Sodium alginate conjugated graphene oxide as a new carrier for drug delivery system

International Journal of Biological Macromolecules 93 (2016) 582–590

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Sodium alginate conjugated graphene oxide as a new carrier for drug delivery system Lihong Fan a , Hongyu Ge a , Shengqiong Zou a , Yao Xiao a , Huigao Wen a , Ya Li a , Han Feng a , Min Nie b,∗ a

College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, China The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China b

a r t i c l e

i n f o

Article history: Received 25 June 2016 Received in revised form 5 September 2016 Accepted 7 September 2016 Available online 9 September 2016 Keywords: Graphene oxide Sodium alginate Doxorubicin hydrochloride Drug delivery Controlled release

a b s t r a c t The biomedical applications of graphene-based materials, including drug delivery, have grown rapidly in the past few years. The aim of this present study is to enhance the efficiency and specificity of anticancer drug delivery and realize intelligently controlled release and targeted delivery. Graphene oxide (GO) was first prepared from purified natural graphite according to a modified Hummers’ method. Then GO was functionalized with adipic acid dihydrazide to introduce amine groups, and sodium alginate (SA) was covalently conjugated to GO by the formation of amide bonds. The resulting GO–SA conjugate was characterized and used as a carrier to encapsulate the anticancer drug doxorubicin hydrochloride (DOX·HCl) to study in vitro release behavior. The maximum loading capacity of DOX on GO–SA was 1.843 mg/mg and the drug release rate under tumor cell microenvironment of pH 5.0 was significantly higher than that under physiological conditions of pH 6.5 and 7.4. Methylthiazol tetrazolium (MTT) assay was applied to evaluate the Hela cells and NIH-3T3 cells cytotoxicity of GO–SA. Results showed that GO–SA had no obvious toxicity and GO–SA/DOX exhibits notable cytotoxicity to Hela cells. Cell uptake studies indicated that GO–SA could specifically transport the DOX into Hela cells over-expressing CD44 receptors and showed enhanced toxicity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction In recent years, design and development of powerful new drug delivery systems has been relentless, with ever more attention devoted to developing new methods for realizing controlled drug release [1,2]. In conventional drug delivery, the drug concentration in the blood rises quickly, and then declines [3,4]. Each drug has a plasma level above which it is toxic and below which it is ineffective [5]. The main aim of an ideal drug delivery system (DDS) is to maintain the drug within a desired therapeutic range after a single dose, and/or target the drug to a specific region while simultaneously lowering the systemic levels of the drug [6–9]. Biopolymers have frequently been used as raw materials for the design of drug delivery formulations owing to their excellent properties [10], such as non-toxicity, biocompatibility, biodegradability and environmental sensitivity, etc [11–13].

∗ Corresponding author. E-mail address: [email protected] (M. Nie). http://dx.doi.org/10.1016/j.ijbiomac.2016.09.026 0141-8130/© 2016 Elsevier B.V. All rights reserved.

Graphene, a two-dimensional nanomaterial reported for the first time in 2004, has been widely investigated for its novel physical properties and potential applications in nanoelectronic devices, transparent conductors, and nanocomposite materials [14,15]. As a graphene derivative, graphene oxide (GO) has been widely explored in the last several years for drug delivery applications by many other research groups [16,17]. GO can be readily exfoliated into monolayer sheets to yield stable suspensions in water because of the hydrophilic oxygenated functional groups on its basal planes and edges [18–20]. These groups enable GO to be functionalized through covalent and noncovalent approaches [21–23], hence making it a building block for synthesizing versatile functional materials [24,25]. Further, the large two-dimensional plane of GO sheets provides large specific surface area to carry drugs via surface adsorption, hydrogen bonding, and other types of interactions [26]. Meanwhile, the excellent biocompatibility and nontoxicity of GO makes it a promising material for drug carrier substances [27–29]. Sodium alginate (SA) is a natural hydrophilic polysaccharide derived from seaweed [30,31]. SA is a water soluble salt of alginic acid, a naturally occurring non-toxic polysaccharide found in all

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species of brown algae [32]. And SA is a biodegradable polymer used extensively in food processing, medical and pharmaceutical industries such as drug carrier [33,34]. In addition, earlier literature cites many applications of SA in drug delivery [35,36], and it has been used to prepare the sustained release particulate systems for a variety of drugs, proteins, and cells [37,38]. In this study, a new anticancer drug carrier system with the abilities of controlled and targeted release was developed. GO was firstly prepared via an improved Hummers’ method and characterized by FTIR, XRD, Raman, TGA, TEM [39]. To integrate the advantages of GO and SA, GO was functionalized with adipic acid dihydrazide (ADH). And then the introduced amino groups were used to conjugate SA. The resulting GO–SA conjugate was characterized by FTIR, TGA, TEM. The in vitro toxicity studies of the GO–SA were carried out by conducting MTT assays. As an anti-tumor drug, DOX was then loaded onto the surface of this conjugate via ␲-␲ stacking and hydrogen-bonding interaction [40], and in vitro release behavior at different pH conditions was monitored via UV–vis spectrometry. The in vitro selective targeting and cytotoxic effect of the DOX–loaded GO–SA (GO–SA/DOX) to HeLa cells over-expressing CD44 receptors and NIH-3T3 cells low-expressing CD44 receptors were examined. 2. Experimental 2.1. Materials Graphite power (D50 < 600 nm) and adipic acid dihydrazide (ADH) were purchased from Aladdin. N-hydroxy sulfosuccinimide (NHS) and 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC) were purchased from Huashun Biological Technology Co. Ltd., Wuhan, China. Sodium alginate (SA) and other reagent used in this article were of analytical grade and without further purification. They were purchased from Sinopharm Group Chemical Reagent Corp. 2.2. Synthesis of the GO–SA conjugate Graphene oxide (GO) was prepared from normal graphite powder by a modified Hummers’ method [39]. To obtain the uniform ultrasmall GO nanosheets, the as-prepared random distributed sized GO nanosheets should be further oxidized twice. Firstly, natural graphite (2 g), K2 S2 O8 (1 g), and P2 O5 (1 g) were put into concentrated H2 SO4 (20 mL). The mixture was strongly magnetic stirred at 80 ◦ C for 5 h, then filtered and washed several times with distilled water, dried in the vacuum oven at 40 ◦ C for 12 h. The resultant mixture was slowly cooled to room temperature over a period of about 6 h. Then filtered and washed several times with distilled water, dried in the vacuum oven at 40 ◦ C for 12 h. Secondly, the preoxidized graphite powder and NaNO3 (2 g) were slowly dispersed into concentrated H2 SO4 (100 mL) in an ice bath with stirring. Meanwhile, KMnO4 (6 g) was gradually added. The dispersion was kept at 35 ◦ C for 5 h and then distilled water (500 mL) and 30% H2 O2 (10 mL) were added gradually to terminate the reaction, after which the color of the mixture changed to bright yellow. The mixture was filtered and washed with 1:10 HCl solution (1000 mL) in order to remove metal ions. The solution was filtered and washed until the filtrate became pH neutral. Finally, the filter cake was sonicated in deionized water for 2–3 h, the single layer GO dispersion was obtained after dialysis and lyophilization. GO (50 mg) was sonicated and dispersed in distilled water (40 mL), and then N-(3-dimethylaminopropyl)hydrochloride (EDC, 50 mg) and N’-ethylcarbodiimide N-hydroxysuccinimide (NHS, 150 mg) were dissolved in distilled water (10 mL), and they were introduced to activate the carboxylic

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acid groups of GO for 30 min [41]. ADH (100 mg) was added to the activated GO solution, followed by 24 h of stirring. The produced GO–ADH was separated by centrifugation and rinsed repeatedly with distilled water. Sodium alginate (15 mg), EDC (30 mg) and NHS (50 mg) were dissolved in distilled water (15 mL), then the above-mentioned GO–ADH was introduced, and the mixture was stirred at room temperature for another 24 h. The resulting GO–SA was collected by centrifugation, washed repeatedly with distilled water, and then obtained after lyophilization. An overview of the preparation of the conjugated GO–SA anticancer drug-carrier with targeting function and pH-sensitivity was shown in Fig. 1. 2.3. Characterization Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-2100F (Japan) transmission electron microscope. Fourier transform infrared (FT-IR) spectra were recorded with a Nicolet 170SX Fourier transform infrared spectrophotometer (USA) in the wavenumber ranging from 400 to 4000 cm−1 . All the test samples were prepared by the KBr disk method. Raman spectroscopy was collected with a RENISHAW INVIA Raman Spectrometer at room temperature with an excitation laser source of 532 nm. Spectra were recorded from 300 to 3300 cm−1 . Xray diffraction (XRD) patterns were obtained on a Rigaku DMAX 2000 diffractometer using Cu-Ka radiation (k = 0.15405 nm) (40 kV, 40 mA). Ultraviolet–visible (UV–vis) absorption spectra were obtained with a BeckMan coulter DU 730 spectrophotometer. Thermogravimetric analysis (TGA) was performed using a TG 209F1 (Netzsch Instruments) thermogravimetric analyzer with a heating rate of 20 ◦ C/min and a temperature range of 30–600 ◦ C in nitrogen. 2.4. Loading of DOX onto GO–SA GO–SA (50 mg) was first dispersed in PBS (pH = 7.4, 50 mL) and then sonicated for a few minutes. The different concentration of DOX·HCl (1 mL) were added into the sonicated GO–SA, respectively. Loading DOX onto GO–SA was performed by stirring for 20 h under dark conditions at room temperature [42]. The DOX drug-loaded GO–SA were collected by centrifugation and dried under vacuum for 24 h, then washed with ionized water three times to remove the excess drug. The loading ratios of DOX were estimated by UV–vis spectroscopy at 490 nm. 2.5. Release of DOX from GO–SA/DOX complex To study the release behavior of DOX from GO–SA, the release behavior of DOX from GO–SA/DOX was investigated at 37 ◦ C at three different pH, using a dialysis bag with 5000 Da molecular cutoff [43]. The GO–SA/DOX complex (5 mg) was suspended in a PBS buffer (10 mL, pH 7.4, 6.5 and 5.0), and the dialysis bags were incubated in PBS buffer (50 mL) of the same pH value at 37 ◦ C under shaking (200 rpm min−1 ). A known quantity (3 mL) of solution from the container was removed after every time step, making sure to replace it with the same amount of fresh PBS solution. The amount of the released DOX was measured at different time points from 0 h to 96 h by a UV–vis spectrophotometer (490 nm). 2.6. Cytotoxicity measurement The in vitro cytotoxicity investigation was conducted using MTT assays [27]. HeLa cell lines and NIH-3T3 cell lines were used to evaluate the in vitro cytotoxicity of the materials [44]. The cells were employed and cultured in DMEM medium supplemented with penicillin (100 units/mL), streptomycin (100 ␮g/mL), and 10% (v/v) heat-inactivated FBS. Hela cells and NIH-3T3 cells were seeded into 96-well plates at a density of 4 × 103 cells/well in 100 ␮L DMEM,

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L. Fan et al. / International Journal of Biological Macromolecules 93 (2016) 582–590 O NH 2

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Fig. 1. Schematic diagram of the synthesis of GO–SA and succedent DOX loading: (a) Functionalizing GO with ADH to produce GO–ADH; (b) conjugation of SA with GO–ADH by the formation of amide bonds to produce GO–SA and (c) loading DOX onto GO–SA.

respectively. After overnight incubation at 37 ◦ C in a humidified 5% CO2 -containing atmosphere, then the medium with different concentrations of GO–SA were added into the 96-well plates and the cells were cultured for 24 h. After extraction of the medium in wells, 20 ␮L of MTT solution was added and the cells continued to be incubated for an additional 4 h. Finally, the medium was removed and 150 ␮L DMSO was added and thoroughly shaken for 15 min, and

the absorption strength of every well at 490 nm was recorded by using a microplate reader. For cell viability measurements, the concentrations of Hela cells and NIH-3T3 cells were 8 × 103 cells/well. The fresh medium containing different concentrations of free DOX (5, 10, 15 and 20 ␮g/mL, respectively) or GO–SA/DOX (with an equivalent concentration of DOX) were added to each well. After incubation for

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Fig. 2. The images of GO for its dispersity (a) a month ago and (b) a month later.

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another 24 h as described above, then the MTT assays were conducted as the above procedures. The relative cell viability was measured by comparing the control wells containing only the cells.

Cell Viability(%) =

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Fig. 5. TGA curves of (a) GO, (b) GO–SA and (c) SA at a heating rate of 20 ◦ C/min in nitrogen.

where ODs , ODc , ODb are optical density values from sample wells, positive control wells and background wells, respectively. 2.7. Cellular uptake The specific cellular uptake of GO–SA/DOX was qualitatively tested in over-expressing CD44 Hela cells and low-expressing CD44 NIH-3T3 cells using confocal microscopy [45]. Cells were seeded in 24-well culture plates containing 5 × 104 cells/well for each cell type. After incubation for 24 h at 37 ◦ C, cells were incubated with DOX, GO–SA/DOX or PBS for 2 h, then the medium was removed

Fig. 4. (a) Raman spectrum of GO and (b) XRD pattern of GO.

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Fig. 6. TEM image of (a) GO and (b) GO–SA.

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and washed with PBS for three times. The cells in the rest one well for each sample were fixed with 500 ␮L of 4% formaldehyde for 15 min at 37 ◦ C and washed twice with PBS again. The nucleus of cells was counterstained with DAPI at 37 ◦ C [46]. Finally, the cells were observed using a confocal laser-scanning microscope (Carl Zeiss LSM 700, Jena, Germany) with an argon blue laser light at 490 nm and a magnification of 63×. 3. Results and discussion 3.1. Synthesis and characterization of GO–SA conjugate First, the GO was prepared by the oxidation of graphite according to a modified Hummers’ method and the following sonication treatment. As shown in Fig. 2, the obtained GO was stable in pure water for over a month and no agglomeration occurred. GO was functionalized with adipic acid dihydrazide (ADH), and then the introduced amino groups were used to conjugate SA. Synthesis of GO and formation of the GO–SA conjugate were confirmed via fourier transform infrared spectroscopy (FT-IR) as shown in Fig. 3. The spectrum of the pristine graphite only showed a weak absorbance centered at 3410 cm−1 , which could be O H stretching bond from absorbed water. The spectrum of GO revealed the presence of C O carbonyl stretching at 1730 cm−1 , the C O epoxy stretching vibration peak at 1230 cm−1 , the C O alkoxy stretching vibration peak at 1060 cm−1 , and the vibration and deformation peaks of O H groups at 3410 cm−1 and 1625 cm−1 ,

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Time (h) Fig. 8. Release profile of DOX from GO–SA/DOX at pH 7.4, 6.5 and 5.0.

respectively. Also, Fig. 3 showed that the FT-IR spectrum of the obtained GO–ADH, after incorporation of gelatin into the GO, the C O vibration peak of GO at 1730 cm−1 became weaker and disappeared completely and a strong band at 1629 cm−1 associated with the amide bonds appeared. Afterwards, the primary amine groups in GO–ADH were allowed to react with SA through an amide linkage. The FT-IR spectrum of the resulting GO–SA was present in Fig. 3. Several new absorbance characteristics of SA appear at 1627 cm−1 (amide I band), 1405 cm−1 (carboxylate symmetric stretching), 1155 cm−1 (skeletal acetal valence band) and 1050 cm−1 (C O stretching), confirming the successful conjugation of SA on GO. Raman spectroscopy was utilized to investigate the carbon structure of graphite during the oxidation process. A typical Raman spectrum of GO was obtained in Fig. 4a. After the oxidation, the G band and D band of graphite become broader in GO. The characteristic peaks of the G band and D band were 1590 cm−1 and 1350 cm−1 , respectively. A significantly increased intensity ratio of the D and G band (ID /IG ) was 1.03, which were similar to the literature values for GO. As shown in Fig. 4b, XRD curves for graphite and GO, again demonstrated that the oxidation process was successful and changed the chemical structure of graphite. A sharp peak was observed at 11.3◦ (d = 0.78 nm), which usually appeared in the XRD patterns of GO samples. This peak was due to the peak position of stacked GO analogs formed during the process of preparation of XRD samples. The characteristic diffraction peak of graphite at 26.5◦ disappeared due to the complete oxidation of graphite. The results indicated that GO had been successfully synthesized and the oxy-

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genated functional groups were present on the surface of graphene sheets expanding the interlayer spacing of graphite. These characterizations confirmed that GO had been successfully prepared. To quantify the compositions of the composites, TGA was performed as shown in Fig. 5. it was observed that GO itself was thermally unstable and starts to lose mass upon heating even below 100 ◦ C. The major mass loss occurred close to 200 ◦ C, which was attributed to the pyrolysis of labile oxygen-containing groups. At 600 ◦ C, GO, GO–SA, and SA had remnant masses of 58%, 39%, and 27%, respectively. Therefore, by comparison of these values, it could be estimated that the weight contents of the grafted GO and SA in GO–SA were 39 wt% and 61 wt%. The morphology of the GO and GO–SA conjugate was characterized with TEM as shown in Fig. 6. Compared with GO (Fig. 6a), the functionalized GO modified with SA (Fig. 6b) retained a lamellar structure and no appreciable aggregation could be observed, suggesting that the modification occurred only on the surface of the GO without changing its intrinsic structure and the conjugation was quite uniform. 3.2. Drug loading and release behavior DOX as an anti-tumor drug with a pH-dependent hydrophilicity is one of the most widely-used chemotherapeutic anticancer drugs, but its administered dosage is strongly limited by the severe side effects [37]. In view of the unique structure and properties of GO, the disadvantages of DOX could be efficaciously resolved by using functionalized GO as drug carriers. Therefore, herein DOX is used

as a model drug to examine the drug loading and release properties of GO–SA [41]. With the large specific surface area, GO is supposed to possess excellent loading behavior. DOX was loaded onto the surface of the GO–SA conjugate via a simple mixture and sonication method by ␲–␲ stacking and hydrophobic interactions between GO and DOX. The unbound drug was removed by centrifugation and the loading efficiency of DOX on the functionalized GO was calculated by measuring the concentration of unbound drug using UV–vis at 490 nm. As indicated in Fig. 7, the loading capacity of DOX on GO–SA increased with the increase of initial drug concentration and finally reached its saturation value when the drug concentration was higher than 1000 ␮g mL−1 . The maximum loading capacity of DOX on GO–SA was up to 1.843 mg/mg. These results indicated that the carrier could load DOX with exceptionally high loading capacity and efficiency. Since in vitro dissolution testing was an important study for drug development and quality control, it had been used to investigate the release rate of DOX from GO–SA/DOX in different pH ranges (pH 7.4, 6.5 and 5.0) at 37 ◦ C to evaluate the best formulations. 37 ◦ C, which was close to the physiological temperature, was chosen for the release response. The release profile of DOX from the GO–SA/DOX in PBS buffer was presented in Fig. 8. DOX was released very slowly from the carrier at neutral conditions (pH = 7.4). There was only about 11.6% of the total bound released for 96 h. However, the release efficiency of DOX enhanced evidently when pH decreased. Especially, in acidic conditions (pH 5.0), DOX was released very quickly in the early stages but the release rate

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Fig. 10. Confocal microscopic images of Hela and NIH-3T3 cells treated with GO–SA/DOX complexes (DOX = 4 ␮g/mL) for 2 h. Hela and NIH-3T3 cells treated with PBS were used as controls. The scale bar in each panel represents 20 ␮m.

gradually declined after 12 h and about 40% of the total bound DOX was released after 96 h. It was known that the release property was related to the nature of interactions between the drug and its carrier. This was attributed to the increased hydrophilicity and solubility of DOX, which was beneficial for its effective ␲–␲ stacking interaction with GO, resulting in an inefficient release. Generally, DOX could be loaded on the functional GO under physiological conditions and released at reduced pH, typical of micro-environments of cancerous tissues, intracellular lysosomes or endosomes, provided an ideal mechanism for selective drug release.

3.3. Therapeutic efficacy of GO–SA/DOX complexes The cytotoxicity of the DOX loaded on the GO–SA complex to tumor cells and normal cells was investigated by the MTT assay. Hela cells and NIH-3T3 cells were incubated with free DOX, GO–SA/DOX and GO–SA for 24 h, respectively. Then they were followed by performing the MTT assay. As shown in Fig. 9a, it was worth to note that the GO–SA without DOX loading did not exhibit cell cytotoxicity even when using relatively high concentrations. As could be seen from Fig. 9b, the GO–SA/DOX complex was able to cause an obvious loss of cell viability when compared

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with the untreated control cells, indicating that the GO–SA/DOX had the potential for selectively killing cancer cells in vitro. However, DOX alone showed the highest toxicity to Hela cells under the same conditions due to the partial inefficient release of DOX from the GO–SA/DOX complex. The targeted therapeutic efficacy of the GO–SA/DOX complex was explored by the MTT assay using Hela cells and NIH-3T3 cells as shown in Fig. 9c. The results of cytotoxicity to NIH-3T3 cells were exhibited that free DOX showed higher toxicity than GO–SA/DOX, which was attributed to the partial inefficient release of DOX from the GO–SA/DOX complex. This indicated that the SA–GO/DOX complex could reduce the damage of the normal cells relative to free DOX to a certain extent. To examine the cellular delivery of Hela cells and NIH-3T3 cells, cell nuclei were stained with DAPI and fluorescence was assessed. Fig. 10 showed the confocal fluorescence images of the two types of cells after being incubated with the GO–SA/DOX complex using the red fluorescence to trace DOX. The cells treated with PBS and NIH-3T3 cells treated with GO–SA/DOX showed no significant fluorescence in the cytoplasm, either. However, the Hela cells incubated with the GO–SA/DOX clearly showed red fluorescence in the cytoplasm than that of the NIH-3T3 cells. The targeting effect of the GO–SA conjugate to tumor cells was attributed to Hela cells with high-level CD44 receptor expression and the normal NIH-3T3 cells with low-level CD44 receptor expression. 4. Conclusion SA has been covalently conjugated to GO using ADH as a linker to produce GO–SA, and this was confirmed by the results from FTIR, XRD, Raman and TEM. The GO–SA carrier was able to complex with DOX through ␲-␲ stacking interactions between DOX and GO, and the whole conjugate displayed a pH-responsive DOX release behavior with a faster rate and a greater amount being released in acidic conditions. The application of GO–SA in cancer treatment has been studied. The in vitro toxicity studies showed that the resulting GO–SA exhibited very low cytotoxicity and no evident toxic effects in NIH-3T3 cells. Cellular uptake experiments demonstrated that the GO–SA could targetedly deliver the anticancer drugs into the cells by receptor-mediated endocytosis. The GO–SA showed a high loading capacity for the anticancer drug DOX, and the resulting GO–SA/DOX exhibited high cytotoxicity to Hela cells. All these positive results suggested that the GO–SA is an ideal drug-carrier for targeted delivery and controlled release of anticancer drugs. Acknowledgements

[3]

[4] [5]

[6]

[7]

[8]

[9]

[10]

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21] [22]

The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, No. 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and Technology of Small and Medium-sized Enterprises (Foundation No.11C26214202642, No. 11C26214212743), Zhuhai Science and Technology Plan Projects (Foundation No. 2011B050102003), Wuhan Science and Technology Development (Foundation No. 201060623262), The Fundamental Research Funds for the Central Universities(Foundation No. 2014-zy-220). Hubei-NOST KLOS & KLOBME.

[23]

[24] [25]

[26]

[27]

[28]

References [1] J. Liu, Y. Huang, A. Kumar, A. Tan, S.B. Jin, X. Liang, et al., pH-sensitive nano-systems for drug delivery in cancer therapy, Biotechnol. Adv. 32 (2014) 693–710. [2] Q.X. Pan, L. Yao, G.R. Williams, L. Tao, H.H. Yang, H.Y. Li, et al., Lactobionic acid and carboxymethyl chitosan functionalized graphene oxide nanocomposites

[29]

[30]

589

as targeted anticancer drug delivery systems, Carbohydr. Polym. 151 (2016) 812–820. L. Zhou, D. Liang, X. He, J. Li, H. Tan, J. Li, et al., The degradation and biocompatibility of pH-sensitive biodegradable polyurethanes for intracellular multifunctional antitumor drug delivery, Biomaterials 33 (2012) 2734–2745. R.M. Cheng, R.T. Zou, S.J. Ou, R. Gou, R.Y. Yan, et al., Graphene oxide complex as a pH-sensitive antitumor drug, Polym. Chem. 6 (2015) 2401–2406. H. Bao, Y. Pan, Y. Ping, N.G. Sahoo, T. Wu, L. Li, et al., Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery, Small 7 (2011) 1569–1578. B. Chen, M. Liu, L. Zhang, J. Huang, J. Yao, Z. Zhang, Polyethylenimine-functionalized graphene oxide as an efficient gene delivery vector, J. Mater. Chem. 21 (2011) 7736–7741. J. Gao, F. Bao, L. Feng, K. Shen, Q. Zhu, D. Wang, et al., Functionalized graphene oxide modified polysebacic anhydride as drug carrier for levofloxacin controlled release, Rsc Adv. 1 (2011) 1737–1744. X. Ma, H. Tao, K. Yang, L. Feng, L. Cheng, X. Shi, et al., A functionalized graphene oxide-iron oxide nanocomposite for magnetically targeted drug delivery, photothermal therapy, and magnetic resonance imaging, Nano Res. 5 (2012) 199–212. D. Ma, J. Lin, Y. Chen, W. Xue, L. Zhang, In situ gelation and sustained release of an antitumor drug by graphene oxide nanosheets, Carbon 50 (2012) 3001–3007. V.K. Rana, M.C. Choi, J.Y. Kong, G.Y. Kim, M.J. Kim, S.H. Kim, et al., Synthesis and drug-delivery behavior of chitosan-gunctionalized graphene oxide hybrid nanosheets, Macromol. Mater. Eng. 296 (2011) 131–140. J. Wang, C. Liu, Y. Shuai, X. Cui, L. Nie, Controlled release of anticancer drug using graphene oxide as a drug-binding effector in konjac glucomannan/sodium alginate hydrogels, Colloids Surf.: B Biointerfaces 113 (2014) 223–229. C.L. Weaver, J.M. Larosa, X. Luo, X.T. Cui, Electrically controlled drug delivery form grapheme oxide nanocomposite films, Acs Nano 8 (2014) 1834–1843. L. Zhang, Z. Lu, Q. Zhao, J. Huang, H. Shen, Z. Zhang, Enhanced chemotherapy efficacy by sequential delivery of siRNA and anticancer drugs using PEI-grafted graphene oxide, Small 7 (2011) 460–464. C. Chung, Y.K. Kim, D. Shin, S.R. Ryoo, B.H. Hong, D.H. Min, Biomedical applications of graphene and graphene oxide, Acc. Chem. Res. 46 (2013) 2211–2224. H. Fan, L. Wang, K. Zhao, N. Li, Z. Shi, Z. Ge, et al., Fabrication, mechanical properties, and biocompatibility of graphene-reinforced chitosan composites, Biomacromolecules 11 (2010) 2345–2351. D. Han, L. Yan, W. Chen, W. Li, Preparation of chitosan/graphene oxide composite film with enhanced mechanical strength in the wet state, Carbohydr. Polym. 83 (2011) 653–658. K. Krishnamoorthy, M. Veerapandian, K. Yun, S.J. Kim, The chemical and structural analysis of graphene oxide with different degrees of oxidation, Carbon 53 (2013) 38–49. L. Liu, C. Li, C. Bao, J. Qiong, P. Xiao, X. Liu, et al., Preparation and characterization of chitosan/graphene oxide composites for the adsorption of Au (III) and Pd(II), Talanta 93 (2012) 350–357. L.C. Tang, Y.J. Wan, D. Yan, Y.B. Pei, L. Zhao, Y.B. Li, et al., The effect of graphene dispersion on the mechanical properties of graphene/epoxy composites, Carbon 60 (2013) 16–27. Y. Zhu, S. Murali, W. Cai, X. Li, J.W. Suk, J.R. Potts, et al., Graphene and graphene oxide: synthesis, properties, and applications, Adv. Mater. 22 (2010) 3906–3924. X. Yang, Y. Tu, L. Li, S. Shang, X. Tao, Well-dispersed chitosan/graphene oxide nanocomposites, Acs Appl. Mater. Interfaces 2 (2010) 1707–1713. Y. Yang, Y.M. Zhang, Y. Chen, D. Zhao, J.T. Chen, Y. Liu, Construction of a graphene oxide based noncovalent multiple nanosupramolecular assembly as a scaffold for drug delivery, Chem. Eur. J. 18 (2012) 4208–4215. H.W. Liu, S.H. Hu, Y.W. Chen, S.Y. Chen, Characterization and drug release behavior of highly responsive chip-like electrically modulated reduced graphene oxide-poly (vinyl alcohol) membranes, J. Mater. Chem. 22 (2012) 17311–17320. Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nanographene oxide for delivery of water-insoluble cancer drugs, J. Am. Chem. Soc. 130 (2008) 10876–10877. I.P.D. Sousa, K. Buttenhauser, W. Suchaoin, A. Partenhauser, et al., Thiolated graphene oxide as promising mucoadhesive carrier for hydrophobic drugs, Int. J. Pharm. 509 (2016) 360–367. X. Yang, Y. Wang, X. Huang, Y. Ma, Y. Huang, R. Yang, et al., Multi-functionalized graphene oxide based anticancer drug-carrier with dual-targeting function and pH-sensitivity, J. Mater. Chem. 21 (2011) 3448–3454. H. Yang, B. David, L. Tao, H. Li, J. Hu, L. Zhu, Carboxymethyl chitosan-mediated synthesis of hyaluronic acid-targeted graphene oxide for cancer drug delivery, Carbohydr. Polym. 135 (2015) 72–78. L. Zhang, J. Xia, Q. Zhao, L. Liu, Z. Zhang, Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs, Small 6 (2010) 537–544. K.H. Liao, Y.S. Lin, C.W. Macosko, C.L. Haynes, Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts, Acs Appl. Mater. Interfaces 3 (2011) 2607–2615. A.D. Augst, H.J. Kong, D.J. Mooney, Alginate hydrogels as biomaterials, Macromol. Biosci. 6 (2006) 623–633.

590

L. Fan et al. / International Journal of Biological Macromolecules 93 (2016) 582–590

[31] V.R. Babu, M. Sairam, K.M. Hosamani, T.M. Aminabhavi, Preparation of sodium alginate-methylcellulose blend microspheres for controlled release of nifedipine, Carbohydr. Polym. 69 (2007) 241–250. [32] L.H. Fan, M. Cao, S. Gao, T. Wang, H. Wu, M. Peng, et al., Preparation and characterization of sodium alginate modified with collagen peptides, Carbohydr. Polym. 93 (2013) 380–385. [33] M. Ionita, M.A. Pandele, H. Iovu, Sodium alginate/graphene oxide composite films with enhanced thermal and mechanical properties, Carbohydr. Polym. 94 (2013) 339–344. [34] S. Hua, H. Ma, X. Li, H. Yang, A. Wang, pH-sensitive sodium alginate/poly (vinyl alcohol) hydrogel beads prepared by combined Ca2+ crosslinking and freeze-thawing cycles for controlled release of diclofenac sodium, Int. J. Biol. Macromol. 46 (2010) 517–523. [35] Z. Li, H.R. Ramay, K.D. Hauch, D. Xiao, M. Zhang, Chitosan-alginate hybrid scaffolds for bone tissue engineering, Biomaterials 26 (2005) 3919–3928. [36] Z. Dong, Q. Wang, Y. Du, Alginate/gelatin blend films and their properties for drug controlled release, Membr. Sci. 280 (2006) 37–44. [37] J. Zhang, Q. Wang, A. Wang, In situ generation of sodium alginate/hydroxyapatite nanocomposite beads as drug-controlled release matrices, Acta Biomater. 6 (2009) 445–454. [38] Q. Wang, X. Hu, Y. Du, J.F. Kennedy, Alginate/starch blend fibers and their properties for drug controlled release, Carbohydr. Polym. 82 (2010) 842–847. [39] D.C. Marcano, D.V. Kosynkin, J.M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, et al., Improved synthesis of graphene oxide, Acs Nano 4 (2010) 4806–4814.

[40] B. Rihova, T. Etrych, M. Pechar, M. Jelinkova, M. Stastny, O. Hovorka, et al., Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes, J. Control. Release 74 (2001) 225–232. [41] H. Wu, H. Shi, Y. Wang, X. Jia, C. Tang, J. Zhang, et al., Hyaluronic acid conjugated graphene oxide for targeted drug delivery, Carbon 69 (2014) 379–389. [42] X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang, Y. Chen, High-efficiency loading and controlled release of doxorubicin hydrochloride on graphene oxide, J. Phys. Chem. C 112 (2008) 17554–17558. [43] R. Venkatesan, A. Pichaimani, K. Hari, P.K. Balasubramanian, J. Kulandaivel, K. Premkumar, Doxorubicin conjugated gold nanorods: a sustained drug delivery carrier for improved anticancer therapy, J. Mater. Chem. B 1 (2013) 1010–1018. [44] W. She, N. Li, K. Luo, C. Guo, G. Wang, Y.Y. Geng, et al., Dendronized heparin-doxorubicin conjugate based nanoparticle as pH-responsive drug delivery system for cancer therapy, Biomaterials 34 (2013) 2252–2264. [45] Y. Tang, T.J. Lei, R. Manchanda, A. Nagesetti, A. Fernandez-Fernandez, S. Srinivasan, et al., Simultaneous delivery of chemotherapeutic and thermal-optical agents to cancer cells by a polymeric (PLGA) nanocarrier: an in vitro study, Pharm. Res. 27 (2010) 2242–2253. [46] C.H. Lu, C.L. Zhu, J. Li, J.J. Liu, X. Chen, H.H. Yang, Using graphene to protect DNA from cleavage during cellular delivery, Chem. Commun. 46 (2010) 3116–3118.