Cellular distribution and cytotoxicity of graphene quantum dots with different functional groups
© Yuan et al.; licensee Springer. 2014
Received: 27 November 2013
Accepted: 19 February 2014
Published: 6 March 2014
Graphene quantum dots (GQDs) have been developed as promising optical probes for bioimaging due to their excellent photoluminescent properties. Additionally, the fluorescence spectrum and quantum yield of GQDs are highly dependent on the surface functional groups on the carbon sheets. However, the distribution and cytotoxicity of GQDs functionalized with different chemical groups have not been specifically investigated. Herein, the cytotoxicity of three kinds of GQDs with different modified groups (NH2, COOH, and CO-N (CH3)2, respectively) in human A549 lung carcinoma cells and human neural glioma C6 cells was investigated using thiazoyl blue colorimetric (MTT) assay and trypan blue assay. The cellular apoptosis or necrosis was then evaluated by flow cytometry analysis. It was demonstrated that the three modified GQDs showed good biocompatibility even when the concentration reached 200 μg/mL. The Raman spectra of cells treated with GQDs with different functional groups also showed no distinct changes, affording molecular level evidence for the biocompatibility of the three kinds of GQDs. The cellular distribution of the three modified GQDs was observed using a fluorescence microscope. The data revealed that GQDs randomly dispersed in the cytoplasm but not diffused into nucleus. Therefore, GQDs with different functional groups have low cytotoxicity and excellent biocompatibility regardless of chemical modification, offering good prospects for bioimaging and other biomedical applications.
KeywordsGraphene quantum dots Chemical modification Cells Intracellular distribution Cytotoxicity
Quantum dots have been widely applied in the biomedical field due to their various advantages such as size-dependent optical properties, high fluorescence quantum yields, and excellent stability against photobleaching [1–3]. However, the biomedical applications of conventional semiconductor quantum dots which generally composed of the elements from the II-VI group or III-V group (e.g., CdSe) have been greatly limited by the release of heavy metals [1–5]. Recently, carbon luminescent nanomaterials have incited great research interest because of their lower toxicity than semiconductor quantum dots and high photostability compared to organic dyes [6–9].
Graphene is a kind of two dimensional honeycomb structure composed by single layer of sp2 carbon atoms, which has been studied in various fields such as optoelectronic devices, energy storage media and drug delivery vectors [10–12]. Graphene quantum dots (GQDs), a kind of zero-dimensional material, have the same single-atom layer as graphene but their lateral dimensions are less than 100 nm [13–16]. Owing to their high surface area and good biocompatibility, GQDs have the potential to be vectors for delivery protein or drug molecules to cells [6, 12, 17–19]. GQDs can also serve as good fluorescent probes for bioimaging due to their excellent luminescent properties [6, 20, 21]. Beyond that, when functionalized with different chemical groups, GQDs can be used to build multifunctional structure through combining with various other materials such as protein, drug molecules, and nanotubes by covalent linkage, which will extend their widespread applications in biomedical field [18, 22, 23]. Jing and his colleagues have fabricated multifunctional core-shell structure capsules composed of olive oil, dual-layer porous TiO2 shell, Fe3O4, and GQDs . In addition, the fluorescence spectrum and the quantum yield (QY) of GQDs also vary with the surface chemical groups on them. Shen and his colleagues have prepared GQDs-PEG with QY as high as 28.0%, which was two times higher than the GQDs (13.1%) without chemical modification [8, 24]. Recently, GQDs with different functional groups have excited extensive and increasing research interest.
Up to now, little effort has been focused on the cytotoxicity and distribution research of GQDs with different functional groups. Wu and his colleagues explored the intracellular distribution and cytotoxicity of GQDs prepared through photo-Fenton reaction of graphene oxide (GO) [25, 26]. The results demonstrated that this kind of GQDs distributed in the cytoplasm, and their cytotoxicity was lower than that of the micrometer-sized GO . Markovic et al. discovered that electrochemically produced GQDs can be used for photodynamic therapy by inducting oxidative stress and activating both apoptosis and autophagy when irradiated with blue light, which raised a concern about their potential toxicity . Zhu and his colleagues reported that the GQDs that they synthesized did not weaken the cell viability significantly . However, the study from Zhang et al. reported that GQDs synthesized by electrochemical means can be used for efficient stem cell labeling with little cytotoxicity, and they dispersed in the cytoplasm . Some of these results were contradictory, and for the newly developed graphene quantum dots and their derivatives, such information was generally lacking.
In this work, we compared the cytotoxicity of three GQDs with different functional groups (NH2, COOH, and CO-N (CH3)2, respectively) and observed their cellular distribution in human A549 lung carcinoma cells and human neural glioma C6 cells. The acquired results will provide valuable information for the GQDs application in biomedical field.
Synthesis of graphene quantum dots
NH2-GQDs (aGQDs) were prepared according to a previous study reported by Jiang et al. . GO stock solution (2.5 mL of 4 mg/mL) was added to a vigorously stirred mixture of 5 mL of ammonia (25% to 28%) and 20 mL of H2O2 (30%). The gray turbid solution was heated to 80°C in a 50-mL conical flask. About 30 min later, the mixed solution became clear and the reaction continued for 24 h. The unreacted H2O2 and ammonia were removed by vacuum drying at 45°C. Finally, the product was dissolved with double-distilled water.
COOH-GQDs (cGQDs) were gained by pyrolyzing 2 g of citric acid at 200°C in a 5-mL beaker . About 30 min later, the liquid became orange, implying the formation of cGQDs. The obtained orange liquid was added to 100 mL of 10 mg/mL−1 NaOH solution drop by drop under vigorous stirring. When the pH was adjusted to 7 with HCl, the resulting yellow-green liquid was dialyzed for 48 h in a 3,500 Da dialysis bag to obtain pure cGQDs.
CO-N (CH3)2-GQDs (dGQDs) were prepared with a top-down method using GO, H2O2, and N,N-dimethylformamide (DMF) as starting materials. The preparing process was similar to that of aGQDs except replacing ammonia with DMF. The unreacted H2O2 and water were removed by vacuum drying, and the residual DMF was removed through dialyzing for 48 h in a 3,500-Da dialysis bag.
Characterization of GQDs
The UV-visible (vis) spectra and fluorescence spectra were obtained using a UV–Vis spectrometer (NanoDrop, Wilmington, DE, USA) and a fluorescence spectrometer (PerkinElmer, Waltham, MA, USA), respectively. Transmission electron microscopy (TEM) observation was performed on a JEM-2100HR transmission electron microscopy (JEOL, Akishima-shi, Japan) operated at 200 kV. Fourier transform infrared (FTIR) spectra were collected using a Tensor 27 FTIR spectrometer (Bruker, Karlsruhe, Germany) in the range 400 to 4,000 cm−1.
A549 and C6 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in an incubator with 5% CO2 and 95% air.
After incubated with GQDs (50 μg/mL) for 12 h, cells adhered on coverslips were washed thoroughly with PBS three times. Formaldehyde (4%) was added to fix the cells for 20 min at room temperature. The cells without GQDs were taken as control. The cell imaging and distribution experiment was conducted by a fluorescence microscope (Leica, Wetzlar, Germany).
The cytotoxicity of three modified GQDs was quantitatively evaluated by thiazoyl blue colorimetric (MTT) assay. Cells seeded in 96-well plates were separately treated with different concentrations (0, 10, 25, 50, 100, and 200 μg/mL) of aGQDs, cGQDs, and GQDs for 24 h. Ten microliters of MTT (5 mg/mL) was added to each well and incubated for another 4 h at 37°C. Next, 100 μL DMSO was added to each well, and the optical density at 490 nm was recorded on a microplate reader (Rayto, Shenzhen, China).
Trypan blue assay
Cells were seeded in 6-well plates and incubated for 24 h. GQDs modified with different functional groups were separately introduced into cells with different concentrations (0, 10, 25, 50, 100, and 200 μg/mL). The cells in the supernatant and the adherent cells were collected and washed with PBS twice after incubation with GQDs for 24 h. Next, the cells were stained with 0.04% trypan blue solution for 3 min. The live and dead cells were counted using a cytometer.
Flow cytometry experiment
Flow cytometry analysis was performed to detect apoptotic and necrotic cells on a FACSCanto™ flow cytometer (BD Biosciences, Heidelberg, Germany). Apoptosis or necrosis was analyzed by double staining with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the instructions of the manufacturer. The FITC positive control was prepared by culturing the control cells in medium containing 1% of H2O2 for 24 h. The PI positive control was designed by keeping the cells in a 70°C water bath for 20 min.
Raman spectra of cells were collected using a Renishaw inVia microspectrometer equipped with a semiconductor laser (785 nm) and a Leica DM2500 microscope (Leica). A × 50 objective was used to focus the laser beam and to collect the Raman signal. The Raman spectra were recorded in the range of 600 to 1,700 cm−1. Before the cell Raman spectra was obtained, the Raman band of silicon wafer at 520 cm−1 was obtained to calibrate the spectrometer and all the data were collected under the same conditions. All experiments were independently carried out at least five times. All the Raman spectra were baseline-corrected, removing the fluorescence background using a Vancouver Raman Algorithm software .
The data of MTT assay, trypan blue assay, and flow cytometry experiment were presented as mean and standard deviation. Independent sample t test was used to analyze the differences between the treated groups and the control groups, and p value less than 0.05 was considered statistically significant.
Results and discussion
Synthesis and characterization of GQDs
As shown in Figure 1b, the fluorescence emission of aGQDs was excitation-dependent. The emission peaks shifted from 470 to 600 nm when the excitation wavelength was changed from 320 to 580 nm in a 20-nm increment. The strongest fluorescence peak was at 500 nm with 420 nm as the excitation wavelength, which was in agreement with a previous report . Whereas, the emission peak of cGQDs and dGQDs were excitation-independent (Figure 1c,d). The maximum excitation wavelength and the maximum emission wavelength were at 400 and 440 nm for cGQDs and 400 and 500 nm for dGQDs, respectively.
The cell uptake and distribution of GQDs
Cell proliferation evaluation
Cell mortality analysis
Flow cytometric analysis of apoptosis or necrosis
Raman spectral analysis
The present study investigated the cell distribution of three GQDs modified with different functional groups and compared their cytotoxicity in A549 and C6 cells. The fluorescent images of cells indicated that the GQDs accumulated in the cytoplasm but not in the nucleus after incubation for 12 h. When the concentration reached 50 μg/mL, three GQDs can illuminate the cells effectively. It was demonstrated that the three GQDs induced slight cell proliferation decreases at high concentrations. However, no visible mortality and apoptosis or necrosis increases resulted from the treatment of the three GQDs even at the concentration of 200 μg/mL. In addition, the Raman spectroscopy experiment provided molecular level evidence for the biocompatibility of the three kinds of GQDs, revealing that no obvious changes in cell Raman spectra were generated by the treatment of 200 μg/mL GQDs. In summary, the results manifested that when modified with different chemical groups, GQDs still possessed excellent biocompatibility and low cytotoxicity to cells, which may make them more promising in bioimaging and other biomedical applications.
XY, MJ, and XW are master’s degree candidates. ZL is a researcher assistant, and YJ is an associate researcher. ZG is a deputy director and professor.
NH2-graphene quantum dots
COOH-graphene quantum dots
CO-N (CH3)2-graphene quantum dots
Dulbecco’s modified Eagle medium
Graphene quantum dots
thiazoyl blue colorimetric
This work was supported by the National Natural Science Foundation of China (No. 61275187, No. 61378089, and No. 61335011), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20114407110001 and No. 200805740003), and the Natural Science Foundation of Guangdong Province (No. 9251063101000009).
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