Preparation of calcium carbonate@graphene oxide ...

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CERAMICS INTERNATIONAL

Ceramics International 42 (2016) 2281–2288 www.elsevier.com/locate/ceramint

Preparation of calcium carbonate@graphene oxide core–shell microspheres in ethylene glycol for drug delivery Zhihang Zhou, Yanbao Lin, Song Yao, Haichen Yan College of Materials Science and Engineering, Nanjing Tech University, Nanjing 210009, China Received 9 August 2015; received in revised form 4 October 2015; accepted 5 October 2015 Available online 21 October 2015

Abstract Calcium carbonate@graphene oxide core–shell microspheres were prepared through encapsulation of graphene oxide (GO) sheets on the surface of calcium carbonate (CaCO3) microspheres. Ethylene glycol was used as encapsulation medium to prevent GO aggregation in the dispersion system. The optimum encapsulation condition was determined through experiments to ensure that GO can be completely encapsulated on the surface of CaCO3 microspheres. The optimum encapsulation temperature was 50 1C, and the optimum encapsulation time was 4 h. The structure of CaCO3@GO microspheres was examined via field emission scanning electron microscopy. Results show that the core–shell microspheres had a uniform size of 3–4 μm when encapsulated under optimum encapsulation condition. The saturated encapsulation ratio of GO, which was calculated via UV–vis spectroscopy, was approximately 28%. The GO sheets were reduced with increasing encapsulation temperature during the preparation of CaCO3@GO microspheres. GO reduction was examined via Raman spectroscopy and UV–vis spectroscopy. The GO sheets in ethylene glycol reduced at high encapsulation temperature. Furthermore, the drug-loading capacity of CaCO3@GO microspheres increased from 38% to 52%, and the drug release time increased from 4.5 h to 6 h. Given these results, CaCO3@GO microspheres have potential application as new drug carriers. & 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Keywords: Calcium carbonate; Graphene oxide; Ethylene glycol; Core–shell structure; Drug carrier

1. Introduction Studies on novel kinds of sustained-release drug carriers have been developed to achieve long-term treatment of lesions and diseases. The structure design of drug carriers is important for their synthesis and affects drug-loading capacity and drug release time. Microspheres are commonly used as drug carriers [1–3] because of their high specific surface area. The application of microspheres as drug carriers began in the last century. Among the several types of microspheres (e.g., solid, porous, mesoporous, and core–shell), core–shell microspheres have attracted considerable attention because of their unique structure. Core–shell microspheres are well-ordered assembly structures that are synthesized through chemical bonding or other forms of interaction between two materials. Core–shell microspheres can be prepared through several methods, such n

Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Li).

http://dx.doi.org/10.1016/j.ceramint.2015.10.022 0272-8842/& 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

as self-assembly [4], surface deposition [5], emulsification [6], and stepwise heterocoagulation methods [7]. The selfassembly method is the most widely used among other methods in the preparation of core–shell microspheres because of its easy operability. Moreover, selecting core and wall materials is important in the preparation of high-performance core–shell microspheres because it can affect both drugloading capacity and drug release time. Calcium carbonate (CaCO3) has been studied for many years as a traditional core material to prepare core–shell microspheres [8,9]. CaCO3 possesses high specific surface area, good biocompatibility, and good dispersion in aqueous solutions. These advantages have considerable potential for industrial, medical, and biological applications [10]. CaCO3 is also a suitable material to be encapsulated with wall materials in the preparation of core–shell microspheres for drug delivery because of their advantages. The utilization of CaCO3 for drug delivery has already been studied. Volodkin et al. [11] employed porous CaCO3 microspheres with a size of 4.75 μm to load

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Z. Zhou et al. / Ceramics International 42 (2016) 2281–2288

lactalbumin. Li et al. [12] examined the drug release profiles of ibuprofen-loaded CaCO3 hollow microspheres. The results of Li et al.'s study showed that the CaCO3 microsphere drug delivery system has high drug-loading capacity and good sustainable release. Although CaCO3 microspheres demonstrate efficient drug delivery performance, one type of suitable material should be considered to encapsulate on the surface of CaCO3 microspheres to improve their drug delivery performance. Graphene oxide (GO) has drawn the interest of several researchers because of its unique properties. GO sheets possess typical 2D structure and numerous oxygen-containing groups, such as hydroxyl, carboxyl, and epoxy groups [13]. Because of these functional groups, GO exhibits good hydrophilic property, which enables dispersion stability in water or in several organic polar solvents. GO has potential applications in electrochemical devices [14,15], energy storage [16,17], catalysis [18], enzyme adsorption [19], cell imaging [20], drug delivery [21,22], biosensors [23], and antibacterial papers [24]. Furthermore, CaCO3 microspheres can be wrapped and interconnected by a GO network because of the oxygen-containing functional groups [25]. However, the dispersion of GO in water containing calcium ions is problematic because of the carboxylic groups that interact with these calcium ions [26,27]. When the GO sheets encapsulate the CaCO3 microspheres with water as the dispersion medium, the GO sheets agglomerate during encapsulation, which ultimately causes the failure of this process. This agglomeration, which comes from the reaction of GO with CaCO3 through the formation of –COOCaOOC–, limits the preparation and application of composite microspheres. Researchers have conducted studies to prevent this problem. Fan et al. [28] initially encapsulated CaCO3 microspheres with polyethylenimine. The surface of the composite microspheres was then encapsulated with the GO sheets. Kurapati et al. [29] used poly(allylamine hydrochloride), which is a stable material, to encapsulate CaCO3 core, and GO sheets were then used to encapsulate the microspheres. Both of these studies used one type of polymer that can provide the positive charge necessary to attach the GO sheets for the preparation of core–shell microspheres via layer by layer self-assembly. Meanwhile, these positively charged materials may prevent the contact of the GO sheets with the CaCO3 core, which can lead to agglomeration. Therefore, the GO sheets can be encapsulated on the microsphere surface, and the redundant GO sheets can be easily separated. However, studies about the direct encapsulation of CaCO3 with GO sheets are rare. The aforementioned issues are resolved using organic solvents as dispersion media in place of water. Organic solvents are common dispersion media; however, most of these solvents are harmful to the environment because of their toxicity and difficulty in recycling. Considering whether or not the dispersion medium can protect drugs, wall materials, and core materials is also an important factor in preparing core– shell drug-loaded microspheres. Ethylene glycol is a common organic solvent and has low toxicity and easy recyclability. Moreover, the CaCO3 microspheres are easily dispersed in ethylene glycol, which can also protect the crystalline phase of vaterite [30]. The GO sheets can also be dispersed in ethylene

glycol better than in other organic solvents [31]. Ethylene glycol is therefore a good choice of dispersion medium in the preparation of CaCO3-based core–shell structure. In this work, the CaCO3 microspheres were encapsulated with the GO sheets in ethylene glycol by the self-assembly method to prepare CaCO3@GO microspheres. This strategy can also prevent GO agglomeration in the presence of calcium ions. The strategy may have potential application in the biomedical and absorption treatment fields. 2. Experimental section 2.1. Materials Graphite powder, hydrogen peroxide (H2O2, 30.0%), tris (hydroxymethyl) aminomethane (Tris), and anhydrous sodium sulfate (Na2SO4) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Sulfuric acid (H2SO4, 98.0%), potassium permanganate (KMnO4), hydrochloric acid (HCl, 37.0%), sodium carbonate (Na2CO3), sodium hydrogen carbonate (NaHCO3), di-potassium hydrogen phosphate trihydrate (K2HPO4 3H2O), and ammonium hydroxide (NH3 H2O, 25–28%) were obtained from Shanghai LingFeng Chemical Reagent Co. Ltd. Sodium nitrate (NaNO3), sodium chloride (NaCl), calcium chloride anhydrous (CaCl2), magnesium chloride hexahydrate (MgCl2 6H2O), and calcium acetate monohydrate (Ca(CH3COO)2 H2O) were acquired from XiLong Chemical Co. Ltd. (Guangzhou, China). Ethylene glycol, ethanol, and n-hexane were purchased from Shanghai No. 4 Reagent & H.V. Chemical Co. Ltd. (Shanghai, China). Ibuprofen and poly(styrene sulfonic acid) sodium salt were obtained from Wuhan Bright Chemical Co. Ltd. (Wuhan, China) and Alfa Aesar Chemical Co. Ltd. (Shanghai, China), respectively. All reagents were of analytical grade and used without further purification. 2.2. Preparation of graphene oxide GO was prepared using a modified Hummers' method [32,33]. Approximately 46 mL of H2SO4 was added into a 500 mL round-bottom flask under agitation. Subsequently, 1 g of graphite powder and 1 g of NaNO3 were successively added into the round-bottom flask in an ice bath. The mixture was stirred for 30 min and 6 g of KMnO4 was then slowly and gradually added. Afterward, the round flask was transferred into a 35 1C water bath, and the suspension was stirred for approximately 1 h. Distilled water (92 mL) was gradually added to the mixture to maintain the temperature below 100 1C. The flask was then placed into an oil bath at 100 1C, and the reaction was maintained at this temperature for 15 min. The mixture was further treated with 150 mL of distilled water and 5 mL of H2O2 solution (30%) until the color changed from brown to bright yellow. The prepared graphite oxide could be easily isolated from the solution through filtration. The product was washed and centrifuged using 5 wt% aqueous hydrochloric acid solution and distilled water to remove metal ions. The washing procedure was repeated until the supernatant was neutral. GO was obtained through ultrasonic drying and freeze drying. GO


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Fig. 1. Schematic of the CaCO3@GO core–shell microsphere preparation.

powders (400 mg) were dispersed in 200 mL of ethylene glycol and ultrasonically treated for 10 min to prepare the GO dispersion.

obtained using a pipette was diluted 100 times with n-hexane. The residual concentration of ibuprofen was determined by measuring its UV–vis spectrum. The absorbance–concentration relation for ibuprofen in hexane was examined.

2.3. Preparation of calcium carbonate The Ca–PSS solution was prepared by dissolution of 0.80 g of PSS into a 200 mL of Ca(CH3COO)2 solution (0.05 M). Thereafter, the 200 mL of Na2CO3 aqueous solution (0.05 M) was poured into the Ca–PSS solution with intense agitation for 30 s, followed by aging of the CaCO3 precipitates for 30 min. Subsequently, the precipitates were filtered using Millipore filters. After a succession of washing with distilled water and ethanol, the precipitates were airdried at 50 1C and reserved at room temperature until use. 2.4. Synthesis of calcium carbonate@graphene oxide core– shell microspheres A dispersion solution of CaCO3 microspheres in 20 mL of ethylene glycol was added drop wise into a 20 mL of GO dispersion (2 mg/mL). Stirring was continued for a given time at a given temperature to ensure homogenous dispersion. The microspheres were separated by centrifugation at 3000 rpm for 5 min and were washed thrice with ethylene glycol. Afterward, the desired microspheres were air-dried at 50 1C and stored at room temperature until use. Fig. 1 shows a schematic of the preparation process of CaCO3@GO core–shell microspheres. 2.5. Drug loading and release 2.5.1. Preparation of simulated body fluid Simulated body fluid (SBF) is an acellular solution with composition and concentration similar to that of human plasma at 36.5 1C. The SBF was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4 3H2O, MgCl2 6H2O, CaCl2, and Na2SO4 in distilled water and buffering at pH 7.40 with Tris and HCl (1 M) at 36.5 1C, as described in a previous study [34]. 2.5.2. Drug loading The 100 mg of microspheres was dispersed in 10 mL of ibuprofen/n-hexane solution (30 mg/mL) in a water bath chader (180 rpm, room temperature) for a given time. The mixture was tightly covered to prevent solvent evaporation. After centrifugation at 7500 rpm for 15 min, the supernatant liquid (1 mL)

2.5.3. Drug release The 100 mg of drug-loaded microspheres was dispersed in 100 mL of SBF. The microspheres were then placed in a plastic bottle and were immersed into a water bath chader (180 rpm, 37 1C) for a given time. The liquid (5 mL) was obtained using a pipette, centrifuged at 9000 rpm for 15 min, and added with another fresh SBF (5 mL) solution. The release amount of ibuprofen was determined by measuring its UV–vis spectrum. This process was repeated until the cumulative release amount exhibited equilibrium. 2.6. Characterization techniques UV–vis spectroscopic analysis was carried out in an absorbance mode using a carry Gold spectrumlab-54 UV–vis spectrometer in the range of 200–600 nm to calculate the adsorption capacity and reduction of GO as well as the drug loading and release. Scanning electron microscopy (SEM) images were obtained using a Hitachi S-4800 at an acceleration voltage of 5.0 kV. X-ray diffraction (XRD) measurements were obtained using an ARL X'TRA X-ray diffractometer with Cu Kα radiation (λ¼ 1.54059 Å) at a scanning rate of 101/min and a scattering angle range of 5–801. Raman spectra were obtained using a Horiba HR 800 spectrometer equipped with a CCD camera detector. As a source of excitation, the 514 nm line of a Spectra Physics 2018 Argon/Krypton Ion Laser System was focused through an Olympus BX41 microscope equipped with a 50 magnification objective. 3. Results and discussion Optimum experimental conditions should be confirmed to prepare the CaCO3@GO microspheres. An apparent color change was observed (Fig. 2) with increasing encapsulation temperature. The color of the powders gradually changed from white to black. The color of the powders encapsulated at 30 1C was white, which is the same color as that of the pure CaCO3 microspheres. As the encapsulation temperature increased to 40 1C, the color of the powders turned gray. However, small amounts of white powders were also be found in the sample

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Fig. 2. Digital images of the CaCO3@GO microspheres prepared in ethylene glycol at varying encapsulation temperatures for 4 h: (A) 30 1C, (B) 40 1C, (C) 50 1C, (D) 60 1C, (E) 70 1C, and (F) 80 1C.

Fig. 3. FESEM images of the CaCO3@GO microspheres prepared in ethylene glycol at varying encapsulation temperatures for 4 h: (A) 30 1C, (B) 40 1C, (C) 50 1C, (D) 60 1C, (E) 70 1C, and (F) 80 1C.

because the CaCO3 microspheres may not have been uniformly encapsulated by the GO sheets. With further increase of the encapsulation temperature to 50 1C, the color of the powders became darker and uniform. When the encapsulation temperature increased to 80 1C, the color of the powders

became completely black, which may be caused by the reduction of the GO sheets. The SEM images of the CaCO3 microspheres after being encapsulated by GO at varying temperatures are shown in Fig. 3. At encapsulation temperature of 30 1C or 40 1C (Fig. 3A and B),


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Fig. 4. FESEM images of the CaCO3@GO microspheres prepared in ethylene glycol at 50 1C for different encapsulation times: (A) 2 h, (B) 4 h, (C) 6 h, and (D) 8 h.

Fig. 5. Encapsulation ratio curve of GO in ethylene glycol at 50 1C for different encapsulation times.

nearly no GO became encapsulated on the surface of the CaCO3 microspheres. With further increase of the encapsulation temperature to 50 1C, the surface of the CaCO3 microspheres became rough. This result indicates that the GO sheets were encapsulated uniformly on the surface of the CaCO3 microspheres. The increase of the encapsulation temperature to 80 1C resulted in smooth surface of CaCO3 microspheres and appearance of GO sheets around the CaCO3 microspheres (Fig. 3F). This phenomenon may be attributed to the reduced GO sheets in ethylene glycol with increasing encapsulation temperature. The oxygencontaining groups of GO were removed when these GO sheets were reduced into RGO sheets. Subsequently, RGO cannot interact with the CaCO3 microspheres, which leads to encapsulation failure in ethylene glycol at 80 1C, (Fig. 3F). These results show that the temperature suitable for the preparation of CaCO3@GO microspheres is 50 1C.

Fig. 4 shows the SEM images of the CaCO3@GO microspheres prepared in ethylene glycol at 50 1C for varying encapsulation times. When the CaCO3 microspheres were encapsulated at 50 1C for 2 h (Fig. 4A), their surface became rough and adsorbed minimal GO. As the encapsulation time increased to 4 h (Fig. 4B), the surface of the CaCO3 microspheres became more rough, which indicates that a high amount of GO sheets is adsorbed on their surface. The surface of the CaCO3@GO microspheres did not exhibit any apparent change with further increases in encapsulation time. Fig. 5 shows the encapsulation ratio curve of GO on the surface of the CaCO3@GO microspheres in ethylene glycol at 50 1C for varying encapsulation times. The encapsulation ratio of GO linearly increased until the encapsulation time reached 4 h. When the encapsulation time was longer than 4 h, the saturated encapsulation ratio of GO was around 28%. With further increase in encapsulation time, the encapsulation ratio of GO remained the same. Therefore, the encapsulation ratio curve of GO exhibited equilibrium when the encapsulation time was 4 h. All diffraction peaks can be indexed with regard to the vaterite structure, which is in good agreement with literature (JCPDS PDF 33-268) [35] from the XRD patterns (Fig. 6). This finding indicates that no phase transformation occurred between the prepared CaCO3 and CaCO3@GO microspheres with the change in experimental conditions. Moreover, GO diffraction peaks were not observed in the pattern, which may be due to the GO sheets that were uniformly and disorderly encapsulated on the surface of the CaCO3 microspheres. The intensity of the characteristic peaks became apparently weak after the GO sheets were encapsulated on the CaCO3 surface, which is attributed to the complete encapsulation of the GO sheets. Unfortunately, some CaCO3 microspheres after being encapsulated by GO sheets (Figs. 3C–F and 4) aggregate together. On

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Fig. 6. XRD patterns of the pure CaCO3 microspheres (A) and CaCO3@GO microspheres prepared in ethylene glycol at 50 1C for 4 h (B).

the one hand, this may be connected by GO sheets. But our GO sheets only have a size of 0.5–1.0 μm as shown in Fig. S1 (supplied as Supplementary material). The size of GO sheets is smaller than that of CaCO3. It is difficult for GO sheets to connect the CaCO3 microspheres. On the other hand, the GO sheets assemble together to form bigger GO film. This GO film can lead to this aggregation through connection of the CaCO3 microspheres. The GO film should have order structure due to the assembly of GO sheets. But there no any diffraction peak of GO in the XRD pattern (Fig. 6B). This indicates that there is not GO assembly with order structure in our samples. Therefore, the aggregation of the CaCO3 microspheres need be investigated further in detail. Moreover, the aggregation of the CaCO3 microspheres should affect their drug loading/release behavior if they are applied as drug carrier. Raman spectroscopy (Fig. 7) and UV–vis spectroscopy (Fig. 8) were carried out to investigate whether or not the GO sheets were reduced by ethylene glycol at high temperature. Fig. 7 shows the characteristic peak for CaCO3 at around 1080 cm 1 [36] and the characteristic peaks for GO at around 1381 cm 1 (D band) and 1594 cm 1 (G band) [37]. These characteristic peaks indicate the coexistence of the CaCO3 microspheres and GO/RGO sheets. The ID/IG ratio (1.003) of the microspheres prepared at 80 1C was slightly higher than that (0.891) of the microspheres prepared at 40 1C. This result confirms that the GO sheets are partly reduced with increasing temperature. The Raman peak at 1125 cm 1 (S¼ O bond) represents the PSS molecule [38], which indicates that several PSS molecules exist in CaCO3 despite several washings. Meanwhile, the characteristic peak at 1080 cm 1 for CaCO3 was strong when the encapsulation temperature was 40 1C because the CaCO3 microspheres were not uniformly encapsulated by the GO sheets, which agrees with the results observed in Figs. 2B and 3B. Notably, the characteristic peak at 1080 cm 1 for CaCO3 became weak when the encapsulation temperature increased to 60 1C. This result may be due to the CaCO3 surface, which was uniformly encapsulated with the GO sheets. This result agrees with those shown in Figs. 2C and 3C. The increase in encapsulation temperature to 80 1C resulted in strong

Fig. 7. Raman spectra of synthesized CaCO3@GO microspheres prepared in ethylene glycol at varying temperatures for 4 h: (A) 40 1C, (B) 60 1C, and (C) 80 1C.

Fig. 8. UV–vis spectra of the GO sheets after soaking in ethylene glycol at 80 1C for 4 h (A) and 50 1C for 4 h (B).

characteristic peak at 1080 cm 1 for CaCO3 because the sheets fell off from the CaCO3 microspheres after GO reduction. This observation supports the idea that high encapsulation temperature is not beneficial in the preparation of CaCO3@GO core– shell microspheres. Fig. 8 shows the absorption peaks of GO in ethylene glycol after being soaked at various temperatures for 4 h. The absorption peak of GO soaked at 80 1C was observed at 235 nm, whereas that of GO soaked at 40 1C was observed at 230 nm. A red-shift was observed after the GO sheets were soaked in ethylene glycol at high temperature. The recovery of double bonds after reduction at high temperature causes an increase in the degree of conjugation, which further leads to the movement of absorption peak of GO to long wavelengths (red-shift). This result shows that the GO sheets were reduced in ethylene glycol with increasing temperature, as reported in a previous study [39]. When the GO sheets were reduced into RGO sheets, most of the oxygen-containing groups disappeared, which caused poor adsorption of these sheets on the CaCO3 surface. Therefore, several RGO sheets were observed around the microspheres, as indicated in Fig. 3F. This result


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Fig. 9. Drug loading (A) and release (B) curves of ibuprofen using the pure CaCO3 microspheres and CaCO3@GO microspheres as drug carriers (DEC: encapsulation efficiency, DLC: drug loading).

shows that GO reduction is unprofitable in the preparation of CaCO3@GO microspheres. Ibuprofen was selected as the model drug to verify the feasibility of CaCO3@GO microspheres as drug carriers. The ibuprofen loading ratios on CaCO3 and CaCO3@GO microspheres were 38% and 52%, respectively (Fig. 9A). This result confirms that the drug-loading capacity of CaCO3@GO microspheres was larger than that of pure CaCO3 microspheres because of the core–shell structure formation in the preparation process of CaCO3@GO microspheres. The presence of GO sheets enlarges the specific surface area of the microspheres such that more drugs can be loaded to the microspheres. The drug release curves show that the release time (6 h) of CaCO3@GO microspheres is longer than that (4.5 h) of CaCO3 microspheres (Fig. 9B). At the beginning of 1 h, the cumulative drug release percentage of CaCO3 and CaCO3@GO microspheres reached about 58%. The initial fast-release may be associated with two factors [40]: (1) The drug concentration in SBF is zero at the early stages of the release process; the large concentration gradient between drug in the microspheres and SBF prompts the fast release of drug. (2) A small amount of drug is easily desorbed from the microsphere surface. After 4.5 h, the drug cumulative release curve of the pure CaCO3 microspheres exhibited equilibrium, whereas that of the CaCO3@GO microspheres continued to grow and only exhibited equilibrium after another 1.5 h. This finding can be explained by the core–shell microsphere structure, which has provided twisted corridors around the CaCO3 core to slow down drug release and prolong the release time. 4. Conclusions The GO sheets were successfully employed in the preparation of CaCO3@GO microspheres through self-assembly on CaCO3 microspheres in ethylene glycol. The optimum encapsulation temperature was 50 1C, and the optimum encapsulation time was 4 h. Ibuprofen was used as the model drug loaded into the CaCO3@GO microspheres and the CaCO3 microspheres. The

CaCO3@GO microspheres had a relatively higher drug loading ratio and longer drug release time than the pure CaCO3 microspheres. The loading ratio and release time of the CaCO3@GO microspheres were 52% and about 6 h, respectively. The drug loading and release property of the CaCO3@GO microspheres indicate that these microspheres may be promising materials for applications in biomedicine and wastewater treatment. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 50802042), the Natural Science Foundation of Jiangsu Province (No. BK2011076), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ceramint. 2015.10.022.

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