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Methotrexate-loaded nitrogen-doped graphene quantum dots nanocarriers as an efficient anticancer drug delivery system
Materials Science and Engineering C 79 (2017) 280–285
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Methotrexate-loaded nitrogen-doped graphene quantum dots nanocarriers as an efficient anticancer drug delivery system Fatemeh Khodadadei a,b, Shahrokh Safarian a,⁎, Narges Ghanbari b a b
Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran 1417614411, Iran School of Chemical Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 21 September 2016 Received in revised form 1 May 2017 Accepted 10 May 2017 Available online 11 May 2017 Keywords: Anticancer drug Graphene quantum dots Methotrexate Nanocarriers
a b s t r a c t Graphene quantum dots (GQDs) are new efficient nanomaterials used in therapeutic applications. In this study, blue fluorescent nitrogen-doped GQDs (N-GQDs) were synthesized by a hydrothermal method via pyrolisis of citric acid as the carbon source and urea as the nitrogen source. The existence of doped nitrogen in GQDs was confirmed by FTIR characterization. Here, for the first time, the N-GQDs were loaded with the anticancer drug, methotrexate (MTX), to prepare MTX-(N-GQDs) as an efficient drug delivery system. The establishment of the strong π-π stacking interaction between MTX and N-GQDs was confirmed by FTIR and UV–vis spectroscopies indicating successful loading of MTX to N-GQDs. The in-vitro cytotoxicity of MTX-(N-GQDs) on human breast cancer cells investigated through MTT assay suggested that the drug-free N-GQDs nanocarriers are highly biocompatible, whereas the MTX-loaded ones are more cytotoxic than the free MTX. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Methotrexate (MTX) is a specific inhibitor of dihydrofolate reductase (DHFR) making essential problems for DNA replication and cell proliferation in cancer cells [1,2]. However, there are some disadvantages in therapeutic usage of this drug such as fast metabolism, fast excretion, and low selectivity for the malignant cells. Coupling of MTX with carriers could solve these problems resulting in enhancement of the delivery and selectivity properties of MTX [3]. Optimizing the efficiency of MTX delivery systems must be provided via sufficiently small size of the drug-carrier complex to achieve efficient penetration into the desired cells affording minimum tendency for excretion. With respect to the extra small size of graphene quantum dots (GQDs), it is used in this study as carriers for MTX to construct a new MTX-nanocarrier system for improving the cytotoxic effects of MTX on cancer cells. GQDs, a new class of carbon-based nanomaterials, show the properties of graphene and QDs, simultaneously. Zero-dimensional GQDs have a hexagonal mono/multilayer structure of graphene sheets with sizes smaller than 30 nm and fluorescence properties, due to quantum confinement and edge effects [4–9]. Regarding the high surface area, good biocompatibility, low-toxicity, abundant surface functional groups, solubility in different solvents, chemical inertness and stable photoluminescence, much attention has been paid to these nanostructures in recent researches. The planar structure of GQDs is filled with delocalized electrons endowing GQDs with the efficient loading ⁎ Corresponding author. E-mail address: [email protected] (S. Safarian).
http://dx.doi.org/10.1016/j.msec.2017.05.049 0928-4931/© 2017 Elsevier B.V. All rights reserved.
capacity for drugs [10–20]. Unlike carbon nanotubes with drug loading capacities limited to their surface and tips, GQDs sheets load drugs via their two faces and edges resulting in up to 200% higher loadings than those of other nanoscale drug carriers [21]. Moreover, due to the layered structure of GQDs, atoms, ions and small molecules can be intercalated between the graphene layers improving its drug loading capacity [22]. Delivery of drug-GQDs complexes can be observed, without further modification with marker dyes, via the photoluminescence properties of GQDs. Furthermore, GQDs have discrete band-gaps and show typical semiconducting properties making them as promising candidates for bioimaging, photocatalysis, photovoltaic and other optoelectronic devices [23–27]. Doping of GQDs with various functional groups is a common strategy for increasing its specific surface area, and decreasing its size and ID/IG ratio as verified for nitrogen-doped graphene through BET, XRD and Raman analysis [28,29]. Thus, nitrogen-doped graphene quantum dots (N-GQDs) offer high surface area and abundant aromatic rings to adsorb MTX molecules through strong π-π stacking interaction leading to enhanced drug loading capacity for drug delivery applications [10,15,18,30]. Synthesis of N-GQDs is carried out using different methods with various carbon and nitrogen sources; for example, the hydrothermal method using citric acid as the carbon source and urea or thiourea as the nitrogen and sulfur sources to produce S and N co-doped GQDs. The synthesized N-GQDs have narrow size distributions in the range of 2– 4 nm and two clear absorption bands at 234 and 337 nm [27]. More recently, dicyandiamide was also used as another nitrogen source to synthesize N-GQDs through hydrothermal method showing an average
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size of 2.3 nm and an absorption band at 333 nm with a small band at 234 nm [31]. In another work, tetrabutylammonium perchlorate (TBAP) in acetontrile was applied as the nitrogen source. TEM images showed that the synthesized N-GQDs have fairly uniform diameters between 2 and 5 nm and an absorption band at 270 nm [32]. There is a theory describing the formation of N-GQDs via self-assembling of citric acid into a nanosheet structure through intermolecular H-bonding, followed by the establishment of the pure graphene core through dehydration between the intermolecular carboxyl and hydroxyl groups under hydrothermal conditions. During this process, the amine (−NH2) groups of the nitrogen source react with the carboxyl or hydroxyl groups of the graphene cores to form N-GQDs [33,34]. In this study, a hydrothermal route was used to synthesize N-GQDs via pyrolisis of citric acid as the carbon source and urea as the nitrogen source. Methotrexate was then loaded to N-GQDs to construct a drug delivery system. The cytotoxic potency of the synthesized nanocarrier system was investigated on MCF-7 as a human breast cancer cell line.
2. Experimental results 2.1. Materials and reagents Citric acid and urea were purchased from Merck. Roswell Park Memorial Institute Medium 1640 (RPMI-1640), fetal bovine serum (FBS), streptomycin and penicillin were purchased from Gibco. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma. The human breast cancer cell line (MCF-7) was obtained from the cell bank of Pasteur Institute, Tehran, Iran. All reagents were of analytical grade and used as received. Deionized water was used throughout the experiments.
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2.2. Synthesis of N-GQDs The N-GQDs were prepared here by paralyzing citric acid in the presence of urea, according to the literature [27]. Briefly, citric acid and urea were dissolved in water and stirred to form a clear solution. Then the solution was heated to 180 °C and kept at the same temperature for 10 h. The suspension was centrifuged and the resulting supernatant was dialyzed in a dialysis bag with molecular weight cutoff of 1000 Da. The preparation of N-GQDs and the mechanism of entrance into the cancer cell accompanied by the MTX release behavior have been illustrated in Scheme 1. 2.3. Loading and release of MTX from N-GQDs MTX release was evaluated by dialysis of MTX-(N-GQDs) against phosphate-buffered saline (PBS) at pH 7.4. The concentration of released MTX was measured using absorbance detection at 304 nm. Briefly, MTX-(N-GQDs) complex was prepared by mixing 30 μg/ml N-GQDs suspension with 10 mg/ml MTX solution in PBS at pH 7.4, while the mixture was stirred for 24 h in the dark. After stirring, most of the MTX molecules are loaded to the N-GQDs. Then, the prepared complex was dialyzed against PBS buffer (pH 7.4) and the concentration of disjunctive MTX was measured outside the dialysis bag at various time intervals. The drug released was calculated according to the following equation: Drug release ð%Þ ¼
Wr 100 Wl
where, Wr is the weight of disjunctive MTX after time “t” and Wl is the weight of loaded MTX at time “t0”.
Scheme 1. Preparation of N-GQDs and subsequent release of MTX from the surface of N-GQDs in a tumor cell environment.
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Fig. 1. Morphology and size distributions of N-GQDs. (a) TEM image; (b) DLS size distribution profile; (c) AFM image; (d) Height profile along the line AB.
2.4. In-vitro cytotoxicity The in-vitro cytotoxicity of MTX-(N-GQDs), N-GQDs and free MTX was evaluated through MTT assay. For cell viability investigations, human breast cancer cells MCF-7 were chosen. The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). The cells were then incubated in a humidified 5% CO2 atmosphere at 37 °C. Cytotoxicity of the treating materials was measured via seeding of 100 μl of cell suspension in each well of a 96-well plate at a density of 2 × 104 cells/well. The cells were incubated for 24 h and then different concentrations of MTX-(NGQDs), N-GQDs and free MTX were added. To evaluate cell killing efficiency of MTX-(N-GQDs), the concentration of its loaded MTX was considered for cell treatments until a better comparison could be achieved with the equivalent concentrations of the free MTX. After additional 12, 24 and 48 h, MTT solution (5 mg/ml in PBS as the 5X stock solution) was added and incubated for 4 h. Finally, the MTT solution was removed and the precipitated formazan crystals were dissolved in 100 μl of dimethyl sulfoxide (DMSO), which was then slowly shaken for 15 min. The absorbance was measured at a wavelength of 570 nm using a microplate reader (Rayto-China). The relative cell viability of each well was then determined relative to the untreated control.
Denmark). Dynamic light scattering (DLS) was performed on a Nanoplus-1 (Micrometrics Co., Norcross, USA) at λ = 656 nm. UV–vis characterizations were accomplished by T90 + UV–vis spectrometer (PG instrument LTD, Leicestershire, UK) to observe the optical absorption edge in the quantum structures. Absorption spectra were obtained in the range of 200–700 nm at 1 nm scan intervals with a scan speed of 600 nm/min. Raman scattering spectrum was recorded from 100 to 4200 cm−1 with an Almega Thermo Nicolet dispersive Raman spectrometer (Thermo Scientific Co. Madison, USA). To identify the surface functional groups of N-GQDs, Fourier Transform Infra-Red (FTIR) spectroscopy (Bruker Vector 22 spectrometer, Germany) was used in transmission mode with a resolution of 5.0 cm− 1. Fluorescence spectrum was recorded on Varian Cary Eclipse luminescence spectrophotometer.
2.5. Instrumentation and characterization Morphological and structural analyses of the samples were carried out using transmission electron microscope (TEM, Model CM30 Philips Electron Optics, Eindhoven, Netherland) operated at 250 kV. Atomic force microscopy (AFM) images were obtained using a DME DualScope™ scanning probe microscope (Model DS 95-200/50, DME,
Fig. 2. Raman spectra of N-GQDs. Disordered carbon (D band) and the graphitic carbon (G band) are detected at 1361 cm−1 and 1573 cm−1, respectively. The ID/IG ratio is 0.96, suggesting the graphitic nature of the synthesized N-GQDs.
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Fig. 3. UV–vis absorption and fluorescence spectra of N-GQDs. π → π* of C_C bonds caused the rising up of the shoulder at 255 nm. Inset: Photograph of the N-GQDs aqueous solution taken under UV light (360 nm). a.u. means Arbitrary Unit.
3. Results and discussion 3.1. Characterization of N-GQDs 3.1.1. Morphological characterization The morphology of the synthesized N-GQDs was characterized by TEM (Fig. 1a) and AFM (Fig. 1c). Drops of dilute aqueous solution of N-GQDs were deposited on carbon-coated gold grid for TEM and on mica substrate for AFM. Moreover, DLS analysis was proformed to determine the size distribution profile of the N-GQDs nanoparticles (Fig. 1b). TEM image of the N-GQDs displays sizes in the range of 7–17 nm. DLS size distribution of the N-GQDs is relatively narrow in the range of 10–14 nm, which is in the same range as that of the TEM. The synthesized N-GQDs size is well-consistent with other reports [5,6,13,20,35]. AFM topographic height of the N-GQDs shows a narrow height distribution range of 2–4 nm (Fig. 1d), which correlates with the overall size obtained from TEM and DLS according to other reports [7,32]. It is reported that the interlayer spacing of graphene and N-GQDs is larger than that of graphite (0.335 nm), and the theoretical thickness of a graphene layer is 0.4 nm [27,28,31,32]. Thus, based on the AFM results showing about 4 nm height for the quantum dots, we estimated that most of the NGQDs consist of 10 graphene layers. This unique structure of N-GQDs offers high surface-to-volume ratio, which is expected to offer higher loading capacity when used as a drug carrier.
Fig. 5. UV–vis absorption spectra of MTX and MTX-(N-GQDs). MTX exhibits three absorption peaks at 380, 305, and 260 nm, which are changed to three shoulders in MTX-(N-GQDs), because of the strong π-π stacking interaction between MTX and NGQDs.
3.1.2. Raman characterization Raman spectroscopy data for N-GQDs nanoparticles is presented in Fig. 2. The disordered carbon (D band) at 1361 cm−1 and the graphitic carbon at 1573 cm− 1 (G band) are detected in the Raman spectrum and are assigned to the presence of sp3 defects as well as in-plane vibration of sp2 carbon, respectively. In graphitic systems, increasing of the ID/IG ratio indicates decreasing of the fraction of sp2 domains so more topological disorder and less crystallinity in the graphite layers. Here, the ID/IG ratio for N-GQDs was found to be around 0.96, which suggests the graphitic nature of the synthesized N-GQDs [36] (see Fig. 2).
3.1.3. UV and fluorescence spectrophotometry To investigate the optical characteristics of N-GQDs, UV–vis absorption and fluorescence analysis were studied as another tool for the confirmation of N-GQDs formation (Fig. 3). The UV–vis absorption
Fig. 4. FTIR spectra of (a) N-GQDs and (b) MTX-(N-GQDs). The functional groups are assigned in the figure based on the position of the vibrational peaks.
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and MTX loading onto N-GQDs. In N-GQDs (Fig. 4a), the broad peak between 3000 and 3500 cm−1 is assigned to O\\H and N\\H stretching vibrations of hydroxyl and amine groups. The peaks at 1640, 1570, 1404 and 1000–1360 cm−1 are attributed to the vibrational bands of C_O in carboxylic group, N\\H, carboxylic O\\H and C\\N, respectively [31,35,37–40]. The presence of N\\H and C\\N groups indicate the successful nitrogen functionalization in N-GQDs. In MTX-(N-GQDs) (Fig. 4b), the spectrum shows not only the characteristic bands of N-GQDs but also the clear characteristic peaks of MTX at 1620 cm−1 (−CONH amide band II) and the intensified N\\H stretching vibrations of amine groups at 3415 and 3475 cm− 1. Besides, the characteristic peaks of MTX at 620 and 480 cm−1 were also observed on the FTIR spectrum of MTX-(N-GQDs). These observations confirm the successful loading of MTX to N-GQDs [41–44]. Fig. 6. In-vitro release profile of MTX from MTX-(N-GQDs) in PBS (pH 7.4) at 37 °C. About 60% of MTX dissociates from the nanocomplex after 9 h. Complete disassembly of the complex has occurred after 48 h.
spectrum of N-GQDs displays a broad absorption shoulder around 255 nm, similar to other absorption bands of N-GQDs prepared by the hydrothermal method, reported in literature [31]. The origin of this shoulder at 255 nm is due to π → π* of C_C bonds. The N-GQDs aqueous solution shows bright blue emission under excitation in UV light. This observation was also verified by fluorescence analysis. The Fluorescence spectrum shows the emission wavelength of the N-GQDs at 443 nm, when excited at 340 nm. 3.1.4. Spectral characterization The nature of surface functional groups was investigated using FTIR spectroscopy. FTIR analysis confirmed effective nitrogen atom doping,
3.2. Loading of MTX to N-GQDs and the MTX releasing properties The states of MTX and N-GQDs in solution are clearly observed from the UV–vis spectrum, as shown in Fig. 5. MTX solution strongly absorbs UV photons at the wavelengths of 380, 305, and 260 nm, due to the existence of heteroaromatic pterine chromophore. The absorbance spectrum of MTX-(N-GQDs) solution exhibits three shoulders at the cited wavelengths with a slight shift to the right (blue-shift) compared with MTX itself, which can be attributed to the vicinity of MTX and N-GQDs in the complex resulting in strong π-π stacking interaction. For example, the peaks of MTX at 305 and 260 nm shifted to 303 and 257 nm after loading onto N-GQDs. Therefore, these results suggest that N-GQDs and MTX molecules formed complex in the solution [18,41,45,46]. The above mentioned hypothesis supportes the FTIR spectrum in Fig. 4, in which it also reveals the strong stacking of MTX onto N-GQDs.
Fig. 7. In-vitro cytotoxicity of MTX-(N-GQDs) after 12 h (a), 24 h (b), 48 h (c) and N-(GQDs) after 48 h (d) incubation time. The MCF-7 cells were exposed to different concentrations (1, 2, 3 and 4 mM) of free MTX (dark bars), and MTX with N-GQDs (grey bars), also different concentrations (10, 20, 30 and 40 mM) of N-GQDs alone. The control samples are the untreated cells. Data are shown as the mean ± SD from three independent experiments. Statistically significance of MTX viability compared to MTX-(N-GQDs) is denoted by “*” and “**”at the level of p b 0.05 and p b 0.01 respectively.
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The in-vitro release profile of MTX from MTX-(N-GQDs) in PBS (pH 7.4) at 37 °C is shown in Fig. 6. About 60% of MTX-(N-GQDs) disassembled after 9 h, followed by the slow release of MTX until all of it dissociated from the complex after 48 h under neutral condition, indicating about 48 h of sustained release at 37 °C in PBS (pH 7.4). 3.3. Cell viability assay The in-vitro cytotoxicity of the synthesized MTX-(N-GQDs) against MCF-7 cells in comparison with those of free MTX or N-GQDs alone was determined through MTT assay (Fig. 7). Viability of the cells in presence of MTX-(N-GQDs) was investigated in those concentrations in which the loaded MTX was equivalent to the free MTX. It is found that the toxicity behavior of the free and loaded MTX depends on the incubation time. For the 12 h incubation time, free MTX shows more toxicity than the loaded one, especially in the lower concentrations, since the free MTX possibly diffuses into the cells rapidly while the loaded MTX takes comparatively longer times to diffuse (Fig. 7a). For the 24 h incubation time, the loaded MTX is gradually showing more cytotoxicity compared to the free MTX (Fig. 7b). This may be occurred because free MTX diffuses out of the cells as easily as it diffuses into the cells, but the loaded MTX remains in the cell more effectively. This could be verified by the 48 h incubation, where the loaded MTX shows significantly more cytotoxicity than the free MTX (Fig. 7c). It was also discovered that N-GQDs exhibits negligible cytotoxicity and is biocompatible (Fig. 7d). Thus, due to the good biocompatibility and high drug loading capacity, the N-GQDs provide an ideal multifunctional delivery vehicle for treatment of cancers [3,34]. 4. Conclusion In summary, we have successfully synthesized about 10 nm nitrogen-doped graphene quantum dots (N-GQDs) with 10 graphitic layers. MTX is loaded to N-GQDs by strong π-π stacking interaction. Furthermore, the layered structure of N-GQDs renders a high specific area to intercalate MTX between the layers creating an inclusive anticancer drug delivery system. In-vitro assays revealed that free MTX rapidly diffuse in and out of the cancer cells, resulting in higher cytotoxicity than MTX(N-GQDs) for incubation times shorter than 24 h. On the other hand, the sluggish diffusion of MTX-(N-GQDs) in and out of the cancer cells leads to its higher cytotoxicity than the free MTX in longer incubation times. Furthermore, the nontoxic properties of N-GQDs assure the biocompatible behavior. All the remarkable findings confirm the accomplishment of GQDs as nanocarriers to prolong cytotoxic effects of its loaded drug for better killing of cancer cells. We expect the subject of future studies to be targeted therapy for cancer treatment aimed at efficient delivery of anti-cancer drugs, with less harm to normal cells. Targeted therapy is usually achieved by conjugating a desired drug to the nanoparticle as targeting ligand, such as monoclonal antibodies, aptamers or folic acid, which provides preferential accumulation of nanoparticles toward cancer cells. Conflict of interest The authors declare no conflict of interest. Acknowledgements Financial support was provided by Iran National Science Foundation (INSF:93002224). The authors would also like to appreciate Research Council of University of Tehran for valuable patronages.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Methotrexate-loaded nitrogen-doped graphene quantum dots nanocarriers as an efficient anticancer drug delivery system Fatemeh Khodadadei a,b, Shahrokh Safarian a,⁎, Narges Ghanbari b a b
Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran 1417614411, Iran School of Chemical Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran, Iran
a r t i c l e
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Article history: Received 21 September 2016 Received in revised form 1 May 2017 Accepted 10 May 2017 Available online 11 May 2017 Keywords: Anticancer drug Graphene quantum dots Methotrexate Nanocarriers
a b s t r a c t Graphene quantum dots (GQDs) are new efficient nanomaterials used in therapeutic applications. In this study, blue fluorescent nitrogen-doped GQDs (N-GQDs) were synthesized by a hydrothermal method via pyrolisis of citric acid as the carbon source and urea as the nitrogen source. The existence of doped nitrogen in GQDs was confirmed by FTIR characterization. Here, for the first time, the N-GQDs were loaded with the anticancer drug, methotrexate (MTX), to prepare MTX-(N-GQDs) as an efficient drug delivery system. The establishment of the strong π-π stacking interaction between MTX and N-GQDs was confirmed by FTIR and UV–vis spectroscopies indicating successful loading of MTX to N-GQDs. The in-vitro cytotoxicity of MTX-(N-GQDs) on human breast cancer cells investigated through MTT assay suggested that the drug-free N-GQDs nanocarriers are highly biocompatible, whereas the MTX-loaded ones are more cytotoxic than the free MTX. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Methotrexate (MTX) is a specific inhibitor of dihydrofolate reductase (DHFR) making essential problems for DNA replication and cell proliferation in cancer cells [1,2]. However, there are some disadvantages in therapeutic usage of this drug such as fast metabolism, fast excretion, and low selectivity for the malignant cells. Coupling of MTX with carriers could solve these problems resulting in enhancement of the delivery and selectivity properties of MTX [3]. Optimizing the efficiency of MTX delivery systems must be provided via sufficiently small size of the drug-carrier complex to achieve efficient penetration into the desired cells affording minimum tendency for excretion. With respect to the extra small size of graphene quantum dots (GQDs), it is used in this study as carriers for MTX to construct a new MTX-nanocarrier system for improving the cytotoxic effects of MTX on cancer cells. GQDs, a new class of carbon-based nanomaterials, show the properties of graphene and QDs, simultaneously. Zero-dimensional GQDs have a hexagonal mono/multilayer structure of graphene sheets with sizes smaller than 30 nm and fluorescence properties, due to quantum confinement and edge effects [4–9]. Regarding the high surface area, good biocompatibility, low-toxicity, abundant surface functional groups, solubility in different solvents, chemical inertness and stable photoluminescence, much attention has been paid to these nanostructures in recent researches. The planar structure of GQDs is filled with delocalized electrons endowing GQDs with the efficient loading ⁎ Corresponding author. E-mail address: [email protected] (S. Safarian).
http://dx.doi.org/10.1016/j.msec.2017.05.049 0928-4931/© 2017 Elsevier B.V. All rights reserved.
capacity for drugs [10–20]. Unlike carbon nanotubes with drug loading capacities limited to their surface and tips, GQDs sheets load drugs via their two faces and edges resulting in up to 200% higher loadings than those of other nanoscale drug carriers [21]. Moreover, due to the layered structure of GQDs, atoms, ions and small molecules can be intercalated between the graphene layers improving its drug loading capacity [22]. Delivery of drug-GQDs complexes can be observed, without further modification with marker dyes, via the photoluminescence properties of GQDs. Furthermore, GQDs have discrete band-gaps and show typical semiconducting properties making them as promising candidates for bioimaging, photocatalysis, photovoltaic and other optoelectronic devices [23–27]. Doping of GQDs with various functional groups is a common strategy for increasing its specific surface area, and decreasing its size and ID/IG ratio as verified for nitrogen-doped graphene through BET, XRD and Raman analysis [28,29]. Thus, nitrogen-doped graphene quantum dots (N-GQDs) offer high surface area and abundant aromatic rings to adsorb MTX molecules through strong π-π stacking interaction leading to enhanced drug loading capacity for drug delivery applications [10,15,18,30]. Synthesis of N-GQDs is carried out using different methods with various carbon and nitrogen sources; for example, the hydrothermal method using citric acid as the carbon source and urea or thiourea as the nitrogen and sulfur sources to produce S and N co-doped GQDs. The synthesized N-GQDs have narrow size distributions in the range of 2– 4 nm and two clear absorption bands at 234 and 337 nm [27]. More recently, dicyandiamide was also used as another nitrogen source to synthesize N-GQDs through hydrothermal method showing an average
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size of 2.3 nm and an absorption band at 333 nm with a small band at 234 nm [31]. In another work, tetrabutylammonium perchlorate (TBAP) in acetontrile was applied as the nitrogen source. TEM images showed that the synthesized N-GQDs have fairly uniform diameters between 2 and 5 nm and an absorption band at 270 nm [32]. There is a theory describing the formation of N-GQDs via self-assembling of citric acid into a nanosheet structure through intermolecular H-bonding, followed by the establishment of the pure graphene core through dehydration between the intermolecular carboxyl and hydroxyl groups under hydrothermal conditions. During this process, the amine (−NH2) groups of the nitrogen source react with the carboxyl or hydroxyl groups of the graphene cores to form N-GQDs [33,34]. In this study, a hydrothermal route was used to synthesize N-GQDs via pyrolisis of citric acid as the carbon source and urea as the nitrogen source. Methotrexate was then loaded to N-GQDs to construct a drug delivery system. The cytotoxic potency of the synthesized nanocarrier system was investigated on MCF-7 as a human breast cancer cell line.
2. Experimental results 2.1. Materials and reagents Citric acid and urea were purchased from Merck. Roswell Park Memorial Institute Medium 1640 (RPMI-1640), fetal bovine serum (FBS), streptomycin and penicillin were purchased from Gibco. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma. The human breast cancer cell line (MCF-7) was obtained from the cell bank of Pasteur Institute, Tehran, Iran. All reagents were of analytical grade and used as received. Deionized water was used throughout the experiments.
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2.2. Synthesis of N-GQDs The N-GQDs were prepared here by paralyzing citric acid in the presence of urea, according to the literature [27]. Briefly, citric acid and urea were dissolved in water and stirred to form a clear solution. Then the solution was heated to 180 °C and kept at the same temperature for 10 h. The suspension was centrifuged and the resulting supernatant was dialyzed in a dialysis bag with molecular weight cutoff of 1000 Da. The preparation of N-GQDs and the mechanism of entrance into the cancer cell accompanied by the MTX release behavior have been illustrated in Scheme 1. 2.3. Loading and release of MTX from N-GQDs MTX release was evaluated by dialysis of MTX-(N-GQDs) against phosphate-buffered saline (PBS) at pH 7.4. The concentration of released MTX was measured using absorbance detection at 304 nm. Briefly, MTX-(N-GQDs) complex was prepared by mixing 30 μg/ml N-GQDs suspension with 10 mg/ml MTX solution in PBS at pH 7.4, while the mixture was stirred for 24 h in the dark. After stirring, most of the MTX molecules are loaded to the N-GQDs. Then, the prepared complex was dialyzed against PBS buffer (pH 7.4) and the concentration of disjunctive MTX was measured outside the dialysis bag at various time intervals. The drug released was calculated according to the following equation: Drug release ð%Þ ¼
Wr 100 Wl
where, Wr is the weight of disjunctive MTX after time “t” and Wl is the weight of loaded MTX at time “t0”.
Scheme 1. Preparation of N-GQDs and subsequent release of MTX from the surface of N-GQDs in a tumor cell environment.
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Fig. 1. Morphology and size distributions of N-GQDs. (a) TEM image; (b) DLS size distribution profile; (c) AFM image; (d) Height profile along the line AB.
2.4. In-vitro cytotoxicity The in-vitro cytotoxicity of MTX-(N-GQDs), N-GQDs and free MTX was evaluated through MTT assay. For cell viability investigations, human breast cancer cells MCF-7 were chosen. The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). The cells were then incubated in a humidified 5% CO2 atmosphere at 37 °C. Cytotoxicity of the treating materials was measured via seeding of 100 μl of cell suspension in each well of a 96-well plate at a density of 2 × 104 cells/well. The cells were incubated for 24 h and then different concentrations of MTX-(NGQDs), N-GQDs and free MTX were added. To evaluate cell killing efficiency of MTX-(N-GQDs), the concentration of its loaded MTX was considered for cell treatments until a better comparison could be achieved with the equivalent concentrations of the free MTX. After additional 12, 24 and 48 h, MTT solution (5 mg/ml in PBS as the 5X stock solution) was added and incubated for 4 h. Finally, the MTT solution was removed and the precipitated formazan crystals were dissolved in 100 μl of dimethyl sulfoxide (DMSO), which was then slowly shaken for 15 min. The absorbance was measured at a wavelength of 570 nm using a microplate reader (Rayto-China). The relative cell viability of each well was then determined relative to the untreated control.
Denmark). Dynamic light scattering (DLS) was performed on a Nanoplus-1 (Micrometrics Co., Norcross, USA) at λ = 656 nm. UV–vis characterizations were accomplished by T90 + UV–vis spectrometer (PG instrument LTD, Leicestershire, UK) to observe the optical absorption edge in the quantum structures. Absorption spectra were obtained in the range of 200–700 nm at 1 nm scan intervals with a scan speed of 600 nm/min. Raman scattering spectrum was recorded from 100 to 4200 cm−1 with an Almega Thermo Nicolet dispersive Raman spectrometer (Thermo Scientific Co. Madison, USA). To identify the surface functional groups of N-GQDs, Fourier Transform Infra-Red (FTIR) spectroscopy (Bruker Vector 22 spectrometer, Germany) was used in transmission mode with a resolution of 5.0 cm− 1. Fluorescence spectrum was recorded on Varian Cary Eclipse luminescence spectrophotometer.
2.5. Instrumentation and characterization Morphological and structural analyses of the samples were carried out using transmission electron microscope (TEM, Model CM30 Philips Electron Optics, Eindhoven, Netherland) operated at 250 kV. Atomic force microscopy (AFM) images were obtained using a DME DualScope™ scanning probe microscope (Model DS 95-200/50, DME,
Fig. 2. Raman spectra of N-GQDs. Disordered carbon (D band) and the graphitic carbon (G band) are detected at 1361 cm−1 and 1573 cm−1, respectively. The ID/IG ratio is 0.96, suggesting the graphitic nature of the synthesized N-GQDs.
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Fig. 3. UV–vis absorption and fluorescence spectra of N-GQDs. π → π* of C_C bonds caused the rising up of the shoulder at 255 nm. Inset: Photograph of the N-GQDs aqueous solution taken under UV light (360 nm). a.u. means Arbitrary Unit.
3. Results and discussion 3.1. Characterization of N-GQDs 3.1.1. Morphological characterization The morphology of the synthesized N-GQDs was characterized by TEM (Fig. 1a) and AFM (Fig. 1c). Drops of dilute aqueous solution of N-GQDs were deposited on carbon-coated gold grid for TEM and on mica substrate for AFM. Moreover, DLS analysis was proformed to determine the size distribution profile of the N-GQDs nanoparticles (Fig. 1b). TEM image of the N-GQDs displays sizes in the range of 7–17 nm. DLS size distribution of the N-GQDs is relatively narrow in the range of 10–14 nm, which is in the same range as that of the TEM. The synthesized N-GQDs size is well-consistent with other reports [5,6,13,20,35]. AFM topographic height of the N-GQDs shows a narrow height distribution range of 2–4 nm (Fig. 1d), which correlates with the overall size obtained from TEM and DLS according to other reports [7,32]. It is reported that the interlayer spacing of graphene and N-GQDs is larger than that of graphite (0.335 nm), and the theoretical thickness of a graphene layer is 0.4 nm [27,28,31,32]. Thus, based on the AFM results showing about 4 nm height for the quantum dots, we estimated that most of the NGQDs consist of 10 graphene layers. This unique structure of N-GQDs offers high surface-to-volume ratio, which is expected to offer higher loading capacity when used as a drug carrier.
Fig. 5. UV–vis absorption spectra of MTX and MTX-(N-GQDs). MTX exhibits three absorption peaks at 380, 305, and 260 nm, which are changed to three shoulders in MTX-(N-GQDs), because of the strong π-π stacking interaction between MTX and NGQDs.
3.1.2. Raman characterization Raman spectroscopy data for N-GQDs nanoparticles is presented in Fig. 2. The disordered carbon (D band) at 1361 cm−1 and the graphitic carbon at 1573 cm− 1 (G band) are detected in the Raman spectrum and are assigned to the presence of sp3 defects as well as in-plane vibration of sp2 carbon, respectively. In graphitic systems, increasing of the ID/IG ratio indicates decreasing of the fraction of sp2 domains so more topological disorder and less crystallinity in the graphite layers. Here, the ID/IG ratio for N-GQDs was found to be around 0.96, which suggests the graphitic nature of the synthesized N-GQDs [36] (see Fig. 2).
3.1.3. UV and fluorescence spectrophotometry To investigate the optical characteristics of N-GQDs, UV–vis absorption and fluorescence analysis were studied as another tool for the confirmation of N-GQDs formation (Fig. 3). The UV–vis absorption
Fig. 4. FTIR spectra of (a) N-GQDs and (b) MTX-(N-GQDs). The functional groups are assigned in the figure based on the position of the vibrational peaks.
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and MTX loading onto N-GQDs. In N-GQDs (Fig. 4a), the broad peak between 3000 and 3500 cm−1 is assigned to O\\H and N\\H stretching vibrations of hydroxyl and amine groups. The peaks at 1640, 1570, 1404 and 1000–1360 cm−1 are attributed to the vibrational bands of C_O in carboxylic group, N\\H, carboxylic O\\H and C\\N, respectively [31,35,37–40]. The presence of N\\H and C\\N groups indicate the successful nitrogen functionalization in N-GQDs. In MTX-(N-GQDs) (Fig. 4b), the spectrum shows not only the characteristic bands of N-GQDs but also the clear characteristic peaks of MTX at 1620 cm−1 (−CONH amide band II) and the intensified N\\H stretching vibrations of amine groups at 3415 and 3475 cm− 1. Besides, the characteristic peaks of MTX at 620 and 480 cm−1 were also observed on the FTIR spectrum of MTX-(N-GQDs). These observations confirm the successful loading of MTX to N-GQDs [41–44]. Fig. 6. In-vitro release profile of MTX from MTX-(N-GQDs) in PBS (pH 7.4) at 37 °C. About 60% of MTX dissociates from the nanocomplex after 9 h. Complete disassembly of the complex has occurred after 48 h.
spectrum of N-GQDs displays a broad absorption shoulder around 255 nm, similar to other absorption bands of N-GQDs prepared by the hydrothermal method, reported in literature [31]. The origin of this shoulder at 255 nm is due to π → π* of C_C bonds. The N-GQDs aqueous solution shows bright blue emission under excitation in UV light. This observation was also verified by fluorescence analysis. The Fluorescence spectrum shows the emission wavelength of the N-GQDs at 443 nm, when excited at 340 nm. 3.1.4. Spectral characterization The nature of surface functional groups was investigated using FTIR spectroscopy. FTIR analysis confirmed effective nitrogen atom doping,
3.2. Loading of MTX to N-GQDs and the MTX releasing properties The states of MTX and N-GQDs in solution are clearly observed from the UV–vis spectrum, as shown in Fig. 5. MTX solution strongly absorbs UV photons at the wavelengths of 380, 305, and 260 nm, due to the existence of heteroaromatic pterine chromophore. The absorbance spectrum of MTX-(N-GQDs) solution exhibits three shoulders at the cited wavelengths with a slight shift to the right (blue-shift) compared with MTX itself, which can be attributed to the vicinity of MTX and N-GQDs in the complex resulting in strong π-π stacking interaction. For example, the peaks of MTX at 305 and 260 nm shifted to 303 and 257 nm after loading onto N-GQDs. Therefore, these results suggest that N-GQDs and MTX molecules formed complex in the solution [18,41,45,46]. The above mentioned hypothesis supportes the FTIR spectrum in Fig. 4, in which it also reveals the strong stacking of MTX onto N-GQDs.
Fig. 7. In-vitro cytotoxicity of MTX-(N-GQDs) after 12 h (a), 24 h (b), 48 h (c) and N-(GQDs) after 48 h (d) incubation time. The MCF-7 cells were exposed to different concentrations (1, 2, 3 and 4 mM) of free MTX (dark bars), and MTX with N-GQDs (grey bars), also different concentrations (10, 20, 30 and 40 mM) of N-GQDs alone. The control samples are the untreated cells. Data are shown as the mean ± SD from three independent experiments. Statistically significance of MTX viability compared to MTX-(N-GQDs) is denoted by “*” and “**”at the level of p b 0.05 and p b 0.01 respectively.
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The in-vitro release profile of MTX from MTX-(N-GQDs) in PBS (pH 7.4) at 37 °C is shown in Fig. 6. About 60% of MTX-(N-GQDs) disassembled after 9 h, followed by the slow release of MTX until all of it dissociated from the complex after 48 h under neutral condition, indicating about 48 h of sustained release at 37 °C in PBS (pH 7.4). 3.3. Cell viability assay The in-vitro cytotoxicity of the synthesized MTX-(N-GQDs) against MCF-7 cells in comparison with those of free MTX or N-GQDs alone was determined through MTT assay (Fig. 7). Viability of the cells in presence of MTX-(N-GQDs) was investigated in those concentrations in which the loaded MTX was equivalent to the free MTX. It is found that the toxicity behavior of the free and loaded MTX depends on the incubation time. For the 12 h incubation time, free MTX shows more toxicity than the loaded one, especially in the lower concentrations, since the free MTX possibly diffuses into the cells rapidly while the loaded MTX takes comparatively longer times to diffuse (Fig. 7a). For the 24 h incubation time, the loaded MTX is gradually showing more cytotoxicity compared to the free MTX (Fig. 7b). This may be occurred because free MTX diffuses out of the cells as easily as it diffuses into the cells, but the loaded MTX remains in the cell more effectively. This could be verified by the 48 h incubation, where the loaded MTX shows significantly more cytotoxicity than the free MTX (Fig. 7c). It was also discovered that N-GQDs exhibits negligible cytotoxicity and is biocompatible (Fig. 7d). Thus, due to the good biocompatibility and high drug loading capacity, the N-GQDs provide an ideal multifunctional delivery vehicle for treatment of cancers [3,34]. 4. Conclusion In summary, we have successfully synthesized about 10 nm nitrogen-doped graphene quantum dots (N-GQDs) with 10 graphitic layers. MTX is loaded to N-GQDs by strong π-π stacking interaction. Furthermore, the layered structure of N-GQDs renders a high specific area to intercalate MTX between the layers creating an inclusive anticancer drug delivery system. In-vitro assays revealed that free MTX rapidly diffuse in and out of the cancer cells, resulting in higher cytotoxicity than MTX(N-GQDs) for incubation times shorter than 24 h. On the other hand, the sluggish diffusion of MTX-(N-GQDs) in and out of the cancer cells leads to its higher cytotoxicity than the free MTX in longer incubation times. Furthermore, the nontoxic properties of N-GQDs assure the biocompatible behavior. All the remarkable findings confirm the accomplishment of GQDs as nanocarriers to prolong cytotoxic effects of its loaded drug for better killing of cancer cells. We expect the subject of future studies to be targeted therapy for cancer treatment aimed at efficient delivery of anti-cancer drugs, with less harm to normal cells. Targeted therapy is usually achieved by conjugating a desired drug to the nanoparticle as targeting ligand, such as monoclonal antibodies, aptamers or folic acid, which provides preferential accumulation of nanoparticles toward cancer cells. Conflict of interest The authors declare no conflict of interest. Acknowledgements Financial support was provided by Iran National Science Foundation (INSF:93002224). The authors would also like to appreciate Research Council of University of Tehran for valuable patronages.
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