Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery Xiaojuan Wang ∗ , Xing Sun, Jun Lao, Hua He, Tiantian Cheng, Mingqing Wang, Shengjie Wang, Fang Huang ∗∗ State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266555, China
a r t i c l e
i n f o
Article history: Received 8 May 2014 Received in revised form 10 July 2014 Accepted 27 July 2014 Available online 7 August 2014 Keywords: Graphene quantum dots Fluorescence Drug delivery Confocal microscopy Selective cytotoxicity
a b s t r a c t This study demonstrates that ligand-modified graphene quantum dots (GQDs) facilitate the simultaneous operation of multiple tasks without the need for external dyes. These tasks include selective cell labeling, targeted drug delivery, and real-time monitoring of cellular uptake. Folic acid (FA)-conjugated GQDs are synthesized and utilized to load the antitumor drug doxorubicin (DOX). The fabricated nanoassembly can unambiguously discriminate cancer cells from normal cells and efficiently deliver the drug to targeted cells. The inherent stable fluorescence of GQDs enables real-time monitoring of the cellular uptake of the DOX–GQD–FA nanoassembly and the consequent release of drugs. The nanoassembly is specifically internalized rapidly by HeLa cells via receptor-mediated endocytosis, whereas DOX release and accumulation are prolonged. In vitro toxicity data suggest that the DOX–GQD–FA nanoassembly can target HeLa cells differentially and efficiently while exhibiting significantly reduced cytotoxicity to non-target cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the past decade, graphene has attracted considerable attention because of its unique physical and chemical properties, such as single-atom-layered structure [1,2], excellent heat and electrical conductivity [3–6], low toxicity [7], and superior mechanical flexibility [8]. The use of graphene has been broadly investigated in various applications, including nanoelectronics [9], energy-storage devices [10,11], and nanocomposite materials [2,12]. Graphene oxide (GO) and its derivatives have also shown considerable potential in biological applications, including molecular imaging [13–15], drug delivery [16], and photothermal therapy [17] due to their hydrophilicity and biocompatibility. GO exhibits an excellent loading capacity for highly aromatic molecules via strong – stacking interactions, which has been employed to deliver various types of water-insoluble drugs into cells [18,19]. Unlike single-walled nanotubes (SWNTs), which load drugs mainly via their surface and tips, GO sheets load drugs via their two faces and edges. The loading ratio of GO (up to 200%) is considerably higher than that of other nanoscale drug carriers [16].
∗ Corresponding author. Tel.: +86 532 86983455. ∗∗ Corresponding author. Tel.: +86 532 86981560; fax: +86 532 86981560. E-mail addresses: [email protected] (X. Wang), [email protected] (F. Huang). http://dx.doi.org/10.1016/j.colsurfb.2014.07.043 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Drug delivery systems have been visualized using organic fluorophores and semiconductor quantum dots to understand the cellular uptake [20–22]. However, the fluorescence of organic dyes, such as rhodamine B and fluorescein isothiocyanate, is significantly quenched by GO after conjugation [21,23,24]. The large size and heavy metal components of semiconductor quantum dots can alter the function of drug delivery systems. Thus, it is desired to develop a functional luminescent nanosystem that can perform imaging and drug delivery tasks without the need for external dyes. In this study, we employed functionalized graphene quantum dots (GQDs) as a novel drug carrier, which has small size and tunable photoluminescence in addition to the remarkable physicochemical properties of GO. Previous studies principally focused on the synthesis and characterization of GQDs [13,15,25–28]. Research on the potential application of GQDs as a cell imaging agent only concentrated on the uptake of bare GQDs by cells without considering the selectivity and function of the agent [13,26,29,30]. In the present study, fluorescent GQDs were synthesized and conjugated with folic acid (FA) to demonstrate highly selective and specific tumor cell imaging. The FA-conjugated GQDs were then employed as a carrier of the antitumor drug doxorubicin (DOX) for targeted cell delivery. The cellular uptake of the DOX–GQD–FA nanoassembly and the consequent drug release were monitored simultaneously in two channels corresponding to GQDs and DOX to reveal their localization and relative internalization kinetics.
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
639
2. Experimental 2.1. Chemicals and materials
Fig. 1. Schematic of the fabricated DOX–GQD–FA nanoassembly for DOX delivery into target cells.
Carbon black (Vulcan CX-72) was obtained from Carbot Co., China. FA was purchased from Sigma–Aldrich, China. 1(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and concentrated nitric acid were obtained from Alfa Aesar Chemical Co., China. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit and DOX were purchased from Shanghai Sangon Biotech Co. High-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Hoechst 33342 were purchased from Life Technologies. All chemicals were used as received without further purification. All solutions were prepared with ultra-pure water (≥18.2 M, Millipore, USA) or 0.01 M phosphate buffer saline (PBS, pH 7.4) unless otherwise specified. 2.2. GQD synthesis and characterization GQDs were prepared as previously described [30]. Briefly, 0.2 g of carbon black was refluxed with nitric acid (50 mL, 6 M)
Fig. 2. Characterization of GQDs. (A) TEM image of GQDs. (B) Size distribution of 120 GQDs, d¯ = 2.3 nm. (C) HRTEM image. (D) AFM image of GQDs. Scale bar is 200 nm. (E) Height profile along the black line in D.
640
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
2.4. Drug loading and release The antitumor drug was loaded by adding 1.0 mL of DOX (100 g/mL) to 4.0 mL of GQD–FA PBS buffer solution (25 g/mL) under stirring for 24 h in the dark at room temperature. The obtained suspension was repeatedly dialyzed using dialysis membrane tubing with a molecular weight cutoff of 2000 Da against PBS buffer for 24 h. The bath solution was changed with fresh PBS buffer every 4 h. All bath solutions were collected, and their absorption at 480 nm was measured to calculate the free drug content. The drug loading efficiency was calculated based on the following equation: drug loading (%) = ((weight of drug added − weight of free drug in the bath solution)/weight of carriers) × 100. The release of DOX from GQD–FA was monitored in PBS buffer at pH 7.4 and 5.5, respectively. The DOX–GQD–FA nanoassembly (1.0 mL, 33.7 g/mL) or free DOX (1.0 mL, 20.0 g/mL) was rapidly mixed with 4.0 mL of PBS buffer and placed in a shaking incubator at 37 ◦ C. The fluorescence spectra of each sample were obtained every 1 h or 2 h within the time course. 2.5. Cell culture Cells were cultured in a DMEM medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere inside an incubator at 37 ◦ C. The cells were harvested from 90% confluent cell culture plates and were resuspended in fresh complete medium before plating. 2.6. Confocal laser microscopy assay
Fig. 3. Characterization of GQD–FA. (A) UV–Vis absorption spectra of GQD, FA, and GQD–FA. (B) FTIR spectra of GQDs and GQD–FA.
for 24 h. The obtained suspension was cooled to room temperature and then centrifuged. The pellet was discarded, and the supernatant was heated and dried to obtain a reddish-brown solid, which was then resuspended in ultra-pure water. The colloidal solution was filtered through a centrifugal filter device using a filtering membrane with a cutoff of 3 kDa (Amicon Ultra4, Millipore). The obtained solution was repeatedly dialyzed against ultra-pure water in a dialysis bag (retaining molecular weight = 1000 Da) for 24 h to obtain a pure GQD solution at neutral pH. The GQD samples were diluted for optical characterization. Ultraviolet–visible (UV–Vis) spectra were obtained using a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were collected using a Hitachi F-2500 fluorescence spectrophotometer. High-resolution transmission electron microscopic (HRTEM) images were recorded on a JEM-2100 electron microscope at 200 kV. Atomic force microscopic (AFM) images were obtained using a Nanoscope IV Multimode AFM (Digital Instruments, Santa Barbara). 2.3. Conjugation of GQDs with FA GQD solution (5.0 mL, 0.3 mg/mL) was allowed to react with EDC (96 mg) and NHS (11.5 mg) in PBS buffer for 30 min at room temperature. FA (10 mM, 1.0 mL) was added, and the mixture was stirred overnight. After 12 h, hydroxylamine (10 mM) was added to quench the reaction. The solution was repeatedly dialyzed using dialysis membrane tubing with a molecular weight cutoff of 1000 Da against PBS buffer for 24 h. After dialysis, the purified GQD–FA solution was stored at 4 ◦ C for further applications. FTIR spectra were obtained using a Nicolet 6700 FTIR spectrometer.
HeLa, A549, and HEK293A were used to verify the selective uptake of GQD–FA. The cells (1 × 105 ) were seeded onto 12 mm sterile coverslips in a 24-well plate. The cells were cultured for 24 h, washed thrice with PBS, and then incubated with GQD–FA (20 g/mL) for 10 min. After washing thrice with cold PBS, the cells were fixed for 20 min in 200 L of 4% paraformaldehyde. After fixation, the cells were washed thrice with PBS, followed by nucleus staining using Hoechst. After 10 min, the cells were washed again with PBS buffer. For the drug delivery assay, the cells were cultured for 24 h, washed with PBS, and then incubated with DOX–GQD–FA containing DMEM medium (33.7 g/mL) at 37 ◦ C for 0.5, 4, 8, and 24 h. After incubation, the cells were washed thrice with PBS and then fixed in 200 L of 4% paraformaldehyde for 20 min. The coverslip with fixed cells was topped by a glass slide with a drop of 10 L of 50% glycerol/PBS (v/v) and placed above the objective on a confocal microscope (Nikon Al, Nikon, Japan). Hoechst was excited with 405 nm laser, and signals were collected from 425 nm to 475 nm. GQDs and DOX were excited with 488 nm laser, and their signals were collected from 500 nm to 530 nm and 552 nm to 617 nm, respectively. 2.7. Cell viability assay The in vitro cytotoxicity of DOX–GQD–FA, free GQDs and DOX was evaluated by MTT cell viability assay. The cells were seeded on 96-well plates at 2 × 104 cells/well in 100 L of medium containing 10% FBS. The plates were incubated for 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. On the day of experiments, the cells were washed with PBS buffer and incubated with fresh medium containing DOX–GQD–FA (33.7 g/mL), GQD (20.0 g/mL), or free DOX (20.0 g/mL). The cells were incubated with the conjugates at 37 ◦ C for 4 h, washed twice with PBS, and then further incubated in fresh growth medium for 24 h. The wells containing cells without GQDs and DOX served as the control. Subsequently, 20 L of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37 ◦ C for 4 h. The precipitated formazan was dissolved
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
641
Fig. 4. Confocal laser scanning microscopy of (A) HeLa human cervical carcinoma cells, (B) A549 adenocarcinomic human alveolar basal epithelial cells, and (C) HEK293A normal human embryonic kidney cells incubated with GQD–FA (20.0 g/mL) at 37 ◦ C for 10 min. Left column: bright field images. Middle column: fluorescence image with Hoechst nuclear stained (blue). Right column: GQD fluorescence image (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
in 150 L of dimethyl sulfoxide. The absorbance of each sample at 570 nm was measured using a microplate autoreader (Molecular Devices, M2e). The cell viability ratio was calculated using A570 nm/A0 570 nm (control). 3. Results and discussion 3.1. Fabrication and characterization of FA-conjugated GQDs The design strategy of the nanoassembly for simultaneous imaging and drug delivery is illustrated in Fig. 1. In the first step, single-layer GQDs with narrow size distribution were prepared, which were then conjugated with FA to facilitate receptor-mediated endocytosis into the target tumor cells. The functionalized GQDs were used as a fluorescent drug carrier to attach DOX for targeted drug delivery. The fluorescence of DOX was quenched after adsorption on the GQD surface and recovered after release in acidic endosome environment. The cellular uptake and consequent drug release processes were monitored simultaneously through two fluorescence channels corresponding to DOX and GQDs. Various methods have been used to cut graphene-based materials through controlled oxidation or reduction processes to fabricate fluorescent GQDs, which show different morphological and optical properties depending on the sizes of the final products and the chemical groups at the edge [13,15,27–29]. The GQDs used in this study were synthesized through a facile method by chemically
oxidizing CX-72 carbon black [30]. The as-prepared GQDs were a mixture of GO sheets with different sizes and ultrafiltration was performed to eliminate large species (>3 kDa). The dried GQDs were dark brown powder, which can easily disperse in water and organic solvents. The GQD water solution was light yellowish under daylight and greenish yellow under UV light (365 nm). HRTEM images show that the diameter of the GQDs ranges from 1 nm to 4 nm, with an average of 2.3 nm (Fig. 2A and B). The crystalline structure of the GQDs is displayed in Fig. 2C. The measured lattice spacing is 0.207 nm, corresponding to the (1 1 0 0) lattice fringe of graphene [31,32]. The GQDs were also observed via AFM to investigate the average height of the products (Fig. 2D and E). Many section lines established on different places reveal that the topographic height of GQDs ranges from 0.5 nm to 0.8 nm, which agrees with the thickness of single-layer graphene [13,30]. The prepared GQDs are planar nanocrystals of graphene with 30 kDa) [14,33]. This unique structure of GQDs offers high surface-to-volume ratio, which is expected to offer higher loading capacity when used as a drug carrier. The GQDs were grafted with FA through amide bond formation via the reaction between the NH2 group of FA and COOH groups of GQDs. The reaction was confirmed by the obtained UV–Vis and FTIR spectra (Fig. 3A and B). In the UV–Vis spectra, a new peak at approximately 280 nm appears after the conjugation,
642
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
indicating the presence of FA. In the FTIR spectra, new peaks at 1271 and 1560 cm−1 appear after EDC/NHS coupling, which are assigned to C N and N H stretching, respectively. The new broad peak in the 3300–3500 cm−1 region corresponds to the N H vibration mode. In addition, characteristic amide–carbonyl ( NH CO ) stretching vibration is observed at 1643 cm−1 , which implies the formation of amide groups in GQD–FA. The fluorescence of GQD is slightly reduced after the conjugation of FA due to the changes of the chemical groups (Fig. S1). GQD–FA was incubated with three cell types that express FA receptor (FR) at different levels to demonstrate that FA retained its binding affinity to FR after conjugation with GQD. As shown in Fig. 4, the fluorescence of the GQDs in the human cervical carcinoma cell line HeLa (Fig. 4A) is considerably stronger than that in the adenocarcinomic human alveolar basal epithelial cell line A549 (Fig. 4B) and human embryonic kidney cell line HEK293A (Fig. 4C). Control experiment showed that no fluorescence was detected in blank HeLa cells, suggesting that the green fluorescence shown in Fig. 4A was not autofluorescence but the internalized GQD–FA (Fig. S2). These results reveal the specific internalization of GQD–FA by HeLa cells, consistent with the fact that HeLa cells overexpress FR while A549 and HEK293A express FR at a low level, indicating that GQD–FA is internalized via FR-induced endocytosis [34,35]. It has been demonstrated that FA-functionalized GQDs can be used as a highly selective cell imaging agent. Since EDC/NHS coupling is a universal route to covalently conjugate amine-bearing molecules, this strategy can be applied to conjugate GQDs with a wide range of biomolecules, including proteins and peptides, and visualize them without interfering with their biological functions. 3.2. Targeted drug delivery with DOX–GQD–FA nanoassembly The antitumor drug DOX was used to evaluate the feasibility of using GQD–FA as a drug carrier. DOX was loaded by mixing with GQD–FA in PBS buffer overnight in the dark, and the unbound DOX was removed by repeated dialysis for 24 h. The single-atom layer of GQDs offers abundant aromatic rings to adsorb DOX through – stacking and hydrophobic interactions [18]. The loading efficiency of DOX on GQD–FA is 68 ± 9 wt% (refer to the equation in the Section 2). The UV–Vis spectrum of the DOX–GQD–FA nanoassembly is shown in Fig. 5A. The featured peak at ∼500 nm indicates the presence of DOX. The absorption peak of DOX attached on the GQDs is bathochromic-shifted by ∼20 nm compared with that of the bare DOX in solution, which can be attributed to the – stacking and hydrophobic interactions between DOX and GQD–FA [16]. The fluorescence spectra (Fig. S3) show that the fluorescence of DOX in the DOX–GQD–FA nanoassembly is considerably weaker than that of the free DOX solution. This phenomenon is likely due to the electron transfer induced fluorescence quench as a consequence of DOX attachment onto the GQD surface, which was also observed previously for dyes adsorbed onto SWNT and GO [36,37]. The fluorescence intensity of DOX–GQD–FA (ex = 488 nm, em = 590 nm) in PBS buffer at pH 5.5 (corresponding to the endosome pH) was recorded at desired time intervals to evaluate the DOX release from the nanoassembly (Fig. 5B). The fluorescence intensities of the nanoassembly in PBS buffer at pH 7.4 and bare DOX in PBS buffer at pH 5.5 and 7.4 served as the control. During the 24 h monitoring period, the fluorescence intensity of bare DOX did not show systematical variation, indicating that the fluorescence of bare DOX is pH inert and stable. The fluorescence intensity of DOX–GQD–FA increased at pH 5.5 but did not change at pH 7.4 during the time course. At the end of the monitoring period, the fluorescence intensity of the nanoassembly at pH 5.5 increased to 3 times higher than that at pH 7.4. This result suggests that some DOX molecules are released at pH 5.5 because of the increased hydrophilicity of the protonated DOX under acidic conditions, which leads to the
Fig. 5. Characterization of DOX–GQD–FA. (A) UV–Vis absorption spectra of DOX, GQD–FA, and DOX–GQD–FA. (B) Time-dependent fluorescence intensity profile of free DOX and DOX–GQD–FA at pH 5.5 and 7.4 (ex = 488 nm, em = 590 nm). The data were displayed as means ± standard deviation with n = 3.
partially recovered fluorescence. Similar fluorescence changes were also observed when DOX was released from SWNTs and GO [18]. Cell imaging assay was performed to evaluate the cell selectivity after drug loading onto GQD–FA. The nanoassembly was incubated with different cells for 4 h instead of 10 min before the cell images were collected to allow the release of DOX. It was found that the fluorescence of DOX was considerably stronger in HeLa cells than in A549 cells (Fig. S4A and B), confirming the target delivery of DOX by the nanoassembly to HeLa cells. When the mixture of DOX–GQD–FA and excessive free FA was applied (Fig. S4C), the fluorescence intensity of DOX in HeLa cells became significantly lower, suggesting a FR-induced uptake mechanism. These results demonstrate that the endocytosis of DOX–GQD–FA is mediated by FR and that the cell selectivity of GQD–FA is effectively maintained after drug loading. 3.3. Real-time monitoring of drug release With the use of fluorescent GQDs, the uptake of the DOX–GQD–FA nanoassembly could be simultaneously monitored through two channels corresponding to GQDs and DOX. As shown in Fig. 6A, DOX–GQD–FA is effectively internalized by HeLa cells within 30 min. The fluorescence of GQDs indicates that they are localized in the cell cytoplasm fraction, which is consistent with the cell imaging of HeLa cells by GQD–FA. However, the fluorescence of DOX could not be observed initially, indicating that most DOX molecules are still adsorbed on the GQD surface and that their fluorescence remains quenched. After 8 h of incubation (Fig. 6B) GQD–FA still localized in the cell cytoplasm, whereas the fluorescence of free DOX was clearly observed in both cytoplasm and
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
643
Fig. 6. Intracellular distribution of internalized DOX–GQD–FA and released free DOX. HeLa cells were incubated with DOX–GQD–FA (33.7 g/mL) at 37 ◦ C for (A) 0.5 h, (B) 8 h, and (C) 24 h. Left to right columns: bright field images, GQD fluorescence images, DOX fluorescence images, overlay of the corresponding images. The GQDs and DOX were excited under 488 nm laser irradiation, and fluorescence signals were collected from 500 nm to 530 nm and 552 nm to 617 nm, respectively.
nuclei. This result demonstrates that DOX is released from the nanoassembly within the acidic endosome, diffuses into the cytoplasm, and then accumulates in the nuclei. After 24 h of incubation with the DOX–GQD–FA nanoassembly (Fig. 6C), HeLa cells showed considerably stronger DOX fluorescence in both cytoplasm and nuclei. This result indicates that DOX has released and accumulated in the cells while the GQDs remain in the cytoplasm. Comparison of the time scale of the fluorescence intensity changes in two channels shows that DOX–GQD–FA endocytosis is rapid while free DOX accumulation is delayed and limited by the drug release step. The different kinetics is exactly the unique character expected for the drug delivery system, where the initial uptake of drug with its carrier is efficient and the subsequent drug release is slow but continuous. 3.4. Cell viability assay The in vitro cytotoxicity of the nanoassembly to HeLa, A549, and HEK293A cells in comparison with DOX or GQD alone was determined by growth inhibition assay to evaluate the efficiency of the targeted delivery of DOX through DOX–GQD–FA nanoassembly. The MTT assay results (Fig. 7) show that the free DOX is taken up by different tumor cells along with the normal cells without obvious selectivity and caused near-equipotent cytotoxicity to all three cell types. After applying the FA-conjugated GQDs as a drug carrier, the DOX–GQD–FA nanoassembly shows a significant specific cytotoxicity to the target HeLa cells compared with the non-target A549 and HEK293A cells. This selectivity is also indicated by the obvious fluorescence signals from HeLa cells in comparison to the blank images of A549 cells after incubation with DOX–GQD–FA at the same condition (Fig. S5). The bare GQDs do not introduce considerable cytotoxicity to all three cell types at our working concentration. Thus, the cytotoxicity of DOX–GQD–FA can mainly be attributed to the free DOX released from the acidic
Fig. 7. Growth inhibition assay (MTT). HeLa, A549, and HEK293A cells were incubated with GQD alone (20.0 g/mL), DOX alone (20.0 g/mL), or DOX–GQD–FA nanoassembly (33.7 g/mL) for 4 h, and the cells were washed with PBS and further incubated for 24 h before measuring cell viability. The data were displayed as means ± standard deviation with n = 6.
endosome after the FR-mediated endocytosis of the nanoassembly. Compared with free GQD, which exhibits no inherent cytotoxicity to all three cell types, the DOX–GQD–FA nanoassembly shows a weak cytotoxicity to FR-negative A549 and HEK293A cells. This weak cytotoxicity can be attributed to the small amount of DOX that is released from the nanoassembly in the culture medium and then diffuses into A549 and HEK293A cells during the incubation period, indicated by the faint DOX fluorescence in A549 cells after 4 h of incubation with DOX–GQD–FA (Fig. S4B). In contrast to free DOX, DOX–GQD–FA shows considerably lower toxicity to the FRnegative A549 and HEK293A cells. These observations suggest that the drug can be delivered with a significant specificity to a certain
644
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
type of target cells with reduced cytotoxicity to the non-target cells by using the functionalized GQDs as a carrier. 4. Conclusion GQDs as a novel drug carrier exhibit several advantages, including small size, excellent biocompatibility, inherent fluorescent property, high surface-to-volume ratio, and abundant carboxylic group rendering conjugation with various anchor molecules. The nanoassembly fabricated by loading drugs onto ligandfunctionalized GQDs enables specific labeling of a certain cell type, thereby effectively delivering drugs to target cells and monitoring the cellular uptake of the drug delivery systems simultaneously. Our results demonstrate that the FA-functionalized GQDs effectively maintain the cell recognition ability of the ligand even after DOX loading, which is crucial for specific cell imaging and target delivery. Given the inherent fluorescence of GQDs, we can easily monitor their movement in the cells in real time without employing external dyes, thereby facilitating simultaneous localization of the drug carrier and the loaded drug. The nanoassembly is internalized by the target cells in a short period via receptormediated endocytosis, whereas the DOX release and accumulation are prolonged. The growth inhibition assay suggests that the DOX–GQD–FA nanoassembly can differentially and efficiently target HeLa cells while exhibiting significantly reduced cytotoxicity to the non-target cells. Thus, the drug-loaded nanoassembly based on GQDs can be used to develop novel targeted therapeutic modalities for effective cancer chemotherapy with improved safety. Furthermore, the FA-functionalized GQDs maintain both the unique optical and morphological properties of GQDs and the bioactivity of FA. By using desired ligands this method could be expanded to build a great variety of functionalized GQDs. Acknowledgements This study was supported by the National Natural Science Foundation of China (21103230, 20905078 and 21033005), the Fundamental Research Funds for the Central Universities (11CX05001A and 12CX04053A), and the Natural Science Foundation for Distinguished Young Scholar of Shandong Province (No. JQ201008). The authors gratefully thank Dr. Hai Xu for his useful suggestions and Dr. Meiwen Cao for his kind assistance with the AFM measurements. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.07.043. References [1] A.K. Geim, K.S. Novoselov, The rise of graphene, Nat. Mater. 6 (2007) 183–191. [2] D. Wu, F. Zhang, H. Liang, X. Feng, Nanocomposites and macroscopic materials assembly of chemically modified graphene sheets, Chem. Soc. Rev. 41 (2012) 6160–6177. [3] A.H. Castro Neto, F. Guinea, N.M.R. Peres, K.S. Novoselov, A.K. Geim, The electronic properties of graphene, Rev. Mod. Phys. 81 (2009) 109–162. [4] H.A. Becerril, J. Mao, Z. Liu, R.M. Stoltenberg, Z. Bao, Y. Chen, Evaluation of solution-processed reduced graphene oxide films as transparent conductors, ACS Nano 2 (2008) 463–470. [5] C. Wang, D. Li, C.O. Too, G.G. Wallace, Electrochemical properties of graphene paper electrodes used in lithium batteries, Chem. Mater. 21 (2009) 2604–2606. [6] H. Ji, L. Zhang, M.T. Pettes, H. Li, S. Chen, L. Shi, et al., Ultrathin graphite foam: a three-dimensional conductive network for battery electrodes, Nano Lett. 12 (2012) 2446–2451. [7] Y. Zhang, T.R. Nayak, H. Hong, W. Cai, Graphene: a versatile nanoplatform for biomedical applications, Nanoscale 4 (2012) 3833–3842.
[8] X. Huang, X. Qi, F. Boey, H. Zhang, Graphene-based composites, Chem. Soc. Rev. 41 (2012) 666–686. [9] R.M. Westervelt, Graphene nanoelectronics, Science 320 (2008) 324–325. [10] Z. Zhang, J. Zhang, N. Chen, L. Qu, Graphene quantum dots: an emerging material for energy-related applications and beyond, Energy Environ. Sci. 5 (2012) 8869–8890. [11] Z.-L. Wang, D. Xu, H.-G. Wang, Z. Wu, X.-B. Zhang, In situ fabrication of porous graphene electrodes for high-performance energy storage, ACS Nano 7 (2013) 2422–2430. [12] S. Stankovich, D.A. Dikin, G.H.B. Dommett, K.M. Kohlhaas, E.J. Zimney, E.A. Stach, et al., Graphene-based composite materials, Nature 442 (2006) 282–286. [13] J. Peng, W. Gao, B.K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, et al., Graphene quantum dots derived from carbon fibers, Nano Lett. 12 (2012) 844–849. [14] X.T. Zheng, A. Than, A. Ananthanaraya, D.-H. Kim, P. Chen, Graphene quantum dots as universal fluorophores and their use in revealing regulated trafficking of insulin receptors in adipocytes, ACS Nano 7 (2013) 6278–6286. [15] M. Zhang, L. Bai, W. Shang, W. Xie, H. Ma, Y. Fu, et al., Facile synthesis of watersoluble, highly fluorescent graphene quantum dots as a robust biological label for stem cells, J. Mater. Chem. 22 (2012) 7461–7467. [16] X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang, Y. Chen, High-efficiency loading and controlled release of doxorubicin hydrochloride on graphene oxide, J. Phys. Chem. C 112 (2008) 17554–17558. [17] J.T. Robinson, S.M. Tabakman, Y. Liang, H. Wang, H. Sanchez Casalongue, D. Vinh, et al., Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy, J. Am. Chem. Soc. 133 (2011) 6825–6831. [18] Z. Liu, X. Sun, N. Nakayama-Ratchford, H. Dai, Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery, ACS Nano 1 (2007) 50–56. [19] Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nanographene oxide for delivery of water-insoluble cancer drugs, J. Am. Chem. Soc. 130 (2008) 10876–10877. [20] M.L. Chen, Y.J. He, X.W. Chen, J.H. Wang, Quantum-dot-conjugated graphene as a probe for simultaneous cancer-targeted fluorescent imaging, tracking, and monitoring drug delivery, Bioconj. Chem. 24 (2013) 387–397. [21] L. Zhang, J. Xia, Q. Zhao, L. Liu, Z. Zhang, Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs, Small 6 (2010) 537–544. [22] Y. Wang, J.T. Chen, X.P. Yan, Fabrication of transferrin functionalized gold nanoclusters/graphene oxide nanocomposite for turn-on near-infrared fluorescent bioimaging of cancer cells and small animals, Anal. Chem. 85 (2013) 2529–2535. [23] Y. He, Y. Lin, H. Tang, D. Pang, A graphene oxide-based fluorescent aptasensor for the turn-on detection of epithelial tumor marker mucin 1, Nanoscale 4 (2012) 2054–2059. [24] C. Zhang, J. Xu, S. Zhang, X. Ji, Z. He, One-pot synthesized DNA-CdTe quantum dots applied in a biosensor for the detection of sequence-specific oligonucleotides, Chemistry 18 (2012) 8296–8300. [25] L. Tang, R. Ji, X. Cao, J. Lin, H. Jiang, X. Li, et al., Deep ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots, ACS Nano 6 (2012) 5102–5110. [26] D. Pan, L. Guo, J. Zhang, C. Xi, Q. Xue, H. Huang, et al., Cutting sp2 clusters in graphene sheets into colloidal graphene quantum dots with strong green fluorescence, J. Mater. Chem. 22 (2012) 3314–3318. [27] H. Tetsuka, R. Asahi, A. Nagoya, K. Okamoto, I. Tajima, R. Ohta, et al., Optically tunable amino-functionalized graphene quantum dots, Adv. Mater. 24 (2012) 5333–5338. [28] F. Yang, M. Zhao, B. Zheng, D. Xiao, L. Wu, Y. Guo, Influence of pH on the fluorescence properties of graphene quantum dots using ozonation pre-oxide hydrothermal synthesis, J. Mater. Chem. 22 (2012) 25471–25479. [29] S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, et al., Strongly greenphotoluminescent graphene quantum dots for bioimaging applications, Chem. Commun. 47 (2011) 6858–6860. [30] Y. Dong, C. Chen, X. Zheng, L. Gao, Z. Cui, H. Yang, et al., One-step and high yield simultaneous preparation of single- and multi-layer graphene quantum dots from CX-72 carbon black, J. Mater. Chem. 22 (2012) 8764–8766. [31] J. Lee, K. Kim, W.I. Park, B.-H. Kim, J.H. Park, T.-H. Kim, et al., Uniform graphene quantum dots patterned from self-assembled silica nanodots, Nano Lett. 12 (2012) 6078–6083. [32] S. Park, J. An, R.D. Piner, I. Jung, D. Yang, A. Velamakanni, et al., Aqueous suspension and characterization of chemically modified graphene sheets, Chem. Mater. 20 (2008) 6592–6594. [33] H. He, M. Feng, J. Hu, C. Chen, J. Wang, X. Wang, et al., Designed short RGD peptides for one-pot aqueous synthesis of integrin-binding CdTe and CdZnTe quantum dots, ACS Appl. Mater. Interfaces 4 (2012) 6362–6370. [34] B. Xue, D.-W. Deng, J. Cao, F. Liu, X. Li, W. Akers, et al., Synthesis of NAC capped near infrared-emitting CdTeS alloyed quantum dots and application for in vivo early tumor imaging, Dalton Trans. 41 (2012) 4935–4947. [35] N. Singh, S. Charan, K. Sanjiv, S.-H. Huang, Y.-C. Hsiao, C.-W. Kuo, et al., Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting, Bioconj. Chem. 23 (2012) 421–430. [36] D.M. Guldi, H. Taieb, G.M.A. Rahman, N. Tagmatarchis, M. Prato, Novel photoactive single-walled carbon nanotube–porphyrin polymer wraps: efficient and long-lived intracomplex charge separation, Adv. Mater. 17 (2005) 871–875. [37] A. Satake, Y. Miyajima, Y. Kobuke, Porphyrin–carbon nanotube composites formed by noncovalent polymer wrapping, Chem. Mater. 17 (2005) 716–724.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery Xiaojuan Wang ∗ , Xing Sun, Jun Lao, Hua He, Tiantian Cheng, Mingqing Wang, Shengjie Wang, Fang Huang ∗∗ State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266555, China
a r t i c l e
i n f o
Article history: Received 8 May 2014 Received in revised form 10 July 2014 Accepted 27 July 2014 Available online 7 August 2014 Keywords: Graphene quantum dots Fluorescence Drug delivery Confocal microscopy Selective cytotoxicity
a b s t r a c t This study demonstrates that ligand-modified graphene quantum dots (GQDs) facilitate the simultaneous operation of multiple tasks without the need for external dyes. These tasks include selective cell labeling, targeted drug delivery, and real-time monitoring of cellular uptake. Folic acid (FA)-conjugated GQDs are synthesized and utilized to load the antitumor drug doxorubicin (DOX). The fabricated nanoassembly can unambiguously discriminate cancer cells from normal cells and efficiently deliver the drug to targeted cells. The inherent stable fluorescence of GQDs enables real-time monitoring of the cellular uptake of the DOX–GQD–FA nanoassembly and the consequent release of drugs. The nanoassembly is specifically internalized rapidly by HeLa cells via receptor-mediated endocytosis, whereas DOX release and accumulation are prolonged. In vitro toxicity data suggest that the DOX–GQD–FA nanoassembly can target HeLa cells differentially and efficiently while exhibiting significantly reduced cytotoxicity to non-target cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the past decade, graphene has attracted considerable attention because of its unique physical and chemical properties, such as single-atom-layered structure [1,2], excellent heat and electrical conductivity [3–6], low toxicity [7], and superior mechanical flexibility [8]. The use of graphene has been broadly investigated in various applications, including nanoelectronics [9], energy-storage devices [10,11], and nanocomposite materials [2,12]. Graphene oxide (GO) and its derivatives have also shown considerable potential in biological applications, including molecular imaging [13–15], drug delivery [16], and photothermal therapy [17] due to their hydrophilicity and biocompatibility. GO exhibits an excellent loading capacity for highly aromatic molecules via strong – stacking interactions, which has been employed to deliver various types of water-insoluble drugs into cells [18,19]. Unlike single-walled nanotubes (SWNTs), which load drugs mainly via their surface and tips, GO sheets load drugs via their two faces and edges. The loading ratio of GO (up to 200%) is considerably higher than that of other nanoscale drug carriers [16].
∗ Corresponding author. Tel.: +86 532 86983455. ∗∗ Corresponding author. Tel.: +86 532 86981560; fax: +86 532 86981560. E-mail addresses: [email protected] (X. Wang), [email protected] (F. Huang). http://dx.doi.org/10.1016/j.colsurfb.2014.07.043 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Drug delivery systems have been visualized using organic fluorophores and semiconductor quantum dots to understand the cellular uptake [20–22]. However, the fluorescence of organic dyes, such as rhodamine B and fluorescein isothiocyanate, is significantly quenched by GO after conjugation [21,23,24]. The large size and heavy metal components of semiconductor quantum dots can alter the function of drug delivery systems. Thus, it is desired to develop a functional luminescent nanosystem that can perform imaging and drug delivery tasks without the need for external dyes. In this study, we employed functionalized graphene quantum dots (GQDs) as a novel drug carrier, which has small size and tunable photoluminescence in addition to the remarkable physicochemical properties of GO. Previous studies principally focused on the synthesis and characterization of GQDs [13,15,25–28]. Research on the potential application of GQDs as a cell imaging agent only concentrated on the uptake of bare GQDs by cells without considering the selectivity and function of the agent [13,26,29,30]. In the present study, fluorescent GQDs were synthesized and conjugated with folic acid (FA) to demonstrate highly selective and specific tumor cell imaging. The FA-conjugated GQDs were then employed as a carrier of the antitumor drug doxorubicin (DOX) for targeted cell delivery. The cellular uptake of the DOX–GQD–FA nanoassembly and the consequent drug release were monitored simultaneously in two channels corresponding to GQDs and DOX to reveal their localization and relative internalization kinetics.
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
639
2. Experimental 2.1. Chemicals and materials
Fig. 1. Schematic of the fabricated DOX–GQD–FA nanoassembly for DOX delivery into target cells.
Carbon black (Vulcan CX-72) was obtained from Carbot Co., China. FA was purchased from Sigma–Aldrich, China. 1(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and concentrated nitric acid were obtained from Alfa Aesar Chemical Co., China. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit and DOX were purchased from Shanghai Sangon Biotech Co. High-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Hoechst 33342 were purchased from Life Technologies. All chemicals were used as received without further purification. All solutions were prepared with ultra-pure water (≥18.2 M, Millipore, USA) or 0.01 M phosphate buffer saline (PBS, pH 7.4) unless otherwise specified. 2.2. GQD synthesis and characterization GQDs were prepared as previously described [30]. Briefly, 0.2 g of carbon black was refluxed with nitric acid (50 mL, 6 M)
Fig. 2. Characterization of GQDs. (A) TEM image of GQDs. (B) Size distribution of 120 GQDs, d¯ = 2.3 nm. (C) HRTEM image. (D) AFM image of GQDs. Scale bar is 200 nm. (E) Height profile along the black line in D.
640
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
2.4. Drug loading and release The antitumor drug was loaded by adding 1.0 mL of DOX (100 g/mL) to 4.0 mL of GQD–FA PBS buffer solution (25 g/mL) under stirring for 24 h in the dark at room temperature. The obtained suspension was repeatedly dialyzed using dialysis membrane tubing with a molecular weight cutoff of 2000 Da against PBS buffer for 24 h. The bath solution was changed with fresh PBS buffer every 4 h. All bath solutions were collected, and their absorption at 480 nm was measured to calculate the free drug content. The drug loading efficiency was calculated based on the following equation: drug loading (%) = ((weight of drug added − weight of free drug in the bath solution)/weight of carriers) × 100. The release of DOX from GQD–FA was monitored in PBS buffer at pH 7.4 and 5.5, respectively. The DOX–GQD–FA nanoassembly (1.0 mL, 33.7 g/mL) or free DOX (1.0 mL, 20.0 g/mL) was rapidly mixed with 4.0 mL of PBS buffer and placed in a shaking incubator at 37 ◦ C. The fluorescence spectra of each sample were obtained every 1 h or 2 h within the time course. 2.5. Cell culture Cells were cultured in a DMEM medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere inside an incubator at 37 ◦ C. The cells were harvested from 90% confluent cell culture plates and were resuspended in fresh complete medium before plating. 2.6. Confocal laser microscopy assay
Fig. 3. Characterization of GQD–FA. (A) UV–Vis absorption spectra of GQD, FA, and GQD–FA. (B) FTIR spectra of GQDs and GQD–FA.
for 24 h. The obtained suspension was cooled to room temperature and then centrifuged. The pellet was discarded, and the supernatant was heated and dried to obtain a reddish-brown solid, which was then resuspended in ultra-pure water. The colloidal solution was filtered through a centrifugal filter device using a filtering membrane with a cutoff of 3 kDa (Amicon Ultra4, Millipore). The obtained solution was repeatedly dialyzed against ultra-pure water in a dialysis bag (retaining molecular weight = 1000 Da) for 24 h to obtain a pure GQD solution at neutral pH. The GQD samples were diluted for optical characterization. Ultraviolet–visible (UV–Vis) spectra were obtained using a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were collected using a Hitachi F-2500 fluorescence spectrophotometer. High-resolution transmission electron microscopic (HRTEM) images were recorded on a JEM-2100 electron microscope at 200 kV. Atomic force microscopic (AFM) images were obtained using a Nanoscope IV Multimode AFM (Digital Instruments, Santa Barbara). 2.3. Conjugation of GQDs with FA GQD solution (5.0 mL, 0.3 mg/mL) was allowed to react with EDC (96 mg) and NHS (11.5 mg) in PBS buffer for 30 min at room temperature. FA (10 mM, 1.0 mL) was added, and the mixture was stirred overnight. After 12 h, hydroxylamine (10 mM) was added to quench the reaction. The solution was repeatedly dialyzed using dialysis membrane tubing with a molecular weight cutoff of 1000 Da against PBS buffer for 24 h. After dialysis, the purified GQD–FA solution was stored at 4 ◦ C for further applications. FTIR spectra were obtained using a Nicolet 6700 FTIR spectrometer.
HeLa, A549, and HEK293A were used to verify the selective uptake of GQD–FA. The cells (1 × 105 ) were seeded onto 12 mm sterile coverslips in a 24-well plate. The cells were cultured for 24 h, washed thrice with PBS, and then incubated with GQD–FA (20 g/mL) for 10 min. After washing thrice with cold PBS, the cells were fixed for 20 min in 200 L of 4% paraformaldehyde. After fixation, the cells were washed thrice with PBS, followed by nucleus staining using Hoechst. After 10 min, the cells were washed again with PBS buffer. For the drug delivery assay, the cells were cultured for 24 h, washed with PBS, and then incubated with DOX–GQD–FA containing DMEM medium (33.7 g/mL) at 37 ◦ C for 0.5, 4, 8, and 24 h. After incubation, the cells were washed thrice with PBS and then fixed in 200 L of 4% paraformaldehyde for 20 min. The coverslip with fixed cells was topped by a glass slide with a drop of 10 L of 50% glycerol/PBS (v/v) and placed above the objective on a confocal microscope (Nikon Al, Nikon, Japan). Hoechst was excited with 405 nm laser, and signals were collected from 425 nm to 475 nm. GQDs and DOX were excited with 488 nm laser, and their signals were collected from 500 nm to 530 nm and 552 nm to 617 nm, respectively. 2.7. Cell viability assay The in vitro cytotoxicity of DOX–GQD–FA, free GQDs and DOX was evaluated by MTT cell viability assay. The cells were seeded on 96-well plates at 2 × 104 cells/well in 100 L of medium containing 10% FBS. The plates were incubated for 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. On the day of experiments, the cells were washed with PBS buffer and incubated with fresh medium containing DOX–GQD–FA (33.7 g/mL), GQD (20.0 g/mL), or free DOX (20.0 g/mL). The cells were incubated with the conjugates at 37 ◦ C for 4 h, washed twice with PBS, and then further incubated in fresh growth medium for 24 h. The wells containing cells without GQDs and DOX served as the control. Subsequently, 20 L of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37 ◦ C for 4 h. The precipitated formazan was dissolved
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
641
Fig. 4. Confocal laser scanning microscopy of (A) HeLa human cervical carcinoma cells, (B) A549 adenocarcinomic human alveolar basal epithelial cells, and (C) HEK293A normal human embryonic kidney cells incubated with GQD–FA (20.0 g/mL) at 37 ◦ C for 10 min. Left column: bright field images. Middle column: fluorescence image with Hoechst nuclear stained (blue). Right column: GQD fluorescence image (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
in 150 L of dimethyl sulfoxide. The absorbance of each sample at 570 nm was measured using a microplate autoreader (Molecular Devices, M2e). The cell viability ratio was calculated using A570 nm/A0 570 nm (control). 3. Results and discussion 3.1. Fabrication and characterization of FA-conjugated GQDs The design strategy of the nanoassembly for simultaneous imaging and drug delivery is illustrated in Fig. 1. In the first step, single-layer GQDs with narrow size distribution were prepared, which were then conjugated with FA to facilitate receptor-mediated endocytosis into the target tumor cells. The functionalized GQDs were used as a fluorescent drug carrier to attach DOX for targeted drug delivery. The fluorescence of DOX was quenched after adsorption on the GQD surface and recovered after release in acidic endosome environment. The cellular uptake and consequent drug release processes were monitored simultaneously through two fluorescence channels corresponding to DOX and GQDs. Various methods have been used to cut graphene-based materials through controlled oxidation or reduction processes to fabricate fluorescent GQDs, which show different morphological and optical properties depending on the sizes of the final products and the chemical groups at the edge [13,15,27–29]. The GQDs used in this study were synthesized through a facile method by chemically
oxidizing CX-72 carbon black [30]. The as-prepared GQDs were a mixture of GO sheets with different sizes and ultrafiltration was performed to eliminate large species (>3 kDa). The dried GQDs were dark brown powder, which can easily disperse in water and organic solvents. The GQD water solution was light yellowish under daylight and greenish yellow under UV light (365 nm). HRTEM images show that the diameter of the GQDs ranges from 1 nm to 4 nm, with an average of 2.3 nm (Fig. 2A and B). The crystalline structure of the GQDs is displayed in Fig. 2C. The measured lattice spacing is 0.207 nm, corresponding to the (1 1 0 0) lattice fringe of graphene [31,32]. The GQDs were also observed via AFM to investigate the average height of the products (Fig. 2D and E). Many section lines established on different places reveal that the topographic height of GQDs ranges from 0.5 nm to 0.8 nm, which agrees with the thickness of single-layer graphene [13,30]. The prepared GQDs are planar nanocrystals of graphene with 30 kDa) [14,33]. This unique structure of GQDs offers high surface-to-volume ratio, which is expected to offer higher loading capacity when used as a drug carrier. The GQDs were grafted with FA through amide bond formation via the reaction between the NH2 group of FA and COOH groups of GQDs. The reaction was confirmed by the obtained UV–Vis and FTIR spectra (Fig. 3A and B). In the UV–Vis spectra, a new peak at approximately 280 nm appears after the conjugation,
642
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
indicating the presence of FA. In the FTIR spectra, new peaks at 1271 and 1560 cm−1 appear after EDC/NHS coupling, which are assigned to C N and N H stretching, respectively. The new broad peak in the 3300–3500 cm−1 region corresponds to the N H vibration mode. In addition, characteristic amide–carbonyl ( NH CO ) stretching vibration is observed at 1643 cm−1 , which implies the formation of amide groups in GQD–FA. The fluorescence of GQD is slightly reduced after the conjugation of FA due to the changes of the chemical groups (Fig. S1). GQD–FA was incubated with three cell types that express FA receptor (FR) at different levels to demonstrate that FA retained its binding affinity to FR after conjugation with GQD. As shown in Fig. 4, the fluorescence of the GQDs in the human cervical carcinoma cell line HeLa (Fig. 4A) is considerably stronger than that in the adenocarcinomic human alveolar basal epithelial cell line A549 (Fig. 4B) and human embryonic kidney cell line HEK293A (Fig. 4C). Control experiment showed that no fluorescence was detected in blank HeLa cells, suggesting that the green fluorescence shown in Fig. 4A was not autofluorescence but the internalized GQD–FA (Fig. S2). These results reveal the specific internalization of GQD–FA by HeLa cells, consistent with the fact that HeLa cells overexpress FR while A549 and HEK293A express FR at a low level, indicating that GQD–FA is internalized via FR-induced endocytosis [34,35]. It has been demonstrated that FA-functionalized GQDs can be used as a highly selective cell imaging agent. Since EDC/NHS coupling is a universal route to covalently conjugate amine-bearing molecules, this strategy can be applied to conjugate GQDs with a wide range of biomolecules, including proteins and peptides, and visualize them without interfering with their biological functions. 3.2. Targeted drug delivery with DOX–GQD–FA nanoassembly The antitumor drug DOX was used to evaluate the feasibility of using GQD–FA as a drug carrier. DOX was loaded by mixing with GQD–FA in PBS buffer overnight in the dark, and the unbound DOX was removed by repeated dialysis for 24 h. The single-atom layer of GQDs offers abundant aromatic rings to adsorb DOX through – stacking and hydrophobic interactions [18]. The loading efficiency of DOX on GQD–FA is 68 ± 9 wt% (refer to the equation in the Section 2). The UV–Vis spectrum of the DOX–GQD–FA nanoassembly is shown in Fig. 5A. The featured peak at ∼500 nm indicates the presence of DOX. The absorption peak of DOX attached on the GQDs is bathochromic-shifted by ∼20 nm compared with that of the bare DOX in solution, which can be attributed to the – stacking and hydrophobic interactions between DOX and GQD–FA [16]. The fluorescence spectra (Fig. S3) show that the fluorescence of DOX in the DOX–GQD–FA nanoassembly is considerably weaker than that of the free DOX solution. This phenomenon is likely due to the electron transfer induced fluorescence quench as a consequence of DOX attachment onto the GQD surface, which was also observed previously for dyes adsorbed onto SWNT and GO [36,37]. The fluorescence intensity of DOX–GQD–FA (ex = 488 nm, em = 590 nm) in PBS buffer at pH 5.5 (corresponding to the endosome pH) was recorded at desired time intervals to evaluate the DOX release from the nanoassembly (Fig. 5B). The fluorescence intensities of the nanoassembly in PBS buffer at pH 7.4 and bare DOX in PBS buffer at pH 5.5 and 7.4 served as the control. During the 24 h monitoring period, the fluorescence intensity of bare DOX did not show systematical variation, indicating that the fluorescence of bare DOX is pH inert and stable. The fluorescence intensity of DOX–GQD–FA increased at pH 5.5 but did not change at pH 7.4 during the time course. At the end of the monitoring period, the fluorescence intensity of the nanoassembly at pH 5.5 increased to 3 times higher than that at pH 7.4. This result suggests that some DOX molecules are released at pH 5.5 because of the increased hydrophilicity of the protonated DOX under acidic conditions, which leads to the
Fig. 5. Characterization of DOX–GQD–FA. (A) UV–Vis absorption spectra of DOX, GQD–FA, and DOX–GQD–FA. (B) Time-dependent fluorescence intensity profile of free DOX and DOX–GQD–FA at pH 5.5 and 7.4 (ex = 488 nm, em = 590 nm). The data were displayed as means ± standard deviation with n = 3.
partially recovered fluorescence. Similar fluorescence changes were also observed when DOX was released from SWNTs and GO [18]. Cell imaging assay was performed to evaluate the cell selectivity after drug loading onto GQD–FA. The nanoassembly was incubated with different cells for 4 h instead of 10 min before the cell images were collected to allow the release of DOX. It was found that the fluorescence of DOX was considerably stronger in HeLa cells than in A549 cells (Fig. S4A and B), confirming the target delivery of DOX by the nanoassembly to HeLa cells. When the mixture of DOX–GQD–FA and excessive free FA was applied (Fig. S4C), the fluorescence intensity of DOX in HeLa cells became significantly lower, suggesting a FR-induced uptake mechanism. These results demonstrate that the endocytosis of DOX–GQD–FA is mediated by FR and that the cell selectivity of GQD–FA is effectively maintained after drug loading. 3.3. Real-time monitoring of drug release With the use of fluorescent GQDs, the uptake of the DOX–GQD–FA nanoassembly could be simultaneously monitored through two channels corresponding to GQDs and DOX. As shown in Fig. 6A, DOX–GQD–FA is effectively internalized by HeLa cells within 30 min. The fluorescence of GQDs indicates that they are localized in the cell cytoplasm fraction, which is consistent with the cell imaging of HeLa cells by GQD–FA. However, the fluorescence of DOX could not be observed initially, indicating that most DOX molecules are still adsorbed on the GQD surface and that their fluorescence remains quenched. After 8 h of incubation (Fig. 6B) GQD–FA still localized in the cell cytoplasm, whereas the fluorescence of free DOX was clearly observed in both cytoplasm and
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
643
Fig. 6. Intracellular distribution of internalized DOX–GQD–FA and released free DOX. HeLa cells were incubated with DOX–GQD–FA (33.7 g/mL) at 37 ◦ C for (A) 0.5 h, (B) 8 h, and (C) 24 h. Left to right columns: bright field images, GQD fluorescence images, DOX fluorescence images, overlay of the corresponding images. The GQDs and DOX were excited under 488 nm laser irradiation, and fluorescence signals were collected from 500 nm to 530 nm and 552 nm to 617 nm, respectively.
nuclei. This result demonstrates that DOX is released from the nanoassembly within the acidic endosome, diffuses into the cytoplasm, and then accumulates in the nuclei. After 24 h of incubation with the DOX–GQD–FA nanoassembly (Fig. 6C), HeLa cells showed considerably stronger DOX fluorescence in both cytoplasm and nuclei. This result indicates that DOX has released and accumulated in the cells while the GQDs remain in the cytoplasm. Comparison of the time scale of the fluorescence intensity changes in two channels shows that DOX–GQD–FA endocytosis is rapid while free DOX accumulation is delayed and limited by the drug release step. The different kinetics is exactly the unique character expected for the drug delivery system, where the initial uptake of drug with its carrier is efficient and the subsequent drug release is slow but continuous. 3.4. Cell viability assay The in vitro cytotoxicity of the nanoassembly to HeLa, A549, and HEK293A cells in comparison with DOX or GQD alone was determined by growth inhibition assay to evaluate the efficiency of the targeted delivery of DOX through DOX–GQD–FA nanoassembly. The MTT assay results (Fig. 7) show that the free DOX is taken up by different tumor cells along with the normal cells without obvious selectivity and caused near-equipotent cytotoxicity to all three cell types. After applying the FA-conjugated GQDs as a drug carrier, the DOX–GQD–FA nanoassembly shows a significant specific cytotoxicity to the target HeLa cells compared with the non-target A549 and HEK293A cells. This selectivity is also indicated by the obvious fluorescence signals from HeLa cells in comparison to the blank images of A549 cells after incubation with DOX–GQD–FA at the same condition (Fig. S5). The bare GQDs do not introduce considerable cytotoxicity to all three cell types at our working concentration. Thus, the cytotoxicity of DOX–GQD–FA can mainly be attributed to the free DOX released from the acidic
Fig. 7. Growth inhibition assay (MTT). HeLa, A549, and HEK293A cells were incubated with GQD alone (20.0 g/mL), DOX alone (20.0 g/mL), or DOX–GQD–FA nanoassembly (33.7 g/mL) for 4 h, and the cells were washed with PBS and further incubated for 24 h before measuring cell viability. The data were displayed as means ± standard deviation with n = 6.
endosome after the FR-mediated endocytosis of the nanoassembly. Compared with free GQD, which exhibits no inherent cytotoxicity to all three cell types, the DOX–GQD–FA nanoassembly shows a weak cytotoxicity to FR-negative A549 and HEK293A cells. This weak cytotoxicity can be attributed to the small amount of DOX that is released from the nanoassembly in the culture medium and then diffuses into A549 and HEK293A cells during the incubation period, indicated by the faint DOX fluorescence in A549 cells after 4 h of incubation with DOX–GQD–FA (Fig. S4B). In contrast to free DOX, DOX–GQD–FA shows considerably lower toxicity to the FRnegative A549 and HEK293A cells. These observations suggest that the drug can be delivered with a significant specificity to a certain
644
X. Wang et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 638–644
type of target cells with reduced cytotoxicity to the non-target cells by using the functionalized GQDs as a carrier. 4. Conclusion GQDs as a novel drug carrier exhibit several advantages, including small size, excellent biocompatibility, inherent fluorescent property, high surface-to-volume ratio, and abundant carboxylic group rendering conjugation with various anchor molecules. The nanoassembly fabricated by loading drugs onto ligandfunctionalized GQDs enables specific labeling of a certain cell type, thereby effectively delivering drugs to target cells and monitoring the cellular uptake of the drug delivery systems simultaneously. Our results demonstrate that the FA-functionalized GQDs effectively maintain the cell recognition ability of the ligand even after DOX loading, which is crucial for specific cell imaging and target delivery. Given the inherent fluorescence of GQDs, we can easily monitor their movement in the cells in real time without employing external dyes, thereby facilitating simultaneous localization of the drug carrier and the loaded drug. The nanoassembly is internalized by the target cells in a short period via receptormediated endocytosis, whereas the DOX release and accumulation are prolonged. The growth inhibition assay suggests that the DOX–GQD–FA nanoassembly can differentially and efficiently target HeLa cells while exhibiting significantly reduced cytotoxicity to the non-target cells. Thus, the drug-loaded nanoassembly based on GQDs can be used to develop novel targeted therapeutic modalities for effective cancer chemotherapy with improved safety. Furthermore, the FA-functionalized GQDs maintain both the unique optical and morphological properties of GQDs and the bioactivity of FA. By using desired ligands this method could be expanded to build a great variety of functionalized GQDs. Acknowledgements This study was supported by the National Natural Science Foundation of China (21103230, 20905078 and 21033005), the Fundamental Research Funds for the Central Universities (11CX05001A and 12CX04053A), and the Natural Science Foundation for Distinguished Young Scholar of Shandong Province (No. JQ201008). The authors gratefully thank Dr. Hai Xu for his useful suggestions and Dr. Meiwen Cao for his kind assistance with the AFM measurements. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.07.043. References [1] A.K. Geim, K.S. Novoselov, The rise of graphene, Nat. Mater. 6 (2007) 183–191. [2] D. Wu, F. Zhang, H. Liang, X. Feng, Nanocomposites and macroscopic materials assembly of chemically modified graphene sheets, Chem. Soc. Rev. 41 (2012) 6160–6177. [3] A.H. Castro Neto, F. Guinea, N.M.R. Peres, K.S. Novoselov, A.K. Geim, The electronic properties of graphene, Rev. Mod. Phys. 81 (2009) 109–162. [4] H.A. Becerril, J. Mao, Z. Liu, R.M. Stoltenberg, Z. Bao, Y. Chen, Evaluation of solution-processed reduced graphene oxide films as transparent conductors, ACS Nano 2 (2008) 463–470. [5] C. Wang, D. Li, C.O. Too, G.G. Wallace, Electrochemical properties of graphene paper electrodes used in lithium batteries, Chem. Mater. 21 (2009) 2604–2606. [6] H. Ji, L. Zhang, M.T. Pettes, H. Li, S. Chen, L. Shi, et al., Ultrathin graphite foam: a three-dimensional conductive network for battery electrodes, Nano Lett. 12 (2012) 2446–2451. [7] Y. Zhang, T.R. Nayak, H. Hong, W. Cai, Graphene: a versatile nanoplatform for biomedical applications, Nanoscale 4 (2012) 3833–3842.
[8] X. Huang, X. Qi, F. Boey, H. Zhang, Graphene-based composites, Chem. Soc. Rev. 41 (2012) 666–686. [9] R.M. Westervelt, Graphene nanoelectronics, Science 320 (2008) 324–325. [10] Z. Zhang, J. Zhang, N. Chen, L. Qu, Graphene quantum dots: an emerging material for energy-related applications and beyond, Energy Environ. Sci. 5 (2012) 8869–8890. [11] Z.-L. Wang, D. Xu, H.-G. Wang, Z. Wu, X.-B. Zhang, In situ fabrication of porous graphene electrodes for high-performance energy storage, ACS Nano 7 (2013) 2422–2430. [12] S. Stankovich, D.A. Dikin, G.H.B. Dommett, K.M. Kohlhaas, E.J. Zimney, E.A. Stach, et al., Graphene-based composite materials, Nature 442 (2006) 282–286. [13] J. Peng, W. Gao, B.K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, et al., Graphene quantum dots derived from carbon fibers, Nano Lett. 12 (2012) 844–849. [14] X.T. Zheng, A. Than, A. Ananthanaraya, D.-H. Kim, P. Chen, Graphene quantum dots as universal fluorophores and their use in revealing regulated trafficking of insulin receptors in adipocytes, ACS Nano 7 (2013) 6278–6286. [15] M. Zhang, L. Bai, W. Shang, W. Xie, H. Ma, Y. Fu, et al., Facile synthesis of watersoluble, highly fluorescent graphene quantum dots as a robust biological label for stem cells, J. Mater. Chem. 22 (2012) 7461–7467. [16] X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang, Y. Chen, High-efficiency loading and controlled release of doxorubicin hydrochloride on graphene oxide, J. Phys. Chem. C 112 (2008) 17554–17558. [17] J.T. Robinson, S.M. Tabakman, Y. Liang, H. Wang, H. Sanchez Casalongue, D. Vinh, et al., Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy, J. Am. Chem. Soc. 133 (2011) 6825–6831. [18] Z. Liu, X. Sun, N. Nakayama-Ratchford, H. Dai, Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery, ACS Nano 1 (2007) 50–56. [19] Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nanographene oxide for delivery of water-insoluble cancer drugs, J. Am. Chem. Soc. 130 (2008) 10876–10877. [20] M.L. Chen, Y.J. He, X.W. Chen, J.H. Wang, Quantum-dot-conjugated graphene as a probe for simultaneous cancer-targeted fluorescent imaging, tracking, and monitoring drug delivery, Bioconj. Chem. 24 (2013) 387–397. [21] L. Zhang, J. Xia, Q. Zhao, L. Liu, Z. Zhang, Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs, Small 6 (2010) 537–544. [22] Y. Wang, J.T. Chen, X.P. Yan, Fabrication of transferrin functionalized gold nanoclusters/graphene oxide nanocomposite for turn-on near-infrared fluorescent bioimaging of cancer cells and small animals, Anal. Chem. 85 (2013) 2529–2535. [23] Y. He, Y. Lin, H. Tang, D. Pang, A graphene oxide-based fluorescent aptasensor for the turn-on detection of epithelial tumor marker mucin 1, Nanoscale 4 (2012) 2054–2059. [24] C. Zhang, J. Xu, S. Zhang, X. Ji, Z. He, One-pot synthesized DNA-CdTe quantum dots applied in a biosensor for the detection of sequence-specific oligonucleotides, Chemistry 18 (2012) 8296–8300. [25] L. Tang, R. Ji, X. Cao, J. Lin, H. Jiang, X. Li, et al., Deep ultraviolet photoluminescence of water-soluble self-passivated graphene quantum dots, ACS Nano 6 (2012) 5102–5110. [26] D. Pan, L. Guo, J. Zhang, C. Xi, Q. Xue, H. Huang, et al., Cutting sp2 clusters in graphene sheets into colloidal graphene quantum dots with strong green fluorescence, J. Mater. Chem. 22 (2012) 3314–3318. [27] H. Tetsuka, R. Asahi, A. Nagoya, K. Okamoto, I. Tajima, R. Ohta, et al., Optically tunable amino-functionalized graphene quantum dots, Adv. Mater. 24 (2012) 5333–5338. [28] F. Yang, M. Zhao, B. Zheng, D. Xiao, L. Wu, Y. Guo, Influence of pH on the fluorescence properties of graphene quantum dots using ozonation pre-oxide hydrothermal synthesis, J. Mater. Chem. 22 (2012) 25471–25479. [29] S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, et al., Strongly greenphotoluminescent graphene quantum dots for bioimaging applications, Chem. Commun. 47 (2011) 6858–6860. [30] Y. Dong, C. Chen, X. Zheng, L. Gao, Z. Cui, H. Yang, et al., One-step and high yield simultaneous preparation of single- and multi-layer graphene quantum dots from CX-72 carbon black, J. Mater. Chem. 22 (2012) 8764–8766. [31] J. Lee, K. Kim, W.I. Park, B.-H. Kim, J.H. Park, T.-H. Kim, et al., Uniform graphene quantum dots patterned from self-assembled silica nanodots, Nano Lett. 12 (2012) 6078–6083. [32] S. Park, J. An, R.D. Piner, I. Jung, D. Yang, A. Velamakanni, et al., Aqueous suspension and characterization of chemically modified graphene sheets, Chem. Mater. 20 (2008) 6592–6594. [33] H. He, M. Feng, J. Hu, C. Chen, J. Wang, X. Wang, et al., Designed short RGD peptides for one-pot aqueous synthesis of integrin-binding CdTe and CdZnTe quantum dots, ACS Appl. Mater. Interfaces 4 (2012) 6362–6370. [34] B. Xue, D.-W. Deng, J. Cao, F. Liu, X. Li, W. Akers, et al., Synthesis of NAC capped near infrared-emitting CdTeS alloyed quantum dots and application for in vivo early tumor imaging, Dalton Trans. 41 (2012) 4935–4947. [35] N. Singh, S. Charan, K. Sanjiv, S.-H. Huang, Y.-C. Hsiao, C.-W. Kuo, et al., Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting, Bioconj. Chem. 23 (2012) 421–430. [36] D.M. Guldi, H. Taieb, G.M.A. Rahman, N. Tagmatarchis, M. Prato, Novel photoactive single-walled carbon nanotube–porphyrin polymer wraps: efficient and long-lived intracomplex charge separation, Adv. Mater. 17 (2005) 871–875. [37] A. Satake, Y. Miyajima, Y. Kobuke, Porphyrin–carbon nanotube composites formed by noncovalent polymer wrapping, Chem. Mater. 17 (2005) 716–724.
