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Highly biocompatible carbon nanocapsules derived from plastic waste for advanced cancer therapy
Accepted Manuscript Highly biocompatible carbon nanocapsules derived from plastic waste for advanced cancer therapy Amine Mezni, Nesrine Ben Saber, A.A. Alhadhrami, Adil Gobouri, Ali Aldalbhi, S. Hay, Abel Santos, Dusan Losic, Tariq Altalhi PII:
S1773-2247(17)30194-6
DOI:
10.1016/j.jddst.2017.08.007
Reference:
JDDST 451
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 2 March 2017 Revised Date:
8 August 2017
Accepted Date: 11 August 2017
Please cite this article as: A. Mezni, N.B. Saber, A.A. Alhadhrami, A. Gobouri, A. Aldalbhi, S. Hay, A. Santos, D. Losic, T. Altalhi, Highly biocompatible carbon nanocapsules derived from plastic waste for advanced cancer therapy, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/ j.jddst.2017.08.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highly Biocompatible Carbon Nanocapsules Derived From Plastic Waste for Advanced Cancer Therapy Amine Mezni a,b,*, Nesrine Ben Saber a, A. A. Alhadhrami b, Adil Gobouri c, Ali
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Aldalbhi d, S. Hay e, Abel Santos b, Dusan Losic b and Tariq Altalhi b,c,* a
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Unite de recherche "Synthese et Structure de Nanomateriaux" UR11ES30, Faculte des Sciences de Bizerte, Universite de Carthage, 7021 Jarzouna, Tunisie b School of Chemical Engineering, University of Adelaide, Adelaide, Australia c Department of Chemistry, Faculty of Science, Taif University, Taif, Saudi Arabia d Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia e Discipline of Surgery, Breast Cancer Research Unit, Basil Hetzel Institute and Centre for Personalised Cancer Medicine, University of Adelaide, Adelaide, Australia
Abstract
Carbon nanotubes (CNT) are increasingly being investigated for their use in biomedical applications and nanomedicine. In order to be used as nano-carriers for delivering anti-cancer drugs (drug delivery), smart free standing carbon nanotubes
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(CNTs) with hydrophilic core were prepared from recycled plastic bags. These CNTs were loaded with doxorubicin (Dox) and its external surface was chemically functionalized with a biodegradable polymer (chitosan) by anchoring its polymeric chains to functional groups on the external surface of CNTs. The obtained Chitosan-
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coated CNTs (Ch-CNCs) nanocomposites were then tested for their localized and slow drug eluting property using the cellular vicinity of MDA-MB-231 TXSA, human
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breast cancer cell line. The preliminary results are very promising and confirm a 500 fold enhanced death rate in case of cells treated with Ch-CNCs compared to the prodrug alone. This work shows that it is possible to develop a highly biocompatible carbon nanocapsules (CNCs) derived from plastic bags and further used them as cargo transporters to study an intra-tumoral delivery of an anti-neoplastic drug is possible. Keywords: Carbon Nanotubes; CVD, Plastic bags, Drug delivery, Cancer therapy * Corresponding author. E-mail address: [email protected] ; [email protected]
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is the leading cause of death worldwide and has enticed researchers worldwide to look for better and advanced treatments as that to chemo and radiation therapy. Lack of specificity and high toxicity has been the major drawback of the general chemotherapeutic procedure. The ideal cancer drug delivery systems combine targeted delivery with controlled release to
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deliver and release in a selective fashion to the target cells [1-4]. Such systems not only improve the efficacy of the drug, but also reduce the toxic side effects of the drug. In recent years, a wide range of different nanoscale drug carriers have been developed and explored [5,6]. Notably, single wall carbon nanotubes (SWCNTs) have emerged as strong potential
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candidate due to their advantages as a high cargo loading, intrinsic stability and structural flexibility, over the more widely studied metal nanoparticle systems, which could prolong the circulation time and enhance the bioavailability of the therapeutic agent [7–16]. Moreover,
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SWCNTs have been shown to enter mammalian cells and thus investigated as potential delivery vehicles for intracellular transport of nucleic acids, proteins and drug molecules. Furthermore, SWCNTs have been functionalized with antibodies as targeting agents providing a high efficiency for nanotube internalization into cells. On the other hand, SWCNTs have also disadvantages relative to MWCNTs such as their higher tendency to
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aggregate into bundles that may be related to cytotoxicity if not controlled [17-23]. However, many groups have reported that the agglomeration of SWCNT in the air-ways is the primary cause of morbidity and granuloma formation. Drug molecules such as doxorubicin (DOX) can be loaded onto CNTs via hydrophobic and π - π stacking interactions and the release rate can
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be controllable by using nanotubes with different diameters [24,25]. However, inherent properties of SWCNTs such as poor aqueous solubility and tendency to form bundles limited their use for pharmacological applications. To overcome these drawbacks, a number of
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synthetic and natural polymers have been used to encase SWCNTs via non-covalent interactions to improve their compatibility with water and physiological environments. On the other hand, beside polymer attachment, also covalent functionalisation is widely used to avoid the cytotoxic effect of CNTs [26-31]. Indeed; surface engineering of CNTs by covalent and noncovalent modifications enables site-specification drug delivery and targeting. The noncovalent functionalization is based on Van Der Waals, hydrophobic or the ability of the extended π-system of the carbon nanotube sidewall to bind guest molecules via π-π-stacking interactions. Its advantage depend on the preservation of the properties of the CNTs, while its disadvantage concerns weak forces between wrapped/coupled molecules that may lower the load transfer in the composite. The covalent functionalization can be performed at the 2
ACCEPTED MANUSCRIPT sidewall site of CNTs or at the defect sites usually located at the tip. New properties can be added by this means of functionalization. Instead, electronic properties of SWCNTs are perturbed by covalent functionalization and double bonds are irreversibly lost. This may affect conductive property, preventing further CNT applications. Despite excellent progress in using SWCNTs as drug carriers, more research is needed to further optimize their ability to
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selectively accumulate in diseased tissues and release the pay load in a controlled manner. Notice that, most of the aforementioned studies using CNTs for drug delivery application are based on single walled CNTs and CNTs prepared using catalyst based CVD methods. This fabrication suffers from the difficulty in controlling the thickness of the CNTs wall and thus
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the nanotube diameter, shape morphology and importantly presence of metal catalyst particles from conventional CNTs (i.e. SWCNTs and catalyst based CNTs) restrict their use as drug delivery systems. Due to the aforementioned reasons, the toxicity of these CNTs is still a
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highly debatable issue. In addition, all the above studies target systemic route of therapeutic administration with no reports on the use of CNTs for local drug administration. In recent studies based on template synthesis of nanotubes for drug delivery, the focus has been on uniformity of these carriers so as to easily internalize into a cell and facilitate an apt calculation for the authenticity of the method. In this work, we have performed an innovative
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CVD approach to synthesize carbon nanotubes by using nanoporous anodic alumina membranes as templates and commercially available plastic bags as a carbon source without need to use any metal catalysts or solvent. This can prevent and reduce many synthesis disadvantages (use of poisonous chemical metal compounds and the expensive chemicals and
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production of commercial products as separation/filtration membranes) which make it a potential nanotechnological recycling approach, which could contribute directly to the conservation of our natural ecosystems in the near future. However, the principal aim of this
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work is to investigate and exploit the role of CNTs as an optimal drug carrier in the field of localized drug delivery. The localized and sustained delivery of an antineoplastic drug, doxorubicin (Dox), has been evaluated in this in vitro study. These chitosan-coated CNTs in their pristine form (i.e. without drug inside) were also tested on both, human breast cancer and human foreskin fibroblast cell lines in order to investigate the toxicity of these carbon based materials where hence concluding them to be an ideal intra-cellular drug transporter for breast cancer treatment.
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ACCEPTED MANUSCRIPT 2. Experimental Section 2.1.
Synthesis of carbon nanotubes (CNTs) or chitosan-coated CNTs
CNTs were prepared as following. 2.1.1. Nanoporous anodic alumina (NAA) Template Fabrication NAATs were prepared using a two-step anodization with a constant voltage of 40V for 20 h
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in 0.3M oxalic acid at 5°C temperature as described in Altalhi et al. [32] Briefly, high purity Al chips (i.e. 1.5 cm in diameter) were cleaned under sonication in ethanol (EtOH) and double distilled water and subsequently electropolished in a mixture of perchloric acid (HClO4) and EtOH 1:4 (v:v) at 20V and 5°C for 3min. Then, the 1st anodization step was carried out in an
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aqueous solution of oxalic acid (H2C2O4) 0.3M at 40V and at 6°C for 20 h. Then, the resulting alumina (Al2O3) layer was selectively dissolved by wet chemical etching in a mixture of phosphoric acid (H3PO4) 0.4M and chromic acid (H2CrO7) 0.2M at 70°C for 3 h. Next, the
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2nd anodization step was performed under the same anodization conditions as the 1st one for 17 h. Once the anodization process finished, the remaining aluminium substrate was removed in a saturated solution of hydrochloric acid and cupric chloride (HCl/CuCl2). Subsequently, a pore opening process was performed by wet chemical etching in an aqueous solution of H3PO4 5wt% at 35°C under current control conditions [33].
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2.1.2. Synthesis of chitosan-coated CNTs
Carbon nanotubes (CNTs) were fabricated as reported by AlTalhi et al. [32] using template and catalyst free CVD synthesis inside nanoporous anodic alumina membranes (NAAMs). The fabrication process was carried out using a CVD system consisting of a two-stage
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furnace equipped with a cylindrical quartz tube with dimensions (43 and 1000 mm in diameter and length, respectively) and temperature and gas flow controllers. Particularly, shopping plastic bags were collected from a local grocery that are typically produced from
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linear low-density polyethylene (LLDPE) and used them as a carbon source to produce CNTs. After collection, these plastic bags were washed with a liquid soap and double distilled water, dried under nitrogen stream, cut into small squares of 1 cm2 and kept in a container with inert atmosphere to prevent them from contaminations before the fabrication process. Then, these small pieces of plastic bag were placed in a ceramic crucible, which was introduced into the pyrolysis zone of the CVD reactor. To ensure the absence of oxygen during the CNTs synthesis, Argon inert (Ar) gas was flowed at 1 dm3 min-1. In the deposition zone of the CVD reactor, prepared NAAMs were placed where the carbon deposition took place. We performed the fabrication process at 850°C and 30 min.
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ACCEPTED MANUSCRIPT After CNTs synthesis, the CVD reactor was cooled down to room temperature. The resulting CNTs–NAAMs were saved in a container under inert conditions. An additional annealing process was used in some CNTs–NAAMs samples by heating under Ar atmosphere at 900°C for 3 h. The obtained CNTs were followed by being taken out and ultrasonicated in HF solution in order to dissolve the alumina membrane. This was followed by centrifugation
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(15000 rpm) and sequential washing with deionized water. The centrifuged pellet hence obtained was then oxidized under air plasma by exposure for 30s and then re-dispersed in phosphate buffer, pH-8.5. The nanotubes remained dispersed for months. 2.2.
Structural Characterisation
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Prepared NAAMs, CNTs and CNTs-NAAMs are characterised by a scanning electron microscope (FEG-SEM FEI Quanta 450) equipped with energy dispersive X-ray spectroscopy (EDXS). The standard image processing package (ImageJ, public domain program developed
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at the RSB of the NIH, USA) was used to carry out the SEM image analysis. Liberated CNTs are analysed by transmission electron microscopy (TEM Philips CM 200). Transmission infrared (IR) spectra are, however, recorded using a Nicolet Fourier Transform Infrared (FTIR) spectrophotometer at a spectral resolution of 2 cm-1 accumulating 128 scans, in a self-supported disk. Thermogravimetric analysis TGA measurements was performed on a SETSYS Evolution 1750 SETARAM instrument under an Ar atmosphere at a heating rate of
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10 K min-1 over a temperature range from 25 to 800°C; the weight of the sample was 20 mg. Drug Loading in chitosan-coated CNTs and Release Study
300µL of 2mg.ml-1 doxorubicin hydrochloride stock solution (Sigma Aldrich) was
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gradually added to 5mL of the alkaline buffer suspended nanotubes and kept under continuous stirring for 24 h at room temperature under dark condition followed by subsequent addition of chitosan solution (1% wt/vol in 3% acetic acid) to form the
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polymeric integument. The drug loaded nanocapsules were isolated through dialysis and then incubated with 1% human serum albumin (HSA) overnight at a constant pH of 8.0. The purification after this again followed the same process of dialysis. The volume of the solution containing the dispersed nanocapsules was thereafter made up to 20 ml. For the drug release study, 10 ml of the buffer containing nanocapsules was taken in a dialysis bag with a threshold of 3000 Daltons and dialyzed against a neutral pH of 7.0 at 37°C. Subsequently, aliquots of the sample were taken at different time intervals to analyze for Dox concentration through UV-visible spectroscopy (at 479 nm). The volume of the aliquot taken out for this study was replaced with fresh buffer
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ACCEPTED MANUSCRIPT (pH 7.0) so as to maintain the concentration gradient at all times. Three replicas were used for each sample composition. 2.4.
Cell Culture
Human breast cancer cell line MDA-MB-231 TXSA and human foreskin fibroblast (HFF) were cultured in DMEM, supplemented with 10% foetal calf serum (FCS) in
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5% CO2 at 37°C in an incubator. Treatment of Cancer Cells with chitosan-coated CNTs
The TXSA breast cancer cells were plated in a 12-well plate and seeded with a concentration of 1.25x104 cells per well. Each of these wells was then dosed with 1ml
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of the varying concentration of drug loaded nanocapsules (100µl/ml and 200µl/ml; prepared in PBS). As a comparative study, the empty chitosan-coated CNTs in similar concentrations as their drug loaded counterparts were added in the adjacent wells. At
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all times, the volume was maintained at 1mL with PBS. Subsequently, as the positive control, the as calculated amount of dox, corresponding to different volume of the drug retained in the varying concentration of nanocapsule dosage, was added. At the end, a well was treated with 1ml of only PBS, which served as the negative control (referred to as blank).
Toxicity Study of chitosan-coated CNTs
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2.6.
As a study for biological toxicity of these chitosan-coated CNTs, their interaction with normal human foreskin fibroblasts (HFF) was tested. Briefly, HFFs were seeded in a similar fashion as above in a 12-well plate with a concentration of 1.25x104 cells per
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well. The wells were dosed with two different concentrations of unloaded chitosancoated CNTs (100µl/ml and 200µl/ml) with dosing volume maintained at 1mL in PBS. As the positive control, a well was treated with blank PBS while another treated with
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corresponding dox concentration (as in the loaded chitosan-coated CNTs) served as the negative control. 2.7.
Cell Viability Study
In each case (with TXSA and HFF cell cultures), the cells were accounted to microscopic observation after every 24 h for 5 days. On the fifth day, the dead cells (now floating up in the medium) were warded off and the adhered alive cells were washed and fixed in 10% formalin. The cells were stained with crystal violet solution followed by subsequent washing with distilled water to remove excess stain and imaged further under microscope. Optical density (OD) for the analysis of percentage viable cells, the stain was dissolved in acetic acid viewed under 96-well plate reader. 6
ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1.
Structural Morphology of NAA Template and CNTs and CNC Fabricated inside NAA Templates
The morphology of the resulting CNTs-NAAMs was analysed by SEM. Figure 1 shows a set of SEM images of NAAMs used as templates and CNTs-NAAMs obtained by the
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afore mentioned CVD process. The different geometric characteristics were established by SEM image analysis. NAAMs feature straight cylindrical pores from top to bottom, the diameter and length of which were 51 ± 4 nm and 52.0 ± 0.2 nm, respectively. The resulting CNTs synthesised inside these NAAMs replicated perfectly the pore geometry
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(i.e. the same outer nanotube diameter and length) with an intertube distance (i.e. distance between centres of adjacent nanotubes) of 102 ± 3 nm (Figures 1b). To establish the density and internal structure of CNTs produced by the proposed CVD method,
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liberated CNTs were analysed by SEM and TEM after dissolving the NAAMs (Figure 1 c-d). Figures 1e shows TEM magnified views of the graphitic structure of the CNTs obtained by the proposed CVD approach. This verifies that the annealing treatment improves the organisation of the graphitic layers in the CNTs structure, which results in a
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subsequent modification of their transport properties.
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Figure 1. Set of SEM images of as-produced NAAMs and CNTs-NAAMs fabricated by CVD synthesis from commercially available non-degradable plastic bags. a) NAAM template top view (scale bar = 500 nm). b) Detail of CNTs embedded in a NAAM template (scale bar = 100 nm). Set of SEM and TEM images of liberated CNTs fabricated at
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different CVD times. c) SEM image of CNTs after dissolution of NAAM (i.e. liberated CNTs). d) TEM image of CNT produced by the proposed CVD for 30 min (scale bar = 10
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nm). e) TEM magnified views of liberated CNTs (scale bar = 5 nm) [with permission from T. Altalhi et al. Carbon, 2013, 63, 423-433, copyright 2013].
The CNTs after treatment was redispersed in buffer of pH 8.5 for drug delivery application and in buffer of pH 7.4 for toxicity analysis. The SEM microscopy images of thus prepared CNTs and chitosan coated CNTs are provided in Figure 2a and b, respectively. A layer of chitosan can be clearly seen in Figure 2b confirming successful coating.
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Figure 2. SEM images of CNTs or CNCs. a) without chitosan coating (high resolution image
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in inset) and b) with chitosan coating.
In order to confirm the successful coating of CNTs by chitosan, these samples are analysed by FTIR spectroscopy and thermogravimetric analysis (TGA). Figure 3 shows the TGA curves of CNTs and Ch-CNTs under Ar atmosphere from 35 to 800°C. For the TGA curve of CNTs, the weight loss of CNTs is negligible before 500°C. For Ch-CNTs, there are three clearly
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separated weight loss stages in the range of 35-130°C, 130-500°C and 500-800°C, which are mainly attributed to the combustion of the absorbed water, grafted chitosan, and CNTs, respectively. Assuming that the grafted chitosan was completely decomposed, the content of
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grafted chitosan was about 37% in the Ch-CNT composites.
Figure 3. Thermogravimetric analysis (TGA) curves of CNTs and Ch-CNT composite. 9
ACCEPTED MANUSCRIPT Figure 4 depicts the FTIR spectra of CNTs and Ch-CNTs. The FTIR spectrum of raw CNTs exhibits a broad absorption peaks in the range of 3450–3460 cm-1 correspond to -OH group, indicating of existence of the hydroxyl groups on the surface of the CNTs. Indeed, it was found that these groups can be attributed to the oxidation of the carbon surface after exposure
structure).
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to air atmosphere and the absence of catalysts during the synthesis process (i.e. less crystalline The two peaks at 2950 and 2850 cm-1 correspond to the C-H stretch vibration.
The C-C characteristic peak can be observed at 1580 cm-1. Another peak at 1650 cm-1 is the C-O stretching mode of the functional groups on the surface of the MWCNTs or arising from the absorption of atmospheric CO2 on the surface of the composites. The peak appeared at
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950 cm-1 can be assigned to the C-O stretching mode. This result is in good agreement with the work reported by mezni et al. [34] Compared to the CNTs FTIR spectrum, the FTIR
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spectrum of chitosan coated CNTs exhibits an important peaks at 1151, 1083, 1034 and 890 cm-1, which are the characteristic peaks of the polysaccharide backbone. Broad peaks around 3000 to 3600 cm-1 can be assigned to OH stretching frequency of chitosan. The peaks around 1640 and 1567 cm-1 correspond to amide I and amide II, respectively. A slight variation in stretching frequency was observed in the Ch-CNTs. This might be because
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of chitosan [35].
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the pi-bonds of the CNTs may be chemically interacting with the amide and OH groups
Figure 4. FTIR spectra of CNTs an Ch-CNTs composite
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ACCEPTED MANUSCRIPT 3.2.
Characterization of Drug Loading and Drug Release
High degree of functionality on the exterior and the interior surface of the nanotubes enabled the successful loading of the drug (Dox) within the nanocapsule cavity. The drug loaded chitosan-coated CNTs (purified through dialysis) were dispersed in a phosphate buffer of pH 8.5. The main aim of coating Chitosan on Dox loaded CNTs or CNCs is to decrease the
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amount of drug release during its burst release period and to allow for sustained release over days. The drug release from the CNCs in both the cases was evaluated against a buffer of pH 7.0 for 10 days at 37°C. The final observed release was considered as the 100% release for further calculations which in this case was observed to be 12 ± 3 µg per 10ml of materials.
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While plotting a cumulative drug release profile against time, the burst release section portrayed a linear profile. Drug released in the case of chitosan coated nanocapsule during the first 6 h was 59%, while 89% of the total drug eluted as a burst release in case of only Dox
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loaded CNTs (Figure 5a). In case of drug loaded CNTs, nearly 100% of the drug exclusion from the tubes was observed in 24 h. Contrary to this, the chitosan coated nanocapsules adopted a slow release profile, extending the 100% drug exclusion to 9 days (216 h) (Figure 5b). This can be accredited to the slow un-entanglement of the chitosan chains at a lower pH (in this case pH 7) and hence controlling the release. This theoretical calculation of drug
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microenvironment.
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release may greatly vary when considering the release in the acidic tumoral
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Figure 5. Drug (Dox) release plots for uncoated and chitosan coated CNTs or CNCs in a buffer of pH 7.0 and at a temperature of 37°C for different time periods. a) Burst release of 360 min. b) Complete release including burst and sustained release for 9 days. Results are presented as the mean ± SD of three repeated measurements.
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Scheme 1. Showing application of liberated CNTs for developing smart nanocarriers for localized drug release over cancer cells.
3.3.
Cell Viability
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The human breast (TXSA) cancer cells, seeded at a concentration of 1.25x104 were treated with two different concentration of drug loaded and unloaded nanocapsules: 100µl (100 ng of Dox) and 200µl (200ng of Dox). The microscopic observations were done for five days before finally discarding the medium and staining it for the study of cell viability. At all times
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during the experiment, the study was based on the comparison between the treated cells and the untreated ones (control). Prima facie, there was no apparent change observed in either the
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physical appearance or the density of the plated cells as an immediate impact of treatment. The multi-well plate was then incubated at 37°C in 5% CO2 atmosphere. The cell cultures were observed under light microscope after every 24 h for the period of 5 days. As observed, cellular infarction resulted in its loss of adhesion from the base, which further resulted in its debris floating up in the medium. After 24 h, the highest cell death observed was in the case of the well treated with Dox alone (Figure 6). This effect however stabilized during the next few observations, as the cells proliferated again and no observable increase in the debris volume. On the other hand, the wells treated with drug loaded CNCs, suffered an inhibited growth rate of the cells when compared to the control. Cellular debris in this case wasn’t observed until after the third day. This effect can be attributed to our hypothesis of slow drug release within the cellular proximity as the release rate being directly proportional to the 13
ACCEPTED MANUSCRIPT acidity of the medium. At low concentration and increased bioavailability, Dox tends to uniformly cease the growth within the treated cells. Over time, as the exposure to the slowly eluting drug increased, Dox mediated cellular apoptosis was observed. The result of the cells treated with empty CNTs, on the other hand, was in direct comparison with the control sample. The cells in the case of both CNT treated and the control, showed a dense vegetative
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growth. This hence confirmed the biocompatibility of the tubes as no signs of toxicity was observed.
The TXSA cancer and HFF cell cultures were discarded off of any floating debris, washed and the remnant cells were then fixed in 10% formalin. This was followed by crystal violet
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staining of the live cells. The cell histology gave a better illustration of the adhered cells and hence simplifying the visual comparison between the various subsets of the experiment. As observed in Fig. 4, the observable cell counts in case of the wells treated with drug loaded
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CNCs are much less than in the ones treated with empty CNTs, which actually is comparable to control. When treated with empty CNTs, there was no visual change in cellular density in either of the cell types (TXSA and HFF), when studied against the control. For an estimate calculation of the percentage viability of cells in each case, the stain was dissolved in acetic acid and studied for optical density in micro plate reader. The data as observed in control was
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calculated against it.
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considered a 100% growth profile. Percentage viability in the case of rest of the samples was
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Figure 6. Light micrograph of TXSA cancer cells treated with drug loaded CNCs, empty
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CNTs and Dox, in two different concentrations. The volume of the dosage in each case was maintained to 1mL in PBS. Measurements were taken after 24 h of incubation with the Doxloaded CNTs/CNCs, empty CNTs and pure Dox (Scale bar = 10µm).
As an effort towards the study of toxicity, the hence synthesized and plasma oxidized CNTs (suspended in a buffer of pH 7.4) were also dosed in the same volume of 100µl (100ng of Dox) and 200µl (200ng of Dox) to the human foreskin fibroblasts (HFF). These fibroblasts were seeded in the same concentration as that of the TXSA cancer cells. As expected, the cells dosed with the CNTs and the one in the control, showed a similar growth curve over the period of 5 days (Figure 7 and Figure 9b).
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Figure 7. Light micrograph of TXSA cancer cells stained in crystal violet after 5 days of
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incubation with the Dox-loaded CNTs/CNCs, empty CNTs and pure Dox (Scale bar = 10µm).
This specific experiment was done to investigate the role of cell type on the vulnerability towards the nanotubes. As seen in Figure 9a it can clearly be noted that the drug loaded CNCs showed an enhanced toxicity as compared to the pure drug. As per the theoretical calculation from the in vitro drug release data, 100µl and 200µl of the sample contains 100 and 200 ng of Dox respectively. When considering the well treated with pure drug, the bioavailability was limited and the drug was retained on the surface. The cell death observed after 24 h was due to this high concentration on the surface. Following this, as the drug activity diminished due to its short half-life, the healthy cancer cells proliferated again, though at a slower pace now. 16
ACCEPTED MANUSCRIPT In case of the cells treated with drug loaded CNCs, however, the drug was retained in its pristine form within the CNT and followed a slow release dependent on the atmospheric acidity. This increased the bioavailability of the drug manifold. The retarded growth in the cancer cells treated with CNCs can be linked back to this. The experiment was conducted at a two different concentration of 100µl and 200µl per ml of dosage. Doubling the concentration
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did not impact the cell viability much, when considering the drug loaded CNCs with a decline rate of only 8.11%. On the other hand, this change in concentration accounted to 16% enhanced toxicity in case of cells treated with Dox alone. This observation indicates a diminished rate of drug toxicity when administered through the CNCs as compared to a
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normal chemotherapeutic procedure, while varying the drug concentration. This data further indicates that it’s easy to attain an extensively high death rate of cancer cells at a much lower concentration of drug when delivered encapsulated inside the CNCs. This opens a remarkable
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opportunity for investigating these CNCs as an optimum delivery vehicle for targeted treatment of cancer and other ailments (Scheme 1). The authenticity of these CNTs as efficient biocompatible cargo transporters was further proven through their stealth property observed in the case of both TXSA breast cancer and HFF cell lines. These cells treated with the empty nanotubes, did not show any variation in the growth profile when compared to the
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control sample (Figure 8). The calculated cell viability of the CNT treated HFF was similar to
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that of control (Figure 9b) with minor deviations.
Figure 8. Light micrograph of empty CNT treated human foreskin fibroblasts (HFF), stained in crystal violet after 5 days of incubation with the CNTs or CNCs. The two different concentration of dosage is compared with control. The dosage volume was maintained at 1 ml in each case.
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Figure 9. Comparative cell viability analysis on the 5th day. a) Drug loaded carbon nanocapsules (CNCs), empty carbon nanotubes (CNTs) and pure drug (Dox) when treated up
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on TXSA cancer cells; studied against control (treated with equal volume of PBS). b) Empty carbon nanotubes (CNTs) on human foreskin fibroblasts (HFF) as an attempt to study CNT
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mediated cytotoxicity. Results are presented as the mean ± SD of three repeated measurements.
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ACCEPTED MANUSCRIPT 4. Conclusions To summarize, this study presents an eco-friendly chemical vapor deposition (CVD) approach to convert commercially non-degradable plastic bags into well-organised carbon nanotube (CNTs). The as-prepared CNTs were coated thereafter by doxorubicin (Dox) and were chemically functionalized with a biodegradable polymer (chitosan) for
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advanced cancer therapy. After functionalization, the chitosan-coated CNTs (ChCNCs) were then tested for their localized and slow drug eluting property within the cellular vicinity of MDA-MB-231 TXSA, human breast cancer cell line. The results confirmed a 500 fold enhanced death rate in case of cells treated with Ch-CNCs as
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compared to the pro-drug alone. The effect of plasma oxidized CNCs with Ch coating alone, was also tested on the TXSA cell line and the normal human foreskin fibroblasts (HFF), further confirming its biocompatibility with no cellular toxicity at all in either
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of the cell lines. This is expected to open a new gateway towards much cheaper and
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advanced therapeutics.
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ACCEPTED MANUSCRIPT Acknowledgements We would like to acknowledge the contribution from Queen Elizabeth Hospital for this cell study. Author acknowledges the financial support by the Deanship of Scientific Research, College of Science Research Center at King Saud University.
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ACCEPTED MANUSCRIPT 1- Synthesis of carbon nanotubes (CNTs) in a custom designed chemical vapor
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deposition (CVD) system using nanoporous anodic alumina (NAA) under different conditions. 2- Fabrication of nanoporous anodic alumina (NAA) 3- Synthesis of smart free standing (CNTs) with hydrophilic core were prepared from recycled plastic bags 4- Role of CNTs prepared as an optimal drug carrier in the field of localized drug delivery
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S1773-2247(17)30194-6
DOI:
10.1016/j.jddst.2017.08.007
Reference:
JDDST 451
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 2 March 2017 Revised Date:
8 August 2017
Accepted Date: 11 August 2017
Please cite this article as: A. Mezni, N.B. Saber, A.A. Alhadhrami, A. Gobouri, A. Aldalbhi, S. Hay, A. Santos, D. Losic, T. Altalhi, Highly biocompatible carbon nanocapsules derived from plastic waste for advanced cancer therapy, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/ j.jddst.2017.08.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highly Biocompatible Carbon Nanocapsules Derived From Plastic Waste for Advanced Cancer Therapy Amine Mezni a,b,*, Nesrine Ben Saber a, A. A. Alhadhrami b, Adil Gobouri c, Ali
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Aldalbhi d, S. Hay e, Abel Santos b, Dusan Losic b and Tariq Altalhi b,c,* a
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Unite de recherche "Synthese et Structure de Nanomateriaux" UR11ES30, Faculte des Sciences de Bizerte, Universite de Carthage, 7021 Jarzouna, Tunisie b School of Chemical Engineering, University of Adelaide, Adelaide, Australia c Department of Chemistry, Faculty of Science, Taif University, Taif, Saudi Arabia d Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia e Discipline of Surgery, Breast Cancer Research Unit, Basil Hetzel Institute and Centre for Personalised Cancer Medicine, University of Adelaide, Adelaide, Australia
Abstract
Carbon nanotubes (CNT) are increasingly being investigated for their use in biomedical applications and nanomedicine. In order to be used as nano-carriers for delivering anti-cancer drugs (drug delivery), smart free standing carbon nanotubes
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(CNTs) with hydrophilic core were prepared from recycled plastic bags. These CNTs were loaded with doxorubicin (Dox) and its external surface was chemically functionalized with a biodegradable polymer (chitosan) by anchoring its polymeric chains to functional groups on the external surface of CNTs. The obtained Chitosan-
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coated CNTs (Ch-CNCs) nanocomposites were then tested for their localized and slow drug eluting property using the cellular vicinity of MDA-MB-231 TXSA, human
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breast cancer cell line. The preliminary results are very promising and confirm a 500 fold enhanced death rate in case of cells treated with Ch-CNCs compared to the prodrug alone. This work shows that it is possible to develop a highly biocompatible carbon nanocapsules (CNCs) derived from plastic bags and further used them as cargo transporters to study an intra-tumoral delivery of an anti-neoplastic drug is possible. Keywords: Carbon Nanotubes; CVD, Plastic bags, Drug delivery, Cancer therapy * Corresponding author. E-mail address: [email protected] ; [email protected]
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is the leading cause of death worldwide and has enticed researchers worldwide to look for better and advanced treatments as that to chemo and radiation therapy. Lack of specificity and high toxicity has been the major drawback of the general chemotherapeutic procedure. The ideal cancer drug delivery systems combine targeted delivery with controlled release to
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deliver and release in a selective fashion to the target cells [1-4]. Such systems not only improve the efficacy of the drug, but also reduce the toxic side effects of the drug. In recent years, a wide range of different nanoscale drug carriers have been developed and explored [5,6]. Notably, single wall carbon nanotubes (SWCNTs) have emerged as strong potential
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candidate due to their advantages as a high cargo loading, intrinsic stability and structural flexibility, over the more widely studied metal nanoparticle systems, which could prolong the circulation time and enhance the bioavailability of the therapeutic agent [7–16]. Moreover,
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SWCNTs have been shown to enter mammalian cells and thus investigated as potential delivery vehicles for intracellular transport of nucleic acids, proteins and drug molecules. Furthermore, SWCNTs have been functionalized with antibodies as targeting agents providing a high efficiency for nanotube internalization into cells. On the other hand, SWCNTs have also disadvantages relative to MWCNTs such as their higher tendency to
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aggregate into bundles that may be related to cytotoxicity if not controlled [17-23]. However, many groups have reported that the agglomeration of SWCNT in the air-ways is the primary cause of morbidity and granuloma formation. Drug molecules such as doxorubicin (DOX) can be loaded onto CNTs via hydrophobic and π - π stacking interactions and the release rate can
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be controllable by using nanotubes with different diameters [24,25]. However, inherent properties of SWCNTs such as poor aqueous solubility and tendency to form bundles limited their use for pharmacological applications. To overcome these drawbacks, a number of
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synthetic and natural polymers have been used to encase SWCNTs via non-covalent interactions to improve their compatibility with water and physiological environments. On the other hand, beside polymer attachment, also covalent functionalisation is widely used to avoid the cytotoxic effect of CNTs [26-31]. Indeed; surface engineering of CNTs by covalent and noncovalent modifications enables site-specification drug delivery and targeting. The noncovalent functionalization is based on Van Der Waals, hydrophobic or the ability of the extended π-system of the carbon nanotube sidewall to bind guest molecules via π-π-stacking interactions. Its advantage depend on the preservation of the properties of the CNTs, while its disadvantage concerns weak forces between wrapped/coupled molecules that may lower the load transfer in the composite. The covalent functionalization can be performed at the 2
ACCEPTED MANUSCRIPT sidewall site of CNTs or at the defect sites usually located at the tip. New properties can be added by this means of functionalization. Instead, electronic properties of SWCNTs are perturbed by covalent functionalization and double bonds are irreversibly lost. This may affect conductive property, preventing further CNT applications. Despite excellent progress in using SWCNTs as drug carriers, more research is needed to further optimize their ability to
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selectively accumulate in diseased tissues and release the pay load in a controlled manner. Notice that, most of the aforementioned studies using CNTs for drug delivery application are based on single walled CNTs and CNTs prepared using catalyst based CVD methods. This fabrication suffers from the difficulty in controlling the thickness of the CNTs wall and thus
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the nanotube diameter, shape morphology and importantly presence of metal catalyst particles from conventional CNTs (i.e. SWCNTs and catalyst based CNTs) restrict their use as drug delivery systems. Due to the aforementioned reasons, the toxicity of these CNTs is still a
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highly debatable issue. In addition, all the above studies target systemic route of therapeutic administration with no reports on the use of CNTs for local drug administration. In recent studies based on template synthesis of nanotubes for drug delivery, the focus has been on uniformity of these carriers so as to easily internalize into a cell and facilitate an apt calculation for the authenticity of the method. In this work, we have performed an innovative
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CVD approach to synthesize carbon nanotubes by using nanoporous anodic alumina membranes as templates and commercially available plastic bags as a carbon source without need to use any metal catalysts or solvent. This can prevent and reduce many synthesis disadvantages (use of poisonous chemical metal compounds and the expensive chemicals and
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production of commercial products as separation/filtration membranes) which make it a potential nanotechnological recycling approach, which could contribute directly to the conservation of our natural ecosystems in the near future. However, the principal aim of this
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work is to investigate and exploit the role of CNTs as an optimal drug carrier in the field of localized drug delivery. The localized and sustained delivery of an antineoplastic drug, doxorubicin (Dox), has been evaluated in this in vitro study. These chitosan-coated CNTs in their pristine form (i.e. without drug inside) were also tested on both, human breast cancer and human foreskin fibroblast cell lines in order to investigate the toxicity of these carbon based materials where hence concluding them to be an ideal intra-cellular drug transporter for breast cancer treatment.
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ACCEPTED MANUSCRIPT 2. Experimental Section 2.1.
Synthesis of carbon nanotubes (CNTs) or chitosan-coated CNTs
CNTs were prepared as following. 2.1.1. Nanoporous anodic alumina (NAA) Template Fabrication NAATs were prepared using a two-step anodization with a constant voltage of 40V for 20 h
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in 0.3M oxalic acid at 5°C temperature as described in Altalhi et al. [32] Briefly, high purity Al chips (i.e. 1.5 cm in diameter) were cleaned under sonication in ethanol (EtOH) and double distilled water and subsequently electropolished in a mixture of perchloric acid (HClO4) and EtOH 1:4 (v:v) at 20V and 5°C for 3min. Then, the 1st anodization step was carried out in an
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aqueous solution of oxalic acid (H2C2O4) 0.3M at 40V and at 6°C for 20 h. Then, the resulting alumina (Al2O3) layer was selectively dissolved by wet chemical etching in a mixture of phosphoric acid (H3PO4) 0.4M and chromic acid (H2CrO7) 0.2M at 70°C for 3 h. Next, the
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2nd anodization step was performed under the same anodization conditions as the 1st one for 17 h. Once the anodization process finished, the remaining aluminium substrate was removed in a saturated solution of hydrochloric acid and cupric chloride (HCl/CuCl2). Subsequently, a pore opening process was performed by wet chemical etching in an aqueous solution of H3PO4 5wt% at 35°C under current control conditions [33].
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2.1.2. Synthesis of chitosan-coated CNTs
Carbon nanotubes (CNTs) were fabricated as reported by AlTalhi et al. [32] using template and catalyst free CVD synthesis inside nanoporous anodic alumina membranes (NAAMs). The fabrication process was carried out using a CVD system consisting of a two-stage
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furnace equipped with a cylindrical quartz tube with dimensions (43 and 1000 mm in diameter and length, respectively) and temperature and gas flow controllers. Particularly, shopping plastic bags were collected from a local grocery that are typically produced from
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linear low-density polyethylene (LLDPE) and used them as a carbon source to produce CNTs. After collection, these plastic bags were washed with a liquid soap and double distilled water, dried under nitrogen stream, cut into small squares of 1 cm2 and kept in a container with inert atmosphere to prevent them from contaminations before the fabrication process. Then, these small pieces of plastic bag were placed in a ceramic crucible, which was introduced into the pyrolysis zone of the CVD reactor. To ensure the absence of oxygen during the CNTs synthesis, Argon inert (Ar) gas was flowed at 1 dm3 min-1. In the deposition zone of the CVD reactor, prepared NAAMs were placed where the carbon deposition took place. We performed the fabrication process at 850°C and 30 min.
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ACCEPTED MANUSCRIPT After CNTs synthesis, the CVD reactor was cooled down to room temperature. The resulting CNTs–NAAMs were saved in a container under inert conditions. An additional annealing process was used in some CNTs–NAAMs samples by heating under Ar atmosphere at 900°C for 3 h. The obtained CNTs were followed by being taken out and ultrasonicated in HF solution in order to dissolve the alumina membrane. This was followed by centrifugation
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(15000 rpm) and sequential washing with deionized water. The centrifuged pellet hence obtained was then oxidized under air plasma by exposure for 30s and then re-dispersed in phosphate buffer, pH-8.5. The nanotubes remained dispersed for months. 2.2.
Structural Characterisation
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Prepared NAAMs, CNTs and CNTs-NAAMs are characterised by a scanning electron microscope (FEG-SEM FEI Quanta 450) equipped with energy dispersive X-ray spectroscopy (EDXS). The standard image processing package (ImageJ, public domain program developed
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at the RSB of the NIH, USA) was used to carry out the SEM image analysis. Liberated CNTs are analysed by transmission electron microscopy (TEM Philips CM 200). Transmission infrared (IR) spectra are, however, recorded using a Nicolet Fourier Transform Infrared (FTIR) spectrophotometer at a spectral resolution of 2 cm-1 accumulating 128 scans, in a self-supported disk. Thermogravimetric analysis TGA measurements was performed on a SETSYS Evolution 1750 SETARAM instrument under an Ar atmosphere at a heating rate of
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10 K min-1 over a temperature range from 25 to 800°C; the weight of the sample was 20 mg. Drug Loading in chitosan-coated CNTs and Release Study
300µL of 2mg.ml-1 doxorubicin hydrochloride stock solution (Sigma Aldrich) was
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gradually added to 5mL of the alkaline buffer suspended nanotubes and kept under continuous stirring for 24 h at room temperature under dark condition followed by subsequent addition of chitosan solution (1% wt/vol in 3% acetic acid) to form the
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polymeric integument. The drug loaded nanocapsules were isolated through dialysis and then incubated with 1% human serum albumin (HSA) overnight at a constant pH of 8.0. The purification after this again followed the same process of dialysis. The volume of the solution containing the dispersed nanocapsules was thereafter made up to 20 ml. For the drug release study, 10 ml of the buffer containing nanocapsules was taken in a dialysis bag with a threshold of 3000 Daltons and dialyzed against a neutral pH of 7.0 at 37°C. Subsequently, aliquots of the sample were taken at different time intervals to analyze for Dox concentration through UV-visible spectroscopy (at 479 nm). The volume of the aliquot taken out for this study was replaced with fresh buffer
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Cell Culture
Human breast cancer cell line MDA-MB-231 TXSA and human foreskin fibroblast (HFF) were cultured in DMEM, supplemented with 10% foetal calf serum (FCS) in
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5% CO2 at 37°C in an incubator. Treatment of Cancer Cells with chitosan-coated CNTs
The TXSA breast cancer cells were plated in a 12-well plate and seeded with a concentration of 1.25x104 cells per well. Each of these wells was then dosed with 1ml
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of the varying concentration of drug loaded nanocapsules (100µl/ml and 200µl/ml; prepared in PBS). As a comparative study, the empty chitosan-coated CNTs in similar concentrations as their drug loaded counterparts were added in the adjacent wells. At
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all times, the volume was maintained at 1mL with PBS. Subsequently, as the positive control, the as calculated amount of dox, corresponding to different volume of the drug retained in the varying concentration of nanocapsule dosage, was added. At the end, a well was treated with 1ml of only PBS, which served as the negative control (referred to as blank).
Toxicity Study of chitosan-coated CNTs
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2.6.
As a study for biological toxicity of these chitosan-coated CNTs, their interaction with normal human foreskin fibroblasts (HFF) was tested. Briefly, HFFs were seeded in a similar fashion as above in a 12-well plate with a concentration of 1.25x104 cells per
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well. The wells were dosed with two different concentrations of unloaded chitosancoated CNTs (100µl/ml and 200µl/ml) with dosing volume maintained at 1mL in PBS. As the positive control, a well was treated with blank PBS while another treated with
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corresponding dox concentration (as in the loaded chitosan-coated CNTs) served as the negative control. 2.7.
Cell Viability Study
In each case (with TXSA and HFF cell cultures), the cells were accounted to microscopic observation after every 24 h for 5 days. On the fifth day, the dead cells (now floating up in the medium) were warded off and the adhered alive cells were washed and fixed in 10% formalin. The cells were stained with crystal violet solution followed by subsequent washing with distilled water to remove excess stain and imaged further under microscope. Optical density (OD) for the analysis of percentage viable cells, the stain was dissolved in acetic acid viewed under 96-well plate reader. 6
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Structural Morphology of NAA Template and CNTs and CNC Fabricated inside NAA Templates
The morphology of the resulting CNTs-NAAMs was analysed by SEM. Figure 1 shows a set of SEM images of NAAMs used as templates and CNTs-NAAMs obtained by the
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afore mentioned CVD process. The different geometric characteristics were established by SEM image analysis. NAAMs feature straight cylindrical pores from top to bottom, the diameter and length of which were 51 ± 4 nm and 52.0 ± 0.2 nm, respectively. The resulting CNTs synthesised inside these NAAMs replicated perfectly the pore geometry
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(i.e. the same outer nanotube diameter and length) with an intertube distance (i.e. distance between centres of adjacent nanotubes) of 102 ± 3 nm (Figures 1b). To establish the density and internal structure of CNTs produced by the proposed CVD method,
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liberated CNTs were analysed by SEM and TEM after dissolving the NAAMs (Figure 1 c-d). Figures 1e shows TEM magnified views of the graphitic structure of the CNTs obtained by the proposed CVD approach. This verifies that the annealing treatment improves the organisation of the graphitic layers in the CNTs structure, which results in a
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subsequent modification of their transport properties.
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Figure 1. Set of SEM images of as-produced NAAMs and CNTs-NAAMs fabricated by CVD synthesis from commercially available non-degradable plastic bags. a) NAAM template top view (scale bar = 500 nm). b) Detail of CNTs embedded in a NAAM template (scale bar = 100 nm). Set of SEM and TEM images of liberated CNTs fabricated at
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different CVD times. c) SEM image of CNTs after dissolution of NAAM (i.e. liberated CNTs). d) TEM image of CNT produced by the proposed CVD for 30 min (scale bar = 10
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nm). e) TEM magnified views of liberated CNTs (scale bar = 5 nm) [with permission from T. Altalhi et al. Carbon, 2013, 63, 423-433, copyright 2013].
The CNTs after treatment was redispersed in buffer of pH 8.5 for drug delivery application and in buffer of pH 7.4 for toxicity analysis. The SEM microscopy images of thus prepared CNTs and chitosan coated CNTs are provided in Figure 2a and b, respectively. A layer of chitosan can be clearly seen in Figure 2b confirming successful coating.
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Figure 2. SEM images of CNTs or CNCs. a) without chitosan coating (high resolution image
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in inset) and b) with chitosan coating.
In order to confirm the successful coating of CNTs by chitosan, these samples are analysed by FTIR spectroscopy and thermogravimetric analysis (TGA). Figure 3 shows the TGA curves of CNTs and Ch-CNTs under Ar atmosphere from 35 to 800°C. For the TGA curve of CNTs, the weight loss of CNTs is negligible before 500°C. For Ch-CNTs, there are three clearly
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separated weight loss stages in the range of 35-130°C, 130-500°C and 500-800°C, which are mainly attributed to the combustion of the absorbed water, grafted chitosan, and CNTs, respectively. Assuming that the grafted chitosan was completely decomposed, the content of
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grafted chitosan was about 37% in the Ch-CNT composites.
Figure 3. Thermogravimetric analysis (TGA) curves of CNTs and Ch-CNT composite. 9
ACCEPTED MANUSCRIPT Figure 4 depicts the FTIR spectra of CNTs and Ch-CNTs. The FTIR spectrum of raw CNTs exhibits a broad absorption peaks in the range of 3450–3460 cm-1 correspond to -OH group, indicating of existence of the hydroxyl groups on the surface of the CNTs. Indeed, it was found that these groups can be attributed to the oxidation of the carbon surface after exposure
structure).
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to air atmosphere and the absence of catalysts during the synthesis process (i.e. less crystalline The two peaks at 2950 and 2850 cm-1 correspond to the C-H stretch vibration.
The C-C characteristic peak can be observed at 1580 cm-1. Another peak at 1650 cm-1 is the C-O stretching mode of the functional groups on the surface of the MWCNTs or arising from the absorption of atmospheric CO2 on the surface of the composites. The peak appeared at
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950 cm-1 can be assigned to the C-O stretching mode. This result is in good agreement with the work reported by mezni et al. [34] Compared to the CNTs FTIR spectrum, the FTIR
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spectrum of chitosan coated CNTs exhibits an important peaks at 1151, 1083, 1034 and 890 cm-1, which are the characteristic peaks of the polysaccharide backbone. Broad peaks around 3000 to 3600 cm-1 can be assigned to OH stretching frequency of chitosan. The peaks around 1640 and 1567 cm-1 correspond to amide I and amide II, respectively. A slight variation in stretching frequency was observed in the Ch-CNTs. This might be because
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of chitosan [35].
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the pi-bonds of the CNTs may be chemically interacting with the amide and OH groups
Figure 4. FTIR spectra of CNTs an Ch-CNTs composite
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Characterization of Drug Loading and Drug Release
High degree of functionality on the exterior and the interior surface of the nanotubes enabled the successful loading of the drug (Dox) within the nanocapsule cavity. The drug loaded chitosan-coated CNTs (purified through dialysis) were dispersed in a phosphate buffer of pH 8.5. The main aim of coating Chitosan on Dox loaded CNTs or CNCs is to decrease the
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amount of drug release during its burst release period and to allow for sustained release over days. The drug release from the CNCs in both the cases was evaluated against a buffer of pH 7.0 for 10 days at 37°C. The final observed release was considered as the 100% release for further calculations which in this case was observed to be 12 ± 3 µg per 10ml of materials.
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While plotting a cumulative drug release profile against time, the burst release section portrayed a linear profile. Drug released in the case of chitosan coated nanocapsule during the first 6 h was 59%, while 89% of the total drug eluted as a burst release in case of only Dox
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loaded CNTs (Figure 5a). In case of drug loaded CNTs, nearly 100% of the drug exclusion from the tubes was observed in 24 h. Contrary to this, the chitosan coated nanocapsules adopted a slow release profile, extending the 100% drug exclusion to 9 days (216 h) (Figure 5b). This can be accredited to the slow un-entanglement of the chitosan chains at a lower pH (in this case pH 7) and hence controlling the release. This theoretical calculation of drug
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microenvironment.
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release may greatly vary when considering the release in the acidic tumoral
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Figure 5. Drug (Dox) release plots for uncoated and chitosan coated CNTs or CNCs in a buffer of pH 7.0 and at a temperature of 37°C for different time periods. a) Burst release of 360 min. b) Complete release including burst and sustained release for 9 days. Results are presented as the mean ± SD of three repeated measurements.
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Scheme 1. Showing application of liberated CNTs for developing smart nanocarriers for localized drug release over cancer cells.
3.3.
Cell Viability
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The human breast (TXSA) cancer cells, seeded at a concentration of 1.25x104 were treated with two different concentration of drug loaded and unloaded nanocapsules: 100µl (100 ng of Dox) and 200µl (200ng of Dox). The microscopic observations were done for five days before finally discarding the medium and staining it for the study of cell viability. At all times
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during the experiment, the study was based on the comparison between the treated cells and the untreated ones (control). Prima facie, there was no apparent change observed in either the
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physical appearance or the density of the plated cells as an immediate impact of treatment. The multi-well plate was then incubated at 37°C in 5% CO2 atmosphere. The cell cultures were observed under light microscope after every 24 h for the period of 5 days. As observed, cellular infarction resulted in its loss of adhesion from the base, which further resulted in its debris floating up in the medium. After 24 h, the highest cell death observed was in the case of the well treated with Dox alone (Figure 6). This effect however stabilized during the next few observations, as the cells proliferated again and no observable increase in the debris volume. On the other hand, the wells treated with drug loaded CNCs, suffered an inhibited growth rate of the cells when compared to the control. Cellular debris in this case wasn’t observed until after the third day. This effect can be attributed to our hypothesis of slow drug release within the cellular proximity as the release rate being directly proportional to the 13
ACCEPTED MANUSCRIPT acidity of the medium. At low concentration and increased bioavailability, Dox tends to uniformly cease the growth within the treated cells. Over time, as the exposure to the slowly eluting drug increased, Dox mediated cellular apoptosis was observed. The result of the cells treated with empty CNTs, on the other hand, was in direct comparison with the control sample. The cells in the case of both CNT treated and the control, showed a dense vegetative
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growth. This hence confirmed the biocompatibility of the tubes as no signs of toxicity was observed.
The TXSA cancer and HFF cell cultures were discarded off of any floating debris, washed and the remnant cells were then fixed in 10% formalin. This was followed by crystal violet
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staining of the live cells. The cell histology gave a better illustration of the adhered cells and hence simplifying the visual comparison between the various subsets of the experiment. As observed in Fig. 4, the observable cell counts in case of the wells treated with drug loaded
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CNCs are much less than in the ones treated with empty CNTs, which actually is comparable to control. When treated with empty CNTs, there was no visual change in cellular density in either of the cell types (TXSA and HFF), when studied against the control. For an estimate calculation of the percentage viability of cells in each case, the stain was dissolved in acetic acid and studied for optical density in micro plate reader. The data as observed in control was
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Figure 6. Light micrograph of TXSA cancer cells treated with drug loaded CNCs, empty
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CNTs and Dox, in two different concentrations. The volume of the dosage in each case was maintained to 1mL in PBS. Measurements were taken after 24 h of incubation with the Doxloaded CNTs/CNCs, empty CNTs and pure Dox (Scale bar = 10µm).
As an effort towards the study of toxicity, the hence synthesized and plasma oxidized CNTs (suspended in a buffer of pH 7.4) were also dosed in the same volume of 100µl (100ng of Dox) and 200µl (200ng of Dox) to the human foreskin fibroblasts (HFF). These fibroblasts were seeded in the same concentration as that of the TXSA cancer cells. As expected, the cells dosed with the CNTs and the one in the control, showed a similar growth curve over the period of 5 days (Figure 7 and Figure 9b).
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Figure 7. Light micrograph of TXSA cancer cells stained in crystal violet after 5 days of
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This specific experiment was done to investigate the role of cell type on the vulnerability towards the nanotubes. As seen in Figure 9a it can clearly be noted that the drug loaded CNCs showed an enhanced toxicity as compared to the pure drug. As per the theoretical calculation from the in vitro drug release data, 100µl and 200µl of the sample contains 100 and 200 ng of Dox respectively. When considering the well treated with pure drug, the bioavailability was limited and the drug was retained on the surface. The cell death observed after 24 h was due to this high concentration on the surface. Following this, as the drug activity diminished due to its short half-life, the healthy cancer cells proliferated again, though at a slower pace now. 16
ACCEPTED MANUSCRIPT In case of the cells treated with drug loaded CNCs, however, the drug was retained in its pristine form within the CNT and followed a slow release dependent on the atmospheric acidity. This increased the bioavailability of the drug manifold. The retarded growth in the cancer cells treated with CNCs can be linked back to this. The experiment was conducted at a two different concentration of 100µl and 200µl per ml of dosage. Doubling the concentration
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did not impact the cell viability much, when considering the drug loaded CNCs with a decline rate of only 8.11%. On the other hand, this change in concentration accounted to 16% enhanced toxicity in case of cells treated with Dox alone. This observation indicates a diminished rate of drug toxicity when administered through the CNCs as compared to a
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normal chemotherapeutic procedure, while varying the drug concentration. This data further indicates that it’s easy to attain an extensively high death rate of cancer cells at a much lower concentration of drug when delivered encapsulated inside the CNCs. This opens a remarkable
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opportunity for investigating these CNCs as an optimum delivery vehicle for targeted treatment of cancer and other ailments (Scheme 1). The authenticity of these CNTs as efficient biocompatible cargo transporters was further proven through their stealth property observed in the case of both TXSA breast cancer and HFF cell lines. These cells treated with the empty nanotubes, did not show any variation in the growth profile when compared to the
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control sample (Figure 8). The calculated cell viability of the CNT treated HFF was similar to
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Figure 8. Light micrograph of empty CNT treated human foreskin fibroblasts (HFF), stained in crystal violet after 5 days of incubation with the CNTs or CNCs. The two different concentration of dosage is compared with control. The dosage volume was maintained at 1 ml in each case.
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Figure 9. Comparative cell viability analysis on the 5th day. a) Drug loaded carbon nanocapsules (CNCs), empty carbon nanotubes (CNTs) and pure drug (Dox) when treated up
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on TXSA cancer cells; studied against control (treated with equal volume of PBS). b) Empty carbon nanotubes (CNTs) on human foreskin fibroblasts (HFF) as an attempt to study CNT
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ACCEPTED MANUSCRIPT 4. Conclusions To summarize, this study presents an eco-friendly chemical vapor deposition (CVD) approach to convert commercially non-degradable plastic bags into well-organised carbon nanotube (CNTs). The as-prepared CNTs were coated thereafter by doxorubicin (Dox) and were chemically functionalized with a biodegradable polymer (chitosan) for
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advanced cancer therapy. After functionalization, the chitosan-coated CNTs (ChCNCs) were then tested for their localized and slow drug eluting property within the cellular vicinity of MDA-MB-231 TXSA, human breast cancer cell line. The results confirmed a 500 fold enhanced death rate in case of cells treated with Ch-CNCs as
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compared to the pro-drug alone. The effect of plasma oxidized CNCs with Ch coating alone, was also tested on the TXSA cell line and the normal human foreskin fibroblasts (HFF), further confirming its biocompatibility with no cellular toxicity at all in either
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ACCEPTED MANUSCRIPT Acknowledgements We would like to acknowledge the contribution from Queen Elizabeth Hospital for this cell study. Author acknowledges the financial support by the Deanship of Scientific Research, College of Science Research Center at King Saud University.
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ACCEPTED MANUSCRIPT 1- Synthesis of carbon nanotubes (CNTs) in a custom designed chemical vapor
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deposition (CVD) system using nanoporous anodic alumina (NAA) under different conditions. 2- Fabrication of nanoporous anodic alumina (NAA) 3- Synthesis of smart free standing (CNTs) with hydrophilic core were prepared from recycled plastic bags 4- Role of CNTs prepared as an optimal drug carrier in the field of localized drug delivery
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