Article pubs.acs.org/Biomac
General and Biomimetic Approach to Biopolymer-Functionalized Graphene Oxide Nanosheet through Adhesive Dopamine Chong Cheng,†,‡ Shuang Li,§,∥ Shengqiang Nie,†,∥ Weifeng Zhao,† Hang Yang,† Shudong Sun,*,† and Changsheng Zhao*,†,‡ †
College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ‡ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China § School of Aeronautics and Astronautics, Shanghai Jiaotong University, Shanghai 200240, China S Supporting Information *
ABSTRACT: Graphene oxide (GO), reduced graphene oxide (rGO), and their derivatives are investigated for various biomedical applications explosively. However, the defective biocompatibility was also recognized, which restricted their potential applications as biomaterials. In this study, a facile biomimetic approach for preparation of biopolymer adhered GO (rGO) with controllable 2D morphology and excellent biocompatibility was proposed. Mussel-inspired adhesive molecule dopamine (DA) was grafted onto heparin backbone to obtain DA grafted heparin (DA-g-Hep) by carbodiimide chemistry method; then, DA-g-Hep was used to prepare heparin-adhered GO (Hep-a-GO) and heparin-adhered rGO (Hep-a-rGO). The obtained heparin-adhered GO (rGO) showed controllable 2D morphology, ultrastable property in aqueous solution, and high drug and dye loading capacity. Furthermore, the biocompatibility of the heparin-adhered GO (rGO) was investigated using human blood cells and human umbilical vein endothelial cells, which indicated that the as-prepared heparin-adhered GO (rGO) exhibited ultralow hemolysis ratio (lower than 1.2%) and high cell viability. Moreover, the highly anticoagulant bioactivity indicated that the adhered heparin could maintain its biological activity after immobilization onto the surface of GO (rGO). The excellent biocompatibility and high bioactivity of the heparin-adhered GO (rGO) might confer its great potentials for various biomedical applications.
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INTRODUCTION Graphene oxide (GO), reduced graphene oxide (rGO), and their derivatives are emerging materials exhibiting attractive electronic, catalytic, mechanical, optical, and magnetic properties, which endow GO and rGO with great potential in various applications ranging from energy storage to biomedical materials.1−4 Recently, much research has been carried out to explore the biomedical benefits of GO and rGO and reveal remarkable performances of GO and rGO in drug delivery, cellular imaging, bone tissues, stem cell differentiation, biosensors, and so on.5−11 However, GO or rGO also showed some fatal defects, such as the cytotoxicity,12,13 hemolysis,12 thrombogenic potential,14 and pulmonary toxicity,15 which limited their applications as biomaterials. Haynes et al.12 revealed that GO would cause serious hemolysis (as high as 90% hemolysis ratio) when exposed to human blood cells. Dash et al.14 found that GO would induce extensive pulmonary thromboembolism and platelet aggregation in mice. Fan and Huang et al.16 revealed that GO nanosheets had direct interaction with the cell membrane, leading to physical damage to the cell membrane. The GO-resulted hemolysis and © 2012 American Chemical Society
cytotoxicity might directly relate to its surfactant-like chemical structure with hydrophilic edges (ionized carboxyl groups) and a more hydrophobic basal plane (polyaromatic islands of unoxidized sp2 carbon skeleton),17,18 which could lead to strong interactions (electrostatic and hydrophobic interactions) between the amphiphilic GO and the lipid bilayer of cell membrane. Most recently, biopolymers such as polysaccharide,10,19−21 protein,16,22,23 and DNA24 have been used to alter the chemical structure of GO. When GO was decorated by negative or positive charged polymer,12,16 the original amphiphilic structure of GO became too hydrophilic to interact with or partition into lipid bilayers;25 then, the strong cell membrane interactions were greatly suppressed. Among them, the simple blending biopolymers as capping agents are the most widely used method to improve the biocompatibility of GO and rGO.16,19,20,24 However, in these studies, the biopolymer Received: September 24, 2012 Revised: November 8, 2012 Published: November 15, 2012 4236
dx.doi.org/10.1021/bm3014999 | Biomacromolecules 2012, 13, 4236−4246
Biomacromolecules
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loading ratio, the elution of the adsorbed biopolymer, and the 2D surface morphology were difficult to be controlled, which might lead to the aggregation and formation of microgel; moreover, heating treatment was usually used, which might affect the biological activity of the biopolymer, such as heparin or enzyme. Grafting hydrophilic polymers,8,26 chitosan,21 and proteins22,23 onto the surface or the edge of GO through the covalent chemistry has been taken for the preparation of water stable and biocompatible GO and rGO to overcome these shortcomings. Whereas these methods also suffer from some inherent drawbacks, for example, some hazardous reagents are used in the grafting process, which are difficult to be removed completely and may contaminate the obtained GO and rGO, moreover, many of these methods are complicated and require additional steps in the preparation process, which restrict its applications in biological and biomedical fields. Hence, producing water stable, green, and biocompatible GO and rGO nanosheets with tunable biopolymer loading ratio and maintained 2D morphology still remains a challenge because most of the unique properties of GO and rGO are only associated with the individual dispersed 2D sheets, which could provide a large specific surface area for the immobilization of biomolecules, fluorescent molecules, drugs, metal nanoparticles, and so on.26 Dopamine (DA), a mussel-adhesive-proteininspired molecule, has drawn strong interest because it could form irreversible covalent bonds to solid surfaces in alkaline aqueous solution.27−32 More importantly, by mimicking the adhesive protein of mussel byssus, it was found that DA could be grafted onto various biopolymers, and the as-prepared biopolymers exhibited excellent adhesive ability to any solid surfaces.33 Meanwhile, recent reports indicated that the catechol groups of DA could also function as the reducing reagent to convert GO into rGO.34,35 In the present study, a facile and green biomimetic approach was proposed to prepare highly morphology controllable and biocompatible 2D biopolymer-adhered GO and rGO nanosheets. Heparin, a highly sulphated linear polysaccharide, was chosen as the model biopolymer due to its versatile ability,36,37 such as remarkable biocompatibility,38,39 excellent anticoagulant ability,37,40 mediating complement activation, vascular regeneration, and antiviral activity.41 First, DA was grafted onto heparin backbone to obtain DA-grafted heparin (DA-g-Hep) by means of carbodiimide chemistry method; then, the DA-g-Hep was used to prepare heparin-adhered GO (Hep-a-GO) and heparin-adhered and -reduced GO (Hep-a-rGO). The 2D surface morphology, long-term stability, hemolysis ratio, and vein endothelial cells viability for the heparin-adhered GO (rGO) nanosheets were investigated. Moreover, the anticoagulant bioactivity for the adhered heparin was also studied. Compared with the other methods, the study presented the following benefits: (1) No toxic reagent was added, the whole process and the obtained heparin-adhered GO (rGO) were absolutely green. (2) The heparin-adhered GO (rGO) exhibited a controllable 2D morphology, aqueous stability, and drug and dye loading capacity. (3) The heparin-adhered GO (rGO) might satisfy various biomedical applications due to their excellent biocompatibility and high bioactivity. (4) A general strategy was proposed for producing biocompatible GO (rGO) nanosheets with tunable heparin-decorated amounts, which might also be extended to the decoration of GO (rGO) with other biopolymers, such as hyaluronic acid, chitosan, polypeptide, enzyme, DNA, and so on.
Article
EXPERIMENTAL SECTION
Preparation of GO Nanosheets and DA Grafted Heparin. GO colloidal suspension was prepared from natural graphite flake according to a previous work.42 The DA grafted heparin was prepared according to previous papers with some minor modifications as mentioned in the Supporting Information.43,44 Hep-a-GO and Hep-a-rGO. We dissolved 40 mg DA-g-Hep in 20 mL of Tris buffer solution (10 mM, pH 8.5); then, 2 mg GO was added to the DA-g-Hep homogeneous solution. The solution was dispersed by sonication for 20 min and then vigorously stirred at 20 °C for 24 h. Then, the obtained Hep-a-GO was centrifuged at 14 800g for three times, followed by dialysis in ultrapure water for 2 days to make sure the ions were removed completely. The Hep-a-rGO was prepared under the same condition as Hep-a-GO, except that the reaction temperature was changed to 60 °C. Characterization. 1H NMR data were obtained with a Bruker spectrometer (400 MHz); D2O was used as the solvent. Atomic force microscopy (AFM) images of the samples were acquired using a Multimode Nanoscope V scanning probe microscopy (SPM) system (Bruker). Transmission electron microscopy (TEM) images were acquired using a Tecnai G2 F20 S-TWIN transmission electron microscope (FEI) operated at 200 kV. Dynamic light scattering (DLS) and zeta potential measurements of the aqueous dispersions for GO and heparin-adhered GO (rGO) were performed using Zetasizer ZS90 (Malvern Instruments). Fourier transform infrared spectroscopy (FTIR) spectra were acquired on a FTIR spectrometer (Nicolet 560, America). Absorption spectra were measured by a UV−vis spectrometer (UV-1750, Shimadzu). X-ray photoelectron spectroscopy (XPS) measurements were performed on an X-ray photoelectron spectrometer (XSAM800, Kratos Analytical). Drug- and Dye-Loading Tests. The doxorubicin hydrochloride (DOX) and methylene blue (MB) were chose as the models to test the loading ability of the heparin-adhered GO (rGO) nanosheets. The experiments were carried out by adding DOX and MB to the heparinadhered GO (rGO) and pristine GO aqueous suspension, respectively. The final concentrations were 0.05 and 0.5 mmol/L for DOX and MB, respectively. Then, the mixed solution was first sonicated for 0.5 h and then stirred overnight at room temperature in dark. All samples were adjusted to pH
