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Biomed. Phys. Eng. Express 2 (2016) 055008 doi:10.1088/2057-1976/2/5/055008

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Non-covalent modifiedmulti-walled carbon nanotubes: dispersioncapabilities and interactions with bacteria

MaryamKhazaee1,4, DanYe1, AnindyaMajumder1, Larysa Baraban1, JoergOpitz1,2 andGianaurelio Cuniberti1,3

1 Institute forMaterials Science andMaxBergmannCenter of Biomaterials, TUDresden, D-01062Dresden,Germany2 Fraunhofer Institute for Ceramic Technologies and Systems IKTSMaterial Diagnostics, D-01109Dresden , Germany3 Division of ITConvergence Engineering, POSTECH, Pohang, Korea4 Current address: Institute forMaterials Science andCenter forNanointegrationDuisburg-Essen (CENIDE), University ofDuisburg-

Essen, Universitätsstraße 15, 45141 Essen, Germany.

E-mail:Maryam.khazaee@uni-due.de

Keywords:multi-walled carbon nanotube, antimicrobial activity, growth curve, dispersion

Supplementarymaterial for this article is available online

AbstractAntimicrobial activity of surfactant-modifiedmulti-walled carbon nanotubes (MWCNTs)wasinvestigated by analyzing the growth curves of anEscherichia coli (E. coli) population in ionic and non-ionic surfactant-modifiedMWCNTs in Luria Bertani (LB) broth. The ionic surfactants (sodiumdodecylbenzenesulfonate (SDBS), sodium cholate (SC), dodecyltrimethylammoniumbromide(DTAB), cetyltrimethylammoniumbromide (CTAB)) as well as a non-ionic polyvinylpyrrolidone(PVP)were used to test the dispersion and resulting antimicrobial effect ofMWCNTs bymeans ofUV–vis spectroscopy and optical density (OD)measurement. Among these surfactants, SDBS andDTABprovidedmaximumandminimumMWCNTdispersion, respectively. Furthermore, thebiocompatibility issues with respect to dispersion capabilities of the ionic and non-ionic surfactants isdiscussed in detail, as a source of potentialmisinterpretation of the obtained growth curves ofE. coliand thus, their antimicrobial effect. Finally, scanning electronmicroscopywas used to study theinteraction of well-dispersedMWCNTswith the E. coli cells.

1. Introduction

The utilization of carbon nanotubes (CNTs) in variousareas of science and technology has been enormouslyenhanced owing to their unique electronic, mechan-ical, and structural properties [1–4] and their potentialapplications in nano-biotechnology [5]. One of themain obstacles encountered for using CNTs in biolo-gical environment is the expected toxicity effects oftubes against biological components e.g., bacterialcells triggering several kinds of potential risks asso-ciated with the consumption of CNTs by humanbeings [6].

The main reported factors which may influencethe toxicity of CNTs against bacteria are diameter [7],length [8, 9], surface functional groups [10, 11], anddegrees of aggregation of tube [12, 13]. This suggeststhat not only physical properties of CNTs are criticalissues in their toxicity but also specific

physicochemical functionalization of tubes can altertheir cytotoxicity in bacterial systems. The pristi-neCNTs form bundles due to the strong π–π interac-tion between tubes directing them into an aggregatedstate in aqueous and organic solutions. Previous stu-dies have proved that the degree of agglomerationplays a critical role in the antimicrobial activity ofCNTs. Liu and coworkers showed that individuallywell-dispersed single walled CNTs exhibit more toxi-city than the aggregated ones. These dispersed tubesact as nano darts against bacterial cells resulting in thedestruction of the lipidmembranes of the cells and thedegradation of the cells integrities and consequentlythe death of the cells [14–16]. As a consequence,proper modification of tubes producing individualtubes from their aggregations is essential for exploringtheir direct interactions with bacteria.

Since the onset of researches on CNTs-bacteriainteraction, various, sometimes even contradictory

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10 February 2016

REVISED

4August 2016

ACCEPTED FOR PUBLICATION

5August 2016

PUBLISHED

8 September 2016

© 2016 IOPPublishing Ltd

observations have been reported. One of themain rea-sons for obtaining these contradictory results may bethe toxicity contribution of the used solution for indi-vidualization of the tubes. Therefore, it is crucial tosearch for biocompatible solutions with excellent sus-ceptibility for individualization of CNTs.

Surfactants are capable of dispersing and stabiliz-ing CNTs in polar solvents which will increase theopportunity contacting with microbial cells, and mayaggravate the toxicity of the tubes. Zhang et al showedthat dispersedmulti-walled carbon nanotubes(MWCNTs) via using synthetic SDBS and octyl phe-noxy polyethoxyethanol (TX100) as ionic surfactantstriggered the toxicity of the tubes by enhancing cellinternalization ofMWCNTs and subsequent oxidativestress in unicellular green alga (Chlorella pyr-enoidosa); while natural originated humic acid alle-viate the toxicity of MWCNTs [17]. In one study,Yadav et al reported that dispersed MWCNTs by nat-ural Acacia concinna could inhibit Escherichia coli(E. coli) growth and damage to the cell membrane ofthe bacteria due to either antibacterial effect of Acaciaconcinna or interaction between well-dispersed tubesand bacteria [18]. Various biomolecules such as pro-teins, amino acids, DNA, and gum Arabic and synth-etic surfactants consisting of SDBS, Triton X100, andCTAB have been evaluated as dispersants for CNTs[19–23]. Such dispersion relies on the non-covalentmodification of tubes. This coverage of the tubes pre-vents their aggregation via steric or electrostatic repul-sion or by incorporation of both. In all of these studies,researchers confirmed that the toxicity of the dis-persant strongly influences the toxicity of the tubesand degree of dispersity of the tubes has substantialeffect on their antibacterial effect.

This study elucidates a strategy to check the anti-microbial activity of well-dispersed MWCNT and toinvestigate the effect of different ionic and non-ionicsurfactants typically used for dispersion of CNT.For these purposes, we implemented into our systemthe ionic surfactants consisting of sodium dode-cylbenzenesulfonate (SDBS), sodium cholate (SC),dodecyltrimethylammonium bromide (DTAB), cetyl-trimethylammonium bromide (CTAB) and poly-vinylpyrrolidone (PVP), as a non-ionic surfactant. Thegram-negative bacteria,E. coliK12 strain, were utilizedfor antimicrobial analysis. Our approach is schemati-cally illustrated in figure 1. Among various methodsfor themodification of CNTs, the non-covalent meth-odology was selected which preserves the inherentstructure of CNTs [24]. In the non-covalent functio-nalization of CNTs, the hydrophobic tail groups ofsurfactants interact with the hydrophobic sidewalls oftubes and the hydrophilic head groups of surfactantsencounter the aqueous environment [25]. Thereafter,this nanotube dispersion was incubated with the bac-teria, grown in the Luria-Bertani (LB) broth medium(figure 1(b)). We showed that the antimicrobial activ-ity of the non-covalently functionalized MWCNTs

was attributable to the coverage of bacteria and sub-sequent penetration of the lipid membrane of the bac-teria via tubes causing their lysis. This research aims tofacilitate the preparation of well-dispersed MWCNTin an aqueous medium and their subsequent applica-tion for ceasing bacterial cell growth.

2. Experimental section

2.1.Materials andmethodsMWCNTs in powder form with 90% carbon purity,produced via the catalytic carbon vapor depositionprocess, were purchased from NanocylTM (Belgium)and used as received. Dispersibility of the tube wasstudied in various surfactants (Sigma Aldrich) consist-ing of SDBS, SC, CTAB, DTAB and PVP dissolved indeionized water (1% wt/wt). Their chemical struc-tures are depicted in figure S1. Dispersion of CNTs inthe aqueous solution was done using a horn sonicatorfor 10 min at 8% amplitude followed by a centrifuga-tion step (Beckman Colter, CTM00M09) for 45 min at15 k rpm. After centrifugation, the upper 80% of thesupernatant was decanted for later investigations.

2.2. Bacteria cultureThe wild type E. coli K12 strain was grown in the LBbroth medium. Small amounts of E. coli colonies werescraped from LB agar plates and cultured in LB brothuntil they reached optical density (OD) 1. They werethenmixed with glycerol (volume ratio glycerol: E. coliOD1 culture=3:17) and freeze-stored at −70 °C. Asmall volume of frozen culture was scraped from the−70 °C storage tube with a sterile pipette tip andtransferred into a small amount of LB broth. Thisculture was activated in an incubator (SartoriusCertomat® IS) at 37 °C and a rotating rate of 170 rpmfor at least 5 h (OD∼1).

2.3. Evaluation of biocompatibility of surfactants,unmodified-MWCNTs and surfactant-MWCNT100 ml of the bacterial culture were applied to thedifferent concentrations of surfactants dissolved in theLB medium to investigate the biocompatibility of thesurfactants. After these treatments, bacterial cellgrowth was monitored by measuring the OD at awavelength of 600 nm at 2 h intervals with an Eppen-dorf®BioPhotometer using standard 1 cmoptical pathlength cuvettes. Interaction of unmodified-MWCNTswith E. coli was studied by treating bacteria withsonicated MWCNTs in the LB medium (seesection 2.1). Furthermore, various concentrationsof SDBS-modified MWCNTs and PVP-modifiedMWCNTs were prepared using 1 wt% dissolvedsurfactant in LB medium as a stock solution. Finally,100 ml of the activated culture was incubated withsurfactant-modified MWCNTs and OD measure-ments were performed. All the antibacterial activity

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Biomed. Phys. Eng. Express 2 (2016) 055008 MKhazaee et al

experiments were carried out at approxi-mately pH=8.

2.4. Characterization of CNTs dispersionsThe characterization of concentration of individuallydispersed nanotube in different suspensions is acritical factor for their biomedical applications (seedetails in the supporting information). In order tovisualize the dispersed tubes in ionic surfactants,atomic force microscopy (AFM) (Nanoscope IIIa ofDigital Instruments) was performed in the tappingmode. In addition, scanning electron microscopy(SEM) was performed in a high vacuum conditionusing DSM 982 GEMINI at 8 kV accelerated voltage.The structure of dispersed CNTs in aqueous solutionswas characterized using high resolution transmissionelectronmicroscopy (HRTEM) (JEOL, 2010F system).The samples for TEM investigations were prepared bydropping 10 μl MWCNT suspensions onto a coppergrid with a carbon film and air-dried before theexamination.

3. Results and discussion

3.1.Dispersion ofMWCNT in surfactantThe dispersion efficiency of MWCNTs in the surfac-tants was characterized by measuring the UV–visspectra of the supernatants, as described in [26, 27].Based on the Beer–Lambert law (supporting informa-tion), absorbance values depend on the concentrationsof the individually dispersed tubes in the suspension.

As a result, the higher the absorbance values are, themore the concentration of the dispersed tubes in thesolution is. In the supporting information, figure S2depicts the UV–vis spectra of the dispersed MWCNTsin these surfactants. The absorbance values of thesespectra at 500 nm were collected to estimate theefficiency of the dispersion of MWCNT by thesesurfactants.

As shown in figure 2(a), SDBS reveals the best cap-ability to solubilize nanotubes. At the same time, thelowest ability to disperse nanotubes in the aqueoussolution was observed for DTAB. This trend betweenused surfactants can be ascribed to their chemicalstructures [26]. Since bundles of CNTs are not activein the UV–visible region and only individual onesabsorb in this region [28], the UV–vis data can give theinsights into dispersed CNTs in the solution resultingin the calculation of MWCNT extinction coefficient.Figure S3 shows the UV–vis spectra of diluted solu-tions of dispersed MWCNTs in PVP. Absorbancevalues of these spectra at 600 nm were collected andthen divided to the optical path, l, to give A600nm l–1

providing the Beer–Lambert correlation plot(figure 2(b)). The linear fitted curve gradient attrib-uted to the extinction coefficient of MWCNTs-PVPsolution is 45 ml mg−1 cm−1, which is in good agree-ment with the previous studies [29, 30]. HRTEMmeasurements were implemented to evaluate theinteraction mechanism of surfactant-nanotubes(figure 2(c)). TEM images of these tubes indicate thatall of the several types of surfactants cover sidewalls ofthe tubes with layers of 2 nm thick. These ionic and

Figure 1. Schematic representation ofMWCNTs, surfactantmolecules and E. coli bacteria (a) and probable interaction of surfactant-modifiedMWCNTswithE. coli bacterial cells (b).

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non-ionic surfactants are wrapped around tubes andthe electrostatic or steric repulsion between their headgroups lead to the individualization of the tubes in theaqueous solution. TEM images confirm that no struc-tural damage is done to the tubes because of the non-covalent modification. It is also feasible to assess theaverage number of walls (5–10) and the average dia-meter of tubes (inner: 3 and outer: 8). Figures S4 andS5 show the AFM and SEM images of the dispersedtubes. As can be seen, the surfactant-assisted disper-sion of the tubes leads to properly solubilized tubes inthe aqueous solution. The analysis of these imagesresulted in the determination of the average length(200–500 nm) and diameter (8.5–9 nm) of the tubesafter applying the above-mentioned horn sonicationemphasizing the shortening of tubes.

3.2. Biocompatibility of surfactantsAlthough the surfactants demonstrated a high suscept-ibility to individualizeMWCNTs, the biocompatibilityof surfactant solutions had to be investigated to ensurethat they were not involved in the probable antibacter-ial activity of CNTs. The antibacterial activity of thesesurfactants was investigated by incubating gram nega-tive E. coli bacterial cells with the following concentra-tion of the surfactants: 0.005 wt% or 0,1 wt%, 0.5 wt%and 1 wt% followed by the examination of their ODgrowth (figure 3). Figure 3(a) illustrates that

antibacterial activity of SDBS depends on concentra-tion and time. For 0.5 and 1 wt% concentrations, aminor antimicrobial activity can be observed after 8 htreatment. However, bacterial treatments with diverseconcentrations of the anionic surfactant SC (figure S6.(a)) have no significant impact on the viability of thecells and the time needed to reach exponential phasegrowth is almost the same as in the control sample.Moreover, the OD growth curves of different concen-trations of PVP in bacteria medium (figure 3(c)) showno toxicity effect against bacterial cells (figure 3(b)).Figures 3(c) and S6.(b) indicate that cationic surfac-tants (DTAB and CTAB) almost inactivate all the cellsin the samples and there is no bacterial growth in thesystems.

Itmay be concluded that positively charged surfac-tants effectively interact with the negatively chargedlipid membrane of E. coli which can increase the per-meability across their lipid membranes and induce therelease of intracellular components such as DNA andRNA resulting in the complete destruction of bacteria.However, electrostatic repulsion between the nega-tively charged lipid membrane of E. coli and negativelycharged surfactants in low concentrations results inkeeping the membrane structures of E. coli intact. Theabsence of the electrostatic forces between non-ionicPVP and the E. colimembrane could be the reason forthe biocompatibility of this surfactant. This findingagrees well with the previous studies by Volkering et al

Figure 2.Comparative evaluation of the solubility ofMWCNTs in SDBS, SC, CTAB, PVP andDTAB (dissolved in distilledwater)solutions collected at 500 nm (a). Beer–Lambert correlation plots collected at 600 nm for dispersedMWCNT in 1 wt%PVP (dissolvedin LB) (b). TEM images ofmodifiedMWCNTs by surfactants in the aqueous solution (c). The red arrows indicate thewrappedsurfactants around tubes.

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[31] and Bai et al [27] reporting cationic surfactants arethemost toxic agents against bacteria at pH�7, whileanionic surfactants exhibit antibacterial activity atpH<7. In general, non-ionic surfactants do notexhibit the antibacterial activity. As a consequence, itcan be claimed that SDBS, SC and PVP in low con-centrations are appropriate solutions for the disper-sion of MWCNTs and further toxicity studies of thesetubes.

3.3. Antibacterial activity ofMWCNTsThe antibacterial activity of CNTs was assessed bymeasuring the growth curve of treated E. coli bacteriaby tubes at OD600 nm. Figure 4(a) compares the ODgrowth curves of treatedE. coliwith various concentra-tions of unmodified dispersed MWCNT in LB. SEMimages in figure 5 verify that incubated bacteria withMWCNTs could grow without any interruption. It isobvious that the unmodifiedMWCNTs does not causeany delay in bacterial growth. This non-antibacterialactivity of MWCNT can be ascribed to the largediameter of the tube compared to SWCNTs and theirpoor solubility in the suspension. This finding suggeststhat the appropriate dispersion of tubes plays a crucialrole in their interactionwith bacteria.

Due to the considerable susceptibility of SDBS todisperse MWCNTs compared to SC, it was chosen forfurther investigation of the antimicrobial activity of

MWCNTs. In addition, PVP as a biocompatible sur-factant was used to check the interaction of tubes withE. coli. E. coli in LB broth was incubated with 0.1, 0.5and 1 mgml−1 concentrations of dispersedMWCNTsin 1 wt% surfactant (dissolved in LB) (figure S7). TheOD growth curves for these samples are depicted infigures 4(b) and (c). As can be seen, the incubation ofE. coliwithMWCNT-SDBS causes the cells lysis for allapplied concentrations after 5 h incubation. However,treated E. coli with PVP-MWCNTs in concentrationsless than 1 mgml−1 does not cause apparent celldeath. The measured OD values higher than the con-trol for 0.5 mg ml−1 of MWCNT-PVP could be due tothe utilization of large amounts of MWCNTs causingan error in the measurement. Figure 5 indicates theSEM images of incubated E. coli bacteria with severalconcentrations of dispersed MWCNTs in LB, SDBS,and PVP. Nomajor damage is observed in the bacteriafor 0.1 and 0.5 mg ml−1 MWCNT-SDBS solutions(figures 5(d) and (e)). However, the destruction of cellmembranes occurred for the incubated E. coli with1 mgml−1 MWCNT-SDBS suspension shown infigure 5(f). It may be concluded that either thesedamages originate from the inherent antibacterialactivity of 1 wt% SDBS or that the penetration of bac-terial cell membranes by nanotubes caused the deathof the bacteria.

Figure 3.ODgrowth curves of incubated E. coliwith 1 wt%SDBS (anionic) (a), 1 wt%PVP (non-ionic) (b), and 1 wt%DTAB(cationic) (c).

Figure 4.ODgrowth curves of incubated E. coliwith pristineMWCNTs (a), SDBS-modifiedMWCNTs (b), and PVP-modifiedMWCNT (c).

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SEM images of the incubated bacteia with 0.1 and0.5 mg ml−1 of MWCNT-PVP depict no destructionof the E. coli membranes (figures 5(g) and (h)),whereas some bacteria membrane damages emergedby applying 1 mgml−1 MWCNTs-PVP to bacteria(figure 5(i)). The observed destruction in the lipidmembrane of bacteria may attribute to the coverage ofbacteria by well-dipersed tubes resulting in the celllysis which is depicted in figures 5(j)–(l). The redarrows in these images show the covered bacteria bydispersed MWCNTs. These results verify that well-dispersed MWCNTs by the biocompatable surfactant(PVP) can generate bacteria cell lysis because of theirdirect contact with the bacteria cell membranes. As aresult, the non-covalent modification of MWCNTsnot only improves the individualization of tubes butalso ameliorates the antimicrobial activity of tubes.

A comprehensive comparision was done betweenthe observed antibacterial activity of surfactants in thisresearch and other ones in the literature. From this

comparison, it can be concluded that non-ionic sur-factants do not show any antimicrobial activity againstgram-positive/negative bacteria. However, there is astrong interaction between ionic surfactants and bac-teria whichmight be due to the existence of an electro-static interaction between the hydrophilic head groupsof the surfactants and charged membranes of bac-teria [14, 27].

Furthermore, the observed nontoxicity effect ofMWCNT on bacteria agrees well with the previouslypublished results [27, 32, 33]. This feature of the tubesmight be attributed to the hydrophobicity of the tubesin polar solvents resulted in the aggregation of thetubes. According to the previous reports [14, 34],direct contact between tubes and bacteria cause thedamage to the membrane of bacteria and subsequentbacteria lysis. Moreover, It has been widely reportedthat pristine SWCNTs have more toxicity against bac-teria than pristine MWCNTs [33, 35, 36]. Thisphenomenon might be attributed to a variety of

Figure 5. SEM images of treated bacteria in 0.1 mg ml−1 (a), 0.5 mg ml−1 (b) and 1 mg ml−1 (c) LB-MWCNT, and treated bacteria in0.1 mg ml−1 (d), 0.5 mg ml−1 (e) and 1 mg ml−1 (f) SDBS-MWCNT, and treated bacteria in 0.1 mg ml−1 (g), 0.5 mg ml−1 (h) and1 mg ml−1 (i)PVP-MWCNT. SEM images of covered bacteria by dispersedMWCNTs in PVP (j), (k) and (l).

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reasons including (1) the large surface area of the tubesfor interacting with bacteria and (2) the smaller dia-meter of the tubes which facilitates piercing the cellmembranes of bacteria and further release of intracel-lular components of the cells.

In addition, modification of the tubes by surfac-tants enhanced the antibacterial activity of MWCNTswhich is in good agreement with the previous reports[27, 32]. This phenomonon might be related to thedevelopment of a direct contact between well dis-persed tubes and bacterial cells leading to the cellsdeath. As a consequence, it can be concluded that dis-persity of the tubes has significant effect on their anti-microbial activity.

4. Conclusion

In this study, the non-covalent functionalization ofMWCNTs using various ionic and non-ionic surfac-tants were successfully investigated. Furthermore, theantibacterial activity of these surfactants on E. coli waschecked. Although cationic surfactants could impress-ively lyse E. coli as a gram negative bacteria, anionicand non-ionic surfactants demonstrated desirablebiocompatibilities. Finally, the antibacterial activity ofwell-dispersed MWCNTs by biocompatable surfac-tants was studied. OD and SEM measurementsconfirmed the toxicity effects of well-dispersedMWCNTs. Anionic surfactant-MWCNTs displayedmore anibacterial activity than non-ionic surfactant-MWCNTs which may be attributed to the cooperatedtoxicity of the surfactant. The antimicrobial activity ofMWCNTs can be explained by the electrostatic forcesbetween the surfactant and the membrane of bacteriain addition to the penetration of the bacteria lipidmembrane by the dispersedMWCNTs.

Acknowledgments

This research was supported by European Center forEmergingMaterials and Processes Dresden. This workhas been partially financed by the Initiative andNetworking Fund of the German Helmholtz Associa-tion, Helmholtz International Research School forNanoelectronic Networks NanoNet (VH-KO-606).Finally, we acknowledge Mark Hermann RümmeliandRafael GregorioMendes for fruitful discussions.

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