Chemical and magnetic functionalization of graphene oxide as a route to enhance its biocompatibility
© Urbas et al.; licensee Springer. 2014
Received: 18 September 2014
Accepted: 26 November 2014
Published: 4 December 2014
The novel approach for deposition of iron oxide nanoparticles with narrow size distribution supported on different sized graphene oxide was reported. Two different samples with different size distributions of graphene oxide (0.5 to 7 μm and 1 to 3 μm) were selectively prepared, and the influence of the flake size distribution on the mitochondrial activity of L929 with WST1 assay in vitro study was also evaluated. Little reduction of mitochondrial activity of the GO-Fe3O4 samples with broader size distribution (0.5 to 7 μm) was observed. The pristine GO samples (0.5 to 7 μm) in the highest concentrations reduced the mitochondrial activity significantly. For GO-Fe3O4 samples with narrower size distribution, the best biocompatibility was noticed at concentration 12.5 μg/mL. The highest reduction of cell viability was noted at a dose 100 μg/mL for GO (1 to 3 μm). It is worth noting that the chemical functionalization of GO and Fe3O4 is a way to enhance the biocompatibility and makes the system independent of the size distribution of graphene oxide.
In recent years, graphene, well-defined 2D honeycomb-like network of carbon atoms, has attracted growing interest owing to its unprecedented combination of unique electrical, thermal, optical, and mechanical properties[1–6].
Graphene derivative, graphene oxide chemically exfoliated from oxidized graphite, is considered as a promising material for biological applications due to its surface functionalizability, amphiphilicity, and excellent aqueous processability. These extraordinary properties are mainly derived from its chemical structures composed of sp3 carbon domains surrounding sp2 carbon domain and a wide range of functional groups such as epoxy, hydroxyl, and carboxyl groups[7–10]. The chemical structure of graphene oxide and large specific surface area enable various chemical modification or functionalization and make graphene oxide an excellent platform for loading magnetic nanoparticles.
Magnetic nanoparticles possessing tailored surface properties and appropriate physicochemistry have been widely investigated for various applications such as hyperthermia, magnetic resonance imaging (MRI), tissue repair, drug delivery, biosensing, and bioanalysis[12–23]. In particular, the magnetite, Fe3O4, has attracted significant attention in the field of biotechnology and medicine because of its strong magnetic properties and low toxicity[24–26]. The properties of nanocrystals strongly depend on the dimension of the nanocrystals; therefore, the control of monodispersed size of nanocrystals plays an important role. Magnetic nanoparticles for the use in biomedical applications are desired to exhibit superparamagnetic properties. The superparamagnetic nature implies that the particles will not be attracted to each other, and so the risk of agglomeration in a medical setting is minimized. Magnetite is traditionally ferromagnetic in nature. However, as the size decreases to 30 nm or smaller, it loses their permanent magnetism and becomes superparamagnetic. Safety concerns could ultimately prevent the adoption of magnetic nanoparticles in medicine. In vitro and in vivo toxicity results often contradict each other hence are an area that needs more research.
Recently, graphene-based materials were extensively investigated for application in biosensing[28–32], imaging[33, 34], and drug delivery[35–38] as vehicles for drugs and as high-performance electrode material for capacitive deionization[39, 40]. Cong et al. report on fabrication of reduced graphene oxide decorated with Fe3O4 nanoparticles through a high-temperature decomposition method. This system could be used as magnetic resonance contrast agent. Shen et al. demonstrated one-step synthesis of GO-Fe3O4 nanoparticle hybrid. He and Gao presented scalable, green, efficient, controllable method of preparation of superparamagnetic, processable, and conductive graphene nanosheets coated with magnetite nanoparticles. He et al. showed attachment of magnetite nanoparticles to GO surface with covalent bonding. Yang et al. described GO-Fe3O4 nanoparticle hybrid supporting doxorubicin hydrochloride (anticancer drug). This system could be easily removed from water by an external magnetic field. Zheng and Li reported on fabrication of a magnetite nanoparticle-decorated graphene oxide (Fe3O4-GO) and reduced graphene oxide (Fe3O4-rGO) loaded with β-lapachone (anticancer drug), in vitro anticancer efficacy and cytotoxicity of obtained materials. Bai et al. presented results of study on the inductive heating property of graphene oxide sheets decorated with magnetite nanoparticles in AC magnetic field. The potential of the obtained nanocomposite was evaluated for localized hyperthermia treatment of cancer cells.
Herein, we present new facile approach for production of the monodispersed Fe3O4 nanoparticles and magnetic attachment of magnetite nanoparticles to graphene oxide sheets with different flake size distributions. The mean size of the obtained magnetite nanoparticles is about 8 nm. Additionally, we performed cytocompatibility study on the influence of these molecular hybrids on the mitochondrial activity of L929 cell line with WST1 assay in respect to the GO and pristine iron oxide nanoparticles. The cellular response was verified with different concentration (0.0, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0 μg/mL) of the nanomaterials.
Preparation of graphene oxide-Fe3O4 nanoparticle hybrid
Synthesis of graphene oxide
Two types of samples of graphene oxide were synthesized by oxidation of graphite with various size of flakes (with narrow and broad size distribution) using the modified Hummer's method fully described elsewhere. To a mixture of 6 g KMnO4 and 1 g graphite, 120 mL of concentrated sulfuric acid and 15 mL of orthophosphoric acid were poured. It was heated to 50°C and stirred for 24 h. The resulting mixture was added to ice (150 mL) with 1 mL of H2O2 (30%) and centrifuged. The separated solid product was washed two times with water and 30% HCl and ethanol and left for vacuum drying for 12 h at 70°C. The sample with broad size distribution is named B-GO, and the sample with narrow size distribution is named N-GO.
Synthesis of magnetite nanoparticles
The magnetite synthesis route was carried out under the action of a rotating magnetic field (RMF). A liquid-filled glass container was placed inside the three-phase stator of an induction squirrel-cage motor which generated the RMF. This kind of the magnetic field might be used to augment the process intensity instead of a mechanical mixing. One of the advantages of the RMF is the possibility to apply it to generation and control of the hydrodynamic states for the magnetic particle mixing systems. In the experimental procedure, the frequency of the RMF was equal to 50 Hz. The intensity of the magnetic field could be 25 mT. The more detailed information about the experimental setup and the measurements of the magnetic field for the tested apparatus may be found here. Finally, the precipitate was collected by filtration and washed three times with deionized water and then dried.
Synthesis of graphene oxide-Fe3O4 nanoparticle hybrid
High-resolution transmission electron microscopy (HRTEM) (FEI Tecnai F30, Frequency Electronics Inc., Mitchel Field, NY, USA) was employed to examine the morphology of the samples and the size and distribution of the magnetite nanoparticles. X-ray diffraction technique (X-ray diffractometer Philips X'Pert PRO, PANalytical B.V., Almelo, The Netherlands, Kα 1 = 1.54056 Å) was used to investigate the structure of the samples and to estimate the average size of magnetite nanoparticles. In order to study the thickness of obtained graphene oxide flakes and nanocomposite, atomic force microscopy (Nanoscope V Multimode 8, Bruker AXS, Mannheim, Germany) was employed. IR absorption spectra were collected on the Nicolet 6700 FTIR spectrometer (Thermo Nicolet Corp., Madison, WI, USA). In order to investigate the thermal behavior of the samples, thermogravimetric analysis was performed on the SDT Q600 simultaneous TGA/DSC (TA Instruments Inc., Milford, MA, USA) under an air flow of 100 mL/min at heating rate of 5°C/min. Raman spectra were acquired on the inVia Raman Microscope (Renishaw PLC, New Mills Wotton-under-Edge, Gloucestershire, UK) at an excitation wavelength of 785 nm.
The cell line of mouse fibroblasts (L929) were seeded on the 96-well plates at the density of 7.4 × 103 per well. Cells were maintained using DMEM cell culture medium (Gibco Corp., Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco Corp., Grand Island, NY, USA), 0.4% streptomycin/penicillin (Sigma-Aldrich Corp., St. Louis, MO, USA), and 2 mM L-glutamine (Sigma-Aldrich Corp., St. Louis, MO, USA) at 37°C, 5% CO2, and 95% humidity. The 200 μL/well final volume of culture medium was used in experiment.
The cytocompatibility study
The cytocompatibility of nanomaterials was tested using WST-1 Cell Proliferation Assay (Roche Applied Science, Penzberg, Germany). The test principle is based on the transformation of WST-1 salt [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] into water-soluble colored formazan by mitochondrial dehydrogenases that are active in rapidly dividing cells. The generation of the dark yellow colored formazan is directly correlated to the number of the metabolically active cells; therefore, the cell number can be quantified by the photometric detection of the formazan. There are several similar proliferation assays using other tetrazolium salts, such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide], and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] available on the market. The main advantage of WST-1 test over those mentioned above is the solubility of reduced WST-1 salt. It also requires no washing, harvesting, or solubilization of cells. To perform the assay, L929 cells were plated in the 96-well plates for 24 h. After incubation period from cells seeding, N-GO, B-GO, N-GO-Fe3O4, B-GO-Fe3O4, and Fe3O4 were introduced separately to cells with different final concentrations (0.0, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0 μg/mL) in culture medium. Cells were incubated with nanomaterial for 24 h. Cells maintained in prepared medium without adding tested samples were taken as a control. To each well, 20 μL of WST-1 solution was added and incubated for additional 30 min at 37°C. After incubation, the absorbance at 450 nm, according to manufacturer's instructions, was recorded on the Sunrise Absorbance Reader (Tecan Group Ltd., Männedorf, Switzerland). All of the experiments were conducted in triplicate.
All experiments were repeated at least three times. The results are given in the form: mean values ± standard deviation (SD). All results were compared using Student's t-test. Differences are considered significant at a level of p < 0.05.
Results and discussion
The effects of GO on mouse fibroblast cells depend on GO dose, and as shown in the study of Wang et al., the effects also depend on the culture time. The most cytotoxic effect of graphene oxide on human fibroblast cells (HDF) was observed on the fifth day of culture at the doses of 50 and 100 μg/mL. Similar results were noticed in tumor cell lines, e.g., human gastric cancer MGC803, human breast cancer MCF-7 and MDA-MB-435, and liver cancer HepG2. In our study, the effects of experimental samples on the cell culture were monitored for 24-h period, but as mentioned earlier, cell viability was reduced the most, to approximately 60%, at GO's concentration of 50 μg/mL. Chang et al. using CCK-8 assay and A549 cells made observation that preparation method of GO has influence of relative cell viability. The influence of different GO samples (s-GO with smaller size, l-GO with larger size, and m-GO mix) on the mitochondria activity may vary. m-GO's effect on cell cultures was insignificant at the concentration range of 100 to 200 μg/mL. When the s-GO was tested, the cell viability was reduced the most at concentration between 50 and 200 μg/mL. It also has been noticed that the difference between some studies might come from the different sample properties and various cell lines. Incubation time can also influence the cell response[55, 57]. In our study, the GO sample with concentration of 100 μg/mL demonstrated weak toxicity. We suggest that higher concentration (100 μg/mL) of graphene oxide may influence on harder GO migration to cell cytoplasm. We also found that GO material shows relatively good cytocompatibility at the concentration of 12.5 μg/mL and that result corresponds to the result obtained by Wojtoniszak et al..
As shown above, some difficulties in the interpretation of the obtained results can arise from variety of the factors that can influence the cell response. Some of the factors are not clearly determined. Regarding the effects of graphene oxide and hybrid GO-Fe3O4 on cell viability, the mechanism is not well explained and still requires further analysis.
The in vitro studies play key role in exploration of the nanomaterial properties in biological environment and interaction with the living matter. The toxicity of the magnetic nanoparticles on biological entities is highly dependent on a range and combination of factors related to the properties of those nanoparticles. The physical properties such as the particle size, shape, and surface coating can evoke a toxic response by aggregating and coagulating according to size and shape. The chemical composition of the particles themselves can be naturally toxic. Here, we clearly demonstrate that the chemical functionalization of GO and Fe3O4 is a way to enhance the biocompatibility of the system and makes the system independent of the size of graphene oxide. Therefore, we believe that the obtained product with high cytocompatibility would be suitable for the application in biomedicine, e.g., as a drug carrier and/or in hyperthermia.
We report a facile method of the preparation of graphene oxide-Fe3O4 nanoparticle hybrid. We prove that it is possible to increase biocompatibility of graphene oxide through the deposition of magnetite nanoparticles on the graphene oxide flakes via chemical interaction. Furthermore, we indicate that the differences in flake size do not result in different cell viability in contact with our systems. These results show the potential application of this hybrid in hyperthermia treatment. Further investigation needs to be performed in order to prove the safety and efficiency of these systems in vivo.
This research was funded by the National Science Center under OPUS Program (Project No. DEC/2011/03/B/ST5/03239).
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