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Carbon dots as Nanodispersants for Multi-walled Carbon Nanotubes: Reduced Cytotoxicity and Metal Nanoparticle Functionalization Sonam Mandani, Prativa Majee, BHAGWATI SHARMA, Daisy Sarma, Neha Thakur, Debasis Nayak, and Tridib K. Sarma Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00557 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017
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Carbon dots as Nanodispersants for Multi-walled Carbon Nanotubes: Reduced Cytotoxicity and Metal Nanoparticle Functionalization Sonam Mandania, Prativa Majeeb, Bhagwati Sharmac, Daisy Sarmaa, Neha Thakura, Debasis Nayakb* and Tridib K. Sarmaa* a
Discipline of Chemistry, Indian Institute of Technology Indore, Simrol, Khandwa Road, Indore
453552, India b
Centre of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore,
Simrol, Khandwa Road, Indore 453552, India c
Institute of Nano Science and Technology, Phase X, Sector-64, Mohali 160062, India
Corresponding author: T. K. Sarma (Email:
[email protected]) KEYWORDS: Carbon nanodots, carbon nanotubes, dispersion, cytotoxicity, Au nanoparticles.
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Abstract
The colloidal stabilization of multi-walled carbon nanotubes (MWCNTs) in aqueous medium through non-covalent interactions has potential benefits towards the practical use of this onedimensional 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 the hydrophilic oxygenated functionalization on C-dot surface provided excellent water dispersibility. The resultant CNT-C-dot composite showed significantly reduced cytotoxicity compared to unmodified or protein coated CNTs, as demonstrated by cell viability and proliferation assays. Further, the reducing capability of C-dots could be envisaged towards the formation of catalytically active metal nanoparticle-CNT-C-dot composite without addition of any external reducing or stabilizing agents that showed excellent catalytic activity towards the reduction of p-nitrophenol in 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 Based on 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 wide exploitation of CNTs in a host of possible applications such as composite reinforcement material, field emission displays, energy storage, sensors, scanning probe tips, drug delivery carriers etc. with newer applications emerging continuously.7-11 However, to realize their full prospect towards development of novel functional high quality nanocomposites for various applications, separation and uniform dispersion of CNTs is a fundamental prerequisite.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 structure, aspect ratio, surface area, degree of aggregation, surface topology, bound functional groups, concentration and dose offered to the cells or organisms.16 The most common mechanisms that lead to cytotoxicity of CNTs include necrosis and apoptosis which are a result of oxidative stress, damage to the DNA and cell membrane, alteration of intracellular metabolic pathways etc.
16-17
Significant efforts have been made to modify CNTs in order to obtain
homogenous 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, non-covalent
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functionalization with various molecules is advantageous that 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 van der Waals force.23 These molecules are amphiphilic in nature and enhance the wetting characteristics of CNTs in water making them less toxic. Several biomolecules (e.g. proteins, DNA, chitosan), polymers (e.g. polyethylene glycol, poly(l-amino acid), pluronics) and surfactants (e.g. sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide (CTAB), Triton-X) have been utilized for the non-covalent functionalization of CNTs which enhance water dispersibility and biocompatibility.23-25 Taking into account the immense potential of CNTs in the field of nanomedicine for bioimaging, delivery of bioactive molecules, biosensors etc., there is still 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 tunability of surface functionalization as desired, makes C-dots vastly applicable in biomedical field such as sensors, imaging, nanovehicles, etc.27 Doping with heteroatoms as well as introducing different surface functionality using diverse precursors during synthesis could direct tunable physicochemical properties.28-29 Also, C-dots 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 surfactant like amphiphilic properties. This property has been exploited for exfoliation and stabilization of graphene in
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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.3234
The surfactant like behavior of C-dots for dispersing CNTs leading to stable C-dot-CNT
composites in water has not been explored yet. Herein, we report C-dots as effective dispersants for debundling multiwalled carbon nanotubes (MWCNTs) that provide efficient separation and high stability in water. The C-dots, synthesized from a commonly available and biocompatible polymer, polyethylene glycol (PEG), could be dispersed in a range of solvents suggesting their amphiphilic nature. 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 non-covalent binding due to their high hydrophilicity. Carbonization of PEG resulted in a conjugated sp2 hybridized network in the C-dot core which 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 a highly dense functionalization on 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 highly stable against blood serum protein, albumin. The reducing capability of C-dots could be exploited further towards the generation of Au nanoparticles on 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: 20 mL of PEG 200 taken in a glass beaker was subjected to heating in a domestic microwave oven (750 W) for 15 minutes upon 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 subjected to dialysis for 48 hours using a cellulose membrane to remove excess PEG. This dialyzed solution was used for further experiments. A 25 mL dialysed solution was lyophilized which 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 minutes and then filtered and washed twice with water to remove any soluble impurities. The resultant CNTs were dried in oven at 80oC for 24 hours and used further. (i)
CNT-C-dots: 2 mg of CNT was added to a 5 mL solution of C-dots (0.52 mg/mL) and sonicated using a probe sonicator (20 kHz, 250 W) for 20 minutes. The sonication experiments were performed in ice water to avoid over-heating of the dispersions. The highly dispersed CNT-C-dots were centrifuged at 2000 rpm for 30 minutes 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 mins. The pellet was
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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/5mL) were first probe sonicated for 20 min in phosphate buffer saline (PBS, pH 7.4) followed by 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 bath sonicator so as to lessen the damage to the protein structure which may be induced by probe sonication. CNTBSA composites were centrifuged at 2000 rpm for 15 min to remove the larger aggregates and the supernatant was for further centrifuged at 12,000 rpm for 15 mins 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 mins and used instantly.
Stability of CNT dispersions and interaction with BSA: To check the stability of the CNT-Cdot dispersion, a 100 µL aliquot of CNT-C-dot dispersion was added to various solvents such as ethanol, acetone, acetonitrile, DMSO and to a 1 mg/mL solution of BSA in PBS. Cell Culture: The HeLa cells (cervical cancer cell line) obtained from National Centre for Cell Science (NCCS), Pune, India were grown in DMEM culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and Pen-Strep solution (100 units/ml penicillin and 100units/ml streptomycin). Media, FBS and Pen-strep solution were purchased from Life Technologies (Gaithersburg, MD, USA). Cells were maintained at 37ºC in a 5% CO2 humidified incubator (New Brunswick- Galaxy 48R).
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24 hours MTT assay: To check the cytotoxicity of different CNTs (CNT-C-dot, CNT-BSA and pCNT), MTT assay was performed. 6 x 103cells were seeded in each well of a 96-well plate and incubated at 37ºC with 5% CO2. Once attached, the cells were treated with the CNTs at various concentrations of 20µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, 100 µg/mL and 150 µg/mL. After 24 hours of treatment, the media was replaced with 100µL of medium containing 0.5mg/mL MTT solution (Alfa Aesar) and incubated in dark for 4 hours. 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 15minutes for complete solubilization of formazan. The absorbance was then measured at 590 nm with the help of a microplate reader (Synergy H1 BioTek multi-mode microplate reader). 24 hours WST-1 assay: In a 96-well plate, 100 µl of media (Opti-MEM, without phenol red, supplemented with 10 % FBS) containing 6 x 103 cells was seeded in each well and incubated at 37ºC with 5% CO2. On adherence of cells to the surface, they were treated with the respective CNTs (CNT-C-dot, CNT-BSA and pCNT) at varied concentrations of 20µg/ml, 40µg/ml, 60µg/ml, 80 µg/ml, 100µg/ml and 150µg/ml, similar to MTT assay. After 24 hours of incubation with the different CNTs, the plate was removed from the incubator and 10µl of activated WST-1 reagent (EZcount™ WST-1 Cell Assay Kit; HiMedia Laboratories Pvt. Ltd) was added to each well in dark. The plate was wrapped with aluminum foil to avoid exposure to light and was incubated for 3 hours. The absorbance was then measured at 450nm wavelength with a reference wavelength of 650nm by the microplate reader. Proliferation Assay: Equal number (19 x 104) of HeLa cells were seeded in each well of a 6well plate and incubated at 37ºC with 5% CO2. They were then treated with CNT-C-dots (80µg/mL) and pCNT (80µg/mL) for 6, 12, 24 and 48 hours respectively and normal cells were
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kept as 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 x 104) were seeded on cover-slips planted on a 6-well plate and incubated at 37ºC with 5% CO2 in humidified incubator. They were then treated with CNT-C-dots (80µg/mL) and pCNT (80µg/mL) for 48 hours. Untreated cells were kept as control. After the treatment, the media was removed and the cover-slips were washed twice with PBS. They were then incubated with propidium iodide solution (20µg/mL, HiMedia Laboratories Pvt. Ltd.) for 10 min in dark. The cells were then fixed with the help of 4% paraformaldehyde in PBS for 20 minutes at room temperature and then washed thrice with PBS. The cells were counterstained with Hoechst 33342 dye (5µg/mL; ThermoFisher) for 5 min. The cells were again washed with PBS. The cover-slips were then 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 10x objective. Hoechst stains nucleus of all cells (both live and dead) making them appear blue (λexc/λems=350/461nm) while Propidium iodide stains the nucleus of dead cells only making them appear red (λexc/λems=535/617nm). Statistical analysis: All the mentioned experiments were carried out in triplicate and the values were represented as mean ± standard deviation. Two-way ANOVA analysis along with Turkey’s multiple comparisons test was performed for all the experiments where P
