Carbon Dots as Nanodispersants for Multiwalled Carbon Nanotubes

Jul 11, 2017 - The chemicals were used as received, and all the solutions were ..... of D and G bands is an important tool in determining the defects ...
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Carbon Dots as Nanodispersants for Multiwalled Carbon Nanotubes: Reduced Cytotoxicity and Metal Nanoparticle Functionalization Sonam Mandani,† Prativa Majee,‡ Bhagwati Sharma,§ Daisy Sarma,† Neha Thakur,† Debasis Nayak,*,‡ and Tridib K. Sarma*,† †

Discipline of Chemistry and ‡Centre of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol, Khandwa Road, Indore 453552, India § Institute of Nano Science and Technology, Phase X, Sector-64, Mohali 160062, India

Langmuir 2017.33:7622-7632. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/12/19. For personal use only.

S Supporting Information *

ABSTRACT: The colloidal stabilization of multiwalled carbon nanotubes (MWCNTs) in an aqueous medium through noncovalent interactions has potential benefits toward the practical use of this one-dimensional carbonaceous material for biomedical applications. Here, we report that fluorescent carbon nanodots can efficiently function as dispersing agents in the preparation of stable aqueous suspensions of CNTs at significant concentrations (0.5 mg/mL). The amphiphilic nature of carbon dots with a hydrophobic graphitic core could effectively interact with the CNT surface, whereas hydrophilic oxygenated functionalization on the C-dot surface provided excellent water dispersibility. The resultant CNT-C-dot composite showed significantly reduced cytotoxicity compared to that of unmodified or protein-coated CNTs, as demonstrated by cell viability and proliferation assays. Furthermore, the reducing capability of C-dots could be envisaged toward the formation of a catalytically active metal nanoparticle-CNT-C-dot composite without the addition of any external reducing or stabilizing agents that showed excellent catalytic activity toward the reduction of p-nitrophenol in the presence of NaBH4. Overall, the present work establishes C-dots as an efficient stabilizer for aqueous dispersions of CNTs, leading to an all-carbon nanocomposite that can be useful for different practical applications.

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INTRODUCTION Carbon nanotubes (CNTs) have emerged as one of the most celebrated carbonaceous nanomaterial and have shown tremendous potential in diverse research fields.1−3 On the basis of their unique one-dimensional nanostructure, CNTs exhibit excellent mechanical, optical, and electronic properties as well as high chemical stability.4−6 These properties have resulted in the wide exploitation of CNTs in a host of possible applications such as composite reinforcement material, field emission displays, energy storage, sensors, scanning probe tips, and drug-delivery carriers, with newer applications emerging continuously.7−11 However, to realize their full prospect toward the development of novel functional high-quality nanocomposites for various applications, separation and a uniform dispersion of CNTs are fundamental prerequisites.12 The high hydrophobicity of CNTs associated with the sp2-hybridized carbon network makes it difficult to disperse them, especially in water.13 Together with this, the associated profound cytotoxicity limits their exploitation in several fields, including biomedical applications.14,15 It has been reported that the toxicity of CNTs is dependent on several factors, including the structure, aspect ratio, surface area, degree of aggregation, surface topology, bound functional © 2017 American Chemical Society

groups, concentration, and dose offered to the cells or organisms.16 The most common mechanisms that lead to the cytotoxicity of CNTs include necrosis and apoptosis, which are results of oxidative stress, damage to the DNA and cell membrane, and alteration of the intracellular metabolic pathways.16,17 Significant efforts have been made to modify CNTs in order to obtain homogeneous dispersions in water for practical utilization in biological systems with high stability and biocomptability.18−20 Surface modification through oxidation under harsh reaction conditions often changes the intrinsic physicochemical properties of CNTs.21 Therefore, noncovalent functionalization with various molecules is advantageous and can enforce not only high stability but also low toxicity without compromising the structural integrity of the nanotubes.22 A variety of molecular systems have been explored that can stabilize CNTs through interactions such as π−π stacking, electrostatic interactions, hydrogen bonding, and the van der Waals force.23 These molecules are amphiphilic in nature and enhance the wetting characteristics of CNTs in water, making Received: February 18, 2017 Revised: July 4, 2017 Published: July 11, 2017 7622

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them less toxic. Several biomolecules [e.g., proteins, DNA, and chitosan], polymers [e.g., poly(ethylene glycol), poly(L-amino acid), and pluronics], and surfactants [e.g., sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide (CTAB), and Triton-X] have been utilized for the noncovalent functionalization of CNTs, which enhances water dispersibility and biocompatibility.23−25 Taking into account the immense potential of CNTs in the field of nanomedicine for bioimaging and the delivery of bioactive molecules and biosensors, there is still a huge scope for designing low-cost, sustainable, and environmentally benign methods for fabricating stable and biocompatible CNT-based nanocomposites. Carbon dots (C-dots) are an emerging class of carbon nanomaterials owing to their splendid emission properties, good biocompatibility, water solubility, photostability, and energy conversion abilities.26 Easy methods of synthesis from low-cost carbon sources coupled with the tunability of surface functionalization as desired makes C-dots vastly applicable in biomedical field such as sensors, imaging, and nanovehicles.27 Doping with heteroatoms as well as introducing different surface functionality using diverse precursors during synthesis could direct tunable physicochemical properties.28,29 Also, Cdots have a graphitic π-conjugated core through which they can interact with hydrophobic materials.30 This hydrophobic core along with hydrophilic functionalities on the surface of C-dots imparts them with surfactant-like amphiphilic properties. This property has been exploited for the exfoliation and stabilization of graphene in water.31 Although recently C-dot-CNT composites have been developed and their applications have been shown in optoelectronic devices and electrochemistry, in most of these methods, either chemically functionalized or polymer-wrapped CNTs were used for dispersion in water. 32−34 The surfactant-like behavior of C-dots for dispersing CNTs, leading to stable C-dot-CNT composites in water, has not yet been explored. Herein, we report C-dots as effective dispersants for debundling multiwalled carbon nanotubes (MWCNTs) that provide efficient separation and high stability in water. The Cdots, synthesized from a commonly available and biocompatible polymer, poly(ethylene glycol) (PEG), could be dispersed in a range of solvents, suggesting their amphiphilic nature. The covalent binding of PEG on chemically modified CNT surfaces is reported to enhance the biocompatibility of CNTs.18 However, PEG alone cannot stabilize pristine CNTs through noncovalent binding as a result of their high hydrophilicity. The carbonization of PEG resulted in a conjugated sp2-hybridized network in the C-dot core that can effectively interact with the CNTs without affecting their intrinsic structure, whereas the oxygenated surface functional groups can render water solubility. The C-dots provided highly dense functionalization on the CNT surface that was instrumental in significantly curtailing the cytoxicity effect inherent to CNTs. The cytotoxicity assay along with cell proliferation studies revealed that the C-dots immobilized on the CNT surface could effectively alter their cellular interaction properties, resulting in decreased cytotoxicity. The resulting all-carbon composite was very stable against blood serum protein, albumin. The reducing capability of C-dots could be exploited further toward the generation of Au nanoparticles on the CNT surface without further need of external reducing and stabilizing agents, thus providing a new method for the generation of metal nanoparticle-CNT composites.

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MATERIALS AND METHODS

Materials. MWCNT (type 2) and p-nitrophenol were obtained from Sisco Research Laboratories Pvt. Ltd., India. PEG 200 and carboxymethyl cellulose (sodium salt) were purchased from Merck, India. Bovine serum albumin (BSA) and hydrogen tetrachloroaurate were obtained from Sigma-Aldrich. The chemicals were used as received, and all the solutions were prepared in Milli-Q water. Synthesis of C-dots. Twenty milliliters of PEG 200 taken in a glass beaker was subjected to heating in a domestic microwave oven (750 W) for 15 min, after which the colorless PEG solution turned brown, indicating the formation of C-dots. The resulting solution was diluted by adding 25 mL of water and then was subjected to dialysis for 48 h using a cellulose membrane to remove excess PEG. This dialyzed solution was used for further experiments. A 25 mL dialyzed solution was lyophilized and yielded a C-dot concentration of 0.52 mg/mL. Dispersion of MWCNTs. The CNTs were first sonicated in water in a bath sonicator for 5 min and then filtered and washed twice with water to remove any soluble impurities. The resultant CNTs were dried in an oven at 80 °C for 24 h and used further. (i) CNT-C-dots: 2 mg of CNT was added to a 5 mL solution of Cdots (0.52 mg/mL) and sonicated using a probe sonicator (20 kHz, 250 W) for 20 min. The sonication experiments were performed in ice water to avoid overheating the dispersions. The highly dispersed CNT-C-dots were centrifuged at 2000 rpm for 30 min to remove any large aggregates. The resultant supernatant had a CNT concentration of 250 μg/mL. For spectroscopic and microscopic analysis of CNT-C-dots, the above supernatant was subjected to centrifugation at 12 000 rpm for 30 min. The pellet was washed twice with water and dried in vacuum. This powder was then redispersed in water by mild sonication in a bath for 10 min and characterized further. (ii) BSA-C-dots: To obtain BSA-coated CNTs, the CNTs (2 mg/5 mL) were first probe sonicated for 20 min in phosphatebuffered saline (PBS, pH 7.4) followed by the instant addition of BSA to achieve a final BSA concentration of 5 mg/mL. The resultant mixture was subjected to sonication for 15 min in a bath sonicator so as to lessen the damage to the protein structure that may be induced by probe sonication. CNT-BSA composites were centrifuged at 2000 rpm for 15 min to remove the larger aggregates, and the supernatant was further centrifuged at 12 000 rpm for 15 min to separate the free BSA. The pellet was redispersed in PBS and used further. (iii) Pristine CNTs (pCNT): The CNTs were probe sonicated in PBS for 30 min and used immediately. Stability of CNT Dispersions and Interaction with BSA. To check the stability of the CNT-C-dot dispersion, a 100 μL aliquot of the CNT-C-dot dispersion was added to various solvents such as ethanol, acetone, acetonitrile, and DMSO and to a 1 mg/mL solution of BSA in PBS. Cell Culture. The HeLa cells (cervical cancer cell line) obtained from the National Centre for Cell Science (NCCS, Pune, India) were grown in DMEM culture medium supplemented with 10% heatinactivated fetal bovine serum (FBS) and Pen-Strep solution (100 units/mL of penicillin and 100 units/mL of streptomycin). Media, FBS, and Pen-strep solution were purchased from Life Technologies (Gaithersburg, MD, USA). Cells were maintained at 37 °C in a 5% CO2 humidified incubator (New Brunswick-Galaxy 48R). Twenty-Four Hour MTT Assay. To check the cytotoxicity of different CNTs (CNT-C-dot, CNT-BSA, and pCNT), an MTT assay was performed. Cells (6 ×103) were seeded in each well of a 96-well plate and incubated at 37 °C with 5% CO2. Once attached, the cells were treated with the CNTs at various concentrations: 20, 40, 60, 80, 100, and 150 μg/mL. After 24 h of treatment, the medium was replaced with 100 μL of a medium containing a 0.5 mg/mL MTT solution (Alfa Aesar) and incubated in the dark for 4 h. Then the medium containing MTT was removed, and 200 μL of DMSO was added to each well to dissolve the MTT product, formazan. The plate was placed in a rocker for 15 min for the complete solubilization of 7623

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Figure 1. (a) Emission spectrum of synthesized C-dots at different excitation wavelengths showing excitation-dependent emission. (Inset) Digital image of a C-dot solution under UV illumination (λex = 365 nm). (b) UV−visible spectrum, (c) TEM image (scale bar 10 nm), and (d) AFM image of the C-dots (scale bar 1 μm). counterstained with Hoechst 33342 dye (5 μg/mL, ThermoFisher) for 5 min and then washed again with PBS. The coverslips were placed on slides with the help of a mounting medium, Floursave (Merck Millipore), and sealed with regular nail polish. The slides were observed under a microscope (Nikon eclipse Ti-U) with a 10× objective. Hoechst stains all cell nuclei (of both live and dead cells), making them appear blue (λexc/λems = 350/461 nm), and propidium iodide stains the nucleus of dead cells only, making them appear red (λexc/λems = 535/617 nm). Statistical Analysis. All of the mentioned experiments were carried out in triplicate, and the values are represented as the mean ± standard deviation. Two-way ANOVA analysis along with Turkey’s multiple comparison test was performed for all of the experiments where P < 0.05 was considered to be statistically significant. Synthesis of the Au-CNT-C-dot Composite. To a 2 mL CNTC-dot dispersion was added 50 μL of a 10 mM aqueous solution of HAuCl4. The mixture was allowed to stand for 24 h, after which it was centrifuged at 8000 rpm and the pellet was washed twice with water to remove excess Au3+. The resulting pellet was redispersed in 2 mL of water by mild sonication in a bath for 10 min and used further. Catalytic Reduction of p-Nitrophenol to p-Aminophenol. To a standard quartz cell of path length 1 cm, 2 mL of 0.1 mM pnitrophenol, 5 mg of NaBH4, and 50 μL of the Au-CNT-C-dot composite catalyst were added, and the UV−visible spectrum kinetics was recorded. For comparative studies, 50 μL of C-dot-CNT was used as a catalyst. Instrumentation. A Varian Cary 100 Bio spectrophotometer was used for UV−visible measurements. Emission spectra were recorded using a Fluoromax-4p fluorometer from Horiba (model FM-100). Powder X-ray diffraction patterns (XRD) were obtained on a Rigaku, Ultima VI powder X-ray diffractometer. FTIR spectra were recorded in a KBr pellet using a Bruker Tensor 27 instrument. An FEI Technai G2, F30 microscope operating at a voltage of 300 kV and a JEOL JEM2100 microscope operating at a voltage of 200 kV were used to obtain

formazan. The absorbance was then measured at 590 nm with the help of a microplate reader (Synergy H1 BioTek multimode microplate reader). Twenty-Four Hour WST-1 Assay. In a 96-well plate, 100 μL of a medium (Opti-MEM, without phenol red, supplemented with 10% FBS) containing 6 × 103 cells was seeded in each well and incubated at 37 °C with 5% CO2. On adherence of the cells to the surface, they were treated with the respective CNTs (CNT-C-dot, CNT-BSA and pCNT) at varied concentrations of 20, 40, 60, 80, 100, and 150 μg/ mL, similar to the MTT assay. After 24 h of incubation with the different CNTs, the plate was removed from the incubator and 10 μL of activated WST-1 reagent (EZcount WST-1 cell assay kit, HiMedia Laboratories Pvt. Ltd.) was added to each well in the dark. The plate was wrapped with aluminum foil to avoid exposure to light and was incubated for 3 h. The absorbance was then measured at 450 nm wavelength with a reference wavelength of 650 nm by the microplate reader. Proliferation Assay. The same number (19 × 104) of HeLa cells was seeded in each well of a 6-well plate and incubated at 37 °C with 5% CO2. They were then treated with CNT-C-dots (80 μg/mL) and pCNT (80 μg/mL) for 6, 12, 24, and 48 h, respectively, and normal cells were kept as a control. After the treatment period, the cells were trypsinized and the number of live cells was counted with the help of a hemocytometer using an inverted cell culture microscope. Live/Dead Staining of Cells. An equal number of cells (30 × 104) were seeded on coverslips planted on a 6-well plate and incubated at 37 °C with 5% CO2 in a humidified incubator. They were then treated with CNT-C-dots (80 μg/mL) and pCNT (80 μg/mL) for 48 h. Untreated cells were kept as a control. After the treatment, the medium was removed and the coverslips were washed twice with PBS. They were then incubated with propidium iodide solution (20 μg/mL, HiMedia Laboratories Pvt. Ltd.) for 10 min in the dark. The cells were then fixed with the help of 4% paraformaldehyde in PBS for 20 min at room temperature and washed three times with PBS. The cells were 7624

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Figure 2. (a) UV−visible spectrum of CNTs (corrected with respect to C-dots). (Inset) Digital image of C-dot solution and CNTs dispersed in Cdots. (b) Sonication-time-dependent absorbance of CNT-C-dots. (c) Emission spectrum of C-dots and CNT-C-dots and (d) lifetime decay curves of C-dots (i) in the absence and (ii) in the presence of CNTs ((iii) instrument response function).

narrow peak at 258 nm that can be ascribed to the π−π* transition of nanocarbon (Figure 1b). The transmission electron microscopy (TEM) image (Figure 1c) validated the formation of well-dispersed spherical nanoparticles, and the average size of C-dots calculated from the particle size histogram was determined to be 4.2 ± 1.5 nm (Figure S1). Atomic force microscopy (AFM) measurements demonstrated the formation of C-dots with particle sizes in the range of 3−6 nm (Figure 1d), with their topographic heights mostly between 1 and 2 nm (Figure S2). The powder X-ray diffraction (PXRD) spectra of C-dots exhibited a broad peak centered at 2θ = 23° that corresponds to a d spacing of 3.8 Å (Figure S3). The Fourier transform infrared spectrum (FTIR) showed prominent peaks at 1106, 1622, 1726, and 2917 cm−1 and a broad peak at 3400 cm−1 that can be assigned to C−O, CC, C−H, and O−H functionalities, respectively (Figure S4). The brown C-dot solution was lyophilized, and the residue obtained could be dispersed in a range of solvents such as ethanol, acetonitrile, dimethyl sulfoxide (DMSO), and ethyl acetate, suggesting that the C-dots are amphiphilic in nature (Figure S5). This could be attributed to the presence of a π-conjugated network of carbon atoms along with surface functional groups such as −COOH and −OH that impart hydrophobic and hydrophilic characteristics to C-dots, respectively, which is similar to the characteristic of a surfactant molecule. The high aspect ratio of CNTs combined with their high flexibility pose the problem of nanotube entanglement and close packing.11 The CNTs are strongly bound by van der Waals forces of attraction, which leads to the tendency of

the transmission electron microscopy (TEM) images. The timeresolved fluorescence studies were performed on a Horiba Yvon (Fluorocube-01-NL model), a nanosecond time-correlated singlephoton counting (TCSPC) system. The dynamic light scattering (DLS) studies were carried out on a Micromeritics Nanoplus 3 instrument. Raman spectra were recorded on LABRAM HR from Horiba with a 632.8 He−Ne laser beam. Atomic force microscopy (AFM) of C-dots was performed on a cleaved mica surface on a Bruker Multimode 8 scanning probe microscope with a silicon cantilever in tapping mode.

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RESULTS AND DISCUSSION Synthesis and Characterization of the CNT-C-dot Composite. The synthesis of carbon dots was carried out using poly(ethylene glycol) (PEG 200) as the carbon precursor with a slight modification from an earlier report.35 PEG is a water-soluble polymer known for its antibiofouling properties and has been approved by the U.S. Food and Drug Administration (FDA) for various biomedical applications owing to its nontoxicity and nonimmunogenicity. A transparent PEG 200 solution when subjected to caramelization in a domestic microwave oven resulted in a brownish solution indicating the formation of C-dots. The dialyzed solution displayed bluish-green fluorescence under UV irradiation (λex = 365 nm, Figure 1a). The C-dots thus obtained showed their maximum emission at 485 nm when excited at 370 nm (Figure 1a). The photoluminescence shifted to longer wavelengths with a decrease in emission intensity upon increasing excitation wavelength, a typical characteristic of C-dots.26 The UV−vis absorption spectrum of a dilute solution of C-dots showed a 7625

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Figure 3. (a) FTIR spectra of pCNTs and the CNT-C-dot composite. (b) TEM image of CNTs dispersed in C-dots (scale bar 200 nm). (c) Highresolution TEM image of the CNT-C-dot composite. (d) Raman spectra of pCNTs and the CNT-C-dot composite.

ratio. For comparison, we studied CNT stabilization using PEG 200, the precursor for C-dots, and found that even at a concentration of 2:1 mass ratio with respect to CNTs, PEG could not stabilize a dispersion of CNTs at a concentration of 0.25 mg/mL. This suggests that the π-conjugated network in Cdots generated during the carbonization of PEG was critical in stabilizing CNTs in water. The interaction between C-dots and CNTs was investigated using fluorescence spectroscopy. The fluorescence intensity of C-dots was quenched upon interaction with CNTs (Figure 2c). It is well established that C-dots are rich in electrons and can act as electron donors.37 On the other hand, CNTs are known electron acceptors in their photoexcited state.32 Therefore, charge transfer might take place from C-dots to CNTs, leading to effective quenching in C-dot emission. It is worth mentioning that the fluorescence lifetime of C-dots as measured by time-resolved single-photon counting (TCSPC) was found to be 3.2 ns, which decreased to 2.33 ns in the CNTC-dot composite, suggesting charge transfer in the excited state (Figure 2d, Table S1) . Furthermore, the FTIR spectrum of CNT-C-dots showed the appearance of several new peaks at 3445, 1714, 1631, and 1074 cm−1 corresponding to O−H, C O, CC, and C−O functionalities, respectively, in comparison to pristine CNTs (pCNTs) (Figure 3a). This could be assigned to the presence of oxygenated functionalities owing to C-dots in the CNT-C-dot composite, suggesting the immobilization of C-dots on the CNT surface. The surface coating of CNTs with C-dots was confirmed using transmission electron microscopy where highly dispersed CNTs were observed to be coated with a low-contrast continuous layer of thickness ca. 4.8 nm on the

CNTs to assemble into bundles, resulting in low dispersibility in both aqueous and nonaquoeus solvents.12 C-dots with a hydrophobic core and a hydrophilic surface decorated with functional groups could potentially act as a dispersing agent as well as a stabilizer for CNTs. For this purpose, we added the Cdot solution to solid CNTs and treated them with ultrasonic irradiation. As shown in Figure 2a, the brownish C-dot solution turned black after the dispersion of CNTs, indicating the efficient deagglomeration of CNTs by C-dots. These dispersions were very stable because they could resist centrifugation at 2000 rpm for at least 15 min without visible sedimentation. Upon standing, the dispersion remained unchanged for several months without any precipitation. For the optimization of the dispersion yield and the study of their stability, the characteristic peak of MWCNTs at 260 nm in the UV−vis spectrum was monitored (Figure 2a). Because C-dots also show absorption around the same wavelength, a stable dispersion of MWCNTs using carboxymethyl cellulose (CMC) as a dispersant was used to calculate the molar extinction coefficient of CNTs.36 The absorption vs concentration curve of MWCNTs displayed a linear relationship at 260 nm, yielding an extinction coefficient of 4.32 mL/mg·mm for MWCNTs (Figure S6). This value was used to measure the concentration of CNTs in all subsequent experiments. The evolution of dispersed CNTs was also dependent on sonication time, as can be seen from Figure 2b where the absorbance at 260 nm increased with longer sonication treatment. The highest CNT concentration in stable dispersions was found to be approximately 0.5 mg/mL using a 2:1 C-dot/CNT mass 7626

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Figure 4. (a) Photographs of CNT-C-dots dispersed in various solvents. (b) UV−visible spectrum of CNT-C-dots upon incubation with BSA. (c) Time-resolved anisotropy decay of C-dots (blue), CNT-C-dots (red), and BSA-CNT-c-dots (black). (d) Dynamic light scattering measurement of CNT-C-dots and BSA-CNT-C-dots.

outer walls of MWCNTs, implying efficient wrapping of CNTs by C-dots (Figure 3b,c). The high density of C-dots wrapped on the CNT surface resulted in the appearance of a continuous carbon layer. Moreover, it can be seen that the structure of CNTs was intact, suggesting that the intrinsic properties of CNTs might be preserved during this modification process. The first-order Raman spectra of pCNTs and the CNT-C-dot composite are shown in Figure 3d. The pCNTs showed two characteristic peaks at 1325.2 and 1586.9 cm_1 that can be assigned to D and G bands of CNTs, respectively. For carbon nanomaterials, the ratio of intensity of D and G bands is an important tool in determining the defects in the material.38 The ratio of intensity of D and G bands in pristine CNT was found to be 1.58, which did not change appreciably after the sonication treatment in the presence of C-dots. This also confirmed that the intrinsic structure of CNTs was not altered during the noncovalent surface functionalization process.39 Stability upon Interaction with External Proteins. The stability of the CNT-C-dot composite was also investigated in a series of solvents such as ethanol, acetone, acetonitrile, and DMSO. Because of the amphiphilic nature of the C-dots, the composite homogeneously diffused in these solvents and remained stable even after a week without any visible agglomeration (Figure 4a). The results indicate the possible processability of the CNT-C-dot composite for thin film deposition and device fabrication via solution processing for which dispersions in low-boiling-point solvents such as ethanol are required. For biomedical applications of CNTs, it is imperative to study the interaction of blood proteins on the CNT-C-dot composite. Because albumin is the most abundant protein in blood, its interaction with the CNT-C-dot composite

was probed. For this, an aliquot of the CNT-C-dot composite was added to the bovine serum albumin (BSA) solution. The resulting solution did not show precipitation for 24 h, pointing to the high stability of the CNT-C-dot composite in biological systems (Figure 4b). Moreover, the UV−vis spectra of the CNT-C-dot composite did not show appreciable changes after 24 h of incubation in a BSA solution, implying that the composite did not aggregate (and eventually did not precipitate) in the presence of BSA (Figure 4b). BSA is also known to stabilize CNTs in water by π−π interactions through its aromatic residues.40 To gain insight into the interaction of BSA with the CNT-C-dot composite, fluorescence anisotropy decay studies were performed (Figure 4c). It is well-known that the fluorescence anisotropy of a fluorophore depends on the molecular rotation in its microenvironment, which depends on the viscosity of the solution, the fluorescence lifetime of the fluorophore, and the size and mass of the molecule to which the fluorophore is attached.41 As observed, the rotational relaxation time of C-dots increased upon dispersing CNTs, implying that the C-dots are immobilized on the CNT surface. Upon incubation of the CNT-C-dot composite with BSA, it was observed that the rotational relaxation time of C-dots increased, further indicating the formation of a larger complex with BSA on the CNT-C-dot surface affecting the rotational diffusion of C-dots. The complexation of BSA with CNT-C-dots was also supplemented by dynamic light scattering (DLS) studies. Although DLS measurements are suitable for determining the diameter of particles that are spherical in shape, the data can be used to compare the relative particle size of suspensions of other nanostructures.42 The mean hydrodynamic radius of the CNT-C-dot composite increased from 388.2 to 471.5 nm upon 7627

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Figure 5. Cytotoxicity of different CNTs on the HeLa cell line after incubation for 24 h at various concentrations as studied by (a) MTT assay and (b) WST-1 assay. Error bars indicate the standard deviation.

Figure 6. Bright-field images and the corresponding fluorescent images after PI/Hoechst staining of (a, d) cells without any treatment, and cells treated with (b, e) CNT-C-dots and (c, f) pCNTs for 48 h (scale bar 50 μm).

cell viability decreased with increases in the amounts of all the three CNTs. However, the CNT-C-dot composite showed significantly lower cytoxicity as compared to that shown by the pCNT and CNT-BSA composites over a range of 20−150 μg/ mL (Figure 5a). When compared to the control, CNT-C-dots exhibited 86.7% viability, whereas pCNT and CNT-BSA showed 55.7 and 75.1% viability, respectively, at a concentration of 20 μg/mL, signifying that the coating of CNTs by Cdots decreases their toxicity by an appreciable extent. According to the literature,43 the MTT assay has certain limitations during cytotoxicity studies with CNTs; therefore, we also performed a WST-1 assay as a check measure. The principle of this assay is similar to that of the MTT assay except for the enzymatic pathway involved. Here, the mitochondrial dehydrogenase of viable cells reduces the WST-1 tetrazolium salt, which can be monitored spectrophotometrically. The results of this assay followed a pattern similar to that of the MTT assay, where the

incubation with BSA, suggesting the binding of BSA on the CNT-C-dot surface (Figure 4d). Toxicity Evaluation of C-dot-Coated CNTs. To evaluate the toxic effects of CNT-C-dots on cells, three independent in vitro experiments (an MTT assay, a WST-1 assay, and cell proliferation studies) were performed on a human cell line (HeLa cells). The MTT assay is based on the principle that cellular NAD(P)H-dependent oxidoreductase enzyme reduces pale-yellow tetrazolium dye MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to a purple-colored compound called formazan, which can be easily monitored through UV−vis spectroscopy. The MTT assay was performed in a dose-dependent manner for a treatment period of 24 h. For comparative studies, we also performed the assay with pristine CNTs (pCNT) and BSA-stabilized CNTs because the binding of blood proteins such as BSA on CNTs is known to reduce their cytotoxicity.40 Upon treatment, it was observed that the 7628

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Figure 7. (a) UV−visible spectrum. (b) High-resolution TEM. (c) PXRD pattern of the Au-CNT-C-dot composite. (d) Time-dependent UV− visible spectrum for the reduction of p-nitrophenol to p-aminophenol by NaBH4 catalyzed by Au-CNT-C-dot composites.

due to which only a few cells (nuclei stained blue) could be seen (Figure 6f). Moreover, a large number of cells with their nuclei stained red implied the cytotoxic effect of pCNTs on cells, which was significantly reduced in the case of CNTs modified with C-dots. Nanoparticle-Functionalized CNTs. The development of CNT-metal nanoparticle composites is an area of immense interest because such materials possess superior properties compared to their individual counterparts.44 Carbon nanotube−Au nanoparticle (CNT-Au) composites have promising applications in several areas, including electronics, optics, and catalysis.45,46 The preparation of CNT-Au nanocomposites has been reported by a variety of methods, such as the direct deposition of Au NPs on the CNT surface by physical methods, covalent and noncovalent linking of Au NPs through various molecules, and direct electroless reduction of gold cations by carbon nanotubes.46 The surface functionalities on C-dots are known to be highly reactive and have been shown to effectively reduce metal ions, leading to the formation of metal nanoparticle-C-dot composites.47,48 In the case of the CNTC-dot composite, C-dots immobilized on the CNT surface could be used as both reducing and stabilizing agents for the growth of Au NPs directly on the surface of CNTs. The reduction of Au3+ was achieved by the simple incubation of a CNT-C-dot dispersion with an aqueous solution of HAuCl4 at room temperature. The UV−visible spectrum of the resultant solution displayed a peak at 542 nm along with the characteristic absorption pattern of CNTs, implying the formation of Au NPs on CNT-C-dot composites (Figure 7a). The formation of the Au-CNT-C-dot composite was further

C-dot-coated CNTs were found to be the least cytotoxic among the CNTs tested (Figure 5b). This observation was further supported by the cell proliferation assay in which CNT-C-dots had a significantly lower impact on cell proliferation compared to the pCNT. On treating the HeLa cells with CNT-C-dots and pCNT for time periods of 6, 12, 24, and 48 h, the proliferation rates decreased promptly in the case of pCNTs after 24 and 48 h, showing the detrimental effect of pristine CNTs over a longer duration. On the other hand, the C-dot-coated CNTs had a very limited effect on cell growth for the same duration (Figure S7). The pCNT-treated cells showed 41.6 and 66.1% less proliferation than the CNT-C-dot-treated cells over periods of 24 and 48 h, respectively. Along with the decrease in the rate of proliferation, there was a prominent change in the morphology of the pCNT-treated cells, as can be seen in the images (Figure S8). The pCNT exposure to HeLa cells changed their shape to spherical, whereas CNT-C-dot proliferated cells retained the characteristic elongated structure typical of these cells. Thus, the immobilization of C-dots on the CNT surface led to a significant decrease in the cytotoxicity inherent to CNTs without much adversarial effect on the structural integrity of the cells, showing potential applicability in biomedical studies. The propidium iodide and Hoechst staining experiment further confirmed the significantly lower toxicity of CNT-C-dot composites. The C-dot-coated CNTs showed significantly less apoptosis of HeLa cells as compared to the pristine CNTs. Figure 6 shows the comparative apoptosis studies of cells stained with propidium iodide (red). The exposure of cells to pCNTs induced damage to cells, ultimately causing cell lysis, 7629

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Langmuir observed by TEM spectroscopy, where Au NPs of sizes in the range of 10−20 nm could be seen on the surfaces of CNTs (Figure S9). The HR-TEM (high-resolution TEM) image shows that the Au NPs are covered with a low contrast layer of C-dots (Figures 7b and S10). The Au-CNT-C-dot composite showed characteristic Bragg’s reflections corresponding to cubic Au along with the reflection of C-dots (Figure 7c). Au-CNT composites have been extensively studied as catalysts for various organic transformations such as the oxidation and hydrogenation of diverse substrates.45 To evaluate the catalytic efficacy of the composite, we studied the reduction of 4-nitrophenol to 4-aminophenol in the presence of borohydride as a model reaction.49 The kinetics of the reduction of 4-nitrophenol was monitored through UV− vis spectroscopy, where the decrease in the absorbance peak at 400 nm signified the conversion of nitrophenol to aminophenol (Figure 7d). A linear relationship between the plot of ln(absorbance) of p-nitrophenol at 400 nm and time was indicative of pseudo-first-order kinetics (Figure S11), which is consistent with earlier reports.50 C-dots embedded on an amorphous carbon matrix are also known to possess catalytic activity toward the reduction of nitrophenol.51 The comparative catalytic study of C-dot-CNT and Au-CNT-C-dot composites showed that the all-carbon composite (C-dot-CNT) could also catalyze the reaction, albeit at a much slower rate as compared to that of Au-C-dot-CNT during a similar time duration (Figure S12). Thus, a simple strategy of dispersing CNTs using C-dots can be exploited to fabricate metal nanoparticle-CNT composites without the use of any external reducing or stabilizing agent at room temperature that showed efficient catalytic activity toward the reduction of organic pollutants.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bhagwati Sharma: 0000-0001-5459-8237 Tridib K. Sarma: 0000-0002-5168-6327 Funding

This research is funded by the Science and Engineering Research Board (SERB), Department of Science and Technology, India (SR/S1/PC-32/2010). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank IIT Indore for providing the infrastructure, experimentation facilities, and financial support. The instrumentation facility from SIC, IIT Indore is duly acknowledged. We are thankful to SAIF, NEHU, Shillong, and SAIF, IIT Bombay for use of the TEM facility. We are also grateful to Central Instrumentation Facility, IISER Bhopal for Raman analysis. The authors thank Dr. Anjan Chakraborty and Mr. Anupam Das for helpful scientific discussions. S.M. acknowledges a research fellowship from CSIR.

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ABBREVIATIONS MWCNT, multiwalled carbon nanotubes; BSA, bovine serum albumin; EtOH, ethanol; ACN, acetonitrile; DMSO, dimethyl sulfoxide; CMC, carboxymethyl cellulose

CONCLUSIONS We have exploited the amphiphilic nature of C-dots to achieve stable dispersions of MWCNTs in water. The microscopic and spectroscopic studies revealed that C-dots are efficiently immobilized on the CNT surface, and this noncovalent modification had a negligible effect on the intrinsic structure of CNTs. The stabilization of CNTs could be accomplished by the π-conjugated network in C-dots generated during the carbonization of PEG, which interacts with the π electrons on the CNT surface. The coating of CNTs by C-dots decreased their cytotoxicity significantly, as was verified by MTT and cell proliferation assays. Moreover, the surface functionalities on the C-dot surface were utilized to fabricate AuNP-CNT-C-dot composites that could effectively catalyze the reduction of nitrophenol to aminophenol. Thus, dispersing CNTs using Cdots provides an easy, one-step, environmentally benign method of generating CNT-C-dot composites that are significantly nontoxic as compared to pristine carbon nanotubes and can be easily functionalized with metal nanoparticles.

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ation studies. TEM images of the Au-CNT-C-dot composite and its catalytic application (PDF)

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b00557. Details of the methods of TCSPC, anisotropy decay, and extinction coefficient studies. Figures regarding the size distribution histrogram, height profile, PXRD, FTIR, and solubility of C-dots in different solvents. Cell prolifer7630

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