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This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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DOI: 10.1039/C4NR07005D
Cite this: DOI: 10.1039/c0xx00000x
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Prathik Roy,a Arun Prakash Periasamy,a Chou-Ya Lin,b Guor-Mour Her,b Wei-Jane Chiu,b Chi-Lin Li,a Chia-Lun Shu,a Chih-Ching Huang,b* Chi-Te Liang,c,d and Huan-Tsung Changa* 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro, however there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescent properties of plant leaf-derived graphene quantum dots (GQDs) modified with Annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish body without affecting its growth significantly at a concentration lower than 2 mg mL−1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of biomedical applications for graphene-based nanomaterials.
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Photoluminescent carbon-based nanomaterials have attracted great attention due to their unique optical properties and high biocompatibility.1 With their high photoluminescence (PL) and biocompatibility, graphene quantum dots (GQDs) and carbon dots (C-dots) have been used for biosensing and cell imaging.1a, 2 In addition, some C-dots have been found to be efficient in inhibiting the growth of cancer cells.1c, 2 GQDs have been used as photodynamic agents for killing human glioma cells through generating reactive oxygen species upon photo-irradiation.3 Recently, we have demonstrated that high-quality GQDs and Cdots can be prepared from plant leaves,1a coffee ground,1b used tea,1c and low-cost organic compounds such as glycine.1b Compared with their semiconductor counterparts, these C-dots and GQDs may have lower quantum yield (Φ) values but possess far greater biocompatibility, photostability and water solubility and easy modification.1a, 1b, 4 Although fluorescent dyes and proteins based fluorescent probes have been widely used in in vivo imaging, they may suffer from photo-decomposition.5 This journal is © The Royal Society of Chemistry [year]
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Recently, noble metal nanoclusters (NCs) such as gold and silver NCs have also been employed for imaging due to its advantages of controllable emission and good solubility.6 However, they are expensive and suffer from lower Φ values and photostability.6a Moreover, the potential toxicity of these metallic NCs is poorly understood. Thus, the development of greatly photostable nanoprobe with high Φ for in vivo imaging is highly desirable. Recently, GQDs have been proposed as a plausible theronastic nanomaterial. The great surface areas of GQDs enables it to be used as a potential drug delivery platform.7 However, there have been few studies regarding the cytotoxicity of GQDs.8 Here we investigate the cytotoxicity of GQDs both in vitro and in vivo. The zebrafish (Danio rerio) embryo has emerged as an invaluable vertebrate model over other systems such as mouse, rat and fly for the purpose of assessing developmental toxicity, studying genetic and acquired diseases, and drug discovery owing to its small size, heavy brooding, and rapid maturation time.9 Furthermore, the zebrafish embryos are transparent and allow visual detection of embryonic cell death and maldevelopment phenotypes in vivo.9c More recently, the zebrafish model have been utilized to study the implications of engineered nanomaterials and nano-related products from the perspective of environmental and human safety.10 Utility of zebrafish for nanotoxicity study is largely based upon the close homology with the human genome.11 Hundreds of transgenic lines of zebrafish have been established until now, each labeling specific types of signaling molecules, cells, tissues, or organs.12 These transgenic lines of zebrafish have been successfully employed for many genetic and molecular analyses as well as the assessment of the biotoxicity of newly developed nanomaterials. Programmed cell death (apoptosis) on the other hand, is a way for multicellular animal to dispose of unwanted and damaged cells. The cell apoptosis has been linked to many debilitating diseases.13 For instance, it has been found that excess apoptosis in mice induces the deficiency of CPP32 caspase enzyme leading to neurodegeneration.14 Moreover, improper regulation of apoptosis has been associated to cancer, neurodegenerative and cardiovascular diseases.15 Nonetheless, how the abnormal cell apoptosis begins and progresses in vivo is not yet fully understood. Understanding various forms of apoptosis could be the key to unlocking the diagnosis and therapy of the related diseases. Several methods such as fluorescent nucleic binding dyes, ie: acridine orange,16 ethidium bromide,16 and propidium [journal], [year], [vol], 00–00 | 1
Nanoscale Accepted Manuscript
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Photoluminescent Graphene Quantum Dots for in vivo Imaging of Apoptotic Cells
Nanoscale
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DOI: 10.1039/C4NR07005D
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Experimental Chemicals Neem leaves were bought from the local (Chennai) market in India. Sodium phosphate monobasic, anhydrous sodium phosphate dibasic, trisodium phosphate, and tris(hydroxymethyl) aminomethane (Tris) were obtained from J.T. Baker (Phillipsburg, NJ, USA). Ultrapure water (18.2 MΩ-cm) was obtained using a Milli-Q ultrapure system (Millipore, Billerica, MA). MCF-7, MCF-10A and HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The pH values of phosphate buffer solutions are 2.0–12.0 prepared from sodium phosphate monobasic, sodium phosphate dibasic, and trisodium phosphate. Phosphate buffered saline (PBS; 1x, 1 L, pH 7.4) contained NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.44 g), and KH2PO4 (0.24 g).
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Synthesis of GQDs GQDs were prepared according to our previous report.[1] In a typical procedure, Neem leaf extract (250 mg) in 20 mL DI water was hydrothermally treated in an autoclave at 300 ºC for 8 h. The reacted solution was centrifuged at a centrifugation force (RCF) of 25,000 g for 20 min to remove the larger particles. The GQDs solution (10 mL) was then dialyzed in ultrapure water (2 L) through a dialysis membrane (MWCO = 3.5–5 kD, Float-A-Lyzer G2, Spectrum Laboratories, Rancho Dominguez, CA, USA) for 24 h. The water was changed with ultrapure water per 4 h. Aliquots (10 mL) of the as-prepared solution containing purified
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GQDs were dried overnight at 60 °C in an oven to obtain pure GQDs. We determined the concentration of purified GQDs solution to be 12 mg mL−1. Characterization of GQDs A double-beam UV–vis spectrophotometer (Cintra 10e, GBC) was used to record the absorption spectra of the GQDs. JEOL JSM-1230 and FEI Tecnai-G2-F20 transmission electron microscopes (TEM) were used to measure the sizes and shapes of the as-prepared GQDs. The re-dispersed GQDs were separately placed on formvar/carbon film Cu grids (200 mesh; Agar Scientific) and dried at ambient temperature. X-ray diffraction (XRD) pattern of GQDs was measured using a PANalytical X’Pert PRO diffractometer (PANalytical B.V., EA Almelo, Netherlands) and Cu-Ka radiation (λ = 0.15418 nm); the samples were prepared on Si substrates. An energy dispersive X-ray spectroscopy (EDS; Inca Energy 200, Oxford) was used to determine the composition of the asprepared GQDs. PL spectra of GQDs were recorded using a Cary Eclipse PL spectrophotometer (Varian CA, USA) operated at excitation wavelengths in the range 350–500 nm. The photostability of GQDs was investigated under continuous illumination of the Xe lamp in Cary Eclipse PL spectrophotometer. Raman spectra of GQDs were recorded using a Raman spectrometer (Dong Woo 500i, Korea) equipped with a 50× objective and a charge-coupled detector. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5000 VersaProbe (Physical Electronics, Eden Prairie, MN, USA). Cellular imaging The human breast cancer cells (MCF-7) were maintained in the RPMI-1640 medium supplemented with fetal bovine serum (FBS, 10%) and 1% Gibco® antibiotic-antimycotic solution in 5% CO2 at 37°C. The human cervical cancer cells (HeLa) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with FBS (10%) and 1% Gibco® antibiotic-antimycotic solution. The normal human mammary epithelial cells (MCF-10A) were maintained in Gibco α-Minimum Essential Media (α-MEM) supplemented with FBS (10%) and 1% Gibco® antibioticantimycotic solution in 5% CO2 at 37 °C. Cells were grown on the glass coverslips to the density of 70 % confluence and then incubated with GQDs (2 mg mL−1) at 37 °C for 4 h. Afterwards, the cells were washed three times with PBS and then fixed in 1% paraformaldehyde for 15 min. Images of the cells were immediately captured at ambient temperature on a fluorescent microscope (BX 61; Olympus, Tokyo, Japan) using a digital camera (DP 71; Olympus). The cell morphology was observed using a differential interference contrast microscope. Cytotoxicity assays Cell viability was determined using the Alamar Blue assay. Following the incubation of HeLa, MCF-7 or MCF-10A cells (~104 cells/0.5 mL/well; 24 well plates) in culture media for 24 h at 37 °C containing 5% CO2, the culture medium was replaced with 0.5 mL of the fresh medium containing GQDs of different concentrations (0−2 mg mL−1). The cells were then incubated for another 24 h. The cells were carefully rinsed with PBS three times followed by treatment with the Alamar Blue reagent (1×,
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Nanoscale Accepted Manuscript
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bromide17 along with standard methods like terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining based on end labeling of enzymatic DNA degradation products exist for labelling apoptotic cells in vitro exist.18 There exist few approaches for labelling apoptotic cells in vivo. Here we describe a method of bio-imaging apoptotic cells in zebrafish in vivo by utilizing GQDs derived from neem (azadirachta indica). The as-prepared GQDs possesses unique properties such as excitation wavelength dependent PL, high photo- and chemical- stability, and biocompatibility. Because zebrafish embryos are more prone to damage by chemical agents than are adult organisms, the plant leaf-derived GQDs were used to evaluate the biosafety of GQDs. The characteristic toxic responses of GQDs to the zebrafish larvae were also studied. Furthermore, we also investigated the mortality rates in zebrafish after treatment of GQDs. The GQDs were also shown to be highly biocompatible for human cell imaging of breast cancer cells (MCF-7), normal human mammary epithelial cells (MCF10A) and human cervical cancer cells (HeLa). The GQDs were further modified using annexin V (A5) to form annexin V (A5)modified GQDs (AbA5-GQDs) for labeling apoptotic cells in zebrafish. The apoptotic cells produced in the zebrafish during the first 4 days of embryonic development have been studied to show transient high level of apoptosis in specific stages of zebrafish development. This method provides great benefit for investigation of apoptotic cell death initiation and progression in in vivo due to advantages of transparent zebrafish and strong PL GQDs. Our result paves a new path for the use of GQDs for in vivo labelling of apoptotic cells using zebrafish as a model and for cell imaging studies.
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0.5 mL/well, BioSource International Inc., Camarillo, CA, USA) for 4 h. Fluorescence due to the reduction of the dye by live cells was measured using a fluorescence microplate reader (Synergy 4) from BioTek (Winooski, VT, USA), with an excitation wavelength at 545 nm and an emission wavelength at 590 nm. Because the fluorescence intensity is directly correlated with cell quantity, cell viability was calculated by assuming 100% viability in the control set (medium containing no GQDs). Effects of GQDs on zebrafish embryos Zebrafish embryos were collected from the zebrafish aquarium at National Taiwan Ocean University. Adult male and female zebrafish were maintained at 28.5 ℃ on a 14 h light/10 h dark cycle in separate tanks. For maximal embryo production, males were transferred into the female tank at a ratio of three males to four females toward the end of the light period. Fertilized eggs were gathered at the beginning of the next light cycle from a collection basket previously placed at the bottom of the tank. After egg collection from the group spawns at 0–2 h postfertilization (hpf), they were washed twice with ultrapure water to remove the surrounding debris, embryos were staged for experimental studies and distributed 12 embryo per well into 24well plates in 2 mL ultrapure water in the absence or in the presence of GQDs. Different concentrations of as-prepared GQDs (0−2 mg mL−1) were dispersed in zebrafish culture water (ultrapure water) and incubated for 72 h at 28.5 °C. Dosing solutions (2 mL) were prepared and renewed every 24 h through 72 hpf. Tests were performed in triplicate. The mortality and malformations of zebrafish embryos at 72 hpf were observed under stereomicroscopes (SZ61, OLYMPUS, Tokyo, Japan) to evaluate the toxicity of the GQDs. In order to observe the release of GQDs from the larvae, we changed the GODs solution to ultrapure water and renewed it every 24 h for a period of 1 week. All animal procedures were in accordance with the regulations approved by the Institution Animal Care and Utilization Committee at National Taiwan Ocean University. This study was carried out in strict accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health, Taiwan. Preparation of annexin V (A5)-modified GQDs (AbA5−GQDs) The AbA5 molecules were modified on GQDs through1-ethyl-3[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)/Nhydroxysulfosuccinimide (sulfo-NHS) coupling protocol.[26] The GQDs (1 mg mL−1) were mixed with 20 mM EDC and 50 mM sulfo-NHS in 5 mM sodium phosphate (pH 7.0, 1.0 mL) solution and incubated for 2 h at room temperature. Subsequently, 1.0 mL of 5 mM sodium phosphate (pH 7.0) solution containing the AbA5 solution (1 mg mL-1) was added and the reaction was allowed to proceed for 2 h at room temperature. Finally, 1 mL of a 100 mM ethanolamine hydrochloride solution was added to quench the reaction. After reaction for 1 h at room temperature, the mixtures were centrifuged for 25 min at a relative centrifugation force of 60,000 g to remove the excess AbA5. Following removal of the supernatants, the oily precipitates were washed with 5 mM sodium phosphate (pH 7.0, 1.0 mL). After three wash/centrifuge cycles, the colloids were resuspended
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separately in 5 mM sodium phosphate and stored in a refrigerator (4 °C). Staining procedures and imaging Embryos (120 hpf) were fixed in fresh 4% paraformaldehyde (PFA) in PBST (PBS solution containing 0.05% Tween-20) overnight at 4 °C. The dehydration was carried out using methanol (3×5 min) and stored overnight at −20 °C. The rehydration was performed using decreasing concentration of methanol in PBST and permeabilized using 0.1% sodium citrate in 150 mM Tris-HCl (pH 9.0) at room temperature for 15 min. The embryos were penetrated with pre-chilled acetone (−20 °C) for 20 min. Fixed and permeabilized embryos were blocked with freshly prepared 10% BSA in PBST for 3 h at 4 °C, followed by several washes in PBST and then incubated the embryo with the AbA5−GQDs (100 µg mL−1) in PBST solution containing 1% BSA at 4 ℃ for 12 h. Embryos that were taken up was fixed by 4% PFA for 20 min at room temperature, and then washed with PBST. Finally, they were replaced with 75% glycerol in PBS (through a series of glycerol/PBS wash: 25% → 50% → 75% glycerol/PBS, 20 min each). Fixed embryos were imaged on a confocal microscope Leica TCS SP2 (Buffalo Grove, IL, USA). Result and Discussion Structural characteristics of GQDs Figure 1A indicates the as-prepared GQDs have size of (5.0 ± 0.4) nm (from 100 counts) with spherical shapes and highly dispersed. The XPS spectrum (Figure 1B) exhibits a typical graphitic C1s peak at 284.8 eV and O1s peak at ca. 532 eV for GQDs. The high-resolution XPS spectra of C1s (Inset to Figure 1B) indicates the presence of carbonyl, hydroxyl, and carboxylic acid groups.20 Figure 1C displays the FTIR spectra of GQDs with C–O (alkoxy) stretching peak at 1129 cm−1, C–H stretching peak at 1420 cm−1, C=O vibrational stretch at 1586 cm−1, and broad O–H vibrational stretch between 3200 and 3500 cm−1. The GQDs have great water solubility largely due to the presence of hydrophilic groups such as –OH and –COOH on their surfaces.20 Raman spectroscopy was further used to characterize the GQDs, showing the D and G bands at 1316 cm−1 (disordered structures of carbon) and 1580 cm−1 (the graphitic structures), respectively.20 The intensity ratio of D and to G band (ID/IG) for the GQDs was found to be 0.95 (Figure 1D). The ratio ID/IG is relatively higher than other reported GQDs which could indicate that it has low defect level in the atomic carbon structure.25,26 On the other hand, the XRD diffraction pattern (Figure S1) illustrates a broader (002) peak centered at around 22.1 degrees resultant to graphitic carbon (JCPDS No. 75-1621).
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Nanoscale Accepted Manuscript
DOI: 10.1039/C4NR07005D
Nanoscale
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Figure 1. (A) TEM image, (B) XPS spectrum, (C) FTIR spectrum, and (D) Raman spectrum of the as-prepared GQDs. The GQD solutions (2 mg mL−1; 50 µL) was deposited on a silicon wafer (1 × 1 cm) for XPS and Raman measurements. Inset to (A): size distribution of GQDs. Inset to (B): de-convoluted XPS spectra of C1s.
Optical characteristics The GQDs shows a typical UV-vis absorption peak at 265 nm, which corresponds to the π–π∗ transition of aromatic sp2 domains (Figure S2A). The GQDs display a typical excitation wavelengthdependent emission wavelength (Figure S2B) similar to that of Cdots in our previous reports with strong PL emission at 440 nm when excited at 365 nm.21 The GQDs (λex = 365 nm; λem = 440 nm) had an Φ of 41.2 % when using quinine (Φ = 0.54) as a reference with intense PL (Inset to Figure S2B). They were also highly stable over a wide range of pH (2.0−12.0; 5 mM phosphate buffer) as reported previously.1a Moreover, the GQDs had great photostability with no change in its PL intensity under continuous excitation with a Xe lamp (450 W cm−2) for 1000 h.10 The results revealed that the GQDs have great potential for labeling and imaging of culture cells in addition to in vivo imaging. Biotoxicity assay by zebrafish model The zebrafish embryo has emerged as a rapid and valuable vertebrate model for assessing developmental toxicity.24,25 The results obtained with zebrafish have relevance to human health and feral fish populations. First, we performed bio-distribution analysis of GQDs in zebrafish at three periods of time (24, 60 and 84 hpf), which were within the chorionic stage, the larvae stage of non open-mouth, and open-mouth, respectively. As shown in Figure 2A, no obvious PL was observed in zebrafish embryos when exposed with GQDs (2 mg mL−1) before 60 hpf. In contrast, we observed strong PL of GQDs in the zebrafish after the larvae stage of open-mouth (84 hpf). These results suggest that GQDs tends to enter zebrafish by mouth, and not through the embryo by chorion and skin.
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Figure 2. (A) TEM image, (B) XPS spectrum, (C) FTIR spectrum, and (D) Raman spectrum of the as-prepared GQDs. The GQD solutions (2 mg mL−1; 50 µL) was deposited on a silicon wafer (1 × 1 cm) for XPS and Raman measurements. Inset to (A): size distribution of GQDs. Inset to (B): de-convoluted XPS spectra of C1s.
The GQDs were found to accumulate in the digestive system, while the blood, muscle and other tissue showed no obvious PL signal. Owing to the GQDs not present in circulatory system, the mortality of zebrafish embryos was very low (Figure S3) in the presence of GQDs (< 2.0 mg mL−1). Screening of phenotypic toxicity might be implemented because of opaque quality of tissues following cell death. Unlike semiconductor QDs (e.g., CdSe, PbTe) whose exposure can cause phenotypically pericardial edema, hypopigmentation and abnormal curvature of the body axis of zebrafish, these were not observed upon GQDs solution exposure at a concentration lower than 2.0 mg mL−1 (Figure S4). However, we observed zebrafish with small hand (SH), albino mutant (AM), stunted growth (SG), nondepleted yolk (YND), pericardial edema (PE), and delayed hatching when they were exposed at a concentration higher than 2 mg mL−1 (Figure S5). The results show that the zebrafish could grow healthily in the presence of GQDs (
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: P. Roy, A. P. Periasamy, C. Lin, G. Her, W. Chiu, C. Li, C. Hsu, C. Huang, C. Liang and H. Chang, Nanoscale, 2014, DOI:
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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DOI: 10.1039/C4NR07005D
Cite this: DOI: 10.1039/c0xx00000x
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Prathik Roy,a Arun Prakash Periasamy,a Chou-Ya Lin,b Guor-Mour Her,b Wei-Jane Chiu,b Chi-Lin Li,a Chia-Lun Shu,a Chih-Ching Huang,b* Chi-Te Liang,c,d and Huan-Tsung Changa* 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro, however there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescent properties of plant leaf-derived graphene quantum dots (GQDs) modified with Annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish body without affecting its growth significantly at a concentration lower than 2 mg mL−1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of biomedical applications for graphene-based nanomaterials.
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Photoluminescent carbon-based nanomaterials have attracted great attention due to their unique optical properties and high biocompatibility.1 With their high photoluminescence (PL) and biocompatibility, graphene quantum dots (GQDs) and carbon dots (C-dots) have been used for biosensing and cell imaging.1a, 2 In addition, some C-dots have been found to be efficient in inhibiting the growth of cancer cells.1c, 2 GQDs have been used as photodynamic agents for killing human glioma cells through generating reactive oxygen species upon photo-irradiation.3 Recently, we have demonstrated that high-quality GQDs and Cdots can be prepared from plant leaves,1a coffee ground,1b used tea,1c and low-cost organic compounds such as glycine.1b Compared with their semiconductor counterparts, these C-dots and GQDs may have lower quantum yield (Φ) values but possess far greater biocompatibility, photostability and water solubility and easy modification.1a, 1b, 4 Although fluorescent dyes and proteins based fluorescent probes have been widely used in in vivo imaging, they may suffer from photo-decomposition.5 This journal is © The Royal Society of Chemistry [year]
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Recently, noble metal nanoclusters (NCs) such as gold and silver NCs have also been employed for imaging due to its advantages of controllable emission and good solubility.6 However, they are expensive and suffer from lower Φ values and photostability.6a Moreover, the potential toxicity of these metallic NCs is poorly understood. Thus, the development of greatly photostable nanoprobe with high Φ for in vivo imaging is highly desirable. Recently, GQDs have been proposed as a plausible theronastic nanomaterial. The great surface areas of GQDs enables it to be used as a potential drug delivery platform.7 However, there have been few studies regarding the cytotoxicity of GQDs.8 Here we investigate the cytotoxicity of GQDs both in vitro and in vivo. The zebrafish (Danio rerio) embryo has emerged as an invaluable vertebrate model over other systems such as mouse, rat and fly for the purpose of assessing developmental toxicity, studying genetic and acquired diseases, and drug discovery owing to its small size, heavy brooding, and rapid maturation time.9 Furthermore, the zebrafish embryos are transparent and allow visual detection of embryonic cell death and maldevelopment phenotypes in vivo.9c More recently, the zebrafish model have been utilized to study the implications of engineered nanomaterials and nano-related products from the perspective of environmental and human safety.10 Utility of zebrafish for nanotoxicity study is largely based upon the close homology with the human genome.11 Hundreds of transgenic lines of zebrafish have been established until now, each labeling specific types of signaling molecules, cells, tissues, or organs.12 These transgenic lines of zebrafish have been successfully employed for many genetic and molecular analyses as well as the assessment of the biotoxicity of newly developed nanomaterials. Programmed cell death (apoptosis) on the other hand, is a way for multicellular animal to dispose of unwanted and damaged cells. The cell apoptosis has been linked to many debilitating diseases.13 For instance, it has been found that excess apoptosis in mice induces the deficiency of CPP32 caspase enzyme leading to neurodegeneration.14 Moreover, improper regulation of apoptosis has been associated to cancer, neurodegenerative and cardiovascular diseases.15 Nonetheless, how the abnormal cell apoptosis begins and progresses in vivo is not yet fully understood. Understanding various forms of apoptosis could be the key to unlocking the diagnosis and therapy of the related diseases. Several methods such as fluorescent nucleic binding dyes, ie: acridine orange,16 ethidium bromide,16 and propidium [journal], [year], [vol], 00–00 | 1
Nanoscale Accepted Manuscript
Published on 16 December 2014. Downloaded by Selcuk University on 21/12/2014 09:07:21.
Photoluminescent Graphene Quantum Dots for in vivo Imaging of Apoptotic Cells
Nanoscale
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DOI: 10.1039/C4NR07005D
Published on 16 December 2014. Downloaded by Selcuk University on 21/12/2014 09:07:21.
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Experimental Chemicals Neem leaves were bought from the local (Chennai) market in India. Sodium phosphate monobasic, anhydrous sodium phosphate dibasic, trisodium phosphate, and tris(hydroxymethyl) aminomethane (Tris) were obtained from J.T. Baker (Phillipsburg, NJ, USA). Ultrapure water (18.2 MΩ-cm) was obtained using a Milli-Q ultrapure system (Millipore, Billerica, MA). MCF-7, MCF-10A and HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The pH values of phosphate buffer solutions are 2.0–12.0 prepared from sodium phosphate monobasic, sodium phosphate dibasic, and trisodium phosphate. Phosphate buffered saline (PBS; 1x, 1 L, pH 7.4) contained NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.44 g), and KH2PO4 (0.24 g).
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Synthesis of GQDs GQDs were prepared according to our previous report.[1] In a typical procedure, Neem leaf extract (250 mg) in 20 mL DI water was hydrothermally treated in an autoclave at 300 ºC for 8 h. The reacted solution was centrifuged at a centrifugation force (RCF) of 25,000 g for 20 min to remove the larger particles. The GQDs solution (10 mL) was then dialyzed in ultrapure water (2 L) through a dialysis membrane (MWCO = 3.5–5 kD, Float-A-Lyzer G2, Spectrum Laboratories, Rancho Dominguez, CA, USA) for 24 h. The water was changed with ultrapure water per 4 h. Aliquots (10 mL) of the as-prepared solution containing purified
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GQDs were dried overnight at 60 °C in an oven to obtain pure GQDs. We determined the concentration of purified GQDs solution to be 12 mg mL−1. Characterization of GQDs A double-beam UV–vis spectrophotometer (Cintra 10e, GBC) was used to record the absorption spectra of the GQDs. JEOL JSM-1230 and FEI Tecnai-G2-F20 transmission electron microscopes (TEM) were used to measure the sizes and shapes of the as-prepared GQDs. The re-dispersed GQDs were separately placed on formvar/carbon film Cu grids (200 mesh; Agar Scientific) and dried at ambient temperature. X-ray diffraction (XRD) pattern of GQDs was measured using a PANalytical X’Pert PRO diffractometer (PANalytical B.V., EA Almelo, Netherlands) and Cu-Ka radiation (λ = 0.15418 nm); the samples were prepared on Si substrates. An energy dispersive X-ray spectroscopy (EDS; Inca Energy 200, Oxford) was used to determine the composition of the asprepared GQDs. PL spectra of GQDs were recorded using a Cary Eclipse PL spectrophotometer (Varian CA, USA) operated at excitation wavelengths in the range 350–500 nm. The photostability of GQDs was investigated under continuous illumination of the Xe lamp in Cary Eclipse PL spectrophotometer. Raman spectra of GQDs were recorded using a Raman spectrometer (Dong Woo 500i, Korea) equipped with a 50× objective and a charge-coupled detector. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5000 VersaProbe (Physical Electronics, Eden Prairie, MN, USA). Cellular imaging The human breast cancer cells (MCF-7) were maintained in the RPMI-1640 medium supplemented with fetal bovine serum (FBS, 10%) and 1% Gibco® antibiotic-antimycotic solution in 5% CO2 at 37°C. The human cervical cancer cells (HeLa) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with FBS (10%) and 1% Gibco® antibiotic-antimycotic solution. The normal human mammary epithelial cells (MCF-10A) were maintained in Gibco α-Minimum Essential Media (α-MEM) supplemented with FBS (10%) and 1% Gibco® antibioticantimycotic solution in 5% CO2 at 37 °C. Cells were grown on the glass coverslips to the density of 70 % confluence and then incubated with GQDs (2 mg mL−1) at 37 °C for 4 h. Afterwards, the cells were washed three times with PBS and then fixed in 1% paraformaldehyde for 15 min. Images of the cells were immediately captured at ambient temperature on a fluorescent microscope (BX 61; Olympus, Tokyo, Japan) using a digital camera (DP 71; Olympus). The cell morphology was observed using a differential interference contrast microscope. Cytotoxicity assays Cell viability was determined using the Alamar Blue assay. Following the incubation of HeLa, MCF-7 or MCF-10A cells (~104 cells/0.5 mL/well; 24 well plates) in culture media for 24 h at 37 °C containing 5% CO2, the culture medium was replaced with 0.5 mL of the fresh medium containing GQDs of different concentrations (0−2 mg mL−1). The cells were then incubated for another 24 h. The cells were carefully rinsed with PBS three times followed by treatment with the Alamar Blue reagent (1×,
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bromide17 along with standard methods like terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining based on end labeling of enzymatic DNA degradation products exist for labelling apoptotic cells in vitro exist.18 There exist few approaches for labelling apoptotic cells in vivo. Here we describe a method of bio-imaging apoptotic cells in zebrafish in vivo by utilizing GQDs derived from neem (azadirachta indica). The as-prepared GQDs possesses unique properties such as excitation wavelength dependent PL, high photo- and chemical- stability, and biocompatibility. Because zebrafish embryos are more prone to damage by chemical agents than are adult organisms, the plant leaf-derived GQDs were used to evaluate the biosafety of GQDs. The characteristic toxic responses of GQDs to the zebrafish larvae were also studied. Furthermore, we also investigated the mortality rates in zebrafish after treatment of GQDs. The GQDs were also shown to be highly biocompatible for human cell imaging of breast cancer cells (MCF-7), normal human mammary epithelial cells (MCF10A) and human cervical cancer cells (HeLa). The GQDs were further modified using annexin V (A5) to form annexin V (A5)modified GQDs (AbA5-GQDs) for labeling apoptotic cells in zebrafish. The apoptotic cells produced in the zebrafish during the first 4 days of embryonic development have been studied to show transient high level of apoptosis in specific stages of zebrafish development. This method provides great benefit for investigation of apoptotic cell death initiation and progression in in vivo due to advantages of transparent zebrafish and strong PL GQDs. Our result paves a new path for the use of GQDs for in vivo labelling of apoptotic cells using zebrafish as a model and for cell imaging studies.
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0.5 mL/well, BioSource International Inc., Camarillo, CA, USA) for 4 h. Fluorescence due to the reduction of the dye by live cells was measured using a fluorescence microplate reader (Synergy 4) from BioTek (Winooski, VT, USA), with an excitation wavelength at 545 nm and an emission wavelength at 590 nm. Because the fluorescence intensity is directly correlated with cell quantity, cell viability was calculated by assuming 100% viability in the control set (medium containing no GQDs). Effects of GQDs on zebrafish embryos Zebrafish embryos were collected from the zebrafish aquarium at National Taiwan Ocean University. Adult male and female zebrafish were maintained at 28.5 ℃ on a 14 h light/10 h dark cycle in separate tanks. For maximal embryo production, males were transferred into the female tank at a ratio of three males to four females toward the end of the light period. Fertilized eggs were gathered at the beginning of the next light cycle from a collection basket previously placed at the bottom of the tank. After egg collection from the group spawns at 0–2 h postfertilization (hpf), they were washed twice with ultrapure water to remove the surrounding debris, embryos were staged for experimental studies and distributed 12 embryo per well into 24well plates in 2 mL ultrapure water in the absence or in the presence of GQDs. Different concentrations of as-prepared GQDs (0−2 mg mL−1) were dispersed in zebrafish culture water (ultrapure water) and incubated for 72 h at 28.5 °C. Dosing solutions (2 mL) were prepared and renewed every 24 h through 72 hpf. Tests were performed in triplicate. The mortality and malformations of zebrafish embryos at 72 hpf were observed under stereomicroscopes (SZ61, OLYMPUS, Tokyo, Japan) to evaluate the toxicity of the GQDs. In order to observe the release of GQDs from the larvae, we changed the GODs solution to ultrapure water and renewed it every 24 h for a period of 1 week. All animal procedures were in accordance with the regulations approved by the Institution Animal Care and Utilization Committee at National Taiwan Ocean University. This study was carried out in strict accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health, Taiwan. Preparation of annexin V (A5)-modified GQDs (AbA5−GQDs) The AbA5 molecules were modified on GQDs through1-ethyl-3[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)/Nhydroxysulfosuccinimide (sulfo-NHS) coupling protocol.[26] The GQDs (1 mg mL−1) were mixed with 20 mM EDC and 50 mM sulfo-NHS in 5 mM sodium phosphate (pH 7.0, 1.0 mL) solution and incubated for 2 h at room temperature. Subsequently, 1.0 mL of 5 mM sodium phosphate (pH 7.0) solution containing the AbA5 solution (1 mg mL-1) was added and the reaction was allowed to proceed for 2 h at room temperature. Finally, 1 mL of a 100 mM ethanolamine hydrochloride solution was added to quench the reaction. After reaction for 1 h at room temperature, the mixtures were centrifuged for 25 min at a relative centrifugation force of 60,000 g to remove the excess AbA5. Following removal of the supernatants, the oily precipitates were washed with 5 mM sodium phosphate (pH 7.0, 1.0 mL). After three wash/centrifuge cycles, the colloids were resuspended
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separately in 5 mM sodium phosphate and stored in a refrigerator (4 °C). Staining procedures and imaging Embryos (120 hpf) were fixed in fresh 4% paraformaldehyde (PFA) in PBST (PBS solution containing 0.05% Tween-20) overnight at 4 °C. The dehydration was carried out using methanol (3×5 min) and stored overnight at −20 °C. The rehydration was performed using decreasing concentration of methanol in PBST and permeabilized using 0.1% sodium citrate in 150 mM Tris-HCl (pH 9.0) at room temperature for 15 min. The embryos were penetrated with pre-chilled acetone (−20 °C) for 20 min. Fixed and permeabilized embryos were blocked with freshly prepared 10% BSA in PBST for 3 h at 4 °C, followed by several washes in PBST and then incubated the embryo with the AbA5−GQDs (100 µg mL−1) in PBST solution containing 1% BSA at 4 ℃ for 12 h. Embryos that were taken up was fixed by 4% PFA for 20 min at room temperature, and then washed with PBST. Finally, they were replaced with 75% glycerol in PBS (through a series of glycerol/PBS wash: 25% → 50% → 75% glycerol/PBS, 20 min each). Fixed embryos were imaged on a confocal microscope Leica TCS SP2 (Buffalo Grove, IL, USA). Result and Discussion Structural characteristics of GQDs Figure 1A indicates the as-prepared GQDs have size of (5.0 ± 0.4) nm (from 100 counts) with spherical shapes and highly dispersed. The XPS spectrum (Figure 1B) exhibits a typical graphitic C1s peak at 284.8 eV and O1s peak at ca. 532 eV for GQDs. The high-resolution XPS spectra of C1s (Inset to Figure 1B) indicates the presence of carbonyl, hydroxyl, and carboxylic acid groups.20 Figure 1C displays the FTIR spectra of GQDs with C–O (alkoxy) stretching peak at 1129 cm−1, C–H stretching peak at 1420 cm−1, C=O vibrational stretch at 1586 cm−1, and broad O–H vibrational stretch between 3200 and 3500 cm−1. The GQDs have great water solubility largely due to the presence of hydrophilic groups such as –OH and –COOH on their surfaces.20 Raman spectroscopy was further used to characterize the GQDs, showing the D and G bands at 1316 cm−1 (disordered structures of carbon) and 1580 cm−1 (the graphitic structures), respectively.20 The intensity ratio of D and to G band (ID/IG) for the GQDs was found to be 0.95 (Figure 1D). The ratio ID/IG is relatively higher than other reported GQDs which could indicate that it has low defect level in the atomic carbon structure.25,26 On the other hand, the XRD diffraction pattern (Figure S1) illustrates a broader (002) peak centered at around 22.1 degrees resultant to graphitic carbon (JCPDS No. 75-1621).
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Nanoscale Accepted Manuscript
DOI: 10.1039/C4NR07005D
Nanoscale
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Figure 1. (A) TEM image, (B) XPS spectrum, (C) FTIR spectrum, and (D) Raman spectrum of the as-prepared GQDs. The GQD solutions (2 mg mL−1; 50 µL) was deposited on a silicon wafer (1 × 1 cm) for XPS and Raman measurements. Inset to (A): size distribution of GQDs. Inset to (B): de-convoluted XPS spectra of C1s.
Optical characteristics The GQDs shows a typical UV-vis absorption peak at 265 nm, which corresponds to the π–π∗ transition of aromatic sp2 domains (Figure S2A). The GQDs display a typical excitation wavelengthdependent emission wavelength (Figure S2B) similar to that of Cdots in our previous reports with strong PL emission at 440 nm when excited at 365 nm.21 The GQDs (λex = 365 nm; λem = 440 nm) had an Φ of 41.2 % when using quinine (Φ = 0.54) as a reference with intense PL (Inset to Figure S2B). They were also highly stable over a wide range of pH (2.0−12.0; 5 mM phosphate buffer) as reported previously.1a Moreover, the GQDs had great photostability with no change in its PL intensity under continuous excitation with a Xe lamp (450 W cm−2) for 1000 h.10 The results revealed that the GQDs have great potential for labeling and imaging of culture cells in addition to in vivo imaging. Biotoxicity assay by zebrafish model The zebrafish embryo has emerged as a rapid and valuable vertebrate model for assessing developmental toxicity.24,25 The results obtained with zebrafish have relevance to human health and feral fish populations. First, we performed bio-distribution analysis of GQDs in zebrafish at three periods of time (24, 60 and 84 hpf), which were within the chorionic stage, the larvae stage of non open-mouth, and open-mouth, respectively. As shown in Figure 2A, no obvious PL was observed in zebrafish embryos when exposed with GQDs (2 mg mL−1) before 60 hpf. In contrast, we observed strong PL of GQDs in the zebrafish after the larvae stage of open-mouth (84 hpf). These results suggest that GQDs tends to enter zebrafish by mouth, and not through the embryo by chorion and skin.
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Figure 2. (A) TEM image, (B) XPS spectrum, (C) FTIR spectrum, and (D) Raman spectrum of the as-prepared GQDs. The GQD solutions (2 mg mL−1; 50 µL) was deposited on a silicon wafer (1 × 1 cm) for XPS and Raman measurements. Inset to (A): size distribution of GQDs. Inset to (B): de-convoluted XPS spectra of C1s.
The GQDs were found to accumulate in the digestive system, while the blood, muscle and other tissue showed no obvious PL signal. Owing to the GQDs not present in circulatory system, the mortality of zebrafish embryos was very low (Figure S3) in the presence of GQDs (< 2.0 mg mL−1). Screening of phenotypic toxicity might be implemented because of opaque quality of tissues following cell death. Unlike semiconductor QDs (e.g., CdSe, PbTe) whose exposure can cause phenotypically pericardial edema, hypopigmentation and abnormal curvature of the body axis of zebrafish, these were not observed upon GQDs solution exposure at a concentration lower than 2.0 mg mL−1 (Figure S4). However, we observed zebrafish with small hand (SH), albino mutant (AM), stunted growth (SG), nondepleted yolk (YND), pericardial edema (PE), and delayed hatching when they were exposed at a concentration higher than 2 mg mL−1 (Figure S5). The results show that the zebrafish could grow healthily in the presence of GQDs (
