nanotechnology

ARTICLEReceived6Mar2012|Accepted29Oct2012|Published27Nov2012Carbonnanotubeliposomesupramolecularnanotrainsforintelligentmolecular-transportsystemsEijiroMiyako1, KenjiKono2, EijiYuba2, ChieHosokawa1, HidenoriNagai1&YoshihisaHagihara1Biological network systems, such as inter- and intra-cellular signalling systems, are handled ina sophisticated manner by the transport of molecular information. Over the past few decades,there has been a growing interest in the development of synthetic molecular-transportsystems. However, several keytechnologieshavenot beensufcientlyrealizedtoachieveoptimumperformanceoftransportationmethods. Hereweshowthatanewtypeofsupra-molecular systemcomprisingof carbonnanotubesandliposomesenablesthedirectionaltransportandcontrolledrelease ofcarriermolecules,andallows anenzymaticreactionatadesiredarea. Thestudy highlights important progress that has beenmade towards thedevelopmentofbiomimeticmolecular-transportsystemsandvariouslab-on-a-chipapplica-tions, such as medical diagnosis, sensors, bionic computers and articial biological networks.OPENDOI:10.1038/ncomms22331HealthResearchInstitute(HRI), National InstituteofAdvancedIndustrial ScienceandTechnology(AIST), 1-8-31Midorigaoka, Ikeda, Osaka563-0026,Japan.2DepartmentofAppliedChemistry, OsakaPrefectureUniversity, 1-1Gakuen-cho, Naka-ku, Sakai, Osaka599-8531, Japan. CorrespondenceandrequestsformaterialsshouldbeaddressedtoE.M. (email:e-miyako@aist.go.jp).NATURECOMMUNICATIONS | 3:1226 | DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications 1&2012MacmillanPublishersLimited.Allrightsreserved.Human activities (for example, thought, emotion andbehaviour) are directly formed by biological networksystems. Biological networksystemssuchasinter/intra-cell-signalling systems are handled in a sophisticated manner bythemoleculartransportofmolecularinformation(low-molecu-lar-weight compounds, proteins and genes and so on)1. Over thepast few decades, there has been a strong interest in the develop-ment of molecular-transport systems using microorganisms2,naturally occurring biomolecular motors3,4and syntheticmicro-nanoscale robots (micro-nanobots)58. However, thefollowing key technologies are not sufciently developed torealizeoptimumperformancefromthetransportationmethod:directional propagationof transporters4,911, controlledrelease8of carrier molecules and transmission/reception systems(articial molecular signalling; expression of signicantbiochemical reactions in the targeted sites) for transferredcarrier molecules3,6.Nanomaterials are potentially promising for a numberof scientic and technological applications12. For the creation offunctional nanomaterialsforvariousnanobiosystems, molecularself-assembly technologies have attracted signicant attention, asthey are easily processed12. In particular, liposomes are one of themost well-knownself-assembledmaterials andhavenumeroususes as biochemical and biophysical tools because of their manyattractive properties, which includes encapsulating a varietyof molecules (drugs, peptides, proteins andgenes andsoon)andexhibitingdramaticconformationchanges duetoexternalphysical or chemical stimulations (pH, light and temperatureandso on)1316. On the other hand, carbon nanotubes (CNTs) offer aunique combination of electrical, mechanical, thermal and opticalproperties that make them highly promising materials fornumerousapplications1724. Wehavebeeninvestigatingtheuseof laser-induced CNTs as a potent photothermal nanomaterial invarious thermal applications1724. Of several photothermallyactive nanomaterials that were considered, CNTs are particularlyinteresting because of the extraordinarily high efciency oftheirphotothermalenergyconversionandhighlight-absorptioncross-section over a wide range of wavelengths1724. Thefunctionalizationof CNTsisthenext keystepfortheintegra-tion of these new materials into different systems fortechnological and biomedical applications25.Inthisstudy, wedemonstratethat(1)anelectrical train-likenanobotbasedonaCNTandtemperature-responsiveliposome(CNTliposome nanotrain) canbe formedby a self-assemblytechnology; (2) the CNTliposome nanotrains successfullyfunction in microdevices for various applications, such asthe solving of mazes and molecular transportations after theapplication of voltage and because of the photothermal propertyof CNT; and(3) theelectric- andlight-drivenCNTliposomenanotrains effectively illustratedthe enzymatic reactionat thetargeted position. Our newly developed molecular-transportsystem, which is based on the photoinduced-CNTliposomenanotrains, can easily trigger the unloading of molecules and anenzymaticreactioninatargetedareainanon-contactmanner.This is performedusingahighlyfocusedexternal laser beam.Our designed supramolecular nanotrains and the intelligentmolecular-transport system provide a starting point for theexplorationofmoresophisticatedbiomimeticmolecular-transportsystems withdirectionallycontrolledmotion, on-timecontrolledrelease of carrier molecules and various biochemical reactionsderived from the receipt of functional molecules at the desired area.ResultsCharacterization of nanotrains. To develop an intelligentmolecular-transport system, wesynthesizedtheCNTliposomenanotrains using a self-assembly technology (Fig. 1) (see Methodsfordetails). However, generally, CNTsarenotdispersedinsol-ventsbecauseoftheir strongtendency toaggregate1724. Hence,we employed a highly water-dispersible avidinpolyethyleneglycol-2,000 (PEG2,000, molecular weight of PEG2,000)phospholipid (PL)-functionalized-single-walled CNT (SWNT)(avidinPEG2,000PLSWNT) complex by the non-covalentapproach1724(Fig. 1a). The hydrocarbonchains of PLs wereadsorbed onto the graphitic surfaces of SWNTs via van der Waalsand hydrophobic interactions, whereas the hydrophilic PEGchains extended into the aqueous phase to impart solubility23. Onthe other hand, generally, the functional liposomes were preparedby labelling 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2,000] (biotinPEG2,000PL) andN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl phosphatidyl-ethanolamine (NBDPE) molecules on the surface of liposomes.In addition, the functional liposomes are composed ofdipalmitoylphosphatidylcholine, hydrogenated soybeanphosphatidylcholine (HSPC) andcholesterol (see Methods fordetails). Furthermore, the functional liposomes spontaneouslybind to SWNTs through the ligandreceptor interaction ofbiotinavidin, andNBDmolecules15onthe liposomes enablethetrackingof theCNTliposomenanotrainmovement inthemicrodevices as a uorescent tracer. Fluorescence microscopyanalysisshowedthattheBrownianmotionofgreenuorescentCNTliposomenanotrains(averagesize r1 mm)isobservedinaqueous solution (Fig. 1b). These visible green uorescentnanotrain particles are observed only when the SWNT andliposomearemixedinapredeterminedstoichiometry(Table1andFig. 2). WeinvestigatedthecompoundingratioofSWNTsand liposome for the synthesis of microscopically detectableuorescent nanotrains. Visible green uorescent particles areobserved only when the SWNT and liposome are mixedinaspeciccompoundingratio(Table1; entry1) (Fig. 2a,h).As a result, the entry 1 sample shows the maximum uorescenceintensity. On the other hand, visible black aggregations resultingfrom CNTs are formed in the cuvette in cases where there is a lowliposome-compoundingratio(Table1; entry2and3). This isbelieved to be caused by the precipitation of CNTs when a smallamount of liposomes form tightly roped bundles via the avidinbiotininteraction(Fig. 2b,c,i andj). Althoughthebackgrounduorescence intensity is relatively high, visible uorescentparticles are not observed in the case of a high liposome-compounding ratio (Table 1; entry 4), because of the lowsyntheticyieldof nanotrains(Fig. 2d,k). WecandetectneithertheavidinPEG2,000PLSWNTnorthefunctional liposomebyuorescence microscopy because of the detection limitationsof microscopy (Table 1; entries 5 and 6) (Fig. 2e,f,l and m). Also, amixedsolutionof thefunctional liposomeandNH2PEG2,000PLSWNTdoesnotexhibituorescencebecauseof incompletesupramolecularcomplexes(Table1; entry7)(Fig. 2g,n). Theseresultsclearlyindicatethat theoptimumconcentrationsof theavidinPEG2,000PLSWNT and the functional liposome are B5and 20,000 nM, respectively. Next, the CNTliposome nanotrainwas structurally characterized by transmission electronmicroscopy (TEM) (Fig. 3). TEM analysis of the CNTliposome nanotrainrevealedthat spherical liposomes (averagediameterE100 nm) are attachedonthe surface of anavidinPEG2,000PLSWNT nanobre (average diameterE10 nm,lengthE2001,000 nm), asshowninFig. 1a. Wealsoobservedthat the lipid-coated breform SWNTs became entangled on thesurfaceofliposomesandresultedintheformationofaciniformstructures. These results clearly show that the synthesizedCNTliposomenanotrainshavesupramolecularnanostructures,and their luminary uorescence facilitates the observationof microscopic uorescence.ARTICLE NATURECOMMUNICATIONS|DOI: 10.1038/ncomms22332 NATURECOMMUNICATIONS | 3:1226| DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications&2012MacmillanPublishersLimited.Allrightsreserved.Nanotrains movement inthemicrodevice. Theabilitytouseelectrical control for the direction and motion of CNTliposomenanotrains ina microdevice is illustratedinFig. 4. First, weprepared a polydimethylsiloxane (PDMS) microdevice based on astraightmicrochannel (width: 100 mm, depth: 50 mm)toinvesti-gate the microscale behaviour of CNTliposome nanotrainsbyapplyinga voltage (Fig. 4a,b). Toprevent the adhesionofnanotrains tothe surface of the microchannels, the fabricatedmicrodevicewastreatedwithahydrophilicpolymer(Methods).In addition, 20 wt% of dextran was introduced into themicrochannel to prevent the undesired permission of nano-trains toenter a microchannel causedby molecular diffusion(Methods). The synthesized CNTliposome nanotrains arenegatively charged in the 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid (HEPES) buffer (pH 7.1) (zetapotential 0.211.6 mV). In this way, the CNTliposomenanotrains can move to the positively charged goal position fromthe negatively charged start position by the application of voltage.Green uorescent CNTliposome nanotrains gradually movestraight ahead to the goal positionafter a voltage is applied(50 V), as indicated by uorescence microscopy (Fig. 4c andSupplementary Movie 1). Figure 2d shows the absolute andrelative speeds of the CNTliposome nanotrains in a micro-channel withthe applicationof different voltages andcurrentoutputs. Under these conditions, the nanotrains can reachabsolute speeds of B700 mms1, which correspond to a relativespeedof B1,200bodylengths(bl)persecond. Thus, wereportthe realization of fast, electric-driven nanobots with speedsthat are comparable to that of catalytic self-propelledmicro-engines (ca. 200 bl s1)5and some bacteria (ca. 50150 bl s1)5.Figure 2e reveals the uorescence intensity curves for themoved CNTliposome nanotrains with the application ofvarious voltages (50400 V). We found that there wasa signicant increase in the uorescence intensity ofnanotrainsasthevoltageoutput increased. Ontheotherhand,it is well known that the electrocataphoresis of DNAin amicro-nanodeviceisrapidbecauseofthesmallcavityinmicro-Table1|SynthesisofCNTliposomenanotrain.Entry Sampleconcentration(nM)FluorescenceintensityVisibleaggregationincuvetteSWNT Liposome1 5 20,000 18.577 2 9 3,600 5.276 3 10 400 7.387 4 1 36,000 14.08 5 10 0 6.714 6 0 40,000 5.244 7* 5 20,000 5.925 *Mixed solution of biotin-functionalized liposome and NH2PEG2,000PLSWNT (without avidinmolecules).2000CH33 CH3CH3H CH3HOHH HN+OPOO O-17H35OO17H35SHNNHOHHHOPOO O-OOOONONNHNO2OPOO O-OOOC15H31C15H31ON+OPO-OOaSWNTca. 1 nmca. 100 nmca. 10 nmAvidinPEG2000PLBiotin and NBD-F labelledtemperature-responsive liposomeca. 1,000~200 nmbCHN+OPOO OOOCCAvidinPEG2000PLDPPCCholesterolStructure of components of the avidinPEG2000PLSWNT:SHNNHOHHHOPOO OO2000PEGHNOOC17H35OC17H35OHNBiotinPEG2000PLHSPC: Positive chargeNBDPE: Negative charge: NBD (fluorescent tracer molecule)SWNTStructure of components of the biotin and NBD-F labelled liposome:: Biotin (ligand molecule)PEGHNOOPOO OOOC17H35C17H35OHNAvidin (receptor protein)NONNHNO2OPOO O15N+OPOOOOOOOC15H31C15H31Figure1|StructuralcharacterizationofCNTliposomenanotrains.(a)SchematicillustrationoftheCNTliposomenanotrain. (b)FluorescencemicroscopicimagesofCNTliposomenanotrains.Magnication: X100.Scalebars,10mm.NATURECOMMUNICATIONS|DOI:10.1038/ncomms2233 ARTICLENATURECOMMUNICATIONS | 3:1226 | DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications 3&2012MacmillanPublishersLimited.Allrightsreserved.nanochannels26. Hence, the speed of the CNTliposomenanotrain was investigated for comparison with that of therepresentative DNAmolecule (l phage DNA), which has anegative charge that is derived from phosphate backbone(zeta potential 16.30.82 mV) (Fig. 4f). In the presentsystem, theaveragespeedof theCNTliposome nanotrains isapproximately two times higher than that of l phage DNAmolecules. This maybe due tothe physical migrationbarrierof molecules in the dextran gel construct (Fig. 4g). Fromuorescencemicroscopy, theparticlesizesofthelphageDNAmolecules intercalatedwithGelRed(average sizeE10 mm) areobviously greater than those of CNTliposome nanotrains(average sizer1 mm) (Fig. 4h and Supplementary Movie 2).Furthermore, to realize the advanced electronic regulation ofnanotrains, we investigated the switchback control of theCNTliposome nanotrain movements in a microchannel(Fig. 4i). The directionof the nanotrains movement is easilycontrolled using this mechanical switchback feature in a mannersimilar tothat of anelectrictrain(Fig. 4j andSupplementaryMovie3). Theseresultscompletelyindicatethat electric-drivenCNTliposome nanotrains can be directionally moved in amicrodevicesimilartoelectrotaxis, whichisthemovement oforganismstowardsorawayfromasourceofelectrostimulationusing a biased-random-walk process27.Navigation system. Next, we investigated about a higher level ofmotion control of CNTliposome nanotrains (Fig. 5). The solvingof maze problems is relevant tothe everydayissues of urbantransportation and experimental psychology and is also one of themodel problems in network and graph theory and robotics2832.Inthis study, weposedseveral problems: ndingtheshortestpaths in the bifurcated microchannel (Fig. 5a,c), the simple maze(Fig. 5b,d and Supplementary Fig. S1) and the complicated maze(Fig. 5e,f) using biomimetic CNTliposome nanotrainelectrotaxis. Thesedevices have100-mm-wideand50-mm-deepmicrochannels (see Methods). Once in the maze, the CNTliposome nanotrains move towards the goal position; that is,towards the source of the electrical potential gradient. In doing so,the shortest path was always found through the bifurcatedmicrochannels (Fig. 5c and Supplementary Movie 4). The speed vof the CNTliposome nanotrains in the microchannel is given bythe following equation33:v mE mV L11wheremisthemobilityofthenanotrains, Eistheelectriceldstrength, Vis the applied voltage and L the length of themicrochannel. Thus, CNTliposomenanotrainsgotheshortestpathbecause vis indirectlyproportional toL. CNTliposomenanotrainscanbequicklymovedtothevarioustargetedgoalsEntry 1 Entry 2 Entry 3 Entry 4 Entry 5 Entry 6Entry 1 Entry 2 Entry 3 Entry 4 Entry 5 Entry 6Entry 7Entry 7Figure 2|Effect of SWNTand liposome concentrations on the nanostructure of CNTliposome nanotrains. (ag) Fluorescence micrographs of variousCNTliposome nanotrains in Table 1 (entry 17). Scale bars, 10mm. (hn) Schematic illustrations of various CNTliposome nanotrains in Table 1 (entry 17).Visible green uorescent nanotrain particles are observed only when the SWNTand liposome are mixed in a specic compounding ratio (entry 1 in Table 1).Graphicsdepicttheexpectednanotrainconformations(Fig.2hn). TheseresultsclearlyindicatethattheoptimumcompoundingratiooftheavidinPEG2,000PLSWNT andfunctionalliposomeis1:4,000forfurtherexperiments(entry1inTable1).Biotin and NBD-F labelled liposomesAbidin-PEG2,000-PL-SWNT complexesFigure3|TEMimagesofCNTliposomenanotrains.Sampleconcentrationfortheseobservations: SWNT5 nM,liposome 20,000nM.Scalebars,100nm.ARTICLE NATURECOMMUNICATIONS|DOI: 10.1038/ncomms22334 NATURECOMMUNICATIONS | 3:1226| DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications&2012MacmillanPublishersLimited.Allrightsreserved.(G1G3) from the start by alteration of the electrode position inthegoal (Fig. 5d; SupplementaryMovie 5andSupplementaryFig. S1). Surprisingly, CNTliposome nanotrains are also able tosolve the complicated maze and migrate smoothly along theshortest path withinB1.5 min without steepness or errors(Fig. 5f and Supplementary Movie 6). These results clearlydemonstrate that CNTliposome nanotrains have intelligentperformance navigation for solving mazes using an electricalpotential gradient.Molecular-transport system. One of the main goals of this studyis todemonstrate for the rst time the controlled(triggered)release of substrates from CNTliposome nanotrains (Fig. 6). Toaccomplishthechallenginggoal of thecontrolledreleasefromsynthetic CNTliposome nanotrains, we functionalized thesurfaceof theCNTliposomenanotrainswithamixed-bindinglissamine rhodamine Bsulfonyl phosphatidylethanolamine(RhPE) as a temperature indicator1821and encapsulated calceinas atransporting modelmolecule into the liposomes(Fig. 6a,b).The present functional liposome has atemperature-responsiveproperty that is designedtostructurally destruct at a tempe-rature of greater than 42 1C by adjusting these chemicalcomponents13,14. Recently, we have demonstratedthat photo-induced CNTs canoperate as exothermic nanomaterials1724.Therefore, laser-induced-CNTliposome nanotrains cantriggerthereleaseof calceinsfromwithintheliposomes, asshowninFig. 6a. When the moving CNTliposome nanotrain in themicrochannel was irradiated by a near-infrared (NIR) laser(wavelength: 1,064 nm, laser power: 700 mW (ca. 69 mWmm2),laser spot diameter: ca. 3.6 mm), we immediatelyobservedtheshiny greenuorescence causedby releasedcalceinmolecules(Fig. 6d and Supplementary Movie 7). The uorescence of calceinis not observed before laser irradiation because calcein moleculesinsidetheliposomesareselfquenchedathighconcentrations16.The controlled release of calceins fromthe CNTliposomenanotrains was achieved by the sequential on and offswitching of the NIRlaser. Figure 6e shows the uorescenceproles at four different laser powers (100, 300, 500 and 700 mW(10, 30, 49 and 69 mWmm2, respectively)). We found that theuorescenceintensityincreasessignicantlywiththeNIRlaserpower. Upon laser NIR irradiation, we estimated the temperatureof nanotrains utilizing Rh molecules on the liposomes (Fig. 6f andSupplementary Movie 8). The uorescence quenching of Rhfor the nanotrains inthe microchannel was observed almostimmediately (below0.03 s) uponNIRlaser irradiationandissimilar to our previous studies1821. In addition, the temperaturesat laser powers of 100, 300, 500and700 mW(10, 30, 49and69 mWmm2, respectively) increased from21 1Cto 22, 36,45and53 1C, respectively(Fig. 6e). TherewerenouorescentObjectiveS GGlass slideG SFluorescent intensity analyzing area45678B A+ B A+ Switch backA ABSwitch backB0100200300400500600700200 300Voltage (V) phage DNA intercalating GelRed(red fluorescence)ca. 2 nmS G27.2 mm+ y = 0.2668x + 5.0194 (400 V)y = 0.199x + 4.9234 (200 V)y = 0.0463x + 5.1004 (50 V): Speed : Current : 400 V : 200 V : 50 V010020030040050060070080050 100 200 300 400050100150200250300S G20.0 mm2.4 mm90 2 4 6 8 10 12Time (s)Direction of movementof CNTliposomenanotrains50 mCNTliposomenanotrainca. 15 mSpeed (m s1)Speed (m s1)Current (A)Fluorescent intensityVoltage (V): CNTliposome nanotrains: phage DNAPDMSFigure4|ElectricalcontrolofCNTliposomenanotrainmovement.(a)Schematicillustrationoftheexperimentsystem.S, start; G, goal. (b)Designdrawing of the microdevice based on a straight microchannel. Red arrows show the direction of CNTliposome nanotrain movement. (c) Movement of theCNTliposome nanotrains in a straight microchannel after applying a voltage. The white square shows the location at which the uorescence intensity ofthemovingCNTliposomenanotrainswasanalysed. Applyingavoltage 50V. Magnicationoftheinsetclose-upgure: X10. Scalebars,100mm.(d)SpeedoftheCNTliposomenanotrainsandcurrentinmicrochannelversusapplyingavoltage. (e)FluorescentintensitycurvesofthemovedCNTliposomenanotrainatvariousappliedvoltages(50400V). (f)Schematicillustrationofthesingle lphageDNAintercalatedwithGelRed molecules.(g) Fluorescence microscopic image of the l phage DNA molecules in a straight microchannel. Applying a voltage 200V. Magnication: X10. (h) Speedof the CNTliposome nanotrains and l phage DNA molecules in a straight microchannel after applying voltage. Scale bar, 100mm. (i) Switchback control ofthe CNTliposome nanotrain movement. Red arrows show the direction of CNTliposome nanotrain movement. (j) A direct observation of the switchbackmovementoftheCNTliposomenanotrainsbyuorescencemicroscopy.Magnication:X10. Applyingvoltage 300V.Scalebars,100mm.NATURECOMMUNICATIONS|DOI:10.1038/ncomms2233 ARTICLENATURECOMMUNICATIONS | 3:1226 | DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications 5&2012MacmillanPublishersLimited.Allrightsreserved.changes illustrating the release of calcein or changes intemperature during the control experiments without CNTs(only functional liposome).Articial molecular signalling. Molecular transportation isinspiredby the observationthat communicationinbiologicalsystems, such as inter/intra-cell signalling, is done throughmolecules1. Toconstruct thesebiomimeticmolecular-transportsystems, themostimportant researchchallengesaredirectionalpropagation, the controlled release of carrier molecules andtransmission/reception systems for carrier molecules. As a result,we investigated a transmission/reception systemarticialmolecular signallingin which the enzymatic reaction wastriggeredbythelaser-induced-CNTliposomenanotrainsatthetargeted position (Fig. 7). The concept of this articial molecularsignalling is illustrated in Fig. 7a. First, the CNTliposomenanotrains encapsulating uorescein digalactoside (FDG) that actas transporters of signal molecules are transferred to the targetedgoal reservoir (site G) fromthe start position (site S) bythe applicationof voltage. Next, directionally migratedCNTliposome nanotrains insite Gare irradiatedbyanNIRlaser(1,064 nm), and FDG molecules are released at the targeted area.It is well known that FDGis a uorogeneic substrate forb-galactosidase34. Hence, non-uorescent FDGis sequentiallyhydrolysed by the enzymes in site G, and it then transformed tohighlyuorescent uorescein34. We canobserve bright-green-coloureduorescenceinsiteGthatiscausedbytheenzymaticreactionas soonas 700-mW(69 mWmm1) NIRirradiationbegins (Fig. 7bandSupplementary Movie 9). There were nochanges in the uorescent observations of the enzymatic reactionwhenthe control experiments without CNTs (onlyfunctionalliposome)wereperformed. Weconcludethat the laser-induced-CNTliposome nanotrains permit the controlled release ofsubstances and the triggered enzymatic reaction through themolecular-transport system, based on the nanotrains electrotaxisand the powerful photothermal property of CNT nanoheaters.DiscussionWe successfully developed a novel supramolecular nanotrainbased on CNTs and liposome using the self-assembly technique.The nanotrain enables both directional migration and the solvingof mazes in an electric eld. In addition, the controlled release ofsubstratesandatriggeredenzymaticreactionweresuccessfullyachieved at a targeted position using laser irradiation. This studyrepresents an important advance in the creation of programmableand adaptable nanobiosystems. The application of micro-nanobots inmicro-nanofabricatedenvironments is anexcitingdevelopment211. Our technology is alsogoing tooffer novelpossibilities for future developments in lab-on-a-chip appli-cations, such as medical diagnosis, sensors, bionic computers andarticial biological networks. In particular, our systemwillprovide a simulation tool for potential in vivo medical2.4 mm2.0 mm8.0 mm1.0 mmG S9.8 mmSG1 G2G3+ +G1S GS2.4 mm4.8 mmSG+2.4 mm11.0 mm15.0 mmSGFigure5|NavigationsbyCNTliposomenanotrains.(a,b,e)Designdrawingsofthemicrodevicesbasedonbifurcated, simple-mazeandcomplicated-maze microchannels, respectively. Red arrows show the direction of CNTliposome nanotrain movement. Green and pink lines indicate the shortest and thelongestpaths,respectively.S, start; G, goal;G1, goal1; G2,goal2; G3, goal3.(c)Adirectobservationofthenanotrainsbehaviourinthebifurcatedmicrochannelafter applyingavoltage(300V). ImageofthenanotrainmovementshowingtheshortestpathbetweenSandG. Scalebar,100mm.(d) A direct observation of the nanotrain behaviour in the simple-maze microchannel after applying a voltage (300V). Image of the nanotrain movementshowingtheroutefortheshortestpathinthemaze. Scalebar,100mm.(f)Adirectobservationofthenanotrainbehaviourinthecomplicated-mazemicrochannelafter applyingvoltage(300V). ImageofthenanotrainmovementshowingthequickestroutefromStoGforamore complexmaze.Scalebar,500mm.ARTICLE NATURECOMMUNICATIONS|DOI: 10.1038/ncomms22336 NATURECOMMUNICATIONS | 3:1226| DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications&2012MacmillanPublishersLimited.Allrightsreserved.applications, suchas remote controlling of untetheredmicro-nanobots in blood vessels. We believe that these kinds ofsimulationareveryuseful for development of advanceddrug-delivery systems and nano-surgery of biological system by the useof micro-nanobots.MethodsSynthesisofCNTliposomenanotrains. The avidinPEG2,000PLSWNT wassynthesized as follows. SWNT (2.5 mg) (high-pressurecarbon monoxide, super-puried SWNTs (purity495%); Unidym) and 3-(N-succinimidylocyglutaryl)aminopropyl, polyethyleneglycol carbamyl distearoylphosphatidylethanolamine(NHSPEG2,000PL) (5 mg) (molecular weight of PEG chain 2,000; NOF) weresonicated for 15 min in PBS buffer (pH 7.3) (5 ml) on ice (o8 1C) in anultrasonication bath (USD-2R; AS ONE) to obtain a uniform dispersion. TheNHSPEG2,000PLSWNT solutions were centrifuged (10,000 g, 15 min, 4 1C)(3500; Kubota). The supernatant was carefully collected after centrifugation.The superuous NHSPEG2,000PL was ltered though a polytetrauoroethylenemembrane (pore size 100 nm; Millipore) washed with PBS buffer (50 ml).The black-coloured NHSPEG2,000PLSWNT sheet on a polytetrauoroethylenemembrane was dispersedin PBS buffer (5 ml) by sonication for a few minutes.Avidin (1 mg) (Wako) was dispersed in a NHSPEG2,000PLSWNT solution(5 ml). After the mixture was vigorously stirred for 3 h at room temperature, it wasltered to remove the unreactedavidin and washed with HEPES buffer (pH 7.1)(50 ml). The obtained avidinPEG2,000PLSWNT sheet was dispersedin theHEPES buffer (5 ml) by sonication. The quantity of SWNTs dispersed in theHEPES buffer by avidinPEG2,000PL molecules was determined through weightmeasurements by the thorough washing and removal of avidinPEG2,000PL andwas found to be ca. 200 mg SWNT per ml HEPES buffer (ca. 1 mM). The amino-terminatedPEG2,000PL-functionalized SWNT (NH2PEG2,000PLSWNT) wassynthesized in the same way as the avidinPEG2,000PLSWNT, with the exceptionof the functionalization of avidin, exceptfor the process by which avidin ismodied. The functional liposomes were synthesized as follows. A mixture ofdipalmitoylphosphatidylcholine (Sigma), HSPC (NOF), cholesterol (Sigma),biotinPEG2,000PL (molecular weight of PEG chain 2,000; Avanti Polar Lipids)and RhPE (Avanti Polar Lipids) or NBDPE (Avanti Polar Lipids) in the molarratio of 58.2:29.1:11.6:0.5:0.6was dissolved in chloroform, and the solvent wasremoved by evaporation to afford a thin mixed-lipid membrane. The HSPC wasgenerously donated by NOF (Tokyo, Japan). The obtained membrane was furtherdried under vacuum conditions and dispersed in a 20 mM HEPES-buffered solu-tion containing 150 mM NaCl (pH 7.4) (HBS) by 10 min sonication using a bath-acbdG S+ e fGBiotin and Rh-labelled temperature-responsive liposome containing calcein: Rh (temperature indicator) : Calcein (transporting molecule)CalceinRhPELaseronLaseroffLaseronLaseronLaseroffLaseroffLaser onLaser off700 mW (69 mW m2): ca. 53 C500 mW (49 mW m2): ca. 45 C300 mW (30 mW m2): ca. 36 C100 mW (10 mW m2): ca. 22 C< With CNT >< Without CNT >700 mW (69 mW m2): ca. 21 CNIR laser (1,064 nm)Laser on2500 5 10 15 20Time (sec)Fluorescent intensityof calceinONN+O OHS O ONHOPOO OO C15H31OOOC15H31OOOHO OHNOOHO OHNHO OOHOGlass slide50 mDirection of movementof CNTliposomenanotrainsObjectivePDMSNIR laser (1,064 nm)CNTliposomenanotrainS21017013090Figure 6|Molecular-transport system by CNTliposome nanotrains. (a) Concept of molecular transportation by CNTliposome nanotrains. (b) Chemicalstructure of calcein and RhPE. (c) Design drawing of the microdevice based on a straight microchannel for molecular transportation. Black arrows showthedirectionofCNTliposomenanotrainmovement.S, start; G, goal. (d)Sequentialcalceinreleasebylaser-induced-CNTliposomenanotrains.Thewhitesquareshowsthelocationatwhichtheuorescenceintensityofreleasedcalceinmoleculeswasanalysed. Magnication:X20. Applyingvoltage 100V. Scalebars, 100mm. (e)Fluorescenceintensitiesofthereleasedcalceinmoleculesatvariouslaserpowers(100700mW(1069mWmm2)).(f)Adirectobservationofultrafasttemperaturechangeinthemicrochannel.ThewhitesquareindicatesthelocationatwhichthetemperaturewasanalysedinFig.4e. ThewhitearrowindicatestheuorescencequenchingofRhbythephotothermalpropertyofCNTs.Magnication: X20.Laserpower: 700mW(69mWmm2). Applyingvoltage 100V.Scalebars,100mm.NATURECOMMUNICATIONS|DOI:10.1038/ncomms2233 ARTICLENATURECOMMUNICATIONS | 3:1226 | DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications 7&2012MacmillanPublishersLimited.Allrightsreserved.type sonicator at B65 1C. The obtained liposome suspension was extruded througha polycarbonate membrane (pore size 100 nm; Advantec). The lipid concentra-tion was determined by the choline oxidase method (Phospholipid C Test Wako;Wako). For the loading of calcein (Wako) or FDG (Molecular Probes), a 63-mMcalcein solution (pH 7.4) or HBS containing FDG (1 mg ml1) was used for thehydration of the lipid membrane. The free calcein or FDG from the liposomesuspension was removed using a Sepharose 4B column (GE Healthcare) withHBS. Finally, the CNTliposome nanotrains were synthesized by mixing theavidinPEG2,000DSPESWNT with the functional liposomes. The compoundingratio of the SWNT and liposome is 1:4,000 (Table 1).CharacterizationofCNTliposomenanotrains. The CNTliposome nanotrainswere characterized using a uorescence microscope (IX71; Olympus) equippedwith an electron-multiplying charge-coupleddevice (EM-CCD) camera(PhotonMAX: 1024B; Princeton Instruments),a TEM (Tecnai G2F20; FEI)(acceleration voltage: 120 kV) and a zeta potential analyser (Nicomp 380 ZLSdynamic light-scattering instrument; Particle Sizing Systems). The TEM observa-tions were supported by the IBEC Innovation Platform, AIST. Before the TEMobservation, the CNTliposome nanotrain in the PBS buffer (pH 7.3) wasdeposited on a carbon-coated support and negatively stained using a 1% uranylacetate solution.Microdevices. The four varieties of microdevices were based on straight,bifurcated, simple-maze and complicated-maze microchannels (width: 100 mm,depth: 50 mm) and fabricated by soft lithography and photolithography methods.First, the PDMS (Sylgard 184; Dow Corning) base and crosslinker(Sylgard 184;Dow Corning) were mixed at a weight ratio of 10:1, as specied by the manu-facturer. Second, the mixture was subjected to shear mixing to ensure the uniformblending of the host matrix and the crosslinker. Next, the mixture was degassed invacuum. A master was prepared by exposing and developing a photoresist patternon a silicon wafer (SU-8 50; MicroChem). The PDMS/crosslinker mixture waspoured onto the master. After curing, the PDMS substrate was peeled away fromthe master. Then, the PDMS sheet was punched with holes (diameter: 3 mm)to produce reservoirs. The PDMS and glass (Matsunami) substrates were closelybonded to each other by plasma treatment using a vacuum plasma coater (PDC210;Yamato). Finally, the microchannels were hydrophilized using a water-solublepolymer (lipidure CR1705; NOF). The lipidure was diluted 50 times with ethanoland introduced into the microchannels with a syringe or by decompressiontreatment. After several minutes, the microchannels were dried using a syringe.Observationofnanotrainmovementinamicrochannel. The 20 wt% dextran(molecular weight 100,000200,000; Polysciences)was dissolved in the HEPESbuffer (pH 7.3), and it was introduced into the microchannels with a syringe or bydecompression treatment. Then, the dextran solution in the start position reservoirwas replaced by the CNTliposome nanotrain solution (ca. 20 ml). The platinumelectrodes (diameter: 600 mm; Nilaco) were placed in both start and goal positions,and a voltage (50400 V) was applied to the microchannel using a high-voltage-power-supply system (HVS448; LabSmith). The nanotrains movement in themicrochannel was observed by uorescence microscopy (IX70; Olympus) with acolour CCD video camera (VB-7010; Keyence). The speed of the nanotrainswas analysedby image-analysis software (ImageJ; NIH). The switchback of thenanotrain movement was controlled by the software (sequence; Labsmith).The movement of l phage DNA (32 mDa, 48502 bp; Toyobo) molecules in amicrochannel was monitored in a manner similar to that in nanotrains. Before theexperiments, the DNA solution (90 ml, 16 mg ml1) was mixed with 1 GelRedsolution (10 mL) (Wako) as an intercalator.Laserexperiments. The directional molecular transport, the temperaturedistribution in the microchannel and the articial molecular-signalling experimentsusing the laser-irradiation set-up were performed as follows. The generalprocedures for these experiments are illustrated in Figs 4a and 5a. A 1,064-nm NIRlaser beam from a continuous-wave YVO4 laser (BL106-C; Spectra Physics)(100700 mW (1069 mWmm2)) was incorporated into the uorescencemicroscopy set-up (IX71; Olympus), as described previously35. The laser beam wasfocused into the moved nanotrains in a straight microchannel with the objective(magnication: X20, numerical aperture 0.4; Olympus) during the application ofvoltage (50300 V). The uorescence images before and during the laser irradiationwere recorded using an EM-CCD camera. The sequential calcein releaseexperiment was performed by manual laser onoff switching. The temperaturedistribution around the laser spot was determined from the uorescence intensitydistribution of Rh molecules on the liposomes. The control experiment for thetemperature assay was also studiedby introducing the biotin- and Rh-labelledliposome containing calcein without the CNT (1 mM) in the microchannel with asyringe. The uorescence intensities of calcein, Rh and uorescein were analysed byImageJ. The articial molecular-signalling experiment was performed by settingb-galactosidase from Escherichia coli (0.5 mg ml1; Roche)/HEPES (pH 7.1)solution (ca. 20 ml) in the goal position reservoir.References1. Alberts, B. et al. Essential Cell Biology,3rd edn (Garland, 2009).2. Weibel, D. B. et al. Microoxen: microorganisms to move microscale loads.Proc. Natl Acad. Sci. USA 102, 1196311967 (2005).3. Fischer, T., Agarwal, A. & Hess, H. A smart dust biosensor powered by kinesinmotors. Nat. Nanotech. 4, 162166 (2009).4. van den Heuvel, M. G. L., de Graaff, M. P. & Dekker, C. Molecular sortingby electrical steering of microtubules in kinesin-coated channels. Science 312,910914 (2006).+ GMovement= :-Gal =HydrolysisFDGO O HOCOOHFluoresceinS O O OOOOHOHHOHOOOHOHOHOHNIR laser (1,064 nm)Ga b= : =Laser onLaser onSSite S Site GSite GFigure 7|Articial molecular signalling by CNTliposome nanotrains. (a) Concept of articial molecular signalling. Black arrows show thedirection ofCNTliposome nanotrain movement. (b) Direct observation of enzymatic reaction triggered by articial molecular signalling. The white arrow indicates theirradiationpositionofthelaserbeam.Magnication:X20. Laserpower: 700mW(69mWmm2). Applyingvoltage 100V. Scalebars, 200mm.ARTICLE NATURECOMMUNICATIONS|DOI: 10.1038/ncomms22338 NATURECOMMUNICATIONS | 3:1226| DOI: 10.1038/ncomms2233 | www.nature.com/naturecommunications&2012MacmillanPublishersLimited.Allrightsreserved.5. Sanchez, S., Ananth, A. N., Fomin, V. M., Viehrig, M. & Schmidt, O. G.Superfast motion of catalytic microjet engines at physiological temperature.J. Am. Chem. Soc. 133, 1486014863 (2011).6. Wu, J. et al. Motion-based DNA detection using catalytic nanomotors. Nat.Commun 1, 36 (2010).7. Paxton, W. F., Sundararajan, S., Mallouk, T. E. & Sen, A. Chemical locomotion.Angew. Chem. Int. Ed. 45, 54205429 (2006).8. Orozco, J. et al. Dynamic isolation and unloading of target proteins byaptamer-modied microtransporters. Anal. Chem. 83, 79627969 (2011).9. Tamaru,S. et al. Fluidic supramolecular nano- and microbres as molecularrails for regulated movement of nanosubstances.Nat. Commun. 1, 20 (2010).10. Wickham, S. F. J. et al. Direct observation of stepwise movement of a syntheticmolecular transporter. Nat. Nanotech. 6, 166169 (2011).11. Kudernac, T. et al. Electrically driven directional motion of a four-wheeledmolecule on a metal surface. Nature 479, 208211 (2011).12. Steed, J. W. & Gale, P. A. Supramolecularchemistry: From molecules tonanomaterials. (Wiley-VCH, 2012).13. Kono, K. Thermosensitive polymer-modied liposomes. Adv. Drug Deliv. Rev.53, 307319 (2001).14. Gaber, M. H., Hong, K., Huang, S. K. & Papahadjoponlos, D. Thermosensitivesterically stabilized liposomes: Formulation and in vitro studies on mechanismof doxorubicin release by bovine serum and human plasma. Pharm. Res. 12,14071416 (1995).15. Yuba, E., Harada, A., Sakanishi, Y. & Kono, K. Carboxylated hyperbranchedpoly(glycidol)s for preparation of pH-sensitive liposomes. J. Control. Release149, 7280 (2011).16. Kono, K., Igawa, T. & Takagishi, T. Cytoplasmic delivery of calcein mediated byliposomes modied with a pH-sensitive poly(ethylene glycol) derivative.Biochim. Biophys. Acta 1325, 143154 (1997).17. Miyako, E. et al. Photothermic regulation of gene expressiontriggered bylaser-induced carbon nanohorns. Proc. Natl Acad. Sci. USA 109, 75237528 (2012).18. Miyako, E. et al. A photo-thermal-electrical converter based on carbonnanotubes for bioelectronic applications. Angew. Chem. Int. Ed. 123,1247412478 (2011).19. Miyako, E., Nagata, H., Hirano, K. & Hirotsu, T. Carbon nanotubepolymercomposite for light-driven microthermal control. Angew. Chem. Int. Ed. 47,36103613 (2008).20. Miyako, E., Nagata, H., Hirano, K. & Hirotsu, T. Micropatterned carbonnanotube-gel composite as photothermal material. Adv. Mater. 21, 28192823(2009).21. Miyako, E., Nagata, H., Hirano, K. & Hirotsu, T. Laser-triggered carbonnanotube microdevice for remote control of biocatalytic reactions. Lab. Chip 9,788794 (2009).22. Miyako, E., Itho, T., Nara, Y. & Hirotsu, T. Ionic liquids on photoinducednanotube composite arrays as a reaction medium. Chem. Eur. J. 15, 75207525(2009).23. Miyako, E., Nagata, H., Hirano, K. & Hirotsu, T. Photodynamicthermoresponsive nanocarbonpolymer gel hybrids. Small 4, 17111715 (2008).24. Miyako, E. et al. Near-infrared laser-triggeredcarbon nanohorns for selectiveelimination of microbes. Nanotechnology 18, 475103 (2007).25. Karchemski,F., Zucker, D., Barenholz, Y. & Regev, O. Carbon nanotubesliposomes conjugate as a platform for drug delivery into cells. J. Control. Release160, 339345 (2012).26. Sparreboom, W., van den Berg, A. & Eijkel, J. C. T. Principles and applicationsof nanouidic transport. Nat. Nanotech. 4, 713720 (2009).27. van West, P., Appiah, A. A. & Gow, N. A. R. Advances in research on oomyceteroot pathogens. Physiol. Mol. Plant. Pathol. 62, 99113 (2003).28. Fuerstman, M. J. et al. Solving mazes using microuidicnetworks. Langmuir19, 47144722 (2003).29. Lagzi, I., Soh, S., Wesson, P. J., Browne, K. P. & Grzybowski, B. A. Maze solvingby chemotactic droplets. J. Am. Chem. Soc. 132, 11981199 (2010).30. Steinbock, O., Toth, A. & Showalter, K. Navigating complex labyrinths: optimalpaths from chemical waves. Nature 267, 868871 (1995).31. Reyes, D. R., Ghanem, M. M., Whitesides, G. M. & Manz, A. Glow discharge inmicrouidic chips for visible analog computing. Lab Chip 2, 113116 (2002).32. Nakagaki, T., Yamada, H. & Toth, A. Maze-solving by an amoeboid organism.Nature 407, 470 (2000).33. Li, S. F. Y. Capillary Electrophoresis: Principles, Practiceand Applications.(Elsevier, 1992).34. Arata, H. F., Rondelez, Y., Noji, H. & Fujita, H. Temperature alternation by anon-chip microheater to reveal enzymatic activity of b-galactosidase at hightemperatures.Anal. Chem 77, 48104814 (2005).35. Hosokawa, C., Kudoh,S. N., Kiyohara, A. & Taguchi, T. Optical trapping ofsynaptic vesicles in neurons. Appl. Phys. Lett. 98, 163705 (2011).AcknowledgementsThis work was supported by The Kao Foundation for Arts and Sciences, The Japan PrizeFoundation and Grant-in-Aidfor Young ScientistsB (23700568). We are grateful toNOF (Tokyo, Japan) for providing the HSPC. We also thank Dr Kazunori Kawasaki(HRI, AIST) and Dr Takeyuki Uchida (IBEC centre, AIST) for TEM observations,Dr Hideya Nagata (Advanced Scientic Technology & Management Research Institute ofKyoto) for fruitful discussions, Mrs Keiko Nishio (HRI, AIST) and Ms Nahoko Naruishi(HRI, AIST) for assisting experiments.AuthorcontributionsE.M. conceived, designed, performedthe experiments and also analysed the data. K.K.and E.Y. synthesizedthe liposomes. C.H. constructed the laser-irradiation system. E.M.prepared the paper. All authors discussed the results and contributed to the manuscript.AdditionalinformationSupplementary Informationaccompanies this paper at http://www.nature.com/naturecommunicationsCompeting nancialinterests: The authors declare no competing nancial interests.Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/How to cite this article: Miyako E. et al. Carbon nanotubeliposome supramolecularnanotrains for intelligent molecular-transportsystems. Nat. Commun. 3:1226doi: 10.1038/ncomms2233 (2012).This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike3.0UnportedLicense. 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