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One-Step In Situ Biosynthesis of Graphene Oxide–Bacterial Cellulose Nanocomposite Hydrogels Hongjuan Si, Honglin Luo, Guangyao Xiong,* Zhiwei Yang, Sudha R. Raman, Ruisong Guo, Yizao Wan* Graphene oxide–bacterial cellulose (GO/BC) nanocomposite hydrogels with well-dispersed GO in the network of BC have been successfully developed using a facile one-step in situ biosynthesis by adding GO suspension into the culture medium of BC. During the biosynthesis process, the crystallinity index of BC decreases and GO is partially reduced. The experimental results indicate that GO nanosheets are uniformly dispersed and well-bound to the BC matrix and that the 3D porous structure of BC is sustained. This is responsible for efficient load transfer between the GO reinforcement and BC matrix. Compared with the pure BC, the tensile strength and Young’s modulus of the GO/BC nanocomposite hydrogel containing 0.48 wt% GO are significantly improved by about 38 and 120%, respectively. The GO/BC nanocomposite hydrogels are promising as a new material for tissue engineering scaffolds.

1. Introduction Two-dimensional carbon-based nanomaterials, including graphene oxide (GO) and graphene, have been considered as candidates for biomedical applications such as artificial scaffolds, sensors, cell labeling, bacterial inhibition, and drug delivery.[1–3] Graphene family materials have also been reported to support and accelerate the adhesion, proliferation, and differentiation of various mammalian cells.[4–7] In particular, GO’s promise as a material for biological applications is due to its good dispersibility

Dr. H. Si, Dr. H. Luo, Dr. Z. Yang, Prof. R. Guo, Prof. Y. Wan School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072, China E-mail: [email protected] Dr. H. Si, Dr. H. Luo, Prof. G. Xiong School of Mechanical and Electrical Engineering, East China Jiaotong University, Nanchang 330013, China E-mail: [email protected] Dr. S. R. Raman Department of Community and Family Medicine, Duke University, NC, USA

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in water and physiological environments, excellent biocompatibility, amphiphilicity, surface functionalizability, surface-enhanced Raman scattering (SERS) property, and fluorescence quenching ability.[8,9] Recently, the incorporation of GO into hydrogels has resulted in improved mechanical properties and cell compatibility.[10] A common strategy to harness the useful properties of graphene and its derivatives is to form nanohybrids or nanocomposites with other materials, in particular polymers.[11] Polymeric materials such as gelatin,[12] poly(vinyl alcohol),[13,14] chitosan,[15] and polyacrylamide[16] have been used to form nanocomposites with GO. In addition, a natural nanofibrous cellulosic material produced by the bacteria Acetobacter xylinum (A. xylinum), namely bacterial cellulose (BC), was also used to form a nanocomposite with GO.[17] However, the intrinsically continuous 3D structure of BC was disrupted during the mechanical mixing and thus not sustained in the GO/BC nanocomposite. It is believed that the continuous 3D structure of BC distinguishes it from other natural polymers. Accordingly, finding an alternative method to fabricate a new GO/BC nanocomposite that maintains the intrinsic structure of BC is of particular importance in the field of tissue engineering and regenerative medicine.

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DOI: 10.1002/marc.201400239

One-Step In Situ Biosynthesis of Graphene Oxide–Bacterial Cellulose Nanocomposite Hydrogels

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Another issue in the processing of graphene-based nanocomposites is the dispersion of graphene materials including GO. The intrinsic van der Waals interactions between layers of graphene easily results in agglomeration, which will inevitably degrade the reinforcement effectiveness of nanofillers. In order to obtain a uniform dispersion of nanosheets in a polymer matrix and achieve high mechanical performance, chemical functionalization is usually needed to induce chemical bonding between graphene materials and polymer matrix.[18] However, in the case of GO/BC composite hydrogels, a uniform dispersion may be obtained even without chemical modification due to the following two characteristics. First, hydrophilic GO can be well dispersed in water and thus, also well dispersed in the culture medium of BC. Second, the hydrophilic functional groups, such as OH, on the surface of GO may form a hydrogen bonding with the OH groups on BC. Thus, when GO is added to the culture medium and is involved in the biosynthesis process, good dispersion of GO in BC is expected. Here, we report the convenient and environmentally friendly one-step in situ biosynthesis of well-dispersed GO/BC nanocomposite hydrogels. It is expected that the GO/BC will be mechanically strong, retain the biocompatibility of GO and BC and retain the advantageous 3D structure of BC, and is thus attractive in the fields of tissue engineering scaffolds and drug delivery.

2. Experimental Section 2.1. Materials Commercially available aqueous dispersion of GO with a concentration of 0.25, 0.5, and 0.75 mg mL−1 was purchased from Nanjing XFNANO Materials Technology Co. Ltd., China. According to the supplier, the GO nanosheets had a layer thickness of 0.7–1.2 nm and a lateral dimension of 200 nm to 10 μm. Reagents for BC production included yeast extract, tryptone, disodium phosphate (Na2HPO4), and acetic acid. All reagents were used without further purification.

2.2. Preparation of BC and GO/BC via In Situ Biosynthesis

GO-dispersed medium. The flasks were incubated under static conditions at 30 °C for 10 d. For comparison purpose, the pristine BC was prepared under the same conditions and also incubated for 10 d. The harvested GO/BC and BC pellicles were purified by soaking in deionized water at 90 °C for 2 h followed by boiling in a 0.5 M NaOH solution for 15 min. The pellicles were then washed several times with deionized water until reaching a neutral pH. The samples were either stored in deionized water at 4 °C for mechanical testing or freeze-dried for 24 h for other characterizations. The GO/BC hydrogels prepared in this work had a GO content of 0.19, 0.29, and 0.48 wt%, respectively, and the GO/BC sample with 0.29 wt% GO was characterized unless otherwise indicated.

2.3. Characterization The morphology of freeze-dried BC and GO/BC samples was observed using a field-emission scanning electron microscope (FE-SEM, Nano 430, FEI, USA) and transmission electron microscope (TEM, Philips Tecnai G2 F20) operating at 200 kV. The Raman spectra of pristine GO and GO/BC were recorded using a Jobin Yvon HR-800 spectrometer with an excitation wavelength of 633 nm. X-ray diffraction (XRD) analysis was conducted to determine the crystalline structure of BC and GO/BC using a Rigaku D/max 2500 X-ray diffractometer. Cu-Kα radiation was utilized (λ = 0.154 nm) to scan the samples from 5° to 30° with a scan speed of 2° min−1. The data were obtained using the MDI/JADE6 software package attached to the Rigaku XRD instrument. The crystallinity index (CI) was calculated by the Segal’s method using the equation CI = (I200−Iam)/I200, where I200 is the intensity at a 2θ value of 22 and Iam denotes the intensity of the baseline at a 2θ value of 18°.[21] Static tensile testing was conducted in accordance with ASTM D 638-98 Type IV specimens by using a universal material testing instrument (Testometric M350–20KN, UK) under ambient temperature and humidity (20 °C, 65% RH) with a cross-head speed of 10 mm min−1. Rectangular specimens with nominal dimensions of 30 × 10 × 2 mm3 were used for tensile tests. The samples were tested in their original hydrogel state. At least five specimens were tested for each sample, and the averages and standard deviations were reported. The electrical conductivity of air-dried BC and GO/BC films was measured using a Keithley 2635 sourcemeter unit as previously reported.[17]

3. Results and Discussion

[19,20]

In accordance to our previous work, the bacterial strain, A. xylinum X-2, was used to produce BC and GO/BC hydrogels. The culture medium was sterilized at 121 °C for 30 min prior to use. The medium for the pristine BC was composed of 2.5% (w/v) glucose, 0.75% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) disodium phosphate (Na2HPO4), and the pH was adjusted to 4.5 by acetic acid. To prepare the GO/BC nanocomposite hydrogels, a GO-dispersed culture medium was prepared. 20 mL GO suspension was added to 200 mL culture medium followed by intense stirring for 60 min. The seed broth (2 mL) was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of the

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3.1. Morphology of BC, GO, and GO/BC Nanocomposite SEM images of pristine BC, GO, and GO/BC samples are shown in Figure 1. Pristine BC has a typical 3D network structure and interconnected pores as is clearly observed in Figure 1a. Additionally, the morphology of GO sheets is shown in Figure 1b, which exhibit a typical folded feature. Figure 1c–e shows several typical SEM images of GO/ BC samples. All the SEM images show a 3D porous web-like

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Figure 2. TEM images of a) GO and b) GO/BC, and HRTEM image of c) GO/BC.

3.2. Structure of GO, BC, and GO/BC

Figure 1. SEM images of a) BC and b–d) GO/BC nanocomposite.

structure. Figure 1c reveals that GO nanosheets are dispersed uniformly within the BC matrix. Notably, the 3D fibrous network and porous structure are still sustained after the incorporation of GO nanosheets. This characteristic could facilitate the movement of cells towards the core of the GO/BC scaffold. Figure 1d shows that GO nanosheets are well bonded to BC fibers. The strong adhesion between GO nanosheets and BC fibers is beneficial to improve the mechanical properties of the GO/BC composites. Furthermore, it seems that GO nanosheets are bound by BC nanofibers (Figure 1e). SEM observation confirms that a good contact between GO and BC is formed by the in situ biosynthesis likely due to the strong interaction between OH groups on BC and GO.[22,23] TEM observation was used to further observe the morphology of GO in a BC network. Figure 2 shows the TEM images of GO and GO/BC. Note that GO nanosheets have a typical crumpled silk wave-like morphology (Figure 2a). Figure 2b reveals that the BC and GO nanosheets are tightly bound together, forming a web-like structure, indicating the good compatibility between GO and BC matrix. Figure 2c shows the crystal lattices of GO, indicating that GO keeps its crystal structure after biosynthesis process.

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The crystalline structure of GO, BC, and GO/BC nanocomposite was characterized by XRD (Figure 3a). The observed XRD spectrum of BC is expected, with three peaks located — at 14.6°, 16.8°, and 22.8°, corresponding to (11 0), (110), and (200) planes of cellulose I, respectively. Similarly, as expected, GO has a sharp peak at around 9.1°. It should be noted that the sharp peak of GO is not observed in the GO/ BC composite, likely because the amount of GO is not sufficient to be measured. The disappearance of GO’s peak was observed in other GO composite systems and was believed to be an indication of uniform dispersion of GO in the matrix.[24,25] In order to evaluate the effect of the addition of GO in the culture medium on the crystal structure, the crystallinity index (CI) of the BC materials produced under the two different conditions was calculated. The CI values calculated based on the XRD profiles (90% for pristine BC versus 85% for GO/BC) indicate that adding GO in the culture medium reduces the crystallinity of BC. Previous studies have noted the decrease of crystallinity caused by addition of foreign substances, for example, adding carbon nanotubes into the culture medium of BC.[17,26,27] It was believed that the variation of viscosity of culture medium due to the addition of foreign supplement disturbed the movement of bacteria, which affected the crystallization process of nascent BC fibrils.[26,28] Therefore, the XRD results clearly suggest that adding GO nanosheets into the culture medium of BC significantly interferes with the crystallization of nascent BC nanofibrils.

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One-Step In Situ Biosynthesis of Graphene Oxide–Bacterial Cellulose Nanocomposite Hydrogels

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GO

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(rGO), during the process of being boiled with 0.5 M NaOH solution for 15 min and cleaned with 1 wt% NaOH for 2 d. The yeast extract in the culture medium may also be responsible for the partial reduction of GO since yeast extract can act as a reducing agent.[34] It should be pointed that the smaller increment (19%) in ID/IG (as compared to that observed by Fan et al.)[32] indicates that the extent of reduction is small. In order to test the hypothesis, the conductivity of air-dried BC and GO/BC films was measured and a substantial increase was observed (from 1.14 × 10−10 S cm−1 of BC to 1.24 × 10−9 S cm−1 of GO/ BC), which confirms the partial reduction of GO to rGO. Although the slight reduction of GO did not enable us to obtain GO/BC materials with high conductivity, this finding might present a new opportunity for modifying the properties of the GO/BC materials (such as electrical conductivity and mechanical performance) by controlling the degree of GO reduction.

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To evaluate the reinforcing effect of GO in the GO/BC nanocomposite hydrogels, tensile testing of neat BC and three GO/BC samples with a GO content of 0.19, 0.29, and 0.48 wt%, respectively, was conducted. The representative stress–strain curves are shown in Figure 4a. As clearly seen

Figure 3. a) XRD patterns of BC, GO, and GO/BC nanocomposite; b) Raman spectra of GO and GO/BC nanocomposite.

Figure 3b presents the Raman spectra of GO and GO/ BC. The Raman spectrum of the pristine GO shows a D-band at 1340 cm−1 and a G-band at 1580 cm−1, which correspond to the disordered structure of GO sheets and the first-order scattering of the E2g vibrational mode, respectively.[29,30] However, there is a significant peak shift (8 cm−1) for the G-band and no obvious shift for the D-band after the in situ biosynthesis process. Previous studies suggest that interaction with other materials might change the Raman peak frequencies.[31] Therefore, we hypothesize that the significant shift in the position of the G band is probably due to the interaction of GO with BC fibrils during the biosynthesis process. Further investigation is needed to clarify this hypothesis. Interestingly, it can be seen that the intensity ratio of D band and G band, ID/IG, of GO is about 0.99, which is comparable with that (0.96) reported in literature[32] and the ID/IG value of GO/ BC is 1.18, which is 19% higher as compared to GO. This increase in ID/IG of GO after the in situ biosynthesis may be due to the removal of some oxygen-containing functional groups on the surface of GO[32] since GO can be deoxygenated in alkaline solutions.[33] In other words, GO may have, to some extent, been converted to reduced GO

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Figure 4. Representative a) stress–strain curves and b) tensile properties of BC and GO/BC nanocomposites with varying GO content.

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in Figure 4b, the incorporation of 0.29 and 0.48 wt% GO nanosheets results in significant enhancements in the tensile properties despite the partial reduction of GO to rGO. The tensile strength and Young’s modulus of the obtained GO/BC are improved by 21% and 60%, respectively, even at a GO content as low as 0.29 wt%. When the GO content is further increased to 0.48 wt%, an improvement of 38% in tensile strength and an improvement of 120% in tensile modulus are noted. Previously, Liang et al.[35] added 0.7 wt% GO into a PVA matrix and a 76% increase in tensile strength and a 62% improvement in Young’s modulus were observed. Therefore, the improvements observed here are notable considering the fact that the GO content is low. These significant enhancements are attributed to the homogeneous dispersion of GO nanosheets and effective load transfer from the matrix to GO through the strong interfacial interactions. Notably, the elongation at break is not decreased markedly, which indicates that the small amount of GO does not lead to brittleness of BC hydrogel. The greatly improved mechanical properties of the GO/BC nanocomposite hydrogels at low GO contents suggest that these materials are attractive tissue engineering scaffolds. However, more extensive investigations regarding the optimization of GO content and its effect on the morphology, structure, mechanical properties, and biological behavior of the nanocomposite hydrogels should be carried out in further work.

4. Conclusions Strong GO/BC nanocomposite hydrogels have been prepared by adding GO suspension into the culture medium of BC. It is noted that the addition of GO reduces the crystallinity of BC and GO experiences a partial reduction to rGO due to the exposure to NaOH solutions and yeast extract in the culture medium. Importantly, GO nanosheets are well dispersed throughout the BC matrix and form a wellbonded 3D network structure. The GO/BC nanocomposite hydrogels with a GO content of 0.29 and 0.48 wt% exhibit significantly higher tensile strength and modulus over pristine BC owing to the uniform dispersion and interaction between GO and BC. These findings may serve as a foundation to the preparation of graphene-based nanocomposite hydrogels with a 3D interconnected porous structure.

Acknowledgements: This work is supported by the National Natural Science Foundation of China (grant No. 51172158) and the Science and Technology Support Program of Tianjin (grant No. 11ZCKFSY01700). Received: April 22, 2014; Revised: June 12, 2014; Published online: September 2, 2014 ; DOI: 10.1002/marc.201400239

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Keywords: cellulose; engineering

hydrogels;

nanocomposites;

tissue

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One-step in situ biosynthesis of graphene oxide-bacterial cellulose nanocomposite hydrogels.

Graphene oxide-bacterial cellulose (GO/BC) nanocomposite hydrogels with well-dispersed GO in the network of BC are successfully developed using a faci...
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