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Simultaneous Reduction and Surface Functionalization of Graphene Oxide for Hydroxyapatite Mineralization Hongyan Liu,† Pinxian Xi,† Guoqiang Xie,† Yanjun Shi,† Fengping Hou,† Liang Huang,† Fengjuan Chen,† Zhengzhi Zeng,*,† Changwei Shao,‡ and Jun Wang‡ †

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Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China ‡ State Key Laboratory of Advanced Ceramic Fibers & Composites, College of Aerospace and Materials Engineering, National University of Defense Technology, Changsha 410073, China S Supporting Information *

ABSTRACT: In this study, we present the synthesis of reduced graphene oxide/hydroxyapatite (RGO/HA) hybrid materials by an environmental-friendly route. Graphene oxide (GO) was first simultaneously reduced and surface functionalized by one-step oxidative polymerization of dopamine (PDA). The bioinspired surface was further used for biomimetic mineralization of hydroxyapatite. When incubated in a simulated body fluid (SBF), the PDA layer enabled efficient interaction between the RGO surface and the mineral ions to improve the bioactivity, promoted the formation of the HA nanoparticles. A detailed structural and morphological characterization of the mineralized composite was performed. The HA-based hybrid materials exhibited no cytotoxic effect on L929 fibroblast cells, showing potential capacity of being a scaffold material for bone tissue regeneration and implantation. This facile strategy also can be a useful platform for other RGO-based nanocomposites.

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INTRODUCTION The synthesis of biocompatible materials to be used in medicine has acquired a great deal of importance in material science.1 Hydroxyapatite (Ca10(PO4)6(OH)2, HA) is the major component of natural bone tissues. It exhibits excellent biocompatibility and bioactivity with human tissues;2,3 as a result, it is currently widely used in various forms and shapes in bone and tissue engineering. Nonetheless, the poor tensile strength and fracture toughness compared with natural bone limit its applications for replacing various parts of the bone systems. To combat this problem, HA has been strengthened with a number of other materials, such as polymers,4 silicon carbide,5 alumina (Al2O3),6,7 titanium (Ti), or Ti alloys.8,9 However, an ideal reinforcement material would impart mechanical integrity to the composite without diminishing its bioactivity. Carbonaceous materials with high chemical inertness are widely recognized to exhibit good biocompatibility.10 A recently discovered carbon-based two-dimensional (2D) nanostructure, known as graphene, is particularly attractive. It has been shown that graphene induces no obvious toxic effects in vivo.11 It has the large aspect ratio, high mechanical strength, and superior electrical conductivity,12,13 as a result, has the potential to strengthen and toughen HA without offsetting its bioactivity. In the biomedical processes, it should be mentioned that graphene has been used along with chitosan in composites that were reported to show a 200 times increase in the elasticity modulus compared to undoped chitosan.14 It is also reported that © 2012 American Chemical Society

graphene papers promote adhesion and proliferation of osteoblasts.15,16 In addition, the superior electrical conductivity of graphene can be used to stimulate osteoblasts electrically during tissue formation.17 Since graphene offer distinct advantages,18 they have emerged as attractive precursors for large-scale synthesis of HA-based composite materials for bone replacement. It remains difficult to accomplish a perfectly homogeneous composition of the graphene−HA composites by traditional mixing technology. Recently, Zhu et al. reported that HA can be reinforced with graphite nanosheets through spark plasma sintering process.19 However, this approach needs high temperature, and the hierarchical structures inside composites are difficult to control. In contrast, biomimetic mineralization, that is, the synthesis of organic−inorganic hybrid materials resembling materials of biological origin has attracted tremendous interest. These bioinspired processes have promoted a great breakthrough on the design of advanced functional materials.20−23 A series of recent studies have extended the bioinspired mineralization process to induce the formation of bone-like hydroxyapatite.24−26 Park et al. reported a polydopamine-assisted hydroxyapatite formation route that can be applied to virtually any type and morphology of scaffold materials.27,28 Additionally, GO nanosheets can be readily Received: October 25, 2011 Revised: December 7, 2011 Published: January 3, 2012 3334

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Figure 1. UV−vis (A) spectra for GO (red), RGO-PDA (blue), and RGO-PDA after removing the PDA layer (black); FTIR (B) spectra for GO (red), PDA (green), RGO-PDA (blue), and RGO-PDA after removing the PDA layer (black); XPS spectra of C1s of GO (C) and RGO-PDA (D). The inset of panel A are photographs of the aqueous colloidal dispersions of GO and RGO-PDA.

membrane filter (0.45 μM pore size, China), washed with deionized water several times, and dried under vacuum. For the mineral coating of HA, each sample was immersed in 1.5× SBF (pH 7.53), in 50 cm3 Nalgen polypropylene centrifugation tubes. The centrifugation tubes were incubated in a thermostatic bath at 37 °C for 2, 7, and 14 days. The composition of 1.5× SBF is as follows: Na+, 213.0 mM; K+, 7.5 mM; Mg2+, 2.25 mM; Ca2+, 3.75 mM; Cl−, 221.7 mM; HCO3−, 6.3 mM; HPO42−, 1.5 mM; SO42−, 0.75 mM. After a thorough washing with water consisting of several centrifugation and ultrafiltration cycles, the product was used for analysis. Characterization. The electronic absorption spectra were recorded on a Varian Cary 100 ultraviolet−visible (UV−vis) spectrophotometer in the wavelength range of 200−800 nm. Fourier transform infrared (FTIR) spectra (4000−400 cm−1) were determined with KBr disks on a Therno Mattson to analyze the surface chemical functionality of the GO. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a PHI-5702 multifunctional spectrometer using Al Kα radiation. The microscopic features were investigated using a Tecnai G2 F30 (FEI) transmission electron microscope at an acceleration voltage of 300 kV. Samples were prepared by placing a drop of a dilute alcohol dispersion of the products on the surface of a copper grid. X-ray diffractometer (XRD) (D/ max-2400pc, Rigaku, Japan) and an energy dispersive X-ray spectrometer (EDX) attached to the TEM were used to investigate the mineral phase and elemental composition of obtained materials. XRD measurements were performed with Cu Kα radiation (λ = 1.54178 Å), with the operation voltage and current at 40 kV and 60 mA, respectively. The 2θ range was from 5 to 90° in steps of 0.02°. Raman spectra were obtained using a confocal microprobe Raman system (Renishaw, RM2000). The thermal characteristics of GO and RGO-PDA were measured by thermogravimetric analysis

reduced by dopamine and the chemical functionalization allows the graphene nanosheets to disperse more easily and enhance interfacial bonding.29 In view of the advantageous properties of the above methods, we developed a simple and efficient route for synthesis of the RGO/HA hybrid materials. The simultaneous surface modification and reduction of GO by polymerization of dopamine resulted in PDA-coated, reduced graphene oxide (RGO-PDA). The bioinspired surface induced the formation of HA when incubated in a SBF under physiological conditions. It was further confirmed that RGO-PDA exhibited a much higher capability for HA mineralization over pristine GO. The biocompatibility of the mineralized RGO-PDA nanomaterials was evaluated using MTT assay.

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EXPERIMENTAL SECTION Materials. Graphite (500 meshes) was purchased from Acros Organic Company. Dopamine-hydrochloride (98.5%) was purchased from Alfa Aesar. All other reagents and solvents were obtained from Guangfu Chemical Co. (Tianjin, China) and used as received without further purification. GO Functionalization for Hydroxyapatite Formation. The aqueous dispersion of GO sheets was performed in accordance with the literature procedure.30 A PDA layer on the surface of GO was formed by an oxidative polymerization of dopamine-hydrochloride. Aqueous suspensions of graphene oxide (10 mL of a 1 mg mL−1 solution) were prepared by 30 min sonication of GO (Ultrasonic cleaner, KQ5200, China). PDA coating was performed by mixing these GO suspensions (0.4 mg mL−1) with a buffer solution (2 mg mL−1 of dopaminehydrochloride in 10 mM Tris buffer, pH 8.5) at room temperature. The mixture was homogenized by a sonic dismembrator for 1 h and then magnetically stirred for an additional 11 h. The sample was collected by a cellulose 3335

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Figure 2. FTIR spectra of RGO-PDA (A) and GO (B) after incubation for 0 (a), 2 (b), 7 (c), and 14 (d) days in SBF.

(TGA ZRY-2P, China) at a heating rate of 10 °C min−1 under a nitrogen atmosphere. All pH measurements were made with a pH-10C digital pH meter. Assessment of Biocompatibility. Cell viability was determined in L929 mouse fibroblast cell lines. The biocompatibility was evaluated using the MTT assay. Basically, cells were plated at a density of 1 × 105 in 96-well plates 24 h prior to the exposure to the different materials. Cells were incubated in the growth medium containing different concentrations of GO, RGO-PDA, or RGO/HA for 48 h. After treatment, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, 5 mg mL−1 in PBS) was added into each well. After 4 h of incubation, culture supernatants were aspirated, and purple insoluble MTT product was redissolved in 150 μL of DMSO in 10 min. The concentration of the reduced MTT in each well was determined spectrophotometrically by subtraction of the absorbance reading at 630 nm from that measured at 570 nm using a microplate reader. Cell viabilities were presented as the percentage of the absorbance of materials treated cells to the absorbance of control cells.

The simultaneous surface functionalization and reduction of GO were also confirmed by FTIR and XPS spectroscopy. FTIR spectrum of RGO-PDA exhibits typical peaks for both GO and PDA (Figure 1B). The peak at 1443 cm−1 corresponds to the C−C vibration of the benzene ring moiety. The peaks at 1506 cm−1 and 1285 cm−1 were assigned to the N−H shearing vibration of the amide group and the phenolic C−OH stretching vibration, respectively,34 confirmed the polymerization of dopamine and presence of the coated PDA layer on RGO. The disappearance of the CO peak at 1727 cm−1 compared to GO provided a solid indication of GO reduction. After treatment with 1 M NaOH to remove the coated PDA layer, the FTIR showed no peaks at 1506 and 1727 cm−1. In addition, the C−OH stretching peak at 1220 cm−1 and the C− O stretching peak at 1046 cm−1 of GO were not detected, strongly indicating that GO was reduced by one-step PDA functionalization (Figure 1B). Meanwhile, the reduction of graphene oxide was characterized by XPS. The C1s band obtained from GO (Figure 1C) can be fitted into three components. The main peak at 284.8 eV is due to graphitic sp2 carbon atoms, and the small ones located at 286.8 and 288.1 eV correspond to carbon atoms connecting with oxygenate groups.35 The C1s band of RGO-PDA (Figure 1D) reveals that, compared to GO (Figure 1C), the oxygenate species is substantially removed, indicating that the PDA-functionalized GO was chemically reduced.36 The mineralization was induced by soaking each composition in SBF designed to have ion concentrations approximate to those of human blood plasma.37 The nucleation of HA on the bioinspired surface was monitored by FITR. Figure 2A showed the FTIR spectra of the RGO-PDA after mineralized for 0, 2, 7, and 14 days. Weak PO43− bands appeared after incubated for 7 days. After further incubation for 14 days, strong PO43− bands were observed. Among these bands, the band at 962 cm−1 is due to the symmetric stretching mode ν1 (PO43−), 1109 and 1037 cm−1 to the vibration mode ν3 (PO43−), 602 and 566 cm−1 to bending modes ν4 (PO43−).38 In order to observe RGOPDA’s capacity to induce HA formation, we compared GO’s degree of mineralization to that of RGO-PDA. It is worth pointing out that the characteristic bands of HA were not observed (Figure 2B), indicating that the PDA layers not only enabled efficient interaction between the RGO surface and mineral ions but also promoted the formation of the HA.

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RESULTS AND DISCUSSION RGO-PDA nanocomposite was produced by mixing an aqueous suspension of GO platelets with a solution of dopaminehydrochloride (2 mg mL−1, 10 mM Tris buffer, pH 8.5). After magnetically stirring for 11 h at room temperature, the yellow brown GO suspension turned into a black solution (inset in Figure 1A). The products were collected and dried under vacuum. The reduction of GO by PDA was demonstrated by the UV−vis absorption spectra (Figure 1A). A successful reduction of GO to RGO by PDA was verified by the sample’s optical absorbance (Figure 1A): a 231 nm absorbance peak characteristic of GO disappeared and a new peak at 280 nm was detected, which is a characteristic absorption of PDA due to the presence of catechols.31 The presence of a PDA layer on the RGO surfaces was further evaluated by the alkaline etching method (1 M NaOH).32 After the removal of PDA, the peak at 280 nm disappeared, while a broad peak with a maximum absorption at 265 nm was observed, suggesting that GO was probably reduced by the release of electrons during the oxidative polymerization of dopamine-hydrochloride.33 3336

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Figure 3. TEM images of GO (A), RGO-PDA (D), GO (B, C), and RGO-PDA after 14 days mineralization (E, F); HRTEM image (G, H) and selected area electron diffraction patterns (I) of RGO/HA nanocomposite.

FITR results. The appearance of lattice orientation of nanoparticles in the HRTEM image in Figure 3G also indicated the formation of crystals, and the lattice fringes with interplanar distance of 2.816 A (Figure 3H) are assigned to the (211) plane of the HA hexagonal structure. The selected area electron diffraction (SAED) patterns from the hybrid materials are shown in Figure 3I; the diffraction dots show the (100) and (110) plane of RGO, the diffraction ring which overlapped with the dots show characteristic phase of the HA hexagonal phase, indicating that the HA are amorphous. Thus, we can conclude that the formation of a PDA layer on the surface of RGO enabled efficient interaction between the RGO and mineral ions, which highly accelerated the formation of HA nanoparticles when incubated in SBF. This process is understandable because polydopamine is a mimic of the specialized adhesive foot protein, Mefp-5 (Mytilus edulis foot protein-5),40 in which the catechol moiety strongly binds to various metal ions.41−43 On the basis of our experimental results, a scheme has been presented to describe such an in situ formation of HA nanoparticles induced by PDA, as illustrated in Scheme 1. In order to confirm that the product is physiological HA, elemental composition of the obtained materials was investigated with XPS and EDX. XPS spectrum of RGO-PDA exhibited C (1s), N (1s), and O (1s) peaks (Figure 4A), indicating the formation of a PDA layer on RGO. After mineralization, a Ca (2p) peak that corresponds to Ca ions in

Further evidence for this conjecture can also be supported by morphological analysis. The changes of microstructure were analyzed by TEM. Figure 3A,D shows the TEM images of the obtained GO before and after PDA coating. At lower magnification, the GO sheet appears as a transparent gray film. The average thickness is about 1.26 nm, and the lateral dimension is about 800 nm, as shown in the AFM measurements (Figure S1, Supporting Information). The scanned height profile is a characteristic thickness from a single layer GO sheet.39 After being functionalized and reduced by PDA, the RGO sheet dispersed well and exhibited the same microscopic features with the pristine GO (Figure 3D), suggesting that the RGO sheets were uniformly covered with the PDA layer by the in situ oxidative polymerization of dopamine. After 14 days of immersion in SBF, both nanoparticles and sheet-like RGO were observed (Figure 3E). From Figure 3F, it can be clearly seen that the grain size of the obtained HA nanoparticles was around 20 nm, evenly formed on the RGO-PDA surface. By comparing the images of the small nanoparticles, we can find that some of the nanoparticles are brighter than the ones that seem to be enveloped by a thin film. This is due to the fact that the PDA layers are coated on both sides of RGO, which induced the formation of HA nanoparticles on both sides. However, no obvious changes were observed on the surface of pristine GO sheet after immersion (Figure 3B,C), in accordance with the 3337

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Scheme 1. Two-Step Method to Synthesize RGO/HA Nanocomposites

Figure 4. XPS spectra of RGO-PDA (A), RGO/HA (B), and EDX spectra acquired from RGO-PDA (C) and GO (D) after 14 days of mineralization.

HA appeared (Figure 4B). This peak was more clearly seen in the high resolution spectrum in inert of Figure 4B. For the EDX analysis, significant Ca and P peaks observed in the spectrum of the mineralized RGO-PDA (Figure 4C) can be attributed to the contribution from the HA. However, only a trace amount of calcium (1.16%) was identified in the samples incubated with pristine GO, besides the C and Cu that

correspond to GO and the TEM grid, respectively (Figure 4D). Chemical composition of the RGO/HA nanocomposite showed a molar ratio of Ca/P = 1.56, which is consistent with that of previous studies of biological apatites and bioinspired mineral coatings.44 According to the literature,45 human biological apatite is always calcium-deficient HA nanoparticles that contains a few percent carbonate, acid 3338

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phosphate, sodium, and magnesium ions due to the continuous contact with a flow of trace ions.46 In this regard, the HA nanoparticles formed with RGO-PDA have a lower Ca/P ratio than pure HA, the chemical composition is much closer to human biological apatite, so they are probably appropriate for applications in the fields of bone tissue engineering. The phase structure and purity of the RGO/HA hybrids was investigated by powder XRD. Figure 5 displays the XRD

Figure 6. Raman spectra of natural graphite (a), GO (b), RGO-PDA (c), and RGO-PDA after 14 days of mineralization (d).

1.1, which suggests the small size of the in-plane sp2 domains of RGO.51,52 After the formation of HA nanoparticles, the ID/IG increased to 1.3. This enhancement could be ascribed to the presence of HA between the RGO sheets. Typically, the Raman spectrum also shows a characteristic peak at 961 cm−1, which is attributed to the symmetric stretching mode ν1 (PO43−).53 Both XRD and Raman analysis demonstrated that the mineral coating belongs to pure HA phase. The content of each component in the prepared nanocomposite can be determined facilely with the TGA technique. Figure 7 shows the representative TGA curves of the GO and

Figure 5. XRD patterns of natural graphite (a), GO (b), and RGOPDA after 14 days of mineralization (c).

patterns of the as-synthesized composite. For GO, the most intense peak at around 2θ = 9.9° corresponds to the (002) reflection. The diffraction patterns of RGO/HA shows characteristic lines of the HA hexagonal phase (JCPDS card, no. 09-0432). The peaks at 31.6°, 32.0°, 33.5°, and 25.9° correspond to the (211), (300), (202), and (002) reflection of HA, while the (002) reflection peak of GO almost disappeared. The peak areas in the XRD spectrum of mineral coated microspheres are broader than the hydroxyapatite power, and this may be due to the small crystal size of the mineral deposited on the RGO-PDA surface, which is also demonstrated by TEM observation (Figure 3E). Raman spectroscopy is a powerful nondestructive tool to characterize carbonaceous materials, particularly for distinguishing ordered and disordered crystal structures of carbon. In our research, Raman spectroscopy was also used to characterize the RGO-PDA before and after mineralization process (Figure 6). For comparison, the Raman spectrum of graphite and GO are also shown. The peak at about 1587 cm−1 (G band), corresponding to an E2g mode of graphite, is usually related to the vibration of the sp2-bonded carbon atoms in a 2dimensional hexagonal lattice, while the peak at about 1325 cm−1 (D band) is an indication of disorder in the Raman of the GO, originating from defects associated with vacancies, grain boundaries,47,48 and amorphous carbon species.49 The intensity ratio of the D to G band (ID/IG) is generally accepted, reflecting the graphitization degree of carbonaceous materials and the defect density.50 ID/IG of pristine GO is about 0.98. For RGO-PDA, the G band was broadened and shifted upward to 1601 cm−1, whereas the intensity of the D band at 1325 cm−1 increased substantially. The PDA coating increased the ID/IG to

Figure 7. TGA curves of GO (a), RGO-PDA (b), and RGO-PDA after 14 days of mineralization (c).

RGO/HA nanocomposite. GO is thermally unstable and starts to lose mass upon heating below 100 °C due to the adsorbed water. There are two significant drops in mass around 200 and 510 °C (Figure 7, trace a). The former is assigned to the evolution of CO and CO2 from GO caused by the destruction of oxygenated functional groups, and the latter is attributed to the combustion of the carbon skeleton of GO.54−56 The weight loss of GO at 200 °C was about 40.2 wt %, which is due to the evaporation of interlamellar water and the decomposition of labile oxygen. By comparison, it was a 24.5 wt % loss at 200 °C for the RGO-PDA (Figure 7, trace b), which was much lower than that of the GO, indicating a decreased amount of 3339

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CONCLUSIONS In summary, we have developed a simple approach for synthesizing RGO/HA hybrid materials by bioinspired mineralization. The structure of the obtained composite was characterized by FITR, XPS, XRD, Raman, TGA, and morphology analyses. The results showed that GO was reduced and uniformly coated by a thin PDA layer through a spontaneous polymerization of dopamine. The functionalized RGO surface was further decorated randomly with HA nanoparticles on both sides by the mineralization in SBF. During these processes, the presence of dopamine (1) reduced GO to RGO, (2) improved the interaction between RGO and calcium ions, and (3) promoted the nucleation of HA nanoparticles under physiological conditions. The biocompatibility of the prepared materials was evaluated using the MTT assay. The RGO-PDA nanocomposite exhibited a high capacity to accelerate HA mineralization, which suggests that RGO-PDA is a promising material for potential use as a bone scaffold. To the best of our knowledge, our study is the first demonstration of several advantages of utilizing PDA coating surface chemistry to RGO for bioinspired mineralization. Further exploration of this route to construct hybrid materials is being explored in our laboratory to improve the technology for RGO-based materials synthesis.

oxygenated functional groups, confirming the chemical reduction of GO by the PDA coating.56 The sharp mass loss at approximately 130 °C is caused by the decomposition of the PDA layer. No significant mass loss is detected when this material is heated up to 650 °C, resulting in decomposition of about 92.2 wt %. For the RGO/HA hybrid materials, it was only a 49.3 wt % loss when heated up to 650 °C (Figure 7, trace c), indicating the formation of thermally stable HA. According to the mass loss of RGO-PDA in RGO/HA composite, it is estimated that about 42.9 wt % of HA was deposited on the surface of functionalized RGO. Biocompatibility is a prerequisite for the use of graphene based materials in biological or medical field. Thus, the biocompatibility of the prepared composite was further investigated on L929 fibroblast cells. The cell viability was evaluated using the MTT assay. We added solutions of GO, RGO-PDA, and RGO/HA to the culture media at different concentrations (i.e., 1, 5, 10, and 20 μg mL−1) to investigate the effects of the amount of materials. Figure 8 shows the results

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ASSOCIATED CONTENT

S Supporting Information *

AFM measurements and bright field microscopy images of cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 931 8610877. Fax: +86 931 8912582. E-mail: [email protected] or [email protected].

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ACKNOWLEDGMENTS This study was supported by the Foundation of Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and the NSFC (20171019). P.X. is thankful for support from the Fundamental Research Funds for the Central University (Lzujbky-2012).

Figure 8. MTT assay of L929 cells cultured for 48 h in media containing pristine GO, RGO-PDA, and RGO/HA.

that indicate that the viability of the cells with the various materials were similar to the control cells, and more than 95% L929 cell viability was observed under varying concentration range. In addition, the viability of cells was not significantly dependent on the different concentrations of materials; in the case of the high concentrations, the cells showed an increase in mitochondrial activity, up to 120% (for RGO-PDA) and 116% (for RGO/HA), respectively, indicating that all three kinds of materials exhibit no cytotoxicity. The increased measured viability seen with increasing concentrations of particles seems to be caused by particles activating cells. The bright field microscopy images of cells grown in the presence and absence of the RGO/HA hybrid material, thus confirming biocompatibility of these structures, are seen in Figure S2 of the Supporting Information. Therefore, the combination of the RGO sheets with HA nanoparticles formed as a nanocomposite with low undesired cytotoxic effects and have major potential applications in nanomedicine. Moreover, the presence of carbon graphene structures that can be easily functionalized with various biomolecules,57 including growth factors, proteins, or drugs, gives these hybrid materials great potential in the development of novel functional scaffolds for fast tissue formation.

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dx.doi.org/10.1021/jp2102226 | J. Phys. Chem. C 2012, 116, 3334−3341