Nonionic, Water Self-Dispersible “Hairy-Rod” Poly( p -phenylene)- g -poly(ethylene glycol) Copolymer/Carbon Nanotube Conjugates for Targeted Cell Imaging

Nonionic, Water Self-Dispersible “Hairy-Rod”Poly(p‑phenylene)‑g‑poly(ethylene glycol) Copolymer/CarbonNanotube Conjugates for Targeted Cell ImagingMerve Yuksel,† Demet Goen Colak,‡ Mehriban Akin,† Ioan Cianga,‡,§ Manolya Kukut,‡ E. Ilker Medine,∥

Mustafa Can,⊥ Serhan Sakarya,# Perihan Unak,∥ Suna Timur,† and Yusuf Yagci*,‡,○

†Department of Biochemistry, Faculty of Science, ∥Institute of Nuclear Sciences, and ⊥Institute of Solar Energy, Ege University, Izmir35100, Turkey‡Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, 34469, Istanbul, Turkey§Petru Poni” Institute of Macromolecular Chemistry, Iasi 700487, Romania#Department of Infectious Diseases and Clinical Microbiology, Adnan Menderes University School of Medicine, 09100, Aydin,Turkey○Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

*S Supporting Information

ABSTRACT: The generation and fabrication of nanoscopic structures are of criticaltechnological importance for future implementations in areas such as nanodevices andnanotechnology, biosensing, bioimaging, cancer targeting, and drug delivery.Applications of carbon nanotubes (CNTs) in biological fields have been impededby the incapability of their visualization using conventional methods. Therefore,fluorescence labeling of CNTs with various probes under physiological conditions hasbecome a significant issue for their utilization in biological processes. Herein, wedemonstrate a facile and additional fluorophore-free approach for cancer cell-imagingand diagnosis by combining multiwalled CNTs with a well-known conjugatedpolymer, namely, poly(p-phenylene) (PP). In this approach, PP decorated withpoly(ethylene glycol) (PEG) was noncovalently (π−π stacking) linked to acid-treatedCNTs. The obtained water self-dispersible, stable, and biocompatible f-CNT/PP-g-PEG conjugates were then bioconjugated to estrogen-specific antibody (anti-ER) via−COOH functionalities present on the side-walls of CNTs. The resulting conjugates were used as an efficient fluorescent probefor targeted imaging of estrogen receptor overexpressed cancer cells, such as MCF-7. In vitro studies and fluorescencemicroscopy data show that these conjugates can specifically bind to MCF-7 cells with high efficiency. The represented resultsimply that CNT-based materials could easily be fabricated by the described approach and used as an efficient “fluorescent probe”for targeting and imaging, thereby providing many new possibilities for various applications in biomedical sensing and diagnosis.

■ INTRODUCTION

The healthcare is one of the most challenging areas for humanbeings and is directly related to our well-being. Every stepattempted toward resolving health-related issues is highly vital.As known, today’s widespread threat for health is the cancerdisease and a great deal of research has been devoted towardthe early detection of cancer cells before the anatomicabnormalities are observed. In this sense, it is important tounderstand the dynamics and mechanisms of cellular processesand detection of targeted molecules in live cells. Locally,biomarking of cells is of great significance for both diagnosisand therapeutic aspects. Therefore, development of newmaterials and methods for early detection and cure of diseasesusing easy and cheap tools are still major challenges. Onepromising strategy to overcome these challenges is the use ofnanomaterials. The intense interest in these materials for theirin vivo utilization is due to their unique properties such as large

specific capacity for drug loading,1 strong superparamagnetism,2

efficient photoluminescence,3 or distinctive Raman signatures.4

Their other fascinating properties are afforded by their smallsize, providing a large specific surface or interface area tovolume ratio. Materials with sizes in the range of 20−200 nmcan avoid renal filtration, leading to extended duration time inthe bloodstream that allows more effective targeting of diseasedtissues.3 They provide an effective bridge between bulkmaterials and atomic or molecular structures. It is reasonableto expect nanoparticles with given sizes and functionalities toperform specific tasks at the subcellular scale in wholeorganism, as long as safety is ensured.5 Moreover, althoughthe development of biomaterials for drug delivery, tissue

Received: April 21, 2012Revised: August 2, 2012Published: August 3, 2012

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engineering, and medical diagnostics is traditionally based onnew chemistries; the physical attributes such as size, shape,surface texture, and compartmentalization are crucial tobiological functions.6

Molecular imaging by optical techniques, especially fluo-rescence monitoring, plays important roles in the study ofcellular processes and complex biological interactions owing tohigh spatial resolution and detection sensitivity.7,8 So, it isessential to design novel nanostructures that possess multiplefunctionalities such as biocompatibility, water solubility/dispersibility, fluorescent signaling, coupling to biologicalmolecules like DNA/RNA, antibodies, and so on, andnontoxicity. Commonly used fluorescent labeling agentsinclude conventional classes of organic fluorophores (organicdyes, fluorescent proteins, etc.) and also newer types ofpolymeric nanoparticles or inorganic nanoparticles such asquantum dots (QDs). Each class has its own advantages anddrawbacks.9 For instance, conventional ones are easily availableat low cost, small in size, and highly water-soluble at certain saltconcentrations. However, they have several limitations due totheir intrinsic properties like broad spectrum profiles, lowphotobleaching thresholds, poor photochemical stability, anddecomposition under repeated excitations along with theirtoxicity. Polymeric π-conjugated fluorophores are moretractable, stable, and less toxic with sufficient fluorescenceefficiencies when compared to the conventional ones. Thenewer classes have the potential to overcome some of theproblems associated with the former class. Presently, bestavailable QDs are made of CdSe cores coated with a layer ofZnS. They feature intense fluorescence emission and highphotostability, but despite these desirable photophysicalproperties, there exist several limitations related to their use.Optical blinking10−12 makes their applications difficult inquantitative assays and their size may affect the function ofattached molecules.13 Furthermore, QDs themselves are notbiocompatible and have to be surface-modified prior to theiruse in live cells.14−16 The most significant challenge thatremains is their disposal and toxicity as they are constructedfrom toxic elemental materials, namely, restricted heavymetals.14,16 As a result, QDs offer superiorities with respectto organic fluorophores but at the cost of decreased safety.The field of polymeric nanoparticles is rapidly expanding and

playing a central role in a wide spectrum of areas ranging fromelectronics to photonics, conducting materials to sensors,medicine to biotechnology, pollution control to environmentaltechnology, and so forth.17 Also, the rise in the interdisciplinarystudy of π-conjugated polymers (CPs), essentially in parallelwith nanoscience, has led to major advances in the fundamentalunderstanding of their diverse electronic, optoelectronic, andphotonic properties while enabling their applications inbiomedical technology.18 Conjugated polyelectrolytes (CPEs)are conjugated polymers decorated with ionic side-chains thatprovide water solubility for the biological applications.However, these charges may cause nonspecific interactionsincreasing the background noise or leading to false readings aswell as affecting the sensor sensivity.19−21 The use of water-soluble/dispersible CPs with nonionic and biocompatible side-chains would significantly reduce such interactions.Among the numerous above-mentioned nanoscale systems,

carbon nanotubes (CNTs) hold great potential for diverseapplications and are becoming a viable component ofbiomedical science. Various biological applications of CNTshave been proposed and actively explored in the past few years

leading to the emergence of a new field in diagnostics andtherapeutics.22−25 One of the key advantages of CNTs’ inbiomedical applications is that they can be easily internalized bycells and, therefore, can act as delivery vehicles for a variety ofmolecules relevant to therapy and diagnosis.24 Moreover, theirunique electrical, thermal, and spectroscopic properties in abiological context offer further advances in the detection,monitoring, and therapy of diseases.26,27 Single-walled (SW) ormultiwalled (MW) carbon nanotubes are now produced insubstantial quantities for a variety of commercial applications.28

As-produced carbon nanotubes are insoluble in most organicsolvents or aqueous media; therefore, for any type of biologicalapplication, the nanotube surface needs to be modified. Theirversatile physicochemical features enable covalent and non-covalent modifications. Different strategies have been devel-oped to make CNTs compatible with the biological environ-ment.28 The two main functionalization methodologies arebased on the noncovalent coating of nanotubes withamphiphilic molecules29−34 and the covalent functionalizationof the nanotube surface by grafting ammonium or carboxylicacid groups directly onto the surface.28 Both types of thestrategies have advantages and disadvantages, but it is sure thatthey remarkably improve the water dispersibility of thenanotubes and, at the same time, offer a flexible platform forfurther derivatization.35−38 Meanwhile the aromatic stackingnature of CNTs surface also provides noncovalent moleculesanchoring by π−π interactions. It is well stated in the literaturethat the conjugated polymers undergo π−π interactions withCNTs.28,39−41 Selective binding of nanotubes with differenttypes of conjugated polymers provides a simple method ofdispersing them in various organic solvents, helps to decreasebundling and, consequently, improves optoelectronic proper-ties.42 When conjugated block copolymers containing non-conjugated blocks with tunable functionality were used,extensively functionalized CNTs were also reported.43−46

In the class of conjugated polymers, the “hairy-rod” conceptis based on the introduction of conformationally mobile,relatively long and flexible side chains to the rigid, conjugatedpolymer backbone.47,48 Such complex macromolecular struc-tures can be prepared by combining the controlled polymer-ization methods such as atom transfer radical polymerization(ATRP) and cationic ring-opening polymerization (ROP) withthe ones specific for the synthesis of conjugated polymers(Suzuki or Yamomoto polycondensation methods). Veryinteresting amphiphilic or amphipolar “hairy-rod” architectures,which undergo organization at the nanoscale by self-assemblingand microphase separation, as the block copolymers exhib-it,49−51 were obtained.52−57 The followed synthetic strategyprovides freedom to design and synthesize diverse macro-molecular architectures of conjugated polymers with different,well-defined macromolecular side-chains that will match up theproperties of required targeted applications.When poly(ethylene glycol) (PEG) side chains are attached

to a conjugated main chain backbone, using the macro-monomer technique, water self-dispersible polymers can beobtained.58−62 In a preceding article, we reported a novelapproach for the synthesis and use of a poly(p-phenylene) (PP)copolymer bearing PEG units as fluorescent probes for in vitroimaging of cancer cells.63 The possibility of bringing togetherseveral different functional components into a single nano-structure proposes a great potential for efficiency and versatilityfor numerous goals. Such hybrid nanostructures unite thespecific functionalities of their component parts in a multi-

Biomacromolecules Article

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functional tool that can execute several tasks simultaneously.Inspired by these considerations, it seemed appropriate tobenefit from both conjugated polymers and CNTs to design anideally suited nanostructure for cancer diagnosis and treatment.In the present approach, we synthesized an amphiphilicfluorescent and water self-dispersible conjugated polymerwith “hairy-rod” architecture, aiming to combine bothfunctionalization methods, namely, noncovalent coating ofCNTs by π−π stacking and COOH surface chemicalfunctionalization of CNTs, for a fluorescent hybrid materialwith improved water self-dispersibility. Moreover, the presenceof PEG brushes attached on the rigid main chain of conjugatedpolymer provides not only a nanomaterial with ultralong bloodcirculation time,64 but also a noncytotoxic one, as recentlyreported for other PEGylated CNTs.65 The promisingadvantage of CNTs is the possibility of crossing biologicalbarriers for the delivery of active molecules. Except theconjugated ionic polyelectrolytes as dispersant agents inaqueous media for CNTs, to the best of our knowledge thisis the first report on the dispersibility of CNTs using a nonionicside-chain decorated conjugated brush polymer.In the frame of our standpoints, synthesized PP-g-PEG was

noncovalently (π−π stacking) linked to acid-treated CNTs.The obtained fluorescent, water self-dispersible, stable, andbiocompatible f-CNT/PP-g-PEG conjugates were then bio-conjugated through −COOH functionalities present on theside-walls of CNTs via EDC/NHS chemistry. The resultingconjugates were used as an efficient fluorescent probe fortargeted imaging of estrogen receptor overexpressed cancercells, such as MCF-7. In vitro studies and fluorescencemicroscopy data show that these conjugates can specificallybind to MCF-7 cells with high efficiency. The representedresults imply that CNT-based materials could easily befabricated by the described approach and used as an efficient“fluorescent probe” for targeting and imaging, therebyproviding many new possibilities for various applications inbiomedical sensing and diagnosis.

■ EXPERIMENTAL SECTION2,5-Dibromo-benzoic acid, 1,4-benzene-diboronic acid, Pd(PPh3)4,dicyclohexylcarbodiimide (DCC), 4-(N,N′-dimethyl)amino pyridine(DMAP), and PEG750 were purchased from Sigma-Aldrich and wereused as received. Solvents were purified and dried, if necessary, byusual methods prior to use. Multiwalled carbon nanotubes producedby chemical vapor deposition (carbon content > 90%; diameter ×length 110−170 nm × 5−9 μm) were purchased from Sigma-AldrichCo. (Sigma-Aldrich Chemie GmbH, Germany). MCF-7 human breastcancer cells were obtained from American Type Culture Collection(Manassas, Va.). PC-3 human prostate carcinoma cells were obtainedfrom Bioengineering Laboratory of Ege University (Izmir, Turkey).Human mammary epithelial cells (HMEC) and mammary epithelialcell growth medium (MEGM) were obtained from Lonza Inc.(Walkersville, MD, U.S.A.). The monoclonal anti-estrogen receptor-βantibody (ER) produced in mouse (E1276) was purchased fromSigma-Aldrich Co. (St. Louis, Missouri). 1-Ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC, E1769), N-hydrox-ysulfo succinimide (sulfo-NHS, 56485), tetrahydrofuran (THF, >99%-inhibitor-free-34865), phosphate-buffered saline powder (pH 7.4-P3813), nitric acid (78%), and sulfuric acid (98%) were purchasedfrom Sigma-Aldrich. A 100 kDa membrane filter was purchased fromMillipore (Tullagreen Carrigtwohill, Ireland).Synthesis of Macromonomer (1,4-Dibromo-2-(Me-PEG750)-

benzene). A 250 mL three-necked round-bottom flask equipped witha dropping funnel, a magnetic stirrer and a nitrogen inlet−outlet wascharged with 7.1 mmol (5.33 g) of mPEG750, 10.7 mmol (3.00 g) 2,5-

dibromobenzoic acid, 1.07 mmol (0.13 g) of DMAP, and dry CH2Cl2(120 mL). To this solution was added 10.7 mmol (2.21 g) of DCC in11 mL of dry CH2Cl2 dropwise under nitrogen, and the reactionmixture was stirred for 48 h at room temperature. The reactionmixture was filtered, and the solution was concentrated and passedthrough a silica-gel column using CH2Cl2 as eluent. Finally, theobtained solution was concentrated and the product was precipitatedin cold diethyl ether. 1H NMR spectra were recorded at roomtemperature on a Bruker Avance DRX-400 spectrometer (400 MHz)in DMSO-d6 and chemical shifts are reported in ppm and referencedto TMS as internal standard (Supporting Information, Figure S1). FT-IR spectra were recorded on KBr pellets using a DIGILAB-FTS 2000spectrometer (Supporting Information, Figure S3). The molecularweights were determined by gel permeation chromatography (GPC)using a PL-EMD instrument, polystyrene standards for the calibrationplot and THF as elution solvent (Mn = 825 g mol−1, Mw/Mn = 1.10).

Synthesis of Poly(p-phenylene-graft-poly(ethylene glycol)(PP-g-PEG). This was achieved according to the following procedureas described previously.64 Thus, a 100 mL three-necked round-bottomflask equipped with a condenser, a rubber septum, a nitrogen inlet−outlet, and a magnetic stirrer was charged with 2.0 M K2CO3 (20 mL)aqueous solution and THF (30 mL). The solvents (THF and K2CO3solution (aq)) were previously bubbled with nitrogen over a period of30 min, and the mixture was refluxed under nitrogen for 4 h.

A 50 mL three-necked round-bottom flask equipped in the sameway as the previous one was charged under inert atmosphere with 1.8mmol (1.8 g) of macromonomer, 1.8 mmol (0.3 g) of 1,4-benzenediboronic acid, and 0.03 mmol (0.03 g) of Pd(PPh3)4. Thesolvent mixture (15 mL) was introduced with a syringe through theseptum. The mixture was refluxed under nitrogen for 4 days undervigorous stirring and in the absence of oxygen and light. The finalpolymer was separated by precipitation in cold diethyl ether, filtrated,and dried. Further purification was performed by passing the polymerthrough a silica-gel column using CH2Cl2 as eluent and reprecipitatingin cold diethyl ether (Mn = 3750 g mol−1, Mw/Mn = 1.20; for 1H NMRand FT-IR spectra, see Supporting Information, Figures S2 and S3,respectively).

Oxidation of CNTs. CNTs need to be in high purity forbiomedical applications, especially if their sidewalls serve as a platformfor noncovalent attachments. There are several types of oxidationmethods used for different purposes. Generally applied methodinvolving acid mixtures was used for the work presented here. In thefirst step, 10 mg of commercial CNTs were incubated in H2SO4 (98%)and HNO3 (68%) 3:1 (v:v) mixture for 7 h at 80 °C. Severalsonication treatments were applied during incubation. This processallows CNTs to disentangle through breaking down bundles that inturn results in the formation of carboxylic acid groups on the sidewalls. After incubation, the mixture was diluted (1:1; (w/w) withbidistilled water.66 To remove dissolved metal catalyst particles,amorphous carbon fragments, and excess acid in the supernatant, themixture was washed with distilled water and centrifuged (5000g) untilthe pH value of the sample became neutral. Afterward, aggregates wereremoved via 0.45 μm pore-sized membrane filter. Finally, resultingcarboxyl-functionalized CNTs were dried overnight at 80 °C in anincubator. Henceforth, this material is referred to as f-CNTs.

Noncovalent Attachment of PP-g-PEG to f-CNTs. A total of 0.1mg/mL of f-CNTs and 0.2 mg/mL of PP-g-PEG fluorescent polymerwere dispersed in 3.0 mL of tetrahydrofuran (THF). The mixture wasshaken overnight at room temperature. During incubation, π−πstacking interactions take place between phenylene groups of thepolymer and hexagonal rings of f-CNTs.67 After incubation, themixture was washed with distilled water to remove THF. The 100 kDacentrifugal membrane filters were used to separate unbound polymer.Resulted conjugate is referred to as f-CNT/PP-g-PEG.

To confirm the solubility, photographic images of the solutions f-CNT and f-CNT/PP-g-PEG at the concentration of labeling andimaging experiments were taken and presented as Figure S4a,b inSupporting Information,

Covalent Modification of f-CNT/PP-g-PEG with Anti-ERAntibody. f-CNT/PP-g-PEG (1.0 mg/mL) conjugate was dispersed



in sodium phosphate buffer (20 mM, pH 5.0). After that, 1.0 mL ofEDC solution (20 mM) and 1.0 mL of 50 mM sulfo-NHS solution inpH 7.0 sodium phosphate buffer were added. The mixture wasincubated for 15 min at room temperature to activate carboxyl groupsof f-CNTs for further coupling to antibody. To quench excess EDC, 2-mercaptoethanol (50 μL) was added. Anti-ER antibody (30 μL, 0.3mg/mL) was added into the reaction mixture and coupling reactionwas allowed to proceed for 2 h at room temperature. Finally, 1.0 mL of250 mM ethanolamine hydrochloride solution was added to themixture to quench the reaction. Uncoupled proteins, excess EDC andNHS were separated via centrifugation with distilled water by using100 kDa membrane filter (5000g) until pH was reached to 7.0.Afterward, the mixture was dialyzed against PBS (pH 7.4). Finally,antibody coupled f-CNT/PP-g-PEG conjugates were washed andfiltered by using 0.2 μM polycarbonate filters (Whatman, NorthAmerica) and resuspended in 10 mL of sodium phosphate buffer (20mM, pH 7.4). Resulted bioconjugates are referred to as f-CNT/PP-g-PEG/ER. Bradford assay was used to estimate the amount of antibodycoupled to PP-g-PEG/f-CNT conjugates. A total of 0.2 mL of sampleswere mixed with 0.8 mL of Bradford reactive and incubated for 15 minat room temperature. After incubation, changes in absorbance wererecorded at 595 nm via microplate reader.68 For the all-cell cultureexperiments and characterizations, only freshly prepared conjugateswere used.Characterization Methods. Optical characteristics of PP-g-PEG,

f-CNT/PP-g-PEG, and f-CNT/PP-g-PEG/ER conjugates were exam-ined with spectrofluorometer (Varian Cary Eclipse, U.S.A.) andstatistical program Graph Pad was used to evaluate the data. Forstructural characterization studies, scanning and transmission electronmicroscopes (SEM and TEM), atomic force microscopy (AFM),Raman spectroscopy, and thermogravimetric analysis (TGA) wereused. Additionally, zeta potential analysis of polymer and conjugateswas carried out.SEM and TEM. SEM images were obtained by using Carl Zeiss Evo

40 (Carl Zeiss NTS GmbH, Germany), and TEM images are taken byusing JEOL type microscopy to obtain high definition images of CNT-based samples. For both analyses, a 2.0 μL drop of f-CNT/PP-g-PEGsolution was placed onto a small piece of gold-covered surface andresidual water was evaporated by using a hot plate. The metal stubsholding the samples were then placed into the vacuum chamber of themicroscope and images acquired using a voltage of 10 kV and a spotsize of 3.0−5.0.MCF-7 cells treated with f-CNT/PP-g-PEG/ER bioconjugates

(0.06 mg/mL) were seeded on one-sided conductive ITO-glass forSEM analysis. First, sterilized glass via UV radiation was populatedwith MCF-7 cells (2.5 × 104). After that, cells were incubated at 37 °C,5.0% CO2, 100% air humidity for 3 days. Then, the slide was washedthree times with PBS before sample (500 μL) was added to the glasssurface covered with cells. Cells were incubated with bioconjugates at37 °C, 5.0% CO2, 100% air humidity for 2 h. Populated glass slideswere incubated with Karnovsky buffer after washing with PBS threetimes. Karnovsky buffer was prepared with 5.0 mg CaCl2, 10 mL ofcacodylate buffer, 20 μL of glutaraldehyde (50%), and 2.48 mL ofdH2O. Here cacodylate buffer was prepared with 0.2 M cacodylacidsodium acid sodium salt trihydrate in dH2O, pH 7.3. Afterward,samples were washed with cacodylate buffer three times. Beforeimaging, the slide surface was dehydrated by incubating samples inacetone/water mixtures (w/w) as follows: 1:9, 1:4, and 2:3 for 15 min;6:3 and 4:1 for 10 min; and finally, 100% acetone two times for 10min. After all, glass slides were left to dry under room temperature. ForSEM images, the glass slide was replaced on the silver-coated surface.The metal stubs holding the sample was then placed into the vacuumchamber of the microscope and images acquired using a voltage of 2.0kV.AFM. AFM measurements were carried out at ambient conditions

by using Ambios Q-Scope 250 instrument. The tapping mode wasused to take topographic images. A 20 μm scanner equipped withsilicon tips with 10 nm tip-curvature and ITO-coated glass substratewas used for measurements. The system is covered with acousticchamber to prevent electromagnetic noises that may affect the

measurements. CNT, f-CNT, and PP-g-PEG/-polymer were spin-coated (WS400B-6NPP-Lite spin processor by Laurel) onto thesubstrate at a spin speed of 1500 rpm for 60 s and dried at 25 °C for 1h in vacuum.

Raman Spectroscopy. Raman spectroscopy was used toinvestigate surface modifications by determining defect points andratio. Thus, samples were prepared by placing a small quantity of CNTin powder or as a droplet of suspension between the glass slide andcoverslip. Measurements were performed using an automatedRenishaw InVia Reflex Raman Microscopy System (U.K) withexcitation at 514 nm and 10× objective, 10% laser power.

TGA. TGA analyses were performed with a Perkin-Elmer TGA-7instrument (Massachusetts, U.S.A.). A 1.0 mg sample was placed in thesample holder in the furnace. As samples were heated up to 800 °C ata rate 10 °C/min, changes in sample weight were measured.

Zeta Potential. The zeta potential values of f-CNT, f-CNT/PP-g-PEG, and f-CNT/PP-g-PEG/ER were measured with a MalvernZetasizer Nano-ZS instrument at 25 °C. Samples (0.1 mg/mL) wereprepared with dH2O (1.0 mL) and transferred into the zeta potentialcuvettes (DTS1060, Malvern). All measurements were repeated threetimes and data presented as means ± SD.

FT-IR Spectroscopy. FT-IR spectra were recorded by a PerkinElmer Spectrum BX-FTIR spectrophotometer by using both ATRsystem (powder form directly usable) and in KBr pellets. IR spectra ofthe compounds CNT and f-CNT as 1.0% suspensions in KBr pelletswere recorded with FTIR spectrophotometer.

Cell Culture Experiments. The MCF-7 (human breastadenocarcinoma cell line) and human mammary epithelial cells(HMEC) were grown in minimum essential medium (Eagle) in Earle’sBSS with 2.0 mM glutamine, 1.5 g/L sodium bicarbonate, 0.1 mMnonessential amino acids, 1.0 mM sodium pyruvate, and 10% fetalbovine serum (FBS). PC-3 (prostate carcinoma cells) were grown inDulbecco’s MEM containing 2.0 mM glutamine, 1.5 g/L sodiumbicarbonate, 1.0 mM sodium pyruvate, and 5.0% fetal bovine serum(FBS). All cell lines were cultured at 37 °C in a moist atmosphere with5.0% CO2. The 25 cm2 cell culture flasks were confluently populatedwith cells and harvested through trypsinization with 0.25% trypsin-0.02% EDTA. Upon proper dilution with fresh culture medium, cells(∼1.0 × 105 cells) were seeded into the wells of a 24-well culture plateand 9.0 cm2 tissue culture plates. After incubation at 37 °C in 5.0%CO2, monolayers were washed three times with PBS and incubatedwith PP-g-PEG, f-CNT/PP-g-PEG, and antibody coupled f-CNT/PP-g-PEG (250 μL, diluted with medium) for 2 h and at 37 °C and 5.0%CO2. After incubation, cells were washed two times with PBS. The 24-well plates were fluorometrically assayed in a multiwell fluorescenceplate reader (Thermo, Milford, MA, U.S.A.) at 410 nm with excitationwavelength 280 nm to determine the relative incorporation of thesamples to the cells (cell incorporation, %). Fluorescence values (%)were estimated regarding the fluorescence signal of the conjugatesbefore they were treated with the cells. After conjugates wereincubated with cells, cells were washed and the remaining fluorescencesignals were calculated according the conjugates’ initial signals beforetreatment. All data were presented as the mean of 3−5 experiments ±SD. For the cell imaging, 9.0 cm2 tissue culture Petri dishes werecovered with a slide and fluorescence mounting medium (Daco,Copenhagen, Denmark) and visualized with 100× magnification andphotographed through epifluorescence microscopy (Olympus, Tokyo,Japan).

In Vitro Cytotoxicity. Worle-Knirsch et al. reported that insolubleformazan crystals that are yielded by MTT assay strongly interact withCNTs, which in turn cause false-positive results.69 Thus, we usedalternative tetrazolium-based cytotoxicity assay, WST-1 for thecytotoxic assessment of CNT-based conjugates. Unlike cell-permeableMTT and its insoluble formazan that accumulate inside the cells,which require a solubilization step for the record, WST assay yieldswater-soluble formazan with a concomitant decreasing toxicity to thecells. WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-yl)-2H-tetrazolium monosodium salt) cell proliferation kit (RocheDiagnostics GmbH Mannheim, Germany) was exposed to MCF-7cells to evaluate toxic effects of f-CNT/PP-g-PEG and f-CNT/PP-g-



PEG/ER conjugates. The dose-course toxic effect of polymerconjugates was examined by treating cells with 1.0, 3.0, 10, 30, 50,and 75 μg/mL sample. Cells were seeded out in 96-well tissue plates(Sarstedt, U.S.A.) in a volume of 100 μL (15 × 103 cells/mL) andcultured to confluence at 37 °C, 5.0% CO2, and 100% air humidity.The medium was removed and the cells were incubated withconjugates for 24 h. Afterward, cells were washed with PBS bufferto remove unbound conjugates prior to WST treatment. Plates werespectrophotometrically assayed with a microplate reader (ThermoScientific Varioskan Flash multimode reader, Vantaa, Finland) after 4 hof treatment with WST-1 dye solution (10 μL) at 450 versus 620 nm.The background absorbance was measured on wells containing onlydye solution and culture medium, which are referred to as control andblank, respectively. The mean optical density values corresponding tonontreated samples were recorded as 100%. The results wereexpressed as percentages of the optical density of treated samplesversus nontreated controls by using the following equation: (Asample −Ablank)/(Acontrol − Ablank) × 100.69

■ RESULTS AND DISCUSSION

Synthesis and Characterization of f-CNT/PP-g-PEGPolymer Conjugates. This study aims to demonstrate the useof f-CNTs acting both as a “carrier platform” and linker for thefluorescent polymer and biomolecules, as well. As presentedbelow, this combination provides possibility of binding polymeras a “targeting agent” to track f-CNTs and to tag the materialwith targeted specific ligand for labeling and imaging of MCF-7cells.The first step in the preparation of CNT conjugates for

targeted cell imaging was the synthesis of poly(p-phenylene)(PP) bearing poly(ethylene glycol) (PEG) side-chains, asshown in Figure 1 (for characterization, see SupportingInformation).The graphitic surface of CNTs needs to be decorated with

hydroxyl, carboxyl, or amine groups to facilitate their use inbiological applications. In our case, CNTs were functionalizedby oxidation through acid treatment to incorporate carboxyl

Figure 1. Synthesis of poly(p-phenylene-graf t-poly(ethylene glycol)) (PP-gPEG).

Figure 2. Overall process for copolymer/CNT conjugates and imaging.



groups as binding sites for the subsequent biomoleculeconjugation. Carboxyl-functionalized CNTs obtained via thisway were exposed to solubility test in water.70 It was observedthat f-CNTs solutions concentrated up to 0.5 mg/mL aretotally soluble in water, whereas concentrations higher than 1.0mg/mL tend to aggregate within one week. Then, PP-g-PEGwas attached to f-CNTs via π−π stacking interactions betweenphenylene groups of polymer and hexagonal rings of nano-tubes. Afterward, an estrogen-specific antibody (ER) wascoupled via EDC/NHS chemistry through the reactionbetween carboxyl groups of f-CNTs and amino groups ofantibody. The overall process is depicted in Figure 2.The intermediates at each stage and obtained bioconjugates

were characterized by various spectroscopic and microscopicinvestigations. Fluorescence properties of PP-g-PEG polymerbefore and after conjugation were evaluated. As can be seenfrom the fluorescence spectra illustrated in Figure 3, PP-g-PEG

has a maximum emission at 410 nm. Upon conjugation, thefluorescence profile of the polymer retained without any majorchanges. However, each modification step caused a significantdecrease in the fluorescence intensity which can be explained interms of fluorescence quenching by CNT through noncovalentinteraction. Regardless of covalent or noncovalent attachments,similar severe or total quenching between fluorescent moleculesand CNTs were previously reported.71 However, the remainedfluorescence signal is still high enough to use the system for invitro cell targeting experiments, which cannot be realized byconventional fluorophores.Raman spectroscopy is another structural characterization

tool for CNTs.72 A large density of electronic states, which aredependent on their unique geometric structures, makes itpossible to examine CNTs with Raman spectroscopy.72 Figure4 shows the Raman spectra of CNT, f-CNT, and f-CNT/PP-g-PEG polymer conjugates. There are three specific bands in theRaman spectra of CNTs: the tangential band (G band) ataround 1400−1600 cm−1, the disorder mode band (D band) ataround 1200−1300 cm−1, and its second-order relatedharmonic band (G′ band) at around 2700 cm−1.72,73 The Gband exhibits graphitic hexagon-pinch mode, while the D bandrepresents the defects in the curved graphene sheets. Thosedefects are attributed to tube ends and sp3-hybridized carbonatoms in the hexagonal framework. Also, in any CNT spectrum,G bands generally appear as multipeak features at around 1580cm−1. In Figure 4a, D bands, G bands, and G′ bands of CNTand f-CNT can be clearly seen at 1350, 1580, and 2700 cm−1,

respectively. In the Raman signals of carbon materials, somephoton frequencies change with the change of laser excitationenergy, which is called “dispersive” behavior. D band, in theradial breathing mode of CNTs, is a dispersive disorder-induced band at 1350 cm−1 whose frequency changes by 53cm−1 as a result of changing the laser energy EL by 1 eV.74

Carboxylation of the CNTs results in an increase in thesubstantial D mode with a concomitant decrease in the G mode(Figure 4b), associated with a rising number of sp3-hybridizedcarbons and a decrease in the sp2-hybridized carbons from thegraphitic sidewalls, respectively. The increase in the ratio ofsp3/sp2 is defined by the ratio of intensity of D and G bands(ID/IG), which is calculated as 0.15 and 0.25 for CNTs and f-CNT, respectively. This increasing ID/IG ratio reflects theincrease in the structural disorder of CNTs that is produced byfunctionalization. In the Raman spectra of f-CNT/PP-g-PEG(Figure 4 c), shifted and broadened D and G bands can benoted at around 1284 and 1606 cm−1, respectively. Theseshifted bands, which are assigned to the sp3- and sp2-hybridizedcarbons, are caused by the presence of the polymer shell on thesurface of the CNTs.The functionalized CNTs and the antibody coupled f-CNT/

PP-g-PEG were further analyzed by FT-IR and compared withthe pristine CNTs (Figure 5). The pristine CNT exhibits acharacteristic absorption band of CC groups at 1560 cm−1. Astrong band of the CO group of f-CNT was observed at1706 cm−1 in KBr pellets, and vibrations of the O−H bonds off-CNT were observed at around 3100−3500 cm−1. Thesehydroxyl groups may arise from either ambient atmosphericmoisture tightly bound to the CNTs or oxidation duringpurification of the pristine material.75 There are also increasedsignals at around 1200 and 1400 cm−1, which are associatedwith C−O stretching and O−H deformation of the carboxylicacid groups.76 These peaks indicate carboxyl groups areincorporated successfully to CNT surfaces (Figure 5a). In thecase of f-CNT/PP-g-PEG (Figure 5b), a broad band wasobserved at 3441 cm−1 due to the presence of −OH group ofcarboxyl functional CNT (f-CNT). Aromatic and aliphatic CHgroups due to the presence of PP-g-PEG exhibited character-istic absorption bands around 2918 and 2848 cm−1. COstretching. Rather lower carbonyl (CO) stretch appears at1708 cm−1 due to the effect of hydrogen bound. f-CNT/PP-g-PEG/ER exhibited a characteristic stretching band at 3431cm−1, indicating the N−H bond in the amide structure. Bandsat 1708 and 1642 cm−1, corresponding to the amide carbonyl(CO) stretch N−H in-plane, clearly confirm the presence ofthe amide group.77 That is to say, FT-IR results prove thatprotein was coupled to f-CNT/PP-g-PEG via amide bondformation.Moreover, the coupling efficiency as calculated according to

Bradford assay was found to be 92%, indicating an almostcoupling efficiency. The assay used for the detection protein inthe conjugates was successfully and practically applied in theprevious works in which QD and conjugated polymers weretargeted with different targeting biomolecules.78,63

Surface morphologies of pristine CNT, f-CNT, and f-CNT/PP-g-PEG were also studied with AFM. The images illustratedin Supporting Information as Figure S5 reveal that acidtreatment caused deformation and reduction in the size. Thepresence of polymer on the f-CNT surfaces causes an increasein the dimensions. This indicates that PP-g-PEG layer isattached to CNT surfaces forming larger CNT conjugates incompared to f-CNT alone.

Figure 3. Fluorescence spectrum of PP-g-PEG, f-CNT/PP-g-PEG, andf-CNT/PP-g-PEG/ER conjugates. (Conjugates were suspended inPBS buffer, pH 7.4, with a concentration 0.2 mg/mL and excited at260 nm.)



Another convincing evidence for the confirmation ofsuccessful functionalization and modification of CNTs wasobtained by thermogravimetric analysis (TGA). Figure 6 showsthe thermal degradation profiles of untreated multiwalled CNT,acid mixture treated CNT (f-CNT), together with that of thepolymer and antibody modified materials. According to TGAthermograms, while pristine CNT is thermally stable up to 800°C, f-CNTs show significant weight losses. Thermal degrada-tion of acid-treated CNTs is a multistage process.79 Attemperatures up to 150 °C, a slight weight loss is detectedregarding the evaporation of the adsorbed water. Degradationbetween 150 and 350 °C is attributed to the decarboxylation ofthe carboxylic groups present on the CNT sidewalls. Additionalweight loss observed from 350 to 500 °C may be ascribed tothe elimination of hydroxyl functionalities attached to the CNTwalls. Finally, at the temperatures higher than 500 °C, theobserved degradation corresponds to the thermal oxidation ofthe remaining disordered carbon.80 The amount of f-CNTresidue after 700 °C corresponds to 77.5%. In general,shortened polymer chains are relatively thermally unstable,which in turn triggers minor weight losses at low temperatureregions.81 Thus, polymer- and antibody-conjugated CNTsshow different weight loss patterns. The rapid weight lossthat occurs after 325 °C for PP-g-PEG attached CNTcorresponds to the polymer chains on the sidewalls of CNT.This observation gives clear evidence that the PP-g-PEG

polymer is adsorbed on the CNT sidewalls. On the other hand,12% of weight loss between 175 and 325 °C is ascribed to thethermal degradation of the covalently coupled antibody. Thisbehavior assures that protein structures are thermally less stablethan polymer material. TGA data verifies successful function-alization of CNTs, noncovalent attachment of polymer to CNTsidewalls, and antibody coupling to carboxyl terminal groups ofCNTs.Surface modification profiles of CNTs were also assessed by

zeta potential analysis (Table 1). Surface potential of thepolymer-attached CNTs (−5.98 mV) became less negative thanthat of the precursor acid-treated CNTs (−13.3 mV). In otherwords, by surface treatment, the potential of hybrid materialbecomes closer to that of the polymer itself (−5.05 mV) due tothe strong stacking interactions between the polymer and theCNT blocking negatively charged carboxyl groups on thesurface. Subsequent covalent attachment of antibody to hybridmaterial neutralizes the surface charge at a potential of −2.95mV due to positive charges of antibody structure. The changesin the zeta-potentials suggest that the surface potentials ofCNTs can be manipulated through covalent and noncovalentmodifications.TEM images were also taken to visualize the morphologies of

CNTs (Figure 7). The reduced dimensions of f-CNTs (Figure7b) compared to pristine CNTs (Figure 7a), as a result ofacidic treatment, have been clearly seen. As it can be noted

Figure 4. Raman spectra of CNT (a), f-CNT (b), and f-CNT/PP-g-PEG (c).



from Figure 7 c, PP-g-PEG polymers are coordinated on CNTssidewalls via π−π stacking interactions that cause differentmorphology.SEM images of CNT, f-CNT, f-CNT/PP-g-PEG, and f-

CNT/PP-g-PEG/antibody covered MCF-7 cell were presentedin Figure 8a−d. Structural defects present in the f-CNTs can be

Figure 5. FT-IR spectra of CNT, f-CNT (a) and f-CNT/PP-g-PEG, f-CNT/PP-g-PEG/ER conjugates (b).

Figure 6. TGA thermograms of CNT (black line), f-CNT (red line), f-CNT/PP-g-PEG (green line), and f-CNT/PP- g-PEG/ER (blue line).

Table 1. Zeta Potential Analysis of f-CNT, f-CNT/PP-g-PEG, and f-CNT/PP-g-PEG/ER

sample zeta potential (mV)

f-CNT −13.3 ± 0.31PP-g-PEG −5.05 ± 0.022f-CNT/PP-g-PEG −5.98 ± 0.027f-CNT/PP-g-PEG/ER −2.95 ± 0.01

Figure 7. TEM images of raw CNT (a), f-CNT (b), and f-CNT/PP-g-PEG (c); Scale bars used in the images are 200 nm for (a) and 50 nmfor (b) and (c).



seen in images (Figure 8a); the curved appearance offunctionalized CNTs comes from structural defects resultingfrom acid mixture treatment forming smaller fibrils comparedto raw CNTs (Figure 8c). As it can be noted from Figure 8b,PP-g-PEG polymers are adsorbed on CNT sidewalls vianoncovalent, attractive interactions of aromatic rings. However,possible aggregations arising from the hydrophobic interactionsof the polymer chains are also distinguished in the SEM images,which correlate with TEM results. Some aggregations wereobserved in SEM images of polymer-modified CNTs. Theseaggregates may be arising from the hydrophobic interactions ofthe polymer chains and blocking of the carboxylic acid groupsby polymer chains, reducing the solubility of the CNTs. Apartfrom that, samples undergo drying prior to SEM analysis, whichmay well cause the aggregation of samples that were well-dispersed while in solution. Yet, the water solubility of thehybrid material is mainly depends on the conjugateconcentration and remains sufficient, especially in the selectedconcentration for the cell culture experiments by virtue of thepresence of PEG chains in the structure of the conjugatedpolymer due to its hydrophilic nature. To investigate theirpotential use in the targeting application, the f-CNT/PP-g-PEGconjugate was applied to MCF-7 cells. After treatment of cellswith conjugates, the localization of the material on the cellsurfaces was clearly observed in Figure 8d. Apparently, the cellsurface was totally covered with the antibody coupled polymer/CNT material.Cytotoxicity Studies. Alteration of CNTs via covalent and

noncovalent attachments allows them to be incorporated intoliving systems for biological applications.82 Untreated CNTstend to accumulate in cells, tissues, and organs, causingdangerous effects as they are not soluble in water.83 However,functionalization of CNTs improves their biocompatibility84,85

and eliminates such toxic effects.86,87 That feature rendersCNTs as possible carrier systems for targeting and therapeuticagents.88−90 Even though CNTs do not affect cell proliferationor viability and are alone nontoxic agents,91 their cellulartoxicity should be considered. To evaluate possible cytotoxiceffects of polymer and antibody conjugated f-CNTs to MCF-7cells, a WST assay was performed. Results displayed in Figure 9

show that CNT conjugates preserve cell functionality withoutsignificantly influencing cell viability. However, in the case ofantibody coupled CNT conjugates, a slightly more toxicbehavior was elicited. Although this effect is more significant,particularly at high concentrations, at least 70% of the cells arestill viable when they are treated with as high as 75 μg/mL ofconjugates for 24 h. The decrease in cell viability is reasonable,hence, the presence of antibody improves cellular attachmentthrough receptor−ligand interactions, thus, enhancing inter-action of conjugates with the cell surface.

Figure 8. SEM images of f-CNT (a), f-CNT/PP-g-PEG (b), raw CNTs (c), and MCF-7 cell treated with f-CNT/PP-g-PEG/ER conjugate (d).

Figure 9. Cell viability data evaluated by WST assay.



Cellular Targeting. CNT/polymer hybrid material wastargeted against MCF-7 cells with estrogen receptor specificantibody to enhance cellular attachment. Cellular targetingefficiency of the conjugate was examined by applyingconjugates to MCF-7, with PC-3 and HMEC cells as negativecontrols. Cellular association results were compared in terms offluorescence intensity of the cells after conjugate treatment. Aspresented in Figure 10, the highest signal was observed with f-

CNT/PP-g-PEG/ER treated MCF-7 cells indicating thatantibody targeted CNT/polymer conjugate binds to theMCF-7 cell surface much more efficiently than that of PC-3and HMEC cells. Also, it is obvious that the presence oftargeting ligand allows specific binding which occurs viareceptor−ligand interactions at a high level. What is more,PEG chains are well-known to be reducing nonspecificbindings.92 The results prove that nevertheless PEG chainsseem to hinder undesired interactions which in turn produceweakened cellular attachment.For further investigation, fluorescence microscopy images of

MCF-7 cells treated with polymer, CNT/polymer, andantibody coupled CNT/polymer conjugates are given in Figure11. Normally, CNTs are engulfed by macrophages. However,the presence of PEG chains on the surface of CNTs comingfrom the polymer structure seems to help CNTs to avoidphagocytosis.4 Hence, CNTs were tracked at the outermembrane of the cells, making cell surface visible byfluorescence. The fluorescence feature of the PP-g-PEGpolymer with a proper carrier agent like CNT and a targetingligand allowed enhanced cellular binding in comparison to

CNT/polymer and polymer alone. The fluorescence images areconsistent with the results of cell association experiments.

■ CONCLUSIONSWe developed a facile approach to solubilize and labelsimultaneously CNTs by the use of a well-known conjugatedpolymer, poly(p-phenylene), bearing PEG units as side-chains.The acid-treated multiwalled CNTs were modified with PP-g-PEG copolymer via π−π stacking, affording water self-dispersible, stable, and biocompatible f-CNT/PP-g-PEGconjugates. After the covalent modification by estrogen-specificantibody (anti-ER) through −COOH functionalities present onthe side-walls, these CNT conjugates were used for targetedimaging of estrogen receptor overexpressed cancer cells,namely, MCF-7. Cellular association and fluorescence micros-copy results show that CNT/PP-g-PEG/ER conjugate binds tothe MCF-7 cell surface specifically and efficiently. Thisinvestigation represents the successful use of macromoleculararchitectures, such as PP-g-PEG for the modification andfunctionalization of CNTs providing higher water solubility,low cytotoxicity, and good fluorescence properties for cancercell targeting and diagnosis. The results presented here mayopen new pathways for the fluorescence labeling of nanotubeswithout necessity of any additional fluorophores, which willallow a wider range for their biological utilizations especially inoptics-based devices for biosensing and bioimaging.

■ ASSOCIATED CONTENT*S Supporting InformationFT-IR spectra of macromonomer (1,4-dibromo-2-(Me-PEG750)-benzene) and poly(p-phenylene-graf t-poly(ethyleneglycol) (PP-g-PEG), photographic images of f-CNT and f-CNT/PP-g-PEG in solution, and AFM/phase mode images ofCNT, f-CNT and f-CNT/PP-g-PEG. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]; [email protected] ContributionsS.T. and Y.Y. conceived the communication strategy, designedexperiments, evaluated the results, and prepared the manu-script; P.U. and S.S. designed cell culture and imagingexperiments and contributed reagents. M.Y., D.G.C., M.A.,I.C., M.K., M.C., and E.I.M. carried out the experiments andcontributed technical expertise.

Figure 10. Cellular binding of PP-g-PEG, f-CNT/PP-g-PEG, and f-CNT/PP-g-PEG/ER to MCF-7, PC-3, and HMEC.

Figure 11. Fluorescence images of MCF-7 cells treated with PP-g-PEG (a), f-CNT/PP-g-PEG (b), and f-CNT/PP-g- PEG/ER for 2 h (c).



http://pubs.acs.org

mailto:[email protected]

mailto:[email protected]

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by TUBITAK (Project No.109T573), EBILTEM (Ege University, Research and Applica-tion Center of Science and Technology, Project No.2010BIL004), Istanbul Technical University and Ege UniversityResearch Funds (Project Nos. 2010 FEN053 and 2010 FEN053). One of the authors (I.C.) thanks TUBITAK for thefinancial support by means of the “Visiting Scientist FellowshipProgram”. Additionally, we appreciate M.Sc. R. Bongartz andM.Sc. C. Baumanis (Gottfried Wilhelm Leibniz University ofHanover, Institute for Technical Chemistry) for operating SEManalysis and C. Geyik (Biochemistry Department of EgeUniversity) for his valuable help.

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