmetaloxide/graphenecomposites
2
hasbeensummarizedinacomprehensivereviewbyUpadhyayetal.[107]. Thecompositeapproach
has also been demonstrated with polymers. For example, Santos et al. combine graphene with
poly(N-vinylcarbazole), again resulting in a higher bacteria toxicity than that of the individual
components[31]. Furthermore,thebiocompatibilityofthiscompositewasshowntobehigherthan
thatofpuregraphene. Asimilarsynergisticincreaseinantibacterialactivitycouldbedemonstrated
byloadinggraphenewithantibioticmolecules. Forexample, Wantetal. describeadrugdelivery
systemofgrapheneandtheantibioticoxide-benzylpenicillin[108],whileAbdelhamidetal. describe
a similar system for gramicidin [109] and Pandey et al. for gentamicin [110]. While chemical
functionalizationofgrapheneiswidelyinvestigated,sofar,toourknowledge,ithasnotbeenapplied
toenhanceantibacterialapplicationsofgraphene,althoughitcouldbeshown,aswealreadydiscussed,
thatdifferentoxidationstatessignificantlyalterbiologicalinteractionsofgraphene[28,44].
3.2. CarbonNanotubes
Analogoustographene,carbonnanotubeshavebeencombinedwithantibacterialnanoparticlesin
ordertoenhancebacteriatoxicityofthebarenanoparticles. Thishasbeen,forexample,demonstrated
byNiuetal. forsilvernanoparticles[111]andbyAkhavenetal. andWoanetal. forTiO [112,113].
2
Likewise, workwithpolymercompositeshasbeendonebyJooetal. andJungetal. whografted
antibacterialpolymerPoly[2-(dimethylamino)ethylmethacrylate]ontoMWNTs[114,115].
Ariasetal.studiedtheantibacterialeffectof–COOH,–OHand–NH functionalizedSWNTs[116].
2
Theynotethat–COOHand–OHterminatedSWNTsaremoretoxicthanonesfunctionalizedwith–NH .
2
However,theauthorsdonotcharacterizethedispersionstateorsurfacechargeofthefunctionalized
SWNTs. Accordingly,itisunclearwhetherthedifferencesinantibacterialactivityaremerelydueto
differentaggregationstatesandthusbiologicalaccessibilityofthenanotubes. Thelattermightexplain
theobservationthattheantibacterialactivityisalsoinfluencedbythepresenceofdifferentbuffers