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graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent
Accepted Manuscript Title: Carboxymethyl cellulose/graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent Authors: Monireh Rasoulzadeh, Hassan Namazi PII: DOI: Reference:
S0144-8617(17)30261-8 http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.014 CARP 12103
To appear in: Received date: Revised date: Accepted date:
14-1-2017 4-3-2017 7-3-2017
Please cite this article as: Rasoulzadeh, Monireh., & Namazi, Hassan., Carboxymethyl cellulose/graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.014 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.
Carboxymethyl cellulose/graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent Monireh Rasoulzadeh, Hassan Namazi*1, 2 1
Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran; Tel.: +98 4133933121, Fax: +98 4133340191, [email protected] 2 Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Science, Tabriz, Iran
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GO nano sheets were successfully embedded on the CMC hydrogel beads. Incorporation of GO nano sheets greatly improved swelling capacity of hydrogels. GO-CMC has high loading and prolonged release for Doxorubicin compare to pure CMC. GO-CMC has no significant toxicity against colon cancer cells (SW480).
Abstract Biodegradable carboxymethyl cellulose/graphene oxide (CMC/GO) nanocomposite hydrogel beads as a drug delivery system were prepared via physically crosslinking with FeCl3.6H2O for controlled release of anticancer drug doxorubicin (DOX). The π-π stacking interaction between DOX and GO resulted in higher loading capacity and controlled release of the DOX loaded from CMC/GO nanocomposites hydrogel. The release profile of DOX from hydrogel beads at pH 6.8 and 7.4 indicated it’s strongly pH dependence. Interaction between GO and DOX with H-bonding could be unstable under acidic conditions which resulted in faster drug release rate in pH 6.8. The formation of GO nanoparticles in the hydrogels was confirmed using X-ray diffraction (XRD), and the chemical structure and morphology of the prepared CMC/GO nanocomposite hydrogel beads were characterized using Fourier transform infrared spectroscopy (FT-IR), SEM and Transmission electron microscopy (TEM). In addition, swelling behavior of nanocomposite hydrogels was investigated in PBS solution. Keywords: Drug delivery; Graphene oxide; Carboxymethyl cellulose; Nanocomposite; Hydrogel. 1. Introduction Super-absorbent polymers have been known as an active compounds in biomedical and engineering fields including in biosensors, tissue implants, drug delivery systems (DDS) (Hoare & Kohane, 2008; Hoffman, 2012). Hydrogel is a macromolecular polymeric gel synthesized by crosslinking of polymer
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chains-through physical, ionic or covalent interactions and are well known for their ability to absorb large amount of water via H-bonding (Hamidi, Azadi & Rafiei, 2008). Hydrogels could be drug delivery agents that can be engineered to have preferred properties. The ideal hydrogels can be designed to release drugs or other agents in response to physical stimuli like temperature and pH (Barkhordari, Yadollahi & Namazi, 2014). The sensitivity of hydrogels is a result of their molecular structure and their polymeric networks (Chang, Duan, Cai & Zhang, 2010).
Nanocomposite hydrogels that described as enormously three-dimensional network polymers which crosslinked chemically or physically with each other or with nanoparticles and nano structures, could imitate tissues inherent properties, structure and microenvironment owing to their hydrated and organized porous structure (Alvarez-Lorenzo, Blanco-Fernandez, Puga & Concheiro, 2013; Haraguchi, 2007; Hoare & Kohane, 2008). However, the requirement of chemical cross-linker as an additional and in some cases toxic agent could adversely affect the loaded drugs and proteins. Therefore, the physically cross-linked hydrogels are more preferred (Abdulkhani, Daliri Sousefi, Ashori & Ebrahimi, 2016; Yadollahi, Farhoudian & Namazi, 2015; Yadollahi, Gholamali, Namazi & Aghazadeh, 2015). Compared to pure polymers, nanocomposites with improved properties can be obtained via nanoparticles containing carbon-based, polymeric, ceramic, and metallic in the polymer structure. Nanocomposites hydrogels have been widely used because of they own higher physical, chemical and biological properties. (Yadollahi & Namazi, 2013; Zhou & Wu, 2011).The amphiphilic characteristics of graphene oxide resulted from its functional groups such as hydroxyl, epoxide and carboxyl groups presents on GO surface (Wang, Li, Wang, Li & Lin, 2011; Zhang, Zhai & He, 2014; Zhu et al., 2010). The unique structure of graphene oxide leads to its wide application in anticancer drugs delivery (Song, Xu, Yang, Cui, Zhang & Liu, 2015; Sun & Wu, 2011; Sun et al., 2008). GO shows good biocompatibility and low toxicity which is obligatory for biomedical applications. Biocompatibility and the anionic-exchange properties of GO make them great candidates to support matrices of cationic drugs in DDS systems (Liu, Cui & Losic, 2013; Liu, Robinson, Tabakman, Yang & Dai, 2011; Rui-Hong, Peng-Gang, Jian, Fang,
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Lian-Zhen & Zhen-Feng, 2016). So far, many types of CMC based hydrogels have been developed due to their low toxicity, biocompatibility and cost-effective advantages. The mechanical properties of a material could greatly enhance by addition of the graphene-family nanomaterials along with even distribution of the filler material (Justin & Chen, 2014). Liu et al. indicated that, with addition of GO the porous structure of the GO/CMC monoliths changed and their compressive strength significantly increased. It is supposed that GO/CMC monoliths not only has a high ability for adsorption of metal ions, but also the biodegradable and non-toxic CMC in the porous GO/CMC monoliths make its potential environmental adsorbents. CMC is a polymer with multiple carboxyl groups, shows outstanding coordination abilities with metal ions (Barkhordari & Yadollahi, 2016; Chang & Zhang, 2011; Hashem, Sharaf, Abd El-Hady & Hebeish, 2013). It can form hydrogels in the presence of metal ions (such as Fe3+) via the coordination between metal ions and the carboxyl groups in the polymer side chains (Abdulkhani, Daliri Sousefi, Ashori & Ebrahimi, 2016; Namazi, Rakhshaei, Hamishehkar & Kafil, 2016). Since the good coordination ability and biodegradability of CMC, it is a good option to prepare double network hydrogels with CMC as the second network which could be cross-linked by simple coordination with metal ions (Ye et al., 2016; Zhou & Wu, 2011). In this regard, CMC/GO hydrogel nanocomposite beads physically crosslinked with FeCl3.6H2O were prepared which are very sensitive to acidic media, leading to complete release of drugs in the acidic media (pH 6.8). Thus, the preparation of GO–drug hybrids coated with a protective polymeric matrix has been recently proposed to sustain the release profile through the digestive tract. In this study, to control the release properties of the prepared GO-Drug nano hybrid, an improved approach was proposed using carboxymethyl cellulose as a pH sensitive polymer. Physically crosslinked CMC-GO composite hydrogel beads were developed for selective drug release at physiological pHs. Doxorubicin, as an anticancer and as a model drug, was loaded on the GO surface through 𝜋 − 𝜋 stacking interaction. In this regard a novel pH sensitive drug delivery system has been synthesized for anti-cancer drug (DOX) based on GO-CMC hydrogel beads. The load and release of
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cytotoxicity profile at pH 6.8 and 7.4 has been studied. An overview of the picture describing the chemical structure of the created dosage is shown in Scheme 1. 2. Materials and Methods 2.1 Materials Sodium carboxymethyl cellulose (CMC, CP, 300–800 mPas (25 g/L, 25 ◦C)) was purchased from Sinopharm Chemical Reagent Co, Ltd, China. FeCl3.6H2O and NaOH were purchased from Merck. DOX was purchased from Danna Pharma Co. (Tabriz, Iran). All the chemicals used as received without further purification. The following chemicals were purchased from sigma Aldrich: sulphuric acid, hydrogen peroxide, potassium permanganate, sodium nitrate, phosphate buffered saline (PBS), and graphite powder (≤20µm). Deionized water used throughout this work 3–(4, 5-dimethylthiazole-2-yl) 2, 2, 5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich. 2.2 Measurement and characterization FT-IR spectra were recorded on a Brucker vector FT-IR spectrometer in 4000-400 cm-1 region using KBr tablets. The X-ray diffraction pattern of the samples was verified with a Siemens-D500 diffractometer using Cu-Kα radiation at 35 kV in the scan range of 2θ from 2 to 70ᵒ (λ = 1.5048 Aᵒ). The morphology of the dried neat hydrogel and nanocomposite hydrogels was examined by Scanning electron microscopy (SEM) (LEO 1430VP) after coating the dried hydrogels with gold. Transmission electron microscopy (TEM) (LEO906E) was used in 100KV to determine the size of GO nanoparticles inside the hydrogel beads. 2.3 Preparation of graphene oxide GO was prepared through oxidation of natural graphite powder according to the modified Hummers’ method (Hummers & Offeman, 1958). Briefly, 1 g of Graphite with 50 g NaCl was finely grounded for 35 minute and rinsed with enough distilled water to remove the excess salt, after filtration the black powder was collected and transferred to a 250 ml balloon, and 50 ml of concentrated sulfuric acid with 0.5 g NaNO3 were added to the reaction mixture in the presence of ice bath to stirred for 24 h then 6 g KMnO4 was slowly added to the solution to prevent the temperature of the suspension
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exceeding from 10 ◦C. The ice-bath was then removed and the suspension was stirred for 48 h at 30 ◦C. Subsequently, 100 ml of deionized water and 30 ml (30%) H2O2 were added separately to obtain yellowish-brown mixture. Finally, the mixture was centrifuged and washed with aqueous HCl solution (10:1, v/v) along with deionized water until the pH of the upper layer of suspension become 6.8. The obtained solution was sonicated for 45 minute. Dried Graphene oxide powder was obtained by drying at 50 ◦C for 24 h under vacuum. 2.4 Preparation of CMC-GO nanocomposite hydrogel beads Firstly, (5, 10, and 15 wt. %) GO was exfoliated and dispersed in 20 ml of water by ultra-sonication for 20 min. afterwards, 1 g CMC was dissolved in the as prepared GO aqueous dispersion. The solutions were transferred in to a syringe to assist the droplet addition of the above mixture into 0.2 M FeCl3 solution. The beads were allowed to crosslink with Fe3+ in solution for 20 min. After that, the beads were filtered and washed with bi-distilled water to remove unreacted FeCl3 on the surface of the beads and dried in 50 ◦C under vacuum for 24 h. 2.5 Swelling measurement The equilibrium swelling (ES) of the CMC/GO nanocomposite hydrogels was determined in distilled water and buffer solutions. Typically, 0.2 g of CMC/GO nanocomposite hydrogel beads was immersed in 50 ml of the prepared buffer solutions with a desired pH at room temperature for 48 h to reach the maximum swelling capacity. The equilibrium swelling of nanocomposites was determined according to Eq. 1. Equilibrium swelling % = (W2-W1)/W1 ×100
Eq. 1
Where, W1 is weight of initial dried samples, and W2 is the weight of samples after swelling for 48 h. The swelling capacity of hydrogels was also investigated in the buffers (pH 6.8 and 7.4) (Yadollahi, Gholamali, Namazi & Aghazadeh, 2015). 2.6 Load and release profile of DOX on to GO-CMC
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Loading of CMC/GO nanocomposite hydrogels with doxorubicin was carried out as follows: 0.2 g of prepared dry bead hydrogels were added to 20 ml of Doxorubicin solution (25 ppm in distilled water) under stirring at room temperature for 72 h in dark conditions. Excess of doxorubicin was washed using distilled water. The quantity of loaded drug in the hydrogels was calculated using UV-VIS spectroscopy at 448 nm by using the Eq. 2. Drug loading (g/g) = (amount of drug in hydrogel)/ (amount of dry hydrogel)
Eq. 2
The drug release properties was studied by transferring 0.2 g of drug-nanocomposite into 20 ml of phosphate buffer (pH 7.4 and 6.8) at 37 ◦C for 72 h under continuous string. In order to measure the amount of released drug at a certain time, adequate amount of sample solution was picked up and its absorption recorded using UV spectroscopy. The same volume of fresh buffer was replaced instead of removed volume in order to maintain the volume of buffer constant. The amount of released doxorubicin from CMC/GO nanocomposite was quantified using UV spectroscopy. 2.7 Cytotoxicity measurement The in vitro cytotoxicity of CMC-GO and CMC-GO/DOX against Human colon cancer cells (SW480) was carried out using MTT assay. Briefly 8000 cell/well were cultured in to 96 well culture plates and incubated in 37 ◦C for 24 h. The obtained cells were incubated with GO-CMC and CMC-GO/DOX at concentration range from 2-32 µM for 72 h. After incubation, the cells were rinsed and treated with 20µL MTT reagent. The media was replaced by 200 µL DMSO in each well and absorbance of the solution at a wavelength of 570 nm can be achieved, and with the help of the standard curve to calculate the number of cells. 3. Results and discussion 3.1 Characterization of GO The successful synthesis of GO, was approved by FT-IR, XRD, SEM and TEM spectrum. To confirm of new chemical bonds presence, morphology and thickness of GO, respectively. Main groups can be noted on FT-IR spectra as shown in Fig. 1(a): The peaks at 1067 cm-1 for C-O bonds; at 1387 cm-1 for
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C=C bonds; at 1667cm-1 for C=O bonds and at 3131 cm-1 for O-H bonds confirm the successful oxidation. Fig. 2 shows the XRD curves for GO, the (002) peak of graphene oxide is at 2θ =11.64 ◦ corresponding to an interlayer spacing of 0.43 nm. 3.2 Characterization of GO/CMC nanocomposite hydrogel beads The IR spectrum of the GO/CMC nanocomposite Fig. 1 (b) demonstrates the characteristic peaks of both CMC and GO. The stretching vibration of the hydroxyl group (from both CMC and GO) identified around at 3375 cm−1 and the peak at 2930 cm−1 is the characteristic C - H stretching of CH2 (from CMC that confirm the formation of the GO and CMC blend through combination. As shown in the Fig. 1(c) the characteristic peaks of CMC at around 3420 and 1608 cm−1 are attributed to the hydroxyl stretching and carboxylate bending modes, respectively. Furthermore, the adsorption bands at 2921 and 1134 cm−1 are related to the C-H stretching and bending modes respectively. To obtain information about the crystalline structure of the GO/CMC hydrogel beads, the XRD patterns of the CMC, GO, and GO/CMC with 5 wt. % GO were measured and are shown in Fig. 2 GO has a characteristic diffraction peak at 11.64◦ with the interlayer spacing of 0.43 nm. The CMC has a broad diffraction peak at 19◦, indicating that CMC is partly crystalline. The CMC/GO has a very small peak in 11.64◦ and an obvious diffraction peak at about 19◦ (Song, Xu, Yang, Cui, Zhang & Liu, 2015). 3.3 Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) One of the most important factors governing drug release behavior is the surface morphology of a carrier. Scanning electron microscopy (SEM) was used to investigate the surface morphology of the samples. GO nano sheets are clearly visible in Fig. 3(a) and (b). SEM images of hydrogel composite beads with different GO contents (0%, 5%, 10%, and 15%) have been shown in Fig. 3(c-f). As shown in SEM image of CMC hydrogel (Fig. 3(c)), the surface morphology showed a rough structure and severe wrinkles. As can be seen from Fig. 3(d-f), with increasing GO content, the surface roughness of CMC/GO hydrogel nanocomposite samples decreases. This can be attributed to the crosslinking effect
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of intercalated GO sheets, which makes strong H-bonding interactions with functional groups of CMC, and thus induces a smooth surface (Ito, 2007). Fig. 4 shows TEM image of CMC/GO hydrogel nanocomposite sample. According to Fig. 4, thin wrinkled sheets of GO are visible, which confirm the well exfoliated structure of GO sheets within hydrogel nanocomposite network without agglomeration. 3.4 Swelling properties of composite hydrogel beads In order to evaluate the sensitivity of the hydrogels to the pH of environment the swelling behavior of the hydrogels was studied in pH 1.2, 6.8 and 7.4 buffer solutions. Fig. 5 shows the effect of buffer on swelling capacity of cross linked CMC. This Fig. shows the significant difference between the swelling ratios of the hydrogel in different pHs , it means that the hydrogel was highly sensitive to pH. As described by Lee and co-workers (Lee, Choi, Paik & Park, 2006), the optimum pH to achieve a maximum swelling ratio of CMC hydrogel is about pH 6–8. Hence, little change in pH in this range leads to significant change in swelling. This is due to the dissociation of CMC and its counter ion concentration as a function of pH. Nevertheless, polymer conformation is greatly affected by ionization(Lee, Lee, Paik & Choi, 2005), under acidic conditions, CMC chains form compact coils. On the other hand, the basic conditions lead to compression of the CMC structure and as the result, the swelling is inhibited. This is because the dissociated sodium ions in basic solutions are abundant enough to compress the structure of the CMC (Lee, Choi, Paik & Park, 2006; Lee, Lee, Paik & Choi, 2005). Therefore, with increasing pH from 1.2 to 7.4, the carboxyl groups on the CMC/GO chains converted to negatively charged carboxylate ions, resulting in higher electrostatic repulsion and water would be taken up and the swelling rate tend to increase with the GO content. The effect of the GO quantity on water absorption of the CMC-hydrogel nanocomposites was investigated by varying the amount of GO from (5%, 10% and 15%). As it is shown in Fig. 5 with increasing the GO amount up to 15%, the swelling capacity was increased. The possible reason for this phenomenon might be correlated with the complex interaction between GO, water molecules and CMC. Theoretically, the hydrophilic groups of GO sheets can simultaneously form various H-bonding with water molecules,
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acid groups in CMC and hydrophilic groups of other GO sheets. At low loading, GO sheets largely interact with water molecules through H-bonding. But as the loading of GO increased, more water molecules were kept in the composite hydrogels, leading to an increase in the swelling ratio. 3.5 Drug loading and release behaviors of nanocomposite With increasing the percentage of graphene oxide nanoparticles, drug loading efficiently increased. (Fig. 6) This could be a result of possible H- bonding between carboxylic groups of the GO and –NH2 groups of the DOX. The drug release behavior was investigated to show the potential of CMC/GO nanocomposites as a drug delivery system. The relation between encapsulation and release of Doxorubicin in nanocomposite hydrogels was examined by altering the amount of GO nanoparticles. The release degree of doxorubicin as a function of time is shown in Fig. 7. The study of drug release from nanocomposite hydrogels and pure hydrogel in pH 6.8 and pH 7.4 for 24 h revealed that, the amount of released drug from pure hydrogel was more than nanocomposite hydrogels. The release amount of DOX from nanocomposite hydrogel beads in acidic conditions pH 6.8 is much higher than pH 7.4 due to the stronger H-bonding interaction under basic conditions than that under acid conditions. It was also observed that as the amount of graphene oxide nanoparticles increased, the drug release reduced. Because the presence of nanoparticles has a significant effect for slow releasing of the drug from nanocomposite hydrogels. Since the swelling of the carrier in pH 1.2 was very low, we avoided release studies in this pH. 3.6 Therapeutic efficacy of GO-CMC/DOX complexes The cytotoxicity of DOX loaded on the GO-CMC complex to SW480 cells was investigated by the MTT assay. SW480 cells were employed and DOX, GO-CMC/DOX were incubated with SW480 for 24 h followed by performing the MTT assay. As shown in Fig. 8(a) no obvious toxicity was observed for the GO-CMC without DOX loading even in relatively high concentrations This confirms the safety of the prepared nanocomposite, as the Fe3+ is potential for cytotoxic. As it can be seen form Fig. 8(b) the GOCMC/DOX complex was able to cause an obvious loss of cell viability compared to the untreated
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control cells, indicating that the GO-CMC/DOX has the potential for selectively killing cancer cells in vitro. However, DOX alone shows the highest toxicity to SW480 cells under the same conditions due to the lower DOX content in GO-CMC/DOX complexes. Moreover, enhancement of GO content in GOCMC/DOX complexes turned out in a greater cell killing property. This arises from the fact that with increasing GO content, loading percent of DOX is also increases. 4. Conclusion In this paper, the synthesis of hydrogel beads from CMC polymer and GO as nanoparticle physically crosslinked by FeCl3.6H2O was investigated. The pH dependent release of DOX from CMC/GO nanocomposite was evaluated. Characteristics and structure of prepared hydrogels were identified using TEM,SEM,XRD and FT-IR. The swelling behaviour of prepared nanocomposite hydrogels was investigated in distilled water and buffers (pH 1.2, 6.8 and 7.4 ). The obtained results showed that the swelling ratio of nanocomposit hydrogels in comparison with neat hydrogel increased by incorporating of graghene oxide nanoparticles due to H-bonding interaction between GO sheets and water molecules and carboxyl groups of CMC hydrogels. Swelling in the buffers showed a significant difference. This means that the hydrogel was sensitive to pH. The loading and releasing behavior of doxorobicin to the nanocomposite hydrogels, showed that nanocomposite hydrogels had better drug loading in comparison to the pure hydrogel. Increasing the amount of nanoparticles increased drug loading. Drug release from pure hydrogel was more than nanocomposite hydrogels and with increasing the amount of GO nanoparticles, drug release reduced due to strong interactions among amine groups of DOX and graghene oxide carboxylic groups. Acknowledgments Authors gratefully acknowledge the financial supports from the University of Tabriz (Grant Number 938645702) and Research Center for Pharmaceutical Nanotechnology (RCPN) of Tabriz University of Medical Science. References
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Scheme 1. The schematic picture describing the chemical structure of the created dosage.
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Fig. 1. FT-IR spectra of CMC/GO nanocomposite hydrogel beads, GO, CMC hydrogel
Fig. 2. XRD pattern of CMC nanocomposite hydrogel beads, GO, CMC hydrogel
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Fig. 3. SEM images of GO (a -b). CMC hydrogel (c), nanocomposite hydrogel beads of CMC/GO (d-5%, e-10% and f-15%).
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Fig. 4. TEM image of CMC/GO nanocomposite hydrogels.
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Fig. 5. Effect of GO concentration on swelling ratio of hydrogel beads in pHs 1.2, 6.8 and 7.4
Fig. 6. Illustrates images of CMC and CMC/GO nanocomposite hydrogel beads with (5, 10 and 15 wt. %) DOX loading.
Fig. 7. Doxorubicin release from the loaded CMC/GO nanocomposite hydrogel beads in PBS.
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Fig. 8. MTT viability assay of SW480 cells treated with : free GO-CMC complexes at concentration of 064µM for 24 h (a) and free DOX and GO-CMC/DOX at corresponding concentrations of the complexes between 0-32 µM (b)
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S0144-8617(17)30261-8 http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.014 CARP 12103
To appear in: Received date: Revised date: Accepted date:
14-1-2017 4-3-2017 7-3-2017
Please cite this article as: Rasoulzadeh, Monireh., & Namazi, Hassan., Carboxymethyl cellulose/graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.014 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.
Carboxymethyl cellulose/graphene oxide bio-nanocomposite hydrogel beads as anticancer drug carrier agent Monireh Rasoulzadeh, Hassan Namazi*1, 2 1
Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran; Tel.: +98 4133933121, Fax: +98 4133340191, [email protected] 2 Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Science, Tabriz, Iran
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GO nano sheets were successfully embedded on the CMC hydrogel beads. Incorporation of GO nano sheets greatly improved swelling capacity of hydrogels. GO-CMC has high loading and prolonged release for Doxorubicin compare to pure CMC. GO-CMC has no significant toxicity against colon cancer cells (SW480).
Abstract Biodegradable carboxymethyl cellulose/graphene oxide (CMC/GO) nanocomposite hydrogel beads as a drug delivery system were prepared via physically crosslinking with FeCl3.6H2O for controlled release of anticancer drug doxorubicin (DOX). The π-π stacking interaction between DOX and GO resulted in higher loading capacity and controlled release of the DOX loaded from CMC/GO nanocomposites hydrogel. The release profile of DOX from hydrogel beads at pH 6.8 and 7.4 indicated it’s strongly pH dependence. Interaction between GO and DOX with H-bonding could be unstable under acidic conditions which resulted in faster drug release rate in pH 6.8. The formation of GO nanoparticles in the hydrogels was confirmed using X-ray diffraction (XRD), and the chemical structure and morphology of the prepared CMC/GO nanocomposite hydrogel beads were characterized using Fourier transform infrared spectroscopy (FT-IR), SEM and Transmission electron microscopy (TEM). In addition, swelling behavior of nanocomposite hydrogels was investigated in PBS solution. Keywords: Drug delivery; Graphene oxide; Carboxymethyl cellulose; Nanocomposite; Hydrogel. 1. Introduction Super-absorbent polymers have been known as an active compounds in biomedical and engineering fields including in biosensors, tissue implants, drug delivery systems (DDS) (Hoare & Kohane, 2008; Hoffman, 2012). Hydrogel is a macromolecular polymeric gel synthesized by crosslinking of polymer
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chains-through physical, ionic or covalent interactions and are well known for their ability to absorb large amount of water via H-bonding (Hamidi, Azadi & Rafiei, 2008). Hydrogels could be drug delivery agents that can be engineered to have preferred properties. The ideal hydrogels can be designed to release drugs or other agents in response to physical stimuli like temperature and pH (Barkhordari, Yadollahi & Namazi, 2014). The sensitivity of hydrogels is a result of their molecular structure and their polymeric networks (Chang, Duan, Cai & Zhang, 2010).
Nanocomposite hydrogels that described as enormously three-dimensional network polymers which crosslinked chemically or physically with each other or with nanoparticles and nano structures, could imitate tissues inherent properties, structure and microenvironment owing to their hydrated and organized porous structure (Alvarez-Lorenzo, Blanco-Fernandez, Puga & Concheiro, 2013; Haraguchi, 2007; Hoare & Kohane, 2008). However, the requirement of chemical cross-linker as an additional and in some cases toxic agent could adversely affect the loaded drugs and proteins. Therefore, the physically cross-linked hydrogels are more preferred (Abdulkhani, Daliri Sousefi, Ashori & Ebrahimi, 2016; Yadollahi, Farhoudian & Namazi, 2015; Yadollahi, Gholamali, Namazi & Aghazadeh, 2015). Compared to pure polymers, nanocomposites with improved properties can be obtained via nanoparticles containing carbon-based, polymeric, ceramic, and metallic in the polymer structure. Nanocomposites hydrogels have been widely used because of they own higher physical, chemical and biological properties. (Yadollahi & Namazi, 2013; Zhou & Wu, 2011).The amphiphilic characteristics of graphene oxide resulted from its functional groups such as hydroxyl, epoxide and carboxyl groups presents on GO surface (Wang, Li, Wang, Li & Lin, 2011; Zhang, Zhai & He, 2014; Zhu et al., 2010). The unique structure of graphene oxide leads to its wide application in anticancer drugs delivery (Song, Xu, Yang, Cui, Zhang & Liu, 2015; Sun & Wu, 2011; Sun et al., 2008). GO shows good biocompatibility and low toxicity which is obligatory for biomedical applications. Biocompatibility and the anionic-exchange properties of GO make them great candidates to support matrices of cationic drugs in DDS systems (Liu, Cui & Losic, 2013; Liu, Robinson, Tabakman, Yang & Dai, 2011; Rui-Hong, Peng-Gang, Jian, Fang,
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Lian-Zhen & Zhen-Feng, 2016). So far, many types of CMC based hydrogels have been developed due to their low toxicity, biocompatibility and cost-effective advantages. The mechanical properties of a material could greatly enhance by addition of the graphene-family nanomaterials along with even distribution of the filler material (Justin & Chen, 2014). Liu et al. indicated that, with addition of GO the porous structure of the GO/CMC monoliths changed and their compressive strength significantly increased. It is supposed that GO/CMC monoliths not only has a high ability for adsorption of metal ions, but also the biodegradable and non-toxic CMC in the porous GO/CMC monoliths make its potential environmental adsorbents. CMC is a polymer with multiple carboxyl groups, shows outstanding coordination abilities with metal ions (Barkhordari & Yadollahi, 2016; Chang & Zhang, 2011; Hashem, Sharaf, Abd El-Hady & Hebeish, 2013). It can form hydrogels in the presence of metal ions (such as Fe3+) via the coordination between metal ions and the carboxyl groups in the polymer side chains (Abdulkhani, Daliri Sousefi, Ashori & Ebrahimi, 2016; Namazi, Rakhshaei, Hamishehkar & Kafil, 2016). Since the good coordination ability and biodegradability of CMC, it is a good option to prepare double network hydrogels with CMC as the second network which could be cross-linked by simple coordination with metal ions (Ye et al., 2016; Zhou & Wu, 2011). In this regard, CMC/GO hydrogel nanocomposite beads physically crosslinked with FeCl3.6H2O were prepared which are very sensitive to acidic media, leading to complete release of drugs in the acidic media (pH 6.8). Thus, the preparation of GO–drug hybrids coated with a protective polymeric matrix has been recently proposed to sustain the release profile through the digestive tract. In this study, to control the release properties of the prepared GO-Drug nano hybrid, an improved approach was proposed using carboxymethyl cellulose as a pH sensitive polymer. Physically crosslinked CMC-GO composite hydrogel beads were developed for selective drug release at physiological pHs. Doxorubicin, as an anticancer and as a model drug, was loaded on the GO surface through 𝜋 − 𝜋 stacking interaction. In this regard a novel pH sensitive drug delivery system has been synthesized for anti-cancer drug (DOX) based on GO-CMC hydrogel beads. The load and release of
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cytotoxicity profile at pH 6.8 and 7.4 has been studied. An overview of the picture describing the chemical structure of the created dosage is shown in Scheme 1. 2. Materials and Methods 2.1 Materials Sodium carboxymethyl cellulose (CMC, CP, 300–800 mPas (25 g/L, 25 ◦C)) was purchased from Sinopharm Chemical Reagent Co, Ltd, China. FeCl3.6H2O and NaOH were purchased from Merck. DOX was purchased from Danna Pharma Co. (Tabriz, Iran). All the chemicals used as received without further purification. The following chemicals were purchased from sigma Aldrich: sulphuric acid, hydrogen peroxide, potassium permanganate, sodium nitrate, phosphate buffered saline (PBS), and graphite powder (≤20µm). Deionized water used throughout this work 3–(4, 5-dimethylthiazole-2-yl) 2, 2, 5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich. 2.2 Measurement and characterization FT-IR spectra were recorded on a Brucker vector FT-IR spectrometer in 4000-400 cm-1 region using KBr tablets. The X-ray diffraction pattern of the samples was verified with a Siemens-D500 diffractometer using Cu-Kα radiation at 35 kV in the scan range of 2θ from 2 to 70ᵒ (λ = 1.5048 Aᵒ). The morphology of the dried neat hydrogel and nanocomposite hydrogels was examined by Scanning electron microscopy (SEM) (LEO 1430VP) after coating the dried hydrogels with gold. Transmission electron microscopy (TEM) (LEO906E) was used in 100KV to determine the size of GO nanoparticles inside the hydrogel beads. 2.3 Preparation of graphene oxide GO was prepared through oxidation of natural graphite powder according to the modified Hummers’ method (Hummers & Offeman, 1958). Briefly, 1 g of Graphite with 50 g NaCl was finely grounded for 35 minute and rinsed with enough distilled water to remove the excess salt, after filtration the black powder was collected and transferred to a 250 ml balloon, and 50 ml of concentrated sulfuric acid with 0.5 g NaNO3 were added to the reaction mixture in the presence of ice bath to stirred for 24 h then 6 g KMnO4 was slowly added to the solution to prevent the temperature of the suspension
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exceeding from 10 ◦C. The ice-bath was then removed and the suspension was stirred for 48 h at 30 ◦C. Subsequently, 100 ml of deionized water and 30 ml (30%) H2O2 were added separately to obtain yellowish-brown mixture. Finally, the mixture was centrifuged and washed with aqueous HCl solution (10:1, v/v) along with deionized water until the pH of the upper layer of suspension become 6.8. The obtained solution was sonicated for 45 minute. Dried Graphene oxide powder was obtained by drying at 50 ◦C for 24 h under vacuum. 2.4 Preparation of CMC-GO nanocomposite hydrogel beads Firstly, (5, 10, and 15 wt. %) GO was exfoliated and dispersed in 20 ml of water by ultra-sonication for 20 min. afterwards, 1 g CMC was dissolved in the as prepared GO aqueous dispersion. The solutions were transferred in to a syringe to assist the droplet addition of the above mixture into 0.2 M FeCl3 solution. The beads were allowed to crosslink with Fe3+ in solution for 20 min. After that, the beads were filtered and washed with bi-distilled water to remove unreacted FeCl3 on the surface of the beads and dried in 50 ◦C under vacuum for 24 h. 2.5 Swelling measurement The equilibrium swelling (ES) of the CMC/GO nanocomposite hydrogels was determined in distilled water and buffer solutions. Typically, 0.2 g of CMC/GO nanocomposite hydrogel beads was immersed in 50 ml of the prepared buffer solutions with a desired pH at room temperature for 48 h to reach the maximum swelling capacity. The equilibrium swelling of nanocomposites was determined according to Eq. 1. Equilibrium swelling % = (W2-W1)/W1 ×100
Eq. 1
Where, W1 is weight of initial dried samples, and W2 is the weight of samples after swelling for 48 h. The swelling capacity of hydrogels was also investigated in the buffers (pH 6.8 and 7.4) (Yadollahi, Gholamali, Namazi & Aghazadeh, 2015). 2.6 Load and release profile of DOX on to GO-CMC
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Loading of CMC/GO nanocomposite hydrogels with doxorubicin was carried out as follows: 0.2 g of prepared dry bead hydrogels were added to 20 ml of Doxorubicin solution (25 ppm in distilled water) under stirring at room temperature for 72 h in dark conditions. Excess of doxorubicin was washed using distilled water. The quantity of loaded drug in the hydrogels was calculated using UV-VIS spectroscopy at 448 nm by using the Eq. 2. Drug loading (g/g) = (amount of drug in hydrogel)/ (amount of dry hydrogel)
Eq. 2
The drug release properties was studied by transferring 0.2 g of drug-nanocomposite into 20 ml of phosphate buffer (pH 7.4 and 6.8) at 37 ◦C for 72 h under continuous string. In order to measure the amount of released drug at a certain time, adequate amount of sample solution was picked up and its absorption recorded using UV spectroscopy. The same volume of fresh buffer was replaced instead of removed volume in order to maintain the volume of buffer constant. The amount of released doxorubicin from CMC/GO nanocomposite was quantified using UV spectroscopy. 2.7 Cytotoxicity measurement The in vitro cytotoxicity of CMC-GO and CMC-GO/DOX against Human colon cancer cells (SW480) was carried out using MTT assay. Briefly 8000 cell/well were cultured in to 96 well culture plates and incubated in 37 ◦C for 24 h. The obtained cells were incubated with GO-CMC and CMC-GO/DOX at concentration range from 2-32 µM for 72 h. After incubation, the cells were rinsed and treated with 20µL MTT reagent. The media was replaced by 200 µL DMSO in each well and absorbance of the solution at a wavelength of 570 nm can be achieved, and with the help of the standard curve to calculate the number of cells. 3. Results and discussion 3.1 Characterization of GO The successful synthesis of GO, was approved by FT-IR, XRD, SEM and TEM spectrum. To confirm of new chemical bonds presence, morphology and thickness of GO, respectively. Main groups can be noted on FT-IR spectra as shown in Fig. 1(a): The peaks at 1067 cm-1 for C-O bonds; at 1387 cm-1 for
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C=C bonds; at 1667cm-1 for C=O bonds and at 3131 cm-1 for O-H bonds confirm the successful oxidation. Fig. 2 shows the XRD curves for GO, the (002) peak of graphene oxide is at 2θ =11.64 ◦ corresponding to an interlayer spacing of 0.43 nm. 3.2 Characterization of GO/CMC nanocomposite hydrogel beads The IR spectrum of the GO/CMC nanocomposite Fig. 1 (b) demonstrates the characteristic peaks of both CMC and GO. The stretching vibration of the hydroxyl group (from both CMC and GO) identified around at 3375 cm−1 and the peak at 2930 cm−1 is the characteristic C - H stretching of CH2 (from CMC that confirm the formation of the GO and CMC blend through combination. As shown in the Fig. 1(c) the characteristic peaks of CMC at around 3420 and 1608 cm−1 are attributed to the hydroxyl stretching and carboxylate bending modes, respectively. Furthermore, the adsorption bands at 2921 and 1134 cm−1 are related to the C-H stretching and bending modes respectively. To obtain information about the crystalline structure of the GO/CMC hydrogel beads, the XRD patterns of the CMC, GO, and GO/CMC with 5 wt. % GO were measured and are shown in Fig. 2 GO has a characteristic diffraction peak at 11.64◦ with the interlayer spacing of 0.43 nm. The CMC has a broad diffraction peak at 19◦, indicating that CMC is partly crystalline. The CMC/GO has a very small peak in 11.64◦ and an obvious diffraction peak at about 19◦ (Song, Xu, Yang, Cui, Zhang & Liu, 2015). 3.3 Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) One of the most important factors governing drug release behavior is the surface morphology of a carrier. Scanning electron microscopy (SEM) was used to investigate the surface morphology of the samples. GO nano sheets are clearly visible in Fig. 3(a) and (b). SEM images of hydrogel composite beads with different GO contents (0%, 5%, 10%, and 15%) have been shown in Fig. 3(c-f). As shown in SEM image of CMC hydrogel (Fig. 3(c)), the surface morphology showed a rough structure and severe wrinkles. As can be seen from Fig. 3(d-f), with increasing GO content, the surface roughness of CMC/GO hydrogel nanocomposite samples decreases. This can be attributed to the crosslinking effect
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of intercalated GO sheets, which makes strong H-bonding interactions with functional groups of CMC, and thus induces a smooth surface (Ito, 2007). Fig. 4 shows TEM image of CMC/GO hydrogel nanocomposite sample. According to Fig. 4, thin wrinkled sheets of GO are visible, which confirm the well exfoliated structure of GO sheets within hydrogel nanocomposite network without agglomeration. 3.4 Swelling properties of composite hydrogel beads In order to evaluate the sensitivity of the hydrogels to the pH of environment the swelling behavior of the hydrogels was studied in pH 1.2, 6.8 and 7.4 buffer solutions. Fig. 5 shows the effect of buffer on swelling capacity of cross linked CMC. This Fig. shows the significant difference between the swelling ratios of the hydrogel in different pHs , it means that the hydrogel was highly sensitive to pH. As described by Lee and co-workers (Lee, Choi, Paik & Park, 2006), the optimum pH to achieve a maximum swelling ratio of CMC hydrogel is about pH 6–8. Hence, little change in pH in this range leads to significant change in swelling. This is due to the dissociation of CMC and its counter ion concentration as a function of pH. Nevertheless, polymer conformation is greatly affected by ionization(Lee, Lee, Paik & Choi, 2005), under acidic conditions, CMC chains form compact coils. On the other hand, the basic conditions lead to compression of the CMC structure and as the result, the swelling is inhibited. This is because the dissociated sodium ions in basic solutions are abundant enough to compress the structure of the CMC (Lee, Choi, Paik & Park, 2006; Lee, Lee, Paik & Choi, 2005). Therefore, with increasing pH from 1.2 to 7.4, the carboxyl groups on the CMC/GO chains converted to negatively charged carboxylate ions, resulting in higher electrostatic repulsion and water would be taken up and the swelling rate tend to increase with the GO content. The effect of the GO quantity on water absorption of the CMC-hydrogel nanocomposites was investigated by varying the amount of GO from (5%, 10% and 15%). As it is shown in Fig. 5 with increasing the GO amount up to 15%, the swelling capacity was increased. The possible reason for this phenomenon might be correlated with the complex interaction between GO, water molecules and CMC. Theoretically, the hydrophilic groups of GO sheets can simultaneously form various H-bonding with water molecules,
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acid groups in CMC and hydrophilic groups of other GO sheets. At low loading, GO sheets largely interact with water molecules through H-bonding. But as the loading of GO increased, more water molecules were kept in the composite hydrogels, leading to an increase in the swelling ratio. 3.5 Drug loading and release behaviors of nanocomposite With increasing the percentage of graphene oxide nanoparticles, drug loading efficiently increased. (Fig. 6) This could be a result of possible H- bonding between carboxylic groups of the GO and –NH2 groups of the DOX. The drug release behavior was investigated to show the potential of CMC/GO nanocomposites as a drug delivery system. The relation between encapsulation and release of Doxorubicin in nanocomposite hydrogels was examined by altering the amount of GO nanoparticles. The release degree of doxorubicin as a function of time is shown in Fig. 7. The study of drug release from nanocomposite hydrogels and pure hydrogel in pH 6.8 and pH 7.4 for 24 h revealed that, the amount of released drug from pure hydrogel was more than nanocomposite hydrogels. The release amount of DOX from nanocomposite hydrogel beads in acidic conditions pH 6.8 is much higher than pH 7.4 due to the stronger H-bonding interaction under basic conditions than that under acid conditions. It was also observed that as the amount of graphene oxide nanoparticles increased, the drug release reduced. Because the presence of nanoparticles has a significant effect for slow releasing of the drug from nanocomposite hydrogels. Since the swelling of the carrier in pH 1.2 was very low, we avoided release studies in this pH. 3.6 Therapeutic efficacy of GO-CMC/DOX complexes The cytotoxicity of DOX loaded on the GO-CMC complex to SW480 cells was investigated by the MTT assay. SW480 cells were employed and DOX, GO-CMC/DOX were incubated with SW480 for 24 h followed by performing the MTT assay. As shown in Fig. 8(a) no obvious toxicity was observed for the GO-CMC without DOX loading even in relatively high concentrations This confirms the safety of the prepared nanocomposite, as the Fe3+ is potential for cytotoxic. As it can be seen form Fig. 8(b) the GOCMC/DOX complex was able to cause an obvious loss of cell viability compared to the untreated
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control cells, indicating that the GO-CMC/DOX has the potential for selectively killing cancer cells in vitro. However, DOX alone shows the highest toxicity to SW480 cells under the same conditions due to the lower DOX content in GO-CMC/DOX complexes. Moreover, enhancement of GO content in GOCMC/DOX complexes turned out in a greater cell killing property. This arises from the fact that with increasing GO content, loading percent of DOX is also increases. 4. Conclusion In this paper, the synthesis of hydrogel beads from CMC polymer and GO as nanoparticle physically crosslinked by FeCl3.6H2O was investigated. The pH dependent release of DOX from CMC/GO nanocomposite was evaluated. Characteristics and structure of prepared hydrogels were identified using TEM,SEM,XRD and FT-IR. The swelling behaviour of prepared nanocomposite hydrogels was investigated in distilled water and buffers (pH 1.2, 6.8 and 7.4 ). The obtained results showed that the swelling ratio of nanocomposit hydrogels in comparison with neat hydrogel increased by incorporating of graghene oxide nanoparticles due to H-bonding interaction between GO sheets and water molecules and carboxyl groups of CMC hydrogels. Swelling in the buffers showed a significant difference. This means that the hydrogel was sensitive to pH. The loading and releasing behavior of doxorobicin to the nanocomposite hydrogels, showed that nanocomposite hydrogels had better drug loading in comparison to the pure hydrogel. Increasing the amount of nanoparticles increased drug loading. Drug release from pure hydrogel was more than nanocomposite hydrogels and with increasing the amount of GO nanoparticles, drug release reduced due to strong interactions among amine groups of DOX and graghene oxide carboxylic groups. Acknowledgments Authors gratefully acknowledge the financial supports from the University of Tabriz (Grant Number 938645702) and Research Center for Pharmaceutical Nanotechnology (RCPN) of Tabriz University of Medical Science. References
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Fig. 5. Effect of GO concentration on swelling ratio of hydrogel beads in pHs 1.2, 6.8 and 7.4
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