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superparamagnetic nanocomposite as a high-efficiency loading nanocarrier for controlled delivery of methotrexate
Accepted Manuscript Title: PEGAylated graphene oxide/superparamagnetic nanocomposite as a high-efficienc loading nanocarrier for controlled delivery of methotrexate Authors: Zahra Abdollahi, Asghar Taheri-Kafrani, Seyed Amir Bahrani, Abolghasem Abbasi Kajani PII: DOI: Reference:
S0168-1656(19)30120-8 https://doi.org/10.1016/j.jbiotec.2019.04.006 BIOTEC 8400
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
1 November 2018 9 March 2019 5 April 2019
Please cite this article as: Abdollahi Z, Taheri-Kafrani A, Bahrani SA, Abbasi Kajani A, PEGAylated graphene oxide/superparamagnetic nanocomposite as a high-efficienc loading nanocarrier for controlled delivery of methotrexate, Journal of Biotechnology (2019), https://doi.org/10.1016/j.jbiotec.2019.04.006 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.
PEGAylated graphene oxide/superparamagnetic nanocomposite as a high-efficiency loading nanocarrier for controlled delivery of
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methotrexate
Zahra Abdollahi a, Asghar Taheri-Kafrani a, Seyed Amir Bahrani b, Abolghasem Abbasi Kajani a
a
: Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of
: IMT Lille Douai, Energy Engineering Department, F-59500 Douai, France.
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b
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Isfahan, Isfahan, 81746-73441, Iran.
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* To whom correspondence should be addressed
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Tel: +98 31 37 93 43 46
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Email: [email protected]
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Highlights
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Fax: +98 31 37 93 23 42
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Graphene
oxide/iron
oxid
nanocomposites
were
functionalized
with
PEGA
(GOMNP/PEGA). The conjugation of methotrexate (MTX) on GOMNP/PEGA was confirmed by different techniques.
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This MTX nanocarrier demonstrated high drug loading and optimum ability for a controlled drug release. The in vitro assay results showed that the drug carrier nanosystem was hemocompatible
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Abstract
Polymer-coated nanocarriers play an important role in targeted drug delivery. The use of
polymers such as polyethylene glycol increases stability, biocompatibility, and blood circulation
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time of the drug, and may consequently improve the success of drug delivery. In the present
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work, a simple approach has been reported for synthesizing polyethylene glycol bis amin
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(PEGA) functionalized graphene oxide/iron oxide nanocomposite as a remarkable unit for
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loading drugs. The biomedical applications of the synthesized nanocomposite were investigated
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by immobilizing methotrexate (MTX), as an anticancer drug. The structural and morphological
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characteristics and the successful synthesis of the nanocomposite were evaluated by different charachterization techniques. The cytotoxicity assay of the nanocarrier showed higher toxicity
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against HeLa and MCF-7 cell lines, compared to free MTX. The drug release experiments in acidic and physiological conditions suggested the first order kinetics model for the release of
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MTX from the nanocomposite. Furthermore, the agglutination, complement activation, and coagulation time experiments demonstrated the blood compatibility of the synthesized
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nanocarrier.
Keywords: Graphene Oxide; Methotrexate; Magnetic nanoparticles; Drug release; Biocompatibility.
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Introduction Cancer is considered as one of the most important reason of death in the world in these years. The different ranges of chemotherapy drugs are used as a significant way to treat various
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cancers. In spite of significant applications, anticancer drugs suffer from some limited effects on target cells due to lack of selectivity, low solubility, uniform tissue distribution, short half-life in the bloodstream and dose-related toxicity (Bao et al., 2015; Rahmanian et al., 2017; Sun et al., 2014). Therefore, exploring an efficient and general approach to overcome drawbacks of
anticancer drugs is quite exigent. Methotrexate (MTX), a chemotherapy drug, is a folic acid
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analog and uses for the treatment of many different kinds of cancers (Chen et al., 2013b;
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Wojtoniszak et al., 2013; Yoon et al., 2010). MTX can mainly blocks the dihydrofolatereductase
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(DHFR) enzyme and subsequently prevents the production of thymidine (Grim et al., 2003;
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Khan et al., 2012; Tian and Cronstein, 2007; Wei et al., 2014). In spite of significant
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applications, this anticancer drug suffers from some limited effects on target cells due to poor
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aqueous solubility, short half-life in plasma, low permeability, and nonspecific drug delivery. Therefore, in modern nanobiotechnology, linking MTX to nanoscale drug carriers in order to
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alter its pharmacokinetic behavior, enhance tumor targeting, reduce toxicity, and overcome drugresistance mechanisms has been noted (Chen et al., 2013b; Khan et al., 2012; Kohler et al., 2006;
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Santos et al., 2007).
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Current advances in biotechnology have improved advanced nanoscale systems for application in drug delivery systems (Barranger et al., 2015). These systems include nanostructures or nanomaterials, such as liposomes (Lokerse et al., 2018), hyperbranched polymers (Shen et al., 2018), microspheres (Vasir et al., 2003), polymeric shells (Haag, 2004), inorganic nanoparticles (Kovalainen et al., 2018) and carbon nanotubes (Da Ros et al., 2018). The loading capacity and
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loading efficiency of the currently developed nanoscale drug delivery systems are still undesirable. In addition, rigorous reaction conditions, long reaction times and expensive reagents are limitations and drawbacks of many of the previously reported methods(Mura et al., 2013).
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Nanoparticles (NPs) have been widely used as nanocarriers for drug delivery due to their small size, release characteristics and targeted delivery of chemotherapeutic drugs (Bao et al., 2015; Barranger et al., 2015; Sun et al., 2014). The development of new strategies to produce high loading drug-containing NPs with appropriate solubility and reaction conditions is still in demand in nanomedicine research area (Shi et al., 2016).
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Nowadays, carbon nanostructures such as graphene and its water-soluble derivative, graphene
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oxide (GO), have attracted more attention than other nanomaterials due to their electronic and
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structural properties, high surface area, adjustable surface chemistry, purity, and two-
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dimensional structure which allows the reactions to occur more efficiently (Amiri et al., 2012; Liu et al., 2013; Ma et al., 2012; Yang et al., 2009; Zhang et al., 2010). Besides, good dispersion
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in water and physiological environments, biocompatibility and lack of obvious toxicity make it
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suitable nanocarriers for drug delivery (Guo et al., 2016; Shi et al., 2013; Yang et al., 2011).
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Also, graphene oxide can be easily functionalized by various agents for better conjugation of drugs (Choi et al., 2010; Shi et al., 2013). One of the promising targeting methods is grafting
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magnetic nanoparticles (MNPs) on the surface of graphene oxide. This conjugate can be rapidly and efficiently separated by an external magnetic field, and therefore, it can be used as the
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support material for biological applications such as targeted drug delivery (Bayramoğlu and Arıca, 2008; Liao et al., 2017; Mody et al., 2014; Reis et al., 2017; Yang et al., 2009). Agglomeration of nanoparticles can reduce efficiency, rapid clearance by macrophages and accumulated in the reticule-endothelial system before reaching the target cells. One way to solve
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this problem is to cover the surface of the nanoparticles with biocompatible polymers such as poly ethylene glycol (PEG). As covering the surface of the nanoparticles with PEG, a flexible hydrophilic polymer, provide to increase nanoparticle circulation time in the blood, improve the
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delivery of nanoparticles to the target tissue and also cause functional groups on the surface capable of conjugating with target agents. Attaching PEG to nanoparticles may also prevent their opsonization and reduce their uptake by the reticuloendothelial system. As a result, PEGylation of nanoparticles enhances bio-compatibility and increases the circulation time of nanoparticles in the blood. The amine groups on the surface of polyethylene glycol bis-amine (PEGA) can be a
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suitable choice as functional groups on the surface of MNPs (Howard et al., 2012; Kohler et al.,
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2004; Reis et al., 2017; Wanna et al., 2016).
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Encouraged by the unique properties and applications of hybrid nanomaterials, we wish to report
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a novel convenient method to prepare high water-soluble superparamagnetic graphene oxide functionalized with PEGA, as a novel functional group. This nanocarrier with high
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biocompatibility was used to improve loading capacity of drug and increase toxicity for tumor
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cells. In addition because of some features such as rapid and easy synthesis and separation in the
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presence of external magnetic field, and modification with PEGA, the GOMNP/PEGA nanocarrier can be a new and appropriate delivery system. The use of this biocompatible
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polymer, in addition, can improve biocompatibility of the nanocarrier, due to the positive charge of bis-amine on the polymer, and increase binding of nanocarrier to negative charged cancer
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cells. This can lead to the unique ability of the system to transfer methotrexate and other cancer drugs to the target tissue. The in vitro behavior of the synthesized nanomaterial was also investigated in order to confirm it as a novel nanocarrier candidate for targeted methotrexate delivery.
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2. Materials and methods 2.1. Materials Methotrexate (MTX) was purchased from Sigma-Aldrich Chemical Co. Graphite powder (300
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mush), iron (II) chloride tetrahydrate (FeCl2.4H2O), iron (III) chloride hexahydrate
(FeCl3.6H2O), sulfuric acid (H2SO4, 98%), potassium permanganate (KMnO4), sodium nitrate
(NaNO3), potassium dihydrogenphosphate (KH2PO4), potassium chloride (KCl), sodium chloride (NaCl) and disodium phosphate (Na2HPO4) were all purchased from Merck Chemical Co.
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2.2. Apparatus
Fourier transform infrared (FTIR) spectra were recorded on a Perkin-Elmer FTIR spectroscopy
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using KBr discs in the 400–4000 cm-1 region to analyze the presence of functional groups and
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the binding of PEGA to the nanocarrier. The surface morphology of samples were observed by scanning electron microscopy (SEM, Hitachi S-4800). Transmission electron microscopy (TEM)
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images were recorded using a Philips CM30 transmission electron microscope operated at 200
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kV and equipped with a LaB6 filament. Powder x-ray diffraction (XRD) patterns were collected on a Bruker D8-advance x-ray diffractometer with Co Kα radiation (λ = 1.7902 A).
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Thermogravimetric analysis experiments (TGA) were carried out using a TGA Q500 V20.10
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Build 36 thermo analysis system (American TA Instruments Cot., USA). The magnetization curves of the as-prepared nanoparticles were determined with a vibrating sample magnetometer
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(VSM, Quantum Design Company, USA). Loading the drug was measured utilizing UV–vis Carry-500 double beam spectrophotometer (USA). The PT/APTT was assessed by coagulation analyzer (IL ACL Elite Pro). The complement system activation was measured by auto analyzer (Hitachi 902). 2.3. Synthesis of nanocarriers 6
2.3.1. Synthesis of graphene oxide Graphene oxide was obtained from graphite using modified Hummer’s method (Liu et al., 2008; Stankovich et al., 2006). Briefly, graphite powder (0.5 g) with an equal amount of NaNO3 (0.5 g)
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in 23 ml of concentrated H2SO4 (12.1 M) were stirred in an ice water bath for 15 minutes. Thereafter, KMnO4 (4.0 g) was added slowly to the mixture in an ice bath until the color turns to purple-green. Then mixture was taken the hot water bath at 40 °C and was stirred for 90 minutes. Afterward, 50 mL of deionized water (DI) was added slowly and stirred for 15 minutes,
following by adding 6 ml of H2O2 until a golden-brown sol appeared. Then, 50 ml of DI water
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was added and the mixture centrifuged with DI water for several times to reduced pH to 6.0.
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The synthesized product, GO, was dried at room temperature under vacuum.
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2.3.2. Preparation of superparamagnetic graphene oxide nanocomposite
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To prepare superparamagnetic graphene oxide nanocomposite (GOMNP), 400 mg of GO was added to 100 mL solution of 0.01 M NaOH, pH 12, and sonicated for 45 min at room
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temperature under nitrogen atmosphere. This can alter the carboxylic acid groups to carboxylate
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anions. The mixture centrifuged and washed with DI water until the pH became neutral. The
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obtained powder was then dispersed in 20 mL DI water and sonicated at room temperature for 30 min. subsequently, 480 mg FeCl3.6H2O and 176 mg FeCl2.4H2O were solved in 5 mL DI water
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and added to the above mixture and sonicated for 1 h at 60 °C. Throughout sonication, ammonium aqueous solution (13 %) was slowly added to mixture and the pH was adjusted to 9–
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12. The product, solid GOMNP, was separated with an external magnet and dried under vacuum. 2.3.3. Functionalization of GOMNP nanocomposite with polyethylene glycol bis amine 8 mg of GOMNP was added to 3 ml of sodium phosphate buffer and sonicated for 20 min following by adding 4 mg of polyethylene glycol bis amine (PEGA) to the mixture and stirring
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for 16 hours at 50 °C. The solution was then separated from the product, GOMNP/PEGA, by a magnet and dried under vacuum. 2.4. Loading of methotrexate onto GOMNP/PEGA nanocomposite
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For methotrexate (MTX) loading onto nanocarrier, the obtained GOMNP/PEGA powder was initially dispersed in 2 ml of DI water by sonication to prepare a homogeneous suspension. Then, 0.25 mg/ml solution of MTX was added to the mixture and stirred at 25 °C overnight. 2.5. In vitro biocompatibility experiments
The relevant guidelines and regulations was used for the in vitro biocompatibility experiments of
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the synthesized nanocomposites. All methods were carried out in accordance with relevant
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guidelines and regulations, informed consent was obtained from all subjects, and the
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experimental protocols were approved by Al-Zahra hospital in Isfahan, Iran. Human cell lines
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were provided from Pasteur Institute of Iran. Red blood cells (RBCs) were provided by healthy volunteers under the approval of Al-Zahra hospital in Isfahan, Iran.
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2.5.1. MTT Assay
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Two cell lines, MCF-7 and HeLa, were seeded to the density of 104 cells in RPMI 1640 medium
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supplemented with 10 % FBS per into a 96-well plate and incubated overnight. The samples including free MTX, GOMNP/PEGA and GOMNP/PEGA/MTX with concentrations of 1, 5, 10,
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20 and 50 μg/ml were added to each well and incubated for 24-48 h. Then the medium was discarded and 100 μl of the culture medium containing MTT (concentration of 0.5 mg/ml) was
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added into each well and incubated at 37 °C for 4 hours. MTT was absorbed by cells and converted to oxalic oxidoreductase by cleavage of the tetrazolium ring using dehydrogenase enzymes. The supernatant was then removed and 100 μl DMSO was added to each well to
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insoluble the formazan crystals. The absorbance was measured at 492 nm using a microplate reader and the percentage of cell viability was obtained. 2.5.2. Hemolysis of red blood cells
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The healthy volunteers are required to perform this experiment. 5 ml of human blood heparinized was centrifuged at 4500 rpm for 10 minutes, a plasma solution was removed, and the RBCs were washed out with PBS until the supernatant was clear. The RBCs were then added to 5 ml of
saline phosphate buffer pH 7.0. For the hemolysis test, 190 μl of diluted RBCs were mixed with 10 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX to reach final concentrations
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of 5, 10, 20, 40, 75, 125 and 200 μg/ml of nanocarriers. Controls were performed with 0% and
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100% hemolysis with PBS (negative control) and Triton X-100 (positive control), respectively.
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The samples were incubated at 37 °C for 30 min and then centrifuged for 5 minutes at 4000 rpm.
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At the end, 100 μl of supernatant solution was taken, diluted with 900 μl PBS buffer, and the absorbance of samples were analyzed at 576 nm for measuring hemoglobin released from
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hemolytic red blood cells. The percentage of hemolysis was measured by comparing the positive
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and negative control absorbance, according to the Eq. 1: 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
(1)
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Hemolysis (%) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑝𝑎𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 2.5.3. Complement activation assay
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The purpose of this experiment was to measure the amount and activity of blood complement
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system in the presence of various concentrations of nanocomposites. The activation of the complement system is made up of the main and secondary pathways, both of which activate C3 and lead to the production of a membrane attack complex. Complementary compounds of the complement (C3 and C4) are quantitatively measurable by immunological methods. For this test, 100 μl of human serum was added to 100 μl of GO, GOMNP, GOMNP/PEGA and 9
GOMNP/PEGA/MTX samples to obtain final concentrations of 10, 50, 100 and 200 μg/ml. samples were incubated at 37 ° C for 30 minutes. For positive control, saline was added to the plasma. Then, the C3 and C4 reaction agents were added to the samples and the activation of C3
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and C4 was measured using a Roche/Hitachi 902 auto analyzer. 2.5.4. Agglutination experiments
In the agglutination test, RBCs were evaluated under the influence of nanocarriers using fresh
blood. 10 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX solutions were added to 40 μl blood to achieve final concentrations of 5, 10, 20, 40, 75, 125 and 200 μg/ml. The
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samples were incubated at 37 ° C for 30 min in an incubator. Then, the samples were centrifuged
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for 5 min at 1500 rpm, the plasma solution was removed and 20 μl of fresh plasma was replaced.
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After that, a drop of sample was placed on the lam and samples were observed using the
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microscopic methods. 2.5.5. Coagulation time
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The fresh blood sample of healthy volunteers contain sodium citrate as anticoagulant were used.
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To isolate the plasma, blood was centrifuged at 1500 rpm for 20 minutes. Prothrombin time (PT)
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measures the external pathway of blood coagulation and factors I, II, V, VII and X. Also, the relative time of thromboplastin (APTT) measures the internal coagulation pathway and factors I,
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II, V, VIII, IX, X, XI and XII. To determine the effect of nanocomposites on PT and APTT, 100 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX samples were added to 900 μl of
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plasma to the final concentrations of 10, 50, 100 and 200 μg/ml. The samples were incubated at 37 ° C for 5 min. For positive control, saline was added to the plasma. Then the PT and APTT agents were added to the samples and their coagulation time were measured by the PT and APTT analyzer.
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2.6. Release of MTX from nanocarrier and kinetic models Dialysis bag (Sigma–Aldrich, USA) with 8 kDa molecular weight cutoff was used to evaluate the release rate of the drug from the nanocarrier. 2 mg of GOMNP/PEGA was dispersed in 5 ml
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DI water, transferred to dialysis bag, and placed in 50 ml phosphate buffer saline pH 5.5 (the cancer cells endosomal pH) and pH 7.4 (physiological pH). The sample was then shaken at 37
°C. Periodically, the absorption of 1 ml of supernatant was read at 304 nm for 60 hours and fresh PBS buffer replaced. The concentration of the MTX released from the surface of the nanocarrier was characterized by UV/Vis spectroscopy at 304 nm. The amount of drug loaded on 1 mg of the
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nanocarrier was 0.42 mg.
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There are various kinetic models available to check the release rate of the drug. The models are
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based on mathematical functions that describe the features of drug recognition. The data obtained
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from drug release during 60 hours were used in 5 kinetic models including zero order, first order, Korsmeyer-Peppas, Hixson-Crowel and Higuchi model. To take the suitable drug release kinetic
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2.7. Statistical analysis
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model, the regression module of each model were compared.
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Statistical analysis was performed via PASW Statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of the obtained data for different compounds was performed
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using One-Way ANOVA followed by Tukey posthoc test. The significance level was set at p > 0.05 and p > 0.001.
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3. Results and discussion 3.1. Synthesis and characterization of GOMNP/PEGA Fig. 1 represents the synthesis of GOMNP/PEGA for the loading and transport of MTX. As shown in this figure, graphene oxide was obtained from modified Hummer’s method and 11
decorated with iron oxide nanoparticles. The as-prepared GOMNP nanocarriers were functionalized with polyethylene glycol bis amine, as a biocompatible polymer, for increasing solubility and biocompatibility. Indeed, the development of GOMNP hybrid by covering with
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hydrophilic polymers is very important to improve its biocompatibility and to reduce toxicity. [Figure 1 to be inserted here]
Fig. 2 shows the TEM images of GO and GOMNP. In Fig. 2a, TEM image of GO demonstrate some few-layered graphene or GO flakes with diameter around 1.2 µm. The changes in the
surface, degradation and the presence of wrinkles in sheets can be considered as a reason of
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oxidation of sheets. In Fig. 2b, which relates to the graphene oxide nanosheet after
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magnetization, TEM image represents completely spherical magnetic nanoparticles on the GO
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nanosheets and the dispersion of iron nanoparticles is relatively uniform on the surface of GO.
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The average dimensions of these nanoparticles were estimated to be less than 20 nm.
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[Figure 2 to be inserted here]
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The FTIR analysis was applied to characterize the samples synthesized in each step. The FTIR spectra of GO, GOMNP and GOMNP/PEGA are shown in Fig. 3. The oxygen containing groups
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in graphene oxide structure showed different peaks in FTIR including a large peak at 3400 cm-1 for the O-H stretching of the hydroxyl group; the C=O bonds of the carboxylic group (O=C-OH)
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in 1734 cm -1; the C=C bond of non-oxidized graphite grains with sp2 hybrid in 1627 cm-1; and C-O-C bond of epoxy groups and C-O bond of carbonyl groups in the range of 1250 cm-1. The
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peak of the hydroxyl groups in 3400 cm-1 corresponds to 3 different O-H transplants including alcoholic and phenolic groups adhering to graphene oxide plates and water molecules surrounded between graphene oxide layers (Acik et al., 2011; Lerf et al., 1998). In the FTIR spectra of GOMNP, the peak of stretching vibration of Fe-O is found in 570 cm-1, which represents the
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functionalization of graphene oxide with iron nanoparticles (Yang et al., 2009). After functionalization of GOMNP with PEGA, the bond corresponds to polyethylene glycol bis amine functionalized GOMNP is detected by an amide bond formed with CH2 in 2900 cm-1 and a
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stretching vibration of NH-CO in 1640 cm-1 (Fig. 3) (Zhang et al., 2011). [Figure 3 to be inserted here]
Fig. 4 shows the SEM images of GO and GOMNP. In Fig. 4a the GO plates have large
dimensions as a result opening of interlayer spacing due to intense oxidation. As can be seen
from Fig. 4b, iron nanoparticles with an average size of less than 20 nm are well grafted to GO
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layers with a narrow size distribution. The SEM results are in agreement with the TEM results
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and also confirm the synthesis of nanocomposite.
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[Figure 4 to be inserted here]
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Fig. S1 shows the magnetization curves of GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX that were measured at room temperature. The amount of saturation magnetization (MS) values of
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GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX were 57.6, 51.8 and 45.2 emu/g,
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respectively. The gradual decline in magnetic properties of both GOMNP/PEGA and
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GOMNP/PEGA/MTX, compared to the GOMNP nanocarrier, is a reason for their magnetic instability. Also, the coating of PEGA has a negligible effect on magnetite properties. In
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addition, the lower saturation magnetism of nanocarrier containing the drug confirms the success of the drug loading process. In addition, the magnetic separability of GOMNP/PEGA/MTX was
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tested by placing an external magnet close to the glass bottle. The particles could be easily collected by an external magnetic field (Fig. S2). X-ray diffraction patterns of GO and GOMNP samples are shown in Fig. S3. As shown in this figure, the graphene oxide synthesized by modified Hummer’s method, has a peak around 2θ =
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12, and also another peak around 2θ = 43, which can be related to the unmodified graphite (Han et al., 2012). In Fig. S3, the XRD pattern of GOMNP matches both the magnetite Fe3O4 and the maghemite c-Fe2O3 structures, according to the joint committee on powder diffraction standards
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(JCPDS) file No. 19-0629 and 39-1346, respectively (Daou et al., 2006). In order to further confirm the bonding of PEGA to GOMNP and loading of drug, the TGA analysis was used. The thermal behaviors of GOMNP, GOMNP/PEGA and
GOMNP/PEGA/MTX are investigated by thermogravimetric analysis at a temperature range of 20-650 ˚C. The temperature range of 0 to 200 ˚C and 200-650 ˚C were related to the removal of
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water and organic moieties on the surface of nanocarriers, respectively. As shown in Fig. S4, the
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weight loss difference between GOMNP/PEGA and GOMNP/PEGA/MTX reflected the amount
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of PEGA and MTX loaded on the GOMNP and GOMNP/PEGA surfaces, respectively.
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3.2. In vitro experiments results 3.2.1. MTT Assay
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MTT assay is commonly used to measure the cellular cytotoxicity affected by anticancer drugs.
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MTT reagent is reduced by succinate dehydrogenase, one of the mitochondrial reductase
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enzymes, and it is then converted to an insoluble purple formazan by cleavage of the tetrazolium ring. The purple colored formazan product is directly correlated to the viable cells with active
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metabolism. The viability of cells in two cell lines MCF-7 and HeLa was investigated after 24h and 48h incubation with various concentrations of 1 to 50 μg/ml of GOMNP/PEGA,
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GOMNP/PEGA/MTX and free MTX. The results are presented in Fig. 5. As shown in this figure, in the MCF-7 cell line, GOMNP/PEGA/MTX killed about 35% of the cells in the highest concentration after 24 hours. The effect of the drug sample on this cell line was about 45% in the first 24 hours. After 48 hours nanoparticles containing the drug caused cell death from 40 to 47
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percent. The effect of GOMNP/PEGA/MTX has increased over a period of 48 hours, indicating a longer release of the drug over time. In the HeLa cell line, with increasing concentrations of GOMNP/PEGA/MTX, 15-40% of the cells were killed. The effect of free drug is similar in
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various concentrations and about it was about 40 to 50% of cell death. After 48 hours, the cell growth was significantly reduced. The percentage of cell viability was about 70 to 53 percent.
The effect of the drug was increased after 48 hours and reached about 50-60% of the cell death. In total, the increasing of GOMNP/PEGA/MTX concentration enhanced the cytotoxic activity. [Figure 5 to be inserted here]
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3.2.2. Hemolysis of RBCs
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Hemolytic analysis of red blood cells was performed to evaluate blood compatibility of
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synthesized nanoparticles and nanocomposites/blood components interactions. In this method,
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nanoparticles at different concentrations were incubated with blood. Accordingly, erythrocytes were isolated as the dominant cells in the blood and combined with nanoparticles with different
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concentrations ranging from 5 to 200 μg/ml. Positive and negative controls were prepared in the
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same way by addition of triton X-100 and PBS, respectively, instead of nanoparticles. Both
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quantitative and qualitative tests were performed to evaluate the hemolytic behavior of nanostructures on erythrocytes. The qualitative results showed that there was no hemolytic
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activity for synthesized nanostructures (Fig. S5). Table 1, demonstrated the quantitative hemolysis results for all samples. The hemolytic activity of the nanoparticles containing the drug
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(GOMNP/PEGA/MTX) illustrated reduction or inactivation of hemolytic activity after sample incubation with RBCs. Also, it can be observed that GOMNP/PEGA/MTX did not induce any undesirable responses and its non-hemolytic property (<2% hemolysis) was confirmed at all concentrations. However, GO and GOMNP nanocarriers exhibited hemolytic activity even at
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low concentrations. GO demonstrate a dose-dependent hemolytic activity which is attributed to the electrostatic interactions between the positively charged of phosphatidylcholine lipids present on the outer surface of the RBCs and the negatively charged oxygen present on GO. (Liao et al.,
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2011). The important elements that determine graphene blood compatibility are surface charge, oxygen content and particle size. Nano-sized GO (350 nm) was found to induce a severe
hemolysis effect (70% hemolysis at 25 μg/mL) compared to micro-sized (3 μm) graphene sheets (less than 10% hemolysis at 100 μg/mL). The aggregated graphene particles had a greater
cytotoxicity than the reversibly aggregated GO, due to forming aggregates which reduces the cell
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contact area. Therefore, when the concentration ranges from 75 μg/ml increase to 125 μg/ml, the
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percentage of hemolysis decreases due to the aggregation of nanocarriers. However, at 200
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μg/ml, the concentration of nanoparticles is so much increased that can enhance collisions of
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aggregates with red blood cells and lead to membrane degradation and subsequently increase hemolytic activity. The presence of carboxylic groups also can lead to direct interactions of GO
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with cell membranes and cause cell destruction or death (Cheng et al., 2012).
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Table 1. Hemolysis percentages of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX at different concentrations of 5-200 μg/ml. Controls were performed with 0% and 100% hemolysis with PBS (negative control) and Triton X-100 (positive control), respectively. Sample
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Concentration
GO
GOMNP
GOMNP/PEGA
GOMNP/PEGA/MTX
1.17±0.09
0.87±0.02
2.50±0.11
2.67±0.13
10 μg/ml
0.08±0.01
0.75±0.04
2.84±0.07
1.88±0.09
20 μg/ml
1.00±0.05
1.67±0.05
2.50±0.09
2.34±0.18
40 μg/ml
2.34±0.10
1.75±0.04
2.00±0.06
0.96±0.03
75 μg/ml
3.59±0.14
2.21±0.11
2.96±0.08
1.19±0.08
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5 μg/ml
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125 μg/ml
1.75±0.02
2.42±0.09
2.04±0.07
1.33±0.11
200 μg/ml
5.26±0.17
4.22±0.13
1.09±0.08
0.37±0.01
GOMNP showed less hemolytic effect at lower concentrations, however, undecorated iron
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nanoparticles caused toxic effects on the cell and cell destruction. The PEGA, a highly biocompatible molecule, was used to cover GOMNPs but the amine group in this polymer could increase the tendency of the system to connect to cell due to its positive charge and subsequently increase hemolysis at lower concentrations. 3.2.3. Complement activation assay
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The complement system consists of a set of plasma proteins and cell surface proteins that interact
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with the external factors to eliminate them. Activation of complement protein components such
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as C3 and C4 and their interaction with nanoparticles is an important parameter for the
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evaluation of biocompatibility of nanoparticles. Fig. 6 shows the complement activity assay in the absence (0 g/mL, as a negative control) and presence of different concentrations (10-200
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g/mL) of nanocarriers. According to this figure, GO, GOMNP, GO/MNP/PEGA and GOMNP/PEGA/MTX samples have normal range of C3 and C4 as compared to the control
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sample. The results demonstrated that the nanocarriers and the nanocarriers containing drug do not activate the complement systems and therefore, the synthesized nanocarriers have biological
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compatibility associated with complement systems. [Figure 6 to be inserted here]
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3.2.4. Agglutination assay The method of hemagglutination of red blood cells was used for the evaluation of agglutination, due to interaction of RBCs with various concentrations of nanoparticles. Using this experiment, the effects of drug-binding nanoparticles on red blood cells and deformation of red blood cells
17
were examined. As shown in Fig. 7, GOMNP/PEGA/MTX samples do not demonstrate any evidence of the hemagglutination of RBCs even at high concentrations. The GOMNP/PEGA samples show less evidence of agglutination than GO and GOMNP samples, while GO and
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GOMNP samples caused RBC agglutination at high concentrations. [Figure 7 to be inserted here] 3.2.5. Coagulation time assay
When the vessel is damaged, platelets accumulate in that area and activate blood factors and
therefore, activate the internal and external pathway of blood coagulation. Prothrombin time (PT)
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and activated partial thromboplastin time (APTT) are medical tests that characterize blood
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coagulation. A normal PT and APTT are approximately 9-15 seconds and 25-35 seconds,
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respectively (Selbi et al., 2013). Coagulation test was performed on the plasma mixed with
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GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX with various concentrations of 10-200
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μg/ml. Samples displayed a normal PT and APTT values of the control experiment. As shown in
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Fig. S6, PT and APTT are within the permissible range for all concentrations of three samples. These results indicated that the internal and external coagulation pathways of the blood are not
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affected by these nanocarrier. Therefore, the synthesized nanocarriers have good
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biocompatibility associated with coagulation assay. 3.2.6. Drug release results
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The drug release experiment was carried out in PBS buffer pH 5.5 at 37 °C through the dialysis setup. The amount of the drug released was obtained using a dialysis bag, by measuring the absorption of 1 ml of the external solution of the dialysis bag at various times. The amount of the drug released at pH values of 5.5 (cancer cells endosomal pH) and 7.4 (physiological pH) was measured over a period of 60 hours. Fig. S7 shows the release amounts of MTX as a function of 18
time under different pH conditions. The results indicated different behavior in the release time profiles of two pHs and the MTX has more efficient release at pH 5.5 compared to pH 7.4. The observed difference of drug release between two pHs can be due to changes in the physical
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interactions between nanocarrier and MTX. In acidic pHs, -NH- group starts to convert into – NH2+. This change can lead to dissociation of hydrogen bonds. So, at lower pHs, the possibility of drug release from the nanocarrier surface is more than that at neutral pHs. The drug release
rate was reached to about 100% drug until 60 hours, indicating complete release of the drug in acidic conditions. According to the acidic microenvironment of cancer cells, the drug delivery
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efficiency was considerably improved by loading MTX in the GOMNP/PEGA nanocarrier.
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3.2.7. The kinetics of drug release
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The data obtained from drug release experiments were investigated using 5 kinetic models including zero order, first order, Korsmeyer-Peppas, Hixson-Crowel and Higuchi model (Fig.
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S8). To obtain a kinetic model related to drug release, the curves of each model were plotted and
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their R2 values were compared (Dash et al., 2010; Gouda et al., 2017; Singhvi and Singh, 2011). By comparing the R2 values of these models and considering that, the R2 values of the first order
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model is higher than other models (Table S1), it can be concluded that the release model of the
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MTX drug may follow the first-order kinetic model. In this model, the graph is based on the log of the concentration ratio of the drug in time t to the initial concentration of drug in the time t0.
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According to this model, the reaction speed is directly related to the concentration of the drug released during the reaction. The higher the initial concentration of the drug, the greater the release rate and therefore, the model follows a linear kinetics. Since the binding of MTX to the nanocarrier is through both covalent and ionic bonds, first the weaker bonds break down and consequently more drugs are released. Hence, in the initial times of drug release assay, an 19
explosive release occurs. Over time, the connections are tightened and the lesser amounts of the drug are released with a very gentle gradient. 4. Conclusions
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In this study the nanoparticles of graphene oxide, decorated with iron nanoparticles and
functionalized with polyethylene glycol bis amine, were used as a nanocarrier for the delivery of methotrexate. Using MTT assay, the cytotoxicity of synthesized nanocomposites on MCF-7 and HeLa cell lines was investigated. The activity of the nanocomposite (GOMNP/PEGA/MTX)
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after incubation with red blood cells reduced hemolytic activity. While the blood is well
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tolerated, the compatibility of the drug containing nanocomposites at all different concentrations
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was confirmed. It has been demonstrated that the synthesized nanocarriers have biological
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compatibility associated with complement systems, agglutination, and coagulation time. The release of the drug from the nanocarrier in acidic conditions was completed after 60 hours.
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Overall, the results of this study indicated high drug loading, high toxicity for tumor cells, proper
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drug release and good biocompatibility of the synthesized nanocarrier, which can increase the therapeutic efficacy. Due to wide range usage of methotrexate in the treatment of different
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diseases, various nano-carriers have been synthesized for the delivery of this drug.
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Ashwanikumar et al. (Ashwanikumar et al., 2014) utilized a random copolymer of poly-lacticco-glycolic acid (PLGA) grafted branched polyethyleneimine for methotrexate delivery. Chen et
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al. (Chen et al., 2013a) synthesized a magnetic targeted drug delivery nanocarrier based on Fe3O4/PLA–PEG. Rahimi et al. (Rahimi et al., 2017) Constructed dendritic chitosan grafted mPEG coated magnetic nanoparticles (DPC@MNPs) as a magnetic nanocarrier for multi drug delivery of doxorubicin (DOX) and methotrexate (MTX). Khodadadei et al. (Khodadadei et al., 2017) designed a nanocarrier with blue fluorescent nitrogen-doped graphene quantum dots an 20
efficient drug delivery system. The synthesized nanocarrier in this study with advantages, including more drug release in acidic conditions, higher toxicity to tumor cells, and blood biocompatibility, has a better performance than other carriers in the delivery of methotrexate.
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Regarding these properties, the GOMNP/PEGA nanocomposite can be considered as a novel candidate for drug delivery due to the features such as easy synthesis and biocompatibility, with controlled target and release of the drug.
Declaration of interests
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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgements
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The authors are grateful to the Center of Excellence of Research Council of the University of
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Isfahan for financial supports of this work.
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Figure legends
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Figure 1. Synthesis of PEGA functionalized GOMNP nanocomposite and MTX loading.
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Figure 2. TEM images of (a) GO and (b) GOMNP.
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Figure 4. SEM images of (a) GO and (b) GOMNP.
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Figure 5. The effect of different concentrations of free MTX, GOMNP/PEGA and GOMNP/PEGA/MTX on viability of (a, c) HeLa and (b, d) MCF-7cell lines after 24 (a, b) and 48
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(c, d) hours incubation.
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of
different
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6.
concentrations
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Figure
of
GOMNP,
GOMNP/PEGA
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GOMNP/PEGA/MTX samples on the complement system; (a) C3 and (b) C4 assays.
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Figure 7. Agglutination of red blood cells in the presence of various concentrations of different
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nanocarriers.
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S0168-1656(19)30120-8 https://doi.org/10.1016/j.jbiotec.2019.04.006 BIOTEC 8400
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
1 November 2018 9 March 2019 5 April 2019
Please cite this article as: Abdollahi Z, Taheri-Kafrani A, Bahrani SA, Abbasi Kajani A, PEGAylated graphene oxide/superparamagnetic nanocomposite as a high-efficienc loading nanocarrier for controlled delivery of methotrexate, Journal of Biotechnology (2019), https://doi.org/10.1016/j.jbiotec.2019.04.006 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.
PEGAylated graphene oxide/superparamagnetic nanocomposite as a high-efficiency loading nanocarrier for controlled delivery of
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methotrexate
Zahra Abdollahi a, Asghar Taheri-Kafrani a, Seyed Amir Bahrani b, Abolghasem Abbasi Kajani a
a
: Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of
: IMT Lille Douai, Energy Engineering Department, F-59500 Douai, France.
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b
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Isfahan, Isfahan, 81746-73441, Iran.
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* To whom correspondence should be addressed
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Tel: +98 31 37 93 43 46
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Email: [email protected]
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Highlights
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Fax: +98 31 37 93 23 42
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Graphene
oxide/iron
oxid
nanocomposites
were
functionalized
with
PEGA
(GOMNP/PEGA). The conjugation of methotrexate (MTX) on GOMNP/PEGA was confirmed by different techniques.
1
This MTX nanocarrier demonstrated high drug loading and optimum ability for a controlled drug release. The in vitro assay results showed that the drug carrier nanosystem was hemocompatible
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Abstract
Polymer-coated nanocarriers play an important role in targeted drug delivery. The use of
polymers such as polyethylene glycol increases stability, biocompatibility, and blood circulation
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time of the drug, and may consequently improve the success of drug delivery. In the present
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work, a simple approach has been reported for synthesizing polyethylene glycol bis amin
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(PEGA) functionalized graphene oxide/iron oxide nanocomposite as a remarkable unit for
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loading drugs. The biomedical applications of the synthesized nanocomposite were investigated
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by immobilizing methotrexate (MTX), as an anticancer drug. The structural and morphological
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characteristics and the successful synthesis of the nanocomposite were evaluated by different charachterization techniques. The cytotoxicity assay of the nanocarrier showed higher toxicity
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against HeLa and MCF-7 cell lines, compared to free MTX. The drug release experiments in acidic and physiological conditions suggested the first order kinetics model for the release of
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MTX from the nanocomposite. Furthermore, the agglutination, complement activation, and coagulation time experiments demonstrated the blood compatibility of the synthesized
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nanocarrier.
Keywords: Graphene Oxide; Methotrexate; Magnetic nanoparticles; Drug release; Biocompatibility.
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Introduction Cancer is considered as one of the most important reason of death in the world in these years. The different ranges of chemotherapy drugs are used as a significant way to treat various
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cancers. In spite of significant applications, anticancer drugs suffer from some limited effects on target cells due to lack of selectivity, low solubility, uniform tissue distribution, short half-life in the bloodstream and dose-related toxicity (Bao et al., 2015; Rahmanian et al., 2017; Sun et al., 2014). Therefore, exploring an efficient and general approach to overcome drawbacks of
anticancer drugs is quite exigent. Methotrexate (MTX), a chemotherapy drug, is a folic acid
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analog and uses for the treatment of many different kinds of cancers (Chen et al., 2013b;
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Wojtoniszak et al., 2013; Yoon et al., 2010). MTX can mainly blocks the dihydrofolatereductase
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(DHFR) enzyme and subsequently prevents the production of thymidine (Grim et al., 2003;
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Khan et al., 2012; Tian and Cronstein, 2007; Wei et al., 2014). In spite of significant
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applications, this anticancer drug suffers from some limited effects on target cells due to poor
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aqueous solubility, short half-life in plasma, low permeability, and nonspecific drug delivery. Therefore, in modern nanobiotechnology, linking MTX to nanoscale drug carriers in order to
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alter its pharmacokinetic behavior, enhance tumor targeting, reduce toxicity, and overcome drugresistance mechanisms has been noted (Chen et al., 2013b; Khan et al., 2012; Kohler et al., 2006;
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Santos et al., 2007).
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Current advances in biotechnology have improved advanced nanoscale systems for application in drug delivery systems (Barranger et al., 2015). These systems include nanostructures or nanomaterials, such as liposomes (Lokerse et al., 2018), hyperbranched polymers (Shen et al., 2018), microspheres (Vasir et al., 2003), polymeric shells (Haag, 2004), inorganic nanoparticles (Kovalainen et al., 2018) and carbon nanotubes (Da Ros et al., 2018). The loading capacity and
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loading efficiency of the currently developed nanoscale drug delivery systems are still undesirable. In addition, rigorous reaction conditions, long reaction times and expensive reagents are limitations and drawbacks of many of the previously reported methods(Mura et al., 2013).
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Nanoparticles (NPs) have been widely used as nanocarriers for drug delivery due to their small size, release characteristics and targeted delivery of chemotherapeutic drugs (Bao et al., 2015; Barranger et al., 2015; Sun et al., 2014). The development of new strategies to produce high loading drug-containing NPs with appropriate solubility and reaction conditions is still in demand in nanomedicine research area (Shi et al., 2016).
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Nowadays, carbon nanostructures such as graphene and its water-soluble derivative, graphene
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oxide (GO), have attracted more attention than other nanomaterials due to their electronic and
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structural properties, high surface area, adjustable surface chemistry, purity, and two-
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dimensional structure which allows the reactions to occur more efficiently (Amiri et al., 2012; Liu et al., 2013; Ma et al., 2012; Yang et al., 2009; Zhang et al., 2010). Besides, good dispersion
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in water and physiological environments, biocompatibility and lack of obvious toxicity make it
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suitable nanocarriers for drug delivery (Guo et al., 2016; Shi et al., 2013; Yang et al., 2011).
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Also, graphene oxide can be easily functionalized by various agents for better conjugation of drugs (Choi et al., 2010; Shi et al., 2013). One of the promising targeting methods is grafting
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magnetic nanoparticles (MNPs) on the surface of graphene oxide. This conjugate can be rapidly and efficiently separated by an external magnetic field, and therefore, it can be used as the
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support material for biological applications such as targeted drug delivery (Bayramoğlu and Arıca, 2008; Liao et al., 2017; Mody et al., 2014; Reis et al., 2017; Yang et al., 2009). Agglomeration of nanoparticles can reduce efficiency, rapid clearance by macrophages and accumulated in the reticule-endothelial system before reaching the target cells. One way to solve
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this problem is to cover the surface of the nanoparticles with biocompatible polymers such as poly ethylene glycol (PEG). As covering the surface of the nanoparticles with PEG, a flexible hydrophilic polymer, provide to increase nanoparticle circulation time in the blood, improve the
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delivery of nanoparticles to the target tissue and also cause functional groups on the surface capable of conjugating with target agents. Attaching PEG to nanoparticles may also prevent their opsonization and reduce their uptake by the reticuloendothelial system. As a result, PEGylation of nanoparticles enhances bio-compatibility and increases the circulation time of nanoparticles in the blood. The amine groups on the surface of polyethylene glycol bis-amine (PEGA) can be a
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suitable choice as functional groups on the surface of MNPs (Howard et al., 2012; Kohler et al.,
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2004; Reis et al., 2017; Wanna et al., 2016).
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Encouraged by the unique properties and applications of hybrid nanomaterials, we wish to report
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a novel convenient method to prepare high water-soluble superparamagnetic graphene oxide functionalized with PEGA, as a novel functional group. This nanocarrier with high
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biocompatibility was used to improve loading capacity of drug and increase toxicity for tumor
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cells. In addition because of some features such as rapid and easy synthesis and separation in the
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presence of external magnetic field, and modification with PEGA, the GOMNP/PEGA nanocarrier can be a new and appropriate delivery system. The use of this biocompatible
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polymer, in addition, can improve biocompatibility of the nanocarrier, due to the positive charge of bis-amine on the polymer, and increase binding of nanocarrier to negative charged cancer
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cells. This can lead to the unique ability of the system to transfer methotrexate and other cancer drugs to the target tissue. The in vitro behavior of the synthesized nanomaterial was also investigated in order to confirm it as a novel nanocarrier candidate for targeted methotrexate delivery.
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2. Materials and methods 2.1. Materials Methotrexate (MTX) was purchased from Sigma-Aldrich Chemical Co. Graphite powder (300
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mush), iron (II) chloride tetrahydrate (FeCl2.4H2O), iron (III) chloride hexahydrate
(FeCl3.6H2O), sulfuric acid (H2SO4, 98%), potassium permanganate (KMnO4), sodium nitrate
(NaNO3), potassium dihydrogenphosphate (KH2PO4), potassium chloride (KCl), sodium chloride (NaCl) and disodium phosphate (Na2HPO4) were all purchased from Merck Chemical Co.
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2.2. Apparatus
Fourier transform infrared (FTIR) spectra were recorded on a Perkin-Elmer FTIR spectroscopy
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using KBr discs in the 400–4000 cm-1 region to analyze the presence of functional groups and
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the binding of PEGA to the nanocarrier. The surface morphology of samples were observed by scanning electron microscopy (SEM, Hitachi S-4800). Transmission electron microscopy (TEM)
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images were recorded using a Philips CM30 transmission electron microscope operated at 200
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kV and equipped with a LaB6 filament. Powder x-ray diffraction (XRD) patterns were collected on a Bruker D8-advance x-ray diffractometer with Co Kα radiation (λ = 1.7902 A).
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Thermogravimetric analysis experiments (TGA) were carried out using a TGA Q500 V20.10
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Build 36 thermo analysis system (American TA Instruments Cot., USA). The magnetization curves of the as-prepared nanoparticles were determined with a vibrating sample magnetometer
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(VSM, Quantum Design Company, USA). Loading the drug was measured utilizing UV–vis Carry-500 double beam spectrophotometer (USA). The PT/APTT was assessed by coagulation analyzer (IL ACL Elite Pro). The complement system activation was measured by auto analyzer (Hitachi 902). 2.3. Synthesis of nanocarriers 6
2.3.1. Synthesis of graphene oxide Graphene oxide was obtained from graphite using modified Hummer’s method (Liu et al., 2008; Stankovich et al., 2006). Briefly, graphite powder (0.5 g) with an equal amount of NaNO3 (0.5 g)
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in 23 ml of concentrated H2SO4 (12.1 M) were stirred in an ice water bath for 15 minutes. Thereafter, KMnO4 (4.0 g) was added slowly to the mixture in an ice bath until the color turns to purple-green. Then mixture was taken the hot water bath at 40 °C and was stirred for 90 minutes. Afterward, 50 mL of deionized water (DI) was added slowly and stirred for 15 minutes,
following by adding 6 ml of H2O2 until a golden-brown sol appeared. Then, 50 ml of DI water
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was added and the mixture centrifuged with DI water for several times to reduced pH to 6.0.
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The synthesized product, GO, was dried at room temperature under vacuum.
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2.3.2. Preparation of superparamagnetic graphene oxide nanocomposite
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To prepare superparamagnetic graphene oxide nanocomposite (GOMNP), 400 mg of GO was added to 100 mL solution of 0.01 M NaOH, pH 12, and sonicated for 45 min at room
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temperature under nitrogen atmosphere. This can alter the carboxylic acid groups to carboxylate
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anions. The mixture centrifuged and washed with DI water until the pH became neutral. The
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obtained powder was then dispersed in 20 mL DI water and sonicated at room temperature for 30 min. subsequently, 480 mg FeCl3.6H2O and 176 mg FeCl2.4H2O were solved in 5 mL DI water
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and added to the above mixture and sonicated for 1 h at 60 °C. Throughout sonication, ammonium aqueous solution (13 %) was slowly added to mixture and the pH was adjusted to 9–
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12. The product, solid GOMNP, was separated with an external magnet and dried under vacuum. 2.3.3. Functionalization of GOMNP nanocomposite with polyethylene glycol bis amine 8 mg of GOMNP was added to 3 ml of sodium phosphate buffer and sonicated for 20 min following by adding 4 mg of polyethylene glycol bis amine (PEGA) to the mixture and stirring
7
for 16 hours at 50 °C. The solution was then separated from the product, GOMNP/PEGA, by a magnet and dried under vacuum. 2.4. Loading of methotrexate onto GOMNP/PEGA nanocomposite
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For methotrexate (MTX) loading onto nanocarrier, the obtained GOMNP/PEGA powder was initially dispersed in 2 ml of DI water by sonication to prepare a homogeneous suspension. Then, 0.25 mg/ml solution of MTX was added to the mixture and stirred at 25 °C overnight. 2.5. In vitro biocompatibility experiments
The relevant guidelines and regulations was used for the in vitro biocompatibility experiments of
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the synthesized nanocomposites. All methods were carried out in accordance with relevant
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guidelines and regulations, informed consent was obtained from all subjects, and the
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experimental protocols were approved by Al-Zahra hospital in Isfahan, Iran. Human cell lines
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were provided from Pasteur Institute of Iran. Red blood cells (RBCs) were provided by healthy volunteers under the approval of Al-Zahra hospital in Isfahan, Iran.
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2.5.1. MTT Assay
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Two cell lines, MCF-7 and HeLa, were seeded to the density of 104 cells in RPMI 1640 medium
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supplemented with 10 % FBS per into a 96-well plate and incubated overnight. The samples including free MTX, GOMNP/PEGA and GOMNP/PEGA/MTX with concentrations of 1, 5, 10,
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20 and 50 μg/ml were added to each well and incubated for 24-48 h. Then the medium was discarded and 100 μl of the culture medium containing MTT (concentration of 0.5 mg/ml) was
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added into each well and incubated at 37 °C for 4 hours. MTT was absorbed by cells and converted to oxalic oxidoreductase by cleavage of the tetrazolium ring using dehydrogenase enzymes. The supernatant was then removed and 100 μl DMSO was added to each well to
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insoluble the formazan crystals. The absorbance was measured at 492 nm using a microplate reader and the percentage of cell viability was obtained. 2.5.2. Hemolysis of red blood cells
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The healthy volunteers are required to perform this experiment. 5 ml of human blood heparinized was centrifuged at 4500 rpm for 10 minutes, a plasma solution was removed, and the RBCs were washed out with PBS until the supernatant was clear. The RBCs were then added to 5 ml of
saline phosphate buffer pH 7.0. For the hemolysis test, 190 μl of diluted RBCs were mixed with 10 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX to reach final concentrations
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of 5, 10, 20, 40, 75, 125 and 200 μg/ml of nanocarriers. Controls were performed with 0% and
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100% hemolysis with PBS (negative control) and Triton X-100 (positive control), respectively.
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The samples were incubated at 37 °C for 30 min and then centrifuged for 5 minutes at 4000 rpm.
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At the end, 100 μl of supernatant solution was taken, diluted with 900 μl PBS buffer, and the absorbance of samples were analyzed at 576 nm for measuring hemoglobin released from
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hemolytic red blood cells. The percentage of hemolysis was measured by comparing the positive
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and negative control absorbance, according to the Eq. 1: 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
(1)
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Hemolysis (%) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑝𝑎𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 2.5.3. Complement activation assay
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The purpose of this experiment was to measure the amount and activity of blood complement
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system in the presence of various concentrations of nanocomposites. The activation of the complement system is made up of the main and secondary pathways, both of which activate C3 and lead to the production of a membrane attack complex. Complementary compounds of the complement (C3 and C4) are quantitatively measurable by immunological methods. For this test, 100 μl of human serum was added to 100 μl of GO, GOMNP, GOMNP/PEGA and 9
GOMNP/PEGA/MTX samples to obtain final concentrations of 10, 50, 100 and 200 μg/ml. samples were incubated at 37 ° C for 30 minutes. For positive control, saline was added to the plasma. Then, the C3 and C4 reaction agents were added to the samples and the activation of C3
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and C4 was measured using a Roche/Hitachi 902 auto analyzer. 2.5.4. Agglutination experiments
In the agglutination test, RBCs were evaluated under the influence of nanocarriers using fresh
blood. 10 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX solutions were added to 40 μl blood to achieve final concentrations of 5, 10, 20, 40, 75, 125 and 200 μg/ml. The
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samples were incubated at 37 ° C for 30 min in an incubator. Then, the samples were centrifuged
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for 5 min at 1500 rpm, the plasma solution was removed and 20 μl of fresh plasma was replaced.
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After that, a drop of sample was placed on the lam and samples were observed using the
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microscopic methods. 2.5.5. Coagulation time
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The fresh blood sample of healthy volunteers contain sodium citrate as anticoagulant were used.
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To isolate the plasma, blood was centrifuged at 1500 rpm for 20 minutes. Prothrombin time (PT)
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measures the external pathway of blood coagulation and factors I, II, V, VII and X. Also, the relative time of thromboplastin (APTT) measures the internal coagulation pathway and factors I,
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II, V, VIII, IX, X, XI and XII. To determine the effect of nanocomposites on PT and APTT, 100 μl of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX samples were added to 900 μl of
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plasma to the final concentrations of 10, 50, 100 and 200 μg/ml. The samples were incubated at 37 ° C for 5 min. For positive control, saline was added to the plasma. Then the PT and APTT agents were added to the samples and their coagulation time were measured by the PT and APTT analyzer.
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2.6. Release of MTX from nanocarrier and kinetic models Dialysis bag (Sigma–Aldrich, USA) with 8 kDa molecular weight cutoff was used to evaluate the release rate of the drug from the nanocarrier. 2 mg of GOMNP/PEGA was dispersed in 5 ml
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DI water, transferred to dialysis bag, and placed in 50 ml phosphate buffer saline pH 5.5 (the cancer cells endosomal pH) and pH 7.4 (physiological pH). The sample was then shaken at 37
°C. Periodically, the absorption of 1 ml of supernatant was read at 304 nm for 60 hours and fresh PBS buffer replaced. The concentration of the MTX released from the surface of the nanocarrier was characterized by UV/Vis spectroscopy at 304 nm. The amount of drug loaded on 1 mg of the
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nanocarrier was 0.42 mg.
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There are various kinetic models available to check the release rate of the drug. The models are
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based on mathematical functions that describe the features of drug recognition. The data obtained
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from drug release during 60 hours were used in 5 kinetic models including zero order, first order, Korsmeyer-Peppas, Hixson-Crowel and Higuchi model. To take the suitable drug release kinetic
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2.7. Statistical analysis
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model, the regression module of each model were compared.
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Statistical analysis was performed via PASW Statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of the obtained data for different compounds was performed
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using One-Way ANOVA followed by Tukey posthoc test. The significance level was set at p > 0.05 and p > 0.001.
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3. Results and discussion 3.1. Synthesis and characterization of GOMNP/PEGA Fig. 1 represents the synthesis of GOMNP/PEGA for the loading and transport of MTX. As shown in this figure, graphene oxide was obtained from modified Hummer’s method and 11
decorated with iron oxide nanoparticles. The as-prepared GOMNP nanocarriers were functionalized with polyethylene glycol bis amine, as a biocompatible polymer, for increasing solubility and biocompatibility. Indeed, the development of GOMNP hybrid by covering with
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hydrophilic polymers is very important to improve its biocompatibility and to reduce toxicity. [Figure 1 to be inserted here]
Fig. 2 shows the TEM images of GO and GOMNP. In Fig. 2a, TEM image of GO demonstrate some few-layered graphene or GO flakes with diameter around 1.2 µm. The changes in the
surface, degradation and the presence of wrinkles in sheets can be considered as a reason of
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oxidation of sheets. In Fig. 2b, which relates to the graphene oxide nanosheet after
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magnetization, TEM image represents completely spherical magnetic nanoparticles on the GO
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nanosheets and the dispersion of iron nanoparticles is relatively uniform on the surface of GO.
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The average dimensions of these nanoparticles were estimated to be less than 20 nm.
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[Figure 2 to be inserted here]
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The FTIR analysis was applied to characterize the samples synthesized in each step. The FTIR spectra of GO, GOMNP and GOMNP/PEGA are shown in Fig. 3. The oxygen containing groups
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in graphene oxide structure showed different peaks in FTIR including a large peak at 3400 cm-1 for the O-H stretching of the hydroxyl group; the C=O bonds of the carboxylic group (O=C-OH)
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in 1734 cm -1; the C=C bond of non-oxidized graphite grains with sp2 hybrid in 1627 cm-1; and C-O-C bond of epoxy groups and C-O bond of carbonyl groups in the range of 1250 cm-1. The
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peak of the hydroxyl groups in 3400 cm-1 corresponds to 3 different O-H transplants including alcoholic and phenolic groups adhering to graphene oxide plates and water molecules surrounded between graphene oxide layers (Acik et al., 2011; Lerf et al., 1998). In the FTIR spectra of GOMNP, the peak of stretching vibration of Fe-O is found in 570 cm-1, which represents the
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functionalization of graphene oxide with iron nanoparticles (Yang et al., 2009). After functionalization of GOMNP with PEGA, the bond corresponds to polyethylene glycol bis amine functionalized GOMNP is detected by an amide bond formed with CH2 in 2900 cm-1 and a
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stretching vibration of NH-CO in 1640 cm-1 (Fig. 3) (Zhang et al., 2011). [Figure 3 to be inserted here]
Fig. 4 shows the SEM images of GO and GOMNP. In Fig. 4a the GO plates have large
dimensions as a result opening of interlayer spacing due to intense oxidation. As can be seen
from Fig. 4b, iron nanoparticles with an average size of less than 20 nm are well grafted to GO
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layers with a narrow size distribution. The SEM results are in agreement with the TEM results
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and also confirm the synthesis of nanocomposite.
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[Figure 4 to be inserted here]
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Fig. S1 shows the magnetization curves of GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX that were measured at room temperature. The amount of saturation magnetization (MS) values of
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GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX were 57.6, 51.8 and 45.2 emu/g,
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respectively. The gradual decline in magnetic properties of both GOMNP/PEGA and
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GOMNP/PEGA/MTX, compared to the GOMNP nanocarrier, is a reason for their magnetic instability. Also, the coating of PEGA has a negligible effect on magnetite properties. In
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addition, the lower saturation magnetism of nanocarrier containing the drug confirms the success of the drug loading process. In addition, the magnetic separability of GOMNP/PEGA/MTX was
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tested by placing an external magnet close to the glass bottle. The particles could be easily collected by an external magnetic field (Fig. S2). X-ray diffraction patterns of GO and GOMNP samples are shown in Fig. S3. As shown in this figure, the graphene oxide synthesized by modified Hummer’s method, has a peak around 2θ =
13
12, and also another peak around 2θ = 43, which can be related to the unmodified graphite (Han et al., 2012). In Fig. S3, the XRD pattern of GOMNP matches both the magnetite Fe3O4 and the maghemite c-Fe2O3 structures, according to the joint committee on powder diffraction standards
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(JCPDS) file No. 19-0629 and 39-1346, respectively (Daou et al., 2006). In order to further confirm the bonding of PEGA to GOMNP and loading of drug, the TGA analysis was used. The thermal behaviors of GOMNP, GOMNP/PEGA and
GOMNP/PEGA/MTX are investigated by thermogravimetric analysis at a temperature range of 20-650 ˚C. The temperature range of 0 to 200 ˚C and 200-650 ˚C were related to the removal of
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water and organic moieties on the surface of nanocarriers, respectively. As shown in Fig. S4, the
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weight loss difference between GOMNP/PEGA and GOMNP/PEGA/MTX reflected the amount
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of PEGA and MTX loaded on the GOMNP and GOMNP/PEGA surfaces, respectively.
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3.2. In vitro experiments results 3.2.1. MTT Assay
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MTT assay is commonly used to measure the cellular cytotoxicity affected by anticancer drugs.
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MTT reagent is reduced by succinate dehydrogenase, one of the mitochondrial reductase
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enzymes, and it is then converted to an insoluble purple formazan by cleavage of the tetrazolium ring. The purple colored formazan product is directly correlated to the viable cells with active
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metabolism. The viability of cells in two cell lines MCF-7 and HeLa was investigated after 24h and 48h incubation with various concentrations of 1 to 50 μg/ml of GOMNP/PEGA,
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GOMNP/PEGA/MTX and free MTX. The results are presented in Fig. 5. As shown in this figure, in the MCF-7 cell line, GOMNP/PEGA/MTX killed about 35% of the cells in the highest concentration after 24 hours. The effect of the drug sample on this cell line was about 45% in the first 24 hours. After 48 hours nanoparticles containing the drug caused cell death from 40 to 47
14
percent. The effect of GOMNP/PEGA/MTX has increased over a period of 48 hours, indicating a longer release of the drug over time. In the HeLa cell line, with increasing concentrations of GOMNP/PEGA/MTX, 15-40% of the cells were killed. The effect of free drug is similar in
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various concentrations and about it was about 40 to 50% of cell death. After 48 hours, the cell growth was significantly reduced. The percentage of cell viability was about 70 to 53 percent.
The effect of the drug was increased after 48 hours and reached about 50-60% of the cell death. In total, the increasing of GOMNP/PEGA/MTX concentration enhanced the cytotoxic activity. [Figure 5 to be inserted here]
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3.2.2. Hemolysis of RBCs
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Hemolytic analysis of red blood cells was performed to evaluate blood compatibility of
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synthesized nanoparticles and nanocomposites/blood components interactions. In this method,
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nanoparticles at different concentrations were incubated with blood. Accordingly, erythrocytes were isolated as the dominant cells in the blood and combined with nanoparticles with different
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concentrations ranging from 5 to 200 μg/ml. Positive and negative controls were prepared in the
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same way by addition of triton X-100 and PBS, respectively, instead of nanoparticles. Both
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quantitative and qualitative tests were performed to evaluate the hemolytic behavior of nanostructures on erythrocytes. The qualitative results showed that there was no hemolytic
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activity for synthesized nanostructures (Fig. S5). Table 1, demonstrated the quantitative hemolysis results for all samples. The hemolytic activity of the nanoparticles containing the drug
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(GOMNP/PEGA/MTX) illustrated reduction or inactivation of hemolytic activity after sample incubation with RBCs. Also, it can be observed that GOMNP/PEGA/MTX did not induce any undesirable responses and its non-hemolytic property (<2% hemolysis) was confirmed at all concentrations. However, GO and GOMNP nanocarriers exhibited hemolytic activity even at
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low concentrations. GO demonstrate a dose-dependent hemolytic activity which is attributed to the electrostatic interactions between the positively charged of phosphatidylcholine lipids present on the outer surface of the RBCs and the negatively charged oxygen present on GO. (Liao et al.,
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2011). The important elements that determine graphene blood compatibility are surface charge, oxygen content and particle size. Nano-sized GO (350 nm) was found to induce a severe
hemolysis effect (70% hemolysis at 25 μg/mL) compared to micro-sized (3 μm) graphene sheets (less than 10% hemolysis at 100 μg/mL). The aggregated graphene particles had a greater
cytotoxicity than the reversibly aggregated GO, due to forming aggregates which reduces the cell
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contact area. Therefore, when the concentration ranges from 75 μg/ml increase to 125 μg/ml, the
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percentage of hemolysis decreases due to the aggregation of nanocarriers. However, at 200
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μg/ml, the concentration of nanoparticles is so much increased that can enhance collisions of
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aggregates with red blood cells and lead to membrane degradation and subsequently increase hemolytic activity. The presence of carboxylic groups also can lead to direct interactions of GO
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with cell membranes and cause cell destruction or death (Cheng et al., 2012).
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Table 1. Hemolysis percentages of GO, GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX at different concentrations of 5-200 μg/ml. Controls were performed with 0% and 100% hemolysis with PBS (negative control) and Triton X-100 (positive control), respectively. Sample
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Concentration
GO
GOMNP
GOMNP/PEGA
GOMNP/PEGA/MTX
1.17±0.09
0.87±0.02
2.50±0.11
2.67±0.13
10 μg/ml
0.08±0.01
0.75±0.04
2.84±0.07
1.88±0.09
20 μg/ml
1.00±0.05
1.67±0.05
2.50±0.09
2.34±0.18
40 μg/ml
2.34±0.10
1.75±0.04
2.00±0.06
0.96±0.03
75 μg/ml
3.59±0.14
2.21±0.11
2.96±0.08
1.19±0.08
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5 μg/ml
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125 μg/ml
1.75±0.02
2.42±0.09
2.04±0.07
1.33±0.11
200 μg/ml
5.26±0.17
4.22±0.13
1.09±0.08
0.37±0.01
GOMNP showed less hemolytic effect at lower concentrations, however, undecorated iron
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nanoparticles caused toxic effects on the cell and cell destruction. The PEGA, a highly biocompatible molecule, was used to cover GOMNPs but the amine group in this polymer could increase the tendency of the system to connect to cell due to its positive charge and subsequently increase hemolysis at lower concentrations. 3.2.3. Complement activation assay
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The complement system consists of a set of plasma proteins and cell surface proteins that interact
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with the external factors to eliminate them. Activation of complement protein components such
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as C3 and C4 and their interaction with nanoparticles is an important parameter for the
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evaluation of biocompatibility of nanoparticles. Fig. 6 shows the complement activity assay in the absence (0 g/mL, as a negative control) and presence of different concentrations (10-200
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g/mL) of nanocarriers. According to this figure, GO, GOMNP, GO/MNP/PEGA and GOMNP/PEGA/MTX samples have normal range of C3 and C4 as compared to the control
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sample. The results demonstrated that the nanocarriers and the nanocarriers containing drug do not activate the complement systems and therefore, the synthesized nanocarriers have biological
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compatibility associated with complement systems. [Figure 6 to be inserted here]
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3.2.4. Agglutination assay The method of hemagglutination of red blood cells was used for the evaluation of agglutination, due to interaction of RBCs with various concentrations of nanoparticles. Using this experiment, the effects of drug-binding nanoparticles on red blood cells and deformation of red blood cells
17
were examined. As shown in Fig. 7, GOMNP/PEGA/MTX samples do not demonstrate any evidence of the hemagglutination of RBCs even at high concentrations. The GOMNP/PEGA samples show less evidence of agglutination than GO and GOMNP samples, while GO and
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GOMNP samples caused RBC agglutination at high concentrations. [Figure 7 to be inserted here] 3.2.5. Coagulation time assay
When the vessel is damaged, platelets accumulate in that area and activate blood factors and
therefore, activate the internal and external pathway of blood coagulation. Prothrombin time (PT)
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and activated partial thromboplastin time (APTT) are medical tests that characterize blood
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coagulation. A normal PT and APTT are approximately 9-15 seconds and 25-35 seconds,
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respectively (Selbi et al., 2013). Coagulation test was performed on the plasma mixed with
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GOMNP, GOMNP/PEGA and GOMNP/PEGA/MTX with various concentrations of 10-200
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μg/ml. Samples displayed a normal PT and APTT values of the control experiment. As shown in
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Fig. S6, PT and APTT are within the permissible range for all concentrations of three samples. These results indicated that the internal and external coagulation pathways of the blood are not
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affected by these nanocarrier. Therefore, the synthesized nanocarriers have good
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biocompatibility associated with coagulation assay. 3.2.6. Drug release results
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The drug release experiment was carried out in PBS buffer pH 5.5 at 37 °C through the dialysis setup. The amount of the drug released was obtained using a dialysis bag, by measuring the absorption of 1 ml of the external solution of the dialysis bag at various times. The amount of the drug released at pH values of 5.5 (cancer cells endosomal pH) and 7.4 (physiological pH) was measured over a period of 60 hours. Fig. S7 shows the release amounts of MTX as a function of 18
time under different pH conditions. The results indicated different behavior in the release time profiles of two pHs and the MTX has more efficient release at pH 5.5 compared to pH 7.4. The observed difference of drug release between two pHs can be due to changes in the physical
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interactions between nanocarrier and MTX. In acidic pHs, -NH- group starts to convert into – NH2+. This change can lead to dissociation of hydrogen bonds. So, at lower pHs, the possibility of drug release from the nanocarrier surface is more than that at neutral pHs. The drug release
rate was reached to about 100% drug until 60 hours, indicating complete release of the drug in acidic conditions. According to the acidic microenvironment of cancer cells, the drug delivery
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efficiency was considerably improved by loading MTX in the GOMNP/PEGA nanocarrier.
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3.2.7. The kinetics of drug release
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The data obtained from drug release experiments were investigated using 5 kinetic models including zero order, first order, Korsmeyer-Peppas, Hixson-Crowel and Higuchi model (Fig.
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S8). To obtain a kinetic model related to drug release, the curves of each model were plotted and
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their R2 values were compared (Dash et al., 2010; Gouda et al., 2017; Singhvi and Singh, 2011). By comparing the R2 values of these models and considering that, the R2 values of the first order
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model is higher than other models (Table S1), it can be concluded that the release model of the
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MTX drug may follow the first-order kinetic model. In this model, the graph is based on the log of the concentration ratio of the drug in time t to the initial concentration of drug in the time t0.
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According to this model, the reaction speed is directly related to the concentration of the drug released during the reaction. The higher the initial concentration of the drug, the greater the release rate and therefore, the model follows a linear kinetics. Since the binding of MTX to the nanocarrier is through both covalent and ionic bonds, first the weaker bonds break down and consequently more drugs are released. Hence, in the initial times of drug release assay, an 19
explosive release occurs. Over time, the connections are tightened and the lesser amounts of the drug are released with a very gentle gradient. 4. Conclusions
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In this study the nanoparticles of graphene oxide, decorated with iron nanoparticles and
functionalized with polyethylene glycol bis amine, were used as a nanocarrier for the delivery of methotrexate. Using MTT assay, the cytotoxicity of synthesized nanocomposites on MCF-7 and HeLa cell lines was investigated. The activity of the nanocomposite (GOMNP/PEGA/MTX)
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after incubation with red blood cells reduced hemolytic activity. While the blood is well
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tolerated, the compatibility of the drug containing nanocomposites at all different concentrations
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was confirmed. It has been demonstrated that the synthesized nanocarriers have biological
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compatibility associated with complement systems, agglutination, and coagulation time. The release of the drug from the nanocarrier in acidic conditions was completed after 60 hours.
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Overall, the results of this study indicated high drug loading, high toxicity for tumor cells, proper
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drug release and good biocompatibility of the synthesized nanocarrier, which can increase the therapeutic efficacy. Due to wide range usage of methotrexate in the treatment of different
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diseases, various nano-carriers have been synthesized for the delivery of this drug.
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Ashwanikumar et al. (Ashwanikumar et al., 2014) utilized a random copolymer of poly-lacticco-glycolic acid (PLGA) grafted branched polyethyleneimine for methotrexate delivery. Chen et
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al. (Chen et al., 2013a) synthesized a magnetic targeted drug delivery nanocarrier based on Fe3O4/PLA–PEG. Rahimi et al. (Rahimi et al., 2017) Constructed dendritic chitosan grafted mPEG coated magnetic nanoparticles (DPC@MNPs) as a magnetic nanocarrier for multi drug delivery of doxorubicin (DOX) and methotrexate (MTX). Khodadadei et al. (Khodadadei et al., 2017) designed a nanocarrier with blue fluorescent nitrogen-doped graphene quantum dots an 20
efficient drug delivery system. The synthesized nanocarrier in this study with advantages, including more drug release in acidic conditions, higher toxicity to tumor cells, and blood biocompatibility, has a better performance than other carriers in the delivery of methotrexate.
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Regarding these properties, the GOMNP/PEGA nanocomposite can be considered as a novel candidate for drug delivery due to the features such as easy synthesis and biocompatibility, with controlled target and release of the drug.
Declaration of interests
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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgements
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The authors are grateful to the Center of Excellence of Research Council of the University of
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Isfahan for financial supports of this work.
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Figure legends
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Figure 1. Synthesis of PEGA functionalized GOMNP nanocomposite and MTX loading.
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Figure 2. TEM images of (a) GO and (b) GOMNP.
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Figure 4. SEM images of (a) GO and (b) GOMNP.
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Figure 5. The effect of different concentrations of free MTX, GOMNP/PEGA and GOMNP/PEGA/MTX on viability of (a, c) HeLa and (b, d) MCF-7cell lines after 24 (a, b) and 48
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GOMNP,
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GOMNP/PEGA/MTX samples on the complement system; (a) C3 and (b) C4 assays.
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Figure 7. Agglutination of red blood cells in the presence of various concentrations of different
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nanocarriers.
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