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Biological and Medical Applications of Materials and Interfaces
Synthesis and Imaging of Biocompatible Graphdiyne Quantum Dots Huan Min, Yingqiu Qi, Yanhuan Chen, Yinlong Zhang, Xuexiang Han, Ying Xu, Ying Liu, Jian-she Hu, Huibiao Liu, Yiye Li, and Guangjun Nie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12801 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019
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Synthesis and Imaging of Biocompatible Graphdiyne Quantum Dots Huan Mina,b,#, Yingqiu Qib,c,#, Yanhuan Chend,e, Yinlong Zhangb,e , Xuexiang Hanb,e, Ying Xub,e,f,g , Ying Liub,e, Jianshe Hua, *, Hiubiao Liud,e,*, Yiye Lib,e,*, Guangjun Nieb,e* a College b CAS
of Science, Northeastern University, Shenyang 110819, P. R. China
Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center
for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P. R. China. c School
of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P. R.
China. d CAS
Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences,
CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China e University
of Chinese Academy of Sciences, Beijing 100049, P. R. China
f Sino-Danish
College, Sino-Danish Center for Education and Research, University of Chinese
Academy of Sciences, Beijing 100049, P. R. China g Department
of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100
Copenhagen, Denmark
*Corresponding authors: Jianshe Hu,
[email protected] Hiubiao Liu, Email:
[email protected] Yiye Li, Email:
[email protected] Guangjun Nie, Email :
[email protected].
# These
authors contributed equally to this work.
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Abstract Graphdiyne has attracted much interest of researchers for their potential applications in energy storage, catalysis and biomedical areas. As one of derivatives of graphdiyne, graphdiyne quantum dots (GDQDs) may possess superior bioactivity due to active acetylene units. However, the biological application of biocompatible GDQDs have not been reported so far. Herein, GDQDs with uniform size and good crystallization were prepared via a classical solvothermal method. The GDQDs exhibit excitation- and pH-dependent fluorescence emission, as well as superior photostability ensuring their potentials in bioimaging. The GDQDs demonstrate efficient cellular uptake and cell imaging without induction of detectable cytotoxic effects in vitro. Systematical safety evaluation further confirmed good biocompatibility of the GDQDs in vivo. Our study preliminarily validates the application of the GDQDs in biomedicine and encourages more thorough studies for better realizing potential of the GDQDs.
Keywords: graphdiyne, quantum dots, nanomaterial, biocompatibility, bioimaging
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Introduction Carbon nanomaterials have attracted tremendous interest in biomedicine for decades by virtue of their great potentials in catalysis,1,2 sensing,3,4 bioimaging,5 drug delivery,6 etc. Carbon-based quantum dots (CQDs), as a “zero-dimension” carbon nanomaterial, possess double superiorities of both quantum dots and carbon nanomaterials, such as excellent physiological stability, large specific surface area, tunable emission wavelengths and superior biocompatibility.7-10 Benefited from the size effect and quantum confinement effect, CQDs exhibit outstanding optical property and have been extensively investigated as new fluorescence reagents for imaging and theranostics.11-14 In addition, CQDs show a high peroxidase-like activity for hydrogen peroxide decomposition and self-targeting for some cancer cells, as well as the capacity of producing singlet oxygen under irradiation.15-17 Considering the high correlation between diverse functions of CQDs and their origins,18 it is meaningful to exploit new CQDs based on specific carbon source to expend the present functions and fulfil various application requirements. Graphdiyne (GDY), a novel allotrope of carbon, is composed of benzene rings and carbon-carbon triple bonds, which forms the large π-conjugated architecture with spand sp2-hybridized carbon atoms.19 The distinctive chemical structure and attractive properties, such as highly conjugated electronic structure and direct natural band gap, make GDY a perfect candidate in catalysis, energy storage and solar cells since its first synthesis on the surface of copper in 2010.20-25 Recently, increasing studies started concentrating on biomedical applications of GDY nanomaterials.26 Owing to the excellent extinction coefficient in near-infrared region of GDY, GDY nanosheets have been explored as good imaging and photothermal conversion agent for cancer therapy.27,28 GDY nanoparticles also possess high free radical scavenging activity for protecting normal tissues from radiation-caused injury.29 It is worth noting that most of 3
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these researches were based on the GDY materials with hundreds of nanometres in size. However, smaller size GDY, especially GDY-based quantum dots (GDQDs), has not been reported in biomedical researches so far. Compared with ordinary GDY nanosheets, small GDQDs may cross the cell membrane more efficiently and exhibit higher bioactivity due to the inherited active acetenyl units and more surface defects.11, 30
Considering bioeffect is a fundamental issue for the application prospect of GDQDs
in biological systems, the fabrication and biocompatibility evaluation of GDQDs are highly desired. Herein, we fabricate GDQDs through a classical solvothermal method, and investigate their physicochemical properties and biocompatibility both in vitro and in vivo. The characterizations of GDQDs indicate the uniformity in size as well as good crystallization. The fluorescence intensity and emission behavior of GDQDs exhibits excitation- and pHdependency, which demonstrate flexible tunability in the optical properties. The resistance to photobleaching greatly facilitates cell imaging of GDQDs, and the spatial distribution of GDQDs is studied in detail. The GDQDs neither disturb cell viability nor lead to oxidative stress in human umbilical vein endothelial (HUVEC) cells. Moreover, systematical safety evaluation in Balb/c mice further validates the biocompatibility of prepared GDQDs. Our results demonstrate feasibility of the GDQD in biomedicine, and verify the biosafety of this new GDY nanomaterial for the first time.
Results and Discussion The GDQDs were prepared by breakage of graphdiyne oxide (GDYO) nanosheets, which was synthesized according to the reported method.26 The characterization of morphology of GDYO nanosheets was performed by the transmission electron microscope (TEM) (Figure S1). To fabricate the GDQDs, the homogeneous aqueous dispersion of GDYO nanosheets was mixed with ammonia solution and underwent a 4
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classical solvothermal reaction (Figure 1a).13 By reacting with some oxygen-containing groups (such as epoxy group and carboxyl group) at the edges of GDYO sheets, the ammonia participates in the breaking of GDYO nanosheets into smaller GDQDs and simultaneous doping into the conjugated system in the form of nitrogen-containing groups. The high content of nitrogen may have a great influence on the quantum yield of GDQDs. The doping of nitrogen would induce the modulation of the chemical and electronic characteristic of prepared GDQDs for the high electronic affinity of N atom.13,31 This modulation would make the GDQDs act as non-radiative electron centres, which leads to the high quantum yield, and facilitates the potential application in bioimaging. The composition of prepared GDQDs was measured using X-ray photoelectron spectroscopy (XPS). The notable N 1s peak of GDQDs, compared with that of GDYO nanosheets, confirmed the doping of nitrogen element (Figure 1b). The characteristic peaks of C=C (284.4 eV), C≡C (284.9 eV), C-O (286.01 eV) and C=O (288.04 eV) in the high-resolution C 1s spectrums of GDYO (Figure 1c) and GDQDs (Figure 1d) suggested their similar carbon skeletons. In Figure 1d, the intensity of the peak at 285.3 eV (corresponding to sp-hybridized carbon) 285.3 eV (corresponding to sp-hybridized carbon) obviously decreased, while the peak at 284.7 eV (corresponding to sp2-hybridized carbon) strengthened, compared with those of GDYO in Figure 1c. These changes reveal the breakage of a certain percentage of the triple bond of carbon and the partial reduction of GDYO during production of the GDQDs. It is worth noting that there was 1.13 % S element in the prepared GDQDs from XPS analysis (Table S1). the S element is most likely derived from the original GDYO, which was synthesized after oxidation of graphdiyne with H2SO4.26 It is reported that the doping of S element within N-doped carbon dots can induce the redistribution of spin and charge densities, and consequently synergistically enhance the quantum yield.32,33 Similar activity of S element may exist in GDQDs. Raman spectroscopy was used to 5
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determine the triple bonds in prepared GDQDs. As shown in Figure S2, the peaks at 1385 cm-1 and 1593 cm-1 correspond to the D band and G band of graphdiyne, respectively. The weak peaks at 1938 cm-1 and 2192 cm-1 can be attributed to the vibration of conjugated diyne linkers, which is consistent with the previous literature.19 Compared with above GDYO, the prepared GDQDs shows similar peaks, indicating the existence of triple bands. The functional groups on the surface of GDQDs were further studied by Fourier transform infrared spectrum (FT-IR). As shown in Figure S3, the characteristic stretching vibration signals of C-N and C=O at 1457 cm-1 and 1656 cm-1 confirm the existence of amide groups on the surface of prepared GDQDs. Morphological characteristic of the GDQDs was investigated using TEM and atomic force microscope (AFM). The GDQDs have a relative narrow size distribution with an average diameter of 4.21 nm (Figure 2a, 2b). The size distribution and zeta potential of GDQDs are measured by dynamic light scatting (DLS) (Figure S5), which is attributed to the existence of abundant hydrophilic groups, such as acylamino and carboxyl groups. Benefited from its negative charged surface, the GDQDs are in low polydispersity and very stable in water, phosphate buffer solution (PBS) and serumcontaining media, existing in a homogeneous state without any precipitation for at least one month (Figure 2b). The high resolution TEM (HRTEM) images of GDQDs demonstrated a typical crystalline structure with a lattice spacing of 0.42 nm (Figure 2c), which is well-consistent with the reported lattice spacing of GDY.19 The corresponding fast Fourier transform pattern indicates the hexagonal carbon network of GDQDs. According to the AFM image (Figure 2d), the average topographic height of GDQDs is about 1.41 nm (Figure 2e), suggesting that the prepared GDQDs are in fewer layers.34 Being one of the best attractions of quantum dots, the optical properties of prepared GDQDs were studied intensively. The UV-visible absorption spectrum 6
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illustrated a broad decreasing absorption band from UV to NIR regions and an observable absorption band at circa 371 nm (Figure 3a), which can be assigned to n-π* (carboxyl and/or C−N) transitions of conjugated domains of GDQDs.35 The photoluminescence spectra of the GDQDs at room temperature were also measured, and the maximum emission was achieved at approximately 495 nm with 371 nm excitation (Figure 3b). The photoluminescence spectra revealed gradually red-shifted emission peaks of GDQDs with the increasing excitation wavelengths (Figure 3c). This excitation-dependent fluorescence emission of the GDQDs is similar to many reported graphene quantum dots, which was attributed to the size-dependent energy gap as well as a variety of surface defected-states or different emissive traps.36 Additionally, the GDQDs exhibited pH-dependent fluorescence emission. Under an excitation of 371 nm, the emission peak of GDQDs reaches the maximum intensity at pH 5, and weakens gradually with the increasing or decreasing pH value (Figure 3d). This pHdependent behaviour may be derived from the protonation or deprotonation of oxygencontaining functional groups on the GDQDs, which affects the centre of the fluorescence emission. The fluorescence quantum yields of GDQDs was calculated to be 17.6 % with 9,10-bis(phenylethynyl)anthracene as a standard (Table S2). To further understand
the
fluorescence
decay
property
of
GDQDs,
the
time-resolved
photoluminescence spectrum was measured with 371 nm excitation at pH 5 by a single photon counting technique. The decay kinetics of GDQDs was fitted very well to a triple-exponential function (Figure 3e), and the time constants and relative amplitudes determined by the fit are summarized in Table S3. The longest lifetime was calculated to be 7.64 ns, and the lifetime in nanosecond suggests the singlet state nature of the GDQDs emission, likely resulting from dipole-allowed recombination across the direct band-gap transition in GDQDs.16,37 Furthermore, the GDQDs exhibited slight photobleaching during continuous excitation at 365 nm for 5 hours (Figure 3f), 7
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indicating their outstanding photostability, which are essential for biomedical applications. The optical properties of GDQDs enable the bioimaging in vitro. To validate the potential of cell imaging and simultaneously study the cellular internalization of GDQDs, HUVEC cells were incubated with GDQDs for different times, and observed by confocal laser scanning microscopy. Plenty of green fluorescence appeared in the cytoplasm, revealing the effective cellular internalization of GDQDs (Figure 4a). The fluorescence intensity increased with the incubation time, and reached the maximum at 24 h (Figure 4b), indicating the cellular uptake of GDQDs reached saturation. The 3D reconstruction of the fluorescence data confirm that the GDQDs disperse throughout the cytoplasm rather than simply adsorption on cytomembrane (Figure 4c, S6). The subcellular localization of the GDQDs was further visualized by Lyso-Tracker staining. Most green fluorescence from GDQDs specifically overlapped with the red fluorescence of Lyso-Tracker, indicating the GDQDs mainly distributed in lysosomes after their internalization (Figure 4d). To investigate the cellular internalization mechanism of GDQDs, HUVEC cells have been pretreated with inhibitors specific to different endocytic pathways before incubating with GDQDs. As shown in Figure S7, the fluorescence intensity in the cells incubated at 4 ℃ is dramatically lower than that at 37 ℃ (control), suggesting an energy-dependent internalization of GDQDs.38,39 The inhibitor of caveolae-mediated endocytosis, nystatin, reduced the cellular uptake over 60 %, more significantly than any other inhibitors. This identified caveolae-mediated endocytosis as the major pathway for GDQDs internalization. In contrast, little impact was detected on the GDQDs uptake after treatments of chlorpromazine (CP) and 5-(Nethyl-N-isopropyl) amiloride (EIPA), suggesting minimal involvement of clathrinmediated endocytosis and micropinocytosis, respectively. This result is consistent with the observation of GDQDs accumulation in lysosome in Figure 4d. Taking together, 8
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cellular uptake of the negatively charged GDQDs is energy-dependent, and caveolaemediated endocytosis is likely the primary mechanism, which was consistent with previous report about cellular uptake of negative charged graphene quantum dots.40 Additionally, the pH-dependency in the cellular internalization of GDQDs was also studied. The fluorescence intensity of HUVEC cells remained unchanged after GDQDs incubation at different pH for 4 h, indicating that the cellular internalization of GDQDs is non-pH dependent (Figure S8). After 12 h and 24 h exposure to different concentrations of GDQDs, no significant reduction in cell viability was observed, even at concentration as high as 200 μg/mL (Figure 4e). Similarly, negligible decrease of cell viability was detected when other two cell lines (human pancreatic cancer Panc-1 cell and human breast cancer MDA-MB-231 cell) were treated with GDQDs for 24 h (Figure S9). It is widely accepted that an increase of intracellular oxidative stress might be induced after exposure of nanomaterials and further lead to cell damages.41 JC-1 was employed here to monitor the depolarization of mitochondrial membrane potential, which is sensitive to cellular oxidative stress. In good agreement with the result of cell viability assay, no obvious membrane potential change was observed between the GDQDs treated cells and the untreated cells (negative control) (Figure 4f), indicating the good biocompatibility of prepared GDQDs in vitro. To confirm the biosafety of the GDQDs, the potential effects of GDQDs in vivo were further investigated. The haemolytic activity of GDQDs was tested firstly to estimate the compatibility of GDQDs with mice red blood cells (RBCs). As shown in Figure 5a, the haemolysis percentage of RBCs increases slowly in a dose-dependent manner after exposure to the GDQDs for 4 h. The GDQDs didn’t cause obvious membrane damage to the RBCs on concentrations less than 100 μg/mL. In the light of the result of haemolysis assay, GDQDs (10 and 20 mg/kg) were intravenously injected to healthy Balb/c mice, and body weights of mice were monitored, no death or 9
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significant body weight loss was observed from the mice treated with the GDQDs (Figure 5b). The mice were then euthanized on day 14 after the injection, and the main organs, including heart, liver, spleen, lung and kidney, were analysed by hematoxylin and eosin (H&E) staining. As presented in Figure 5c, no obvious morphological differences were observed between saline and the GDQD group. Consistent with this, the GDQDs didn’t disturb hepatic or renal function of the mice, revealed by different serum biochemical indicators (Figure 5d). These results confirm the good safety of GDQDs under the experimental conditions.
Experimental section Materials. Ammonium hydroxide (28 %), Heparin were purchased from Sigma-Aldrich. ultrafiltration device with 10 KDa molecular weight cut-off membrane (Millipore) Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Tokyo, Japan) were purchased from Hualide Biotechnology Co., Ltd. 35 mm confocal petri dishes (Cellvis, Mountain View, CA) were purchased from Hualide Technologies Co Ltd. PBS, Fetal Bovine Serum (FBS), RPMI-1640 medium, Kaighn's Modification of Ham's F-12 Medium (F-12K), and dulbecco's modified eagle medium (DMEM) were purchased from Wisent Corporation. Lyso-Tracker red and JC-1 kit were obtained from Beyotime Institute of Biotechnology. Endothelial cell growth supplement was purchased from BD Biosciences.
Preparation of GDQDs. GDYO was prepared as previously described.19,27 Briefly, GDY was synthesized by hexaethynylbenzene on the surface of copper, and the GDYO was prepared from GDY by a modified Hummer’s method. GDQDs were obtained by sonication (120 W, 100 kHz) of a mixture of GDYO/water suspension (5 mL, 1 mg/mL) and ammonia solution (28 wt. %) for 30 min. Subsequently the mixture was transferred to a Teflon autoclave and heated at 200 °C. 5 h later, the product was cooled naturally and ultrafiltered with a 10 kDa 10
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molecular weight cut-off membrane. The residual ammonia of the solution was removed with the aid of a rotary evaporator. The obtained suspension was further dialyzed using dialysis tubing membrane with a molecular weight cut-off of 500 Da for 3 days. Then the obtained pure GDQDs were stored at 4 ℃ for further characterization and use. To accurately control the concentration of GDQDs we used, the prepared GDQDs have been lyophilized and resuspended in water, and then the corresponding UV-vis spectra were collected (shown in Figure S4). The standard curve of GDQDs at 600 nm has been plotted with a linear fitting determination coefficient R2= 0.997. We have controlled the accurate concentration of GDQDs based on this standard curve after measuring the UV-vis absorbance at 600 nm.
Characterization. XPS was performed on an ESCA Lab 220i-XL electron spectrometer after the GDQDs were freezed-dried. The binding energies of C1s and N1s was performed with a Gaussian-Lorentzian peak shape. TEM images and HR-TEM images of GDYOs or GDQDs were obtained with F-20 S-TWIN electron microscope (TecnaiG2, FEI Company) for morphology analysis. AFM images of GDQDs deposited on a silicon wafer were collected on a AFM (Dimension 3100, Veeco, USA) in a tapping mode. Dynamic light scattering (Malvern Zetasizer Nano ZS90, UK) was used to characterize the zeta potentials of prepared samples. UV-vis spectrophotometer (Lambda 650, PerkinElmer, USA) was used to measure the absorbance spectra of prepared samples in the range of 200-700 nm. Fluorescence spectra were acquired using a spectrofluorometer (FS5 spectrofluorometer). FT-IR spectrometer (Spectrum One, Perkin Elmer, USA) was used to obtained the FT-IR spectroscopy from 4000 to 400 cm-1 at a solution of 2 cm-1. Time-correlated single photon counting technique was used to measure the time resolved photoluminescence (TRPL) on an Edinburgh-spectrometer (Model No. FLSP-920) at room temperature. All spectra were recorded with quartz tube of 10 mm path length at room temperature. 11
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Quantum yields (QY) measurement. The quantum yield of GDQDs (in water) was calculated according to the equation as shown below.11 Briefly, 9,10-Bis (phenylethynyl) anthracene (BPEA) in cyclohexane (QY = 1) was chosen as standard. 2
Ix x Ast Yx Yst* * * Ist st Ax
Where Y is the quantum yield, Ι is the collected fluorescence emission intensity, η is the refractive index of the solvent, and A is the indicated absorbance intensity. The subscript "st" is the standard with known quantum yield and "x" means samples. To minimize re-absorption effects, the absorbance intensity of sample at 360 nm was kept below 0.10.
Cell culture. HUVEC cells were cultured in F-12K medium with 0.1 g/mL heparin, 5 % endothelial cell growth supplement and containing 10 % FBS. MDA-MB-231 cells and Panc1 cells were cultured in DMEM with 10 % FBS. All cells were cultured at 37 °C in a humidified 5 % CO2 atmosphere.
Cell viability assay. Cells (HUVEC, Panc-1 and MDA-MB-231) were seeded in 96-well plates at a density of 105 cells/mL medium. After cultured for 12 h, cell medium was replaced with fresh medium containing indicated concentrations (0, 5, 10, 20, 50, 100, 200 μg/mL) of GDQDs, and continuously cultured for 12 or 24 h. The proportion of viable cells was evaluated using a CCK-8 assay. Briefly, the medium in each well was replaced with 100 μL medium containing 10 μL CCK-8. After 2 h incubation at 37 °C, the absorbance of each well was measured using a microplate reader (TECAN Infinite M200, Austria) at 450 nm.
Cellular uptake and distribution in vitro. To observe the cellular uptake of GDQDs, HUVEC cells were seeded in confocal petri dishes (35 mm) at a density of 5 × 103 cells/mL 12
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medium, and cultured for 12 h. For time-dependent cellular uptake, cells were incubated with GDQDs for 1, 6, 12 and 24 h and then washed with PBS for three times. For tracking the intracellular distribution, cells were stained with Lyso-Tracker red and then observed using a confocal microscopy (Zeiss 710, Carl Zeiss AG). Fluorescence signals of GDQDs were detected at the excitation wavelength of 405 nm.
Detection of cellular internalization pathway: The cellular internalization pathway was studied according to the previous article.38 Briefly, HUVEC cells were seeded in 6 wellplates at a density of 2 × 104 cells/mL medium. After cultured for 12 h, medium was replaced with fresh medium. Subsequently, cells were treated with different inhibitors: 30 μM chlorpromazine (CP) for clathrin-mediated endocytosis, 30 μM nystatin for caveolaemediated
endocytosis
or
100
μM
5-(N-ethyl-N-isopropyl)
amiloride
(EIPA)
for
micropinocytosis. Cells were incubated at 4 ℃ to minimize energy mediated pathway. 2 h later, cells were washed with PBS and treated with GDQDs. Another 4 h later, cells were washed with PBS and collected for quantitatively fluorescence analysis with flow cytometry. HUVEC cells treated without inhibitor was used as negative control. To evaluate the pHdependency in the cellular internalization of GDQDs, HUVEC cells were incubated with GDQDs (100 μg/mL) at pH 5.0, 6.0, 6.5 and 7.4 for 4 h. Subsequently, cells were wshed with PBS and collected for quantitatively fluorescence analysis with flow cytometry
Detection of mitochondrial membrane potential. HUVEC cells were seeded in 35 mm petri dishes at a density of 5×103 cells/mL for 12 h, and then incubated with GDQDs for 12 h and 24 h. As for positive control, cells were treated with 0.3 % H2O2 diluted in medium for 20 min. After being washed with PBS for three times, cells were stained with JC-1 (10 μg/mL) at
13
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37 °C for 20 min. Then cells were washed with PBS for 2 times for detection with confocal microscopy (Zeiss 710, Carl Zeiss AG).
Haemolysis assay. 1 mL of fresh blood was collected from healthy BALB/c white mice in a centrifuge tube containing 2 mL PBS with 1% heparin sodium, and centrifuged at 1000 g for 10 min at 37 ℃. After washed with PBS for 3 times, the obtained red blood cells (RBCs) was diluted 10 times with PBS. 0.5 mL of RBCs suspension was mixed with 1 mL of PBS containing different concentration of GDQDs. RBCs suspension (0.5 mL) with PBS (1.0 mL) was used as negative control, and RBCs suspension (0.5 mL) with diluted water (1.0 mL) was used as positive control (haemolysis percentage = 100 %). After 4 h incubation at 37 ℃, the mixtures were centrifuged at 2000 g for 10 min, and the resulting haemolysis was determined by measuring the absorbance of sample at 540 nm with UV-vis spectrophotometer. The hemolysis percentage was calculated as follows: Haemolysis percent (%) = (As -An -AGDQDs) / (Ap-An) × 100 Where As, An, Ap and AGDQDs are the absorbance at 540 nm of the supernatants of the sample, negative control, positive control and the GDQDs at the corresponding concentration, respectively.
Biosafety of GDQDs in vivo. BALB/c female mice (6-8 week age, 18-20 g body weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal protocols were approved by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology. The mice were divided into 3 groups (n = 5), and then intravenously administrated with: (i) Saline; (ii) GDQDs (10 mg/kg); (iii) GDQDs (20 mg/kg). The body weights were measured every other day. The mice were then euthanized on day 14 after the injection. Approximately 500 μL of blood was collected from each mouse of different groups and subsequently centrifuged at 2000 g for 15 min to obtain blood plasma for 14
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blood chemistry examination. The major organs (including heart, liver, spleen, lung and kidney) of mice were harvested, fixed with 4 % paraformaldehyde and sectioned into slices. Then sections were stained with H&E, and examined by a light microscopy (AMG EVOS xl core, Life Technologies, USA).
Statistical analysis. All experiments were performed in triplicate unless otherwise indicated. Results were expressed as means (standard deviation). ANOVA was used for statistical analysis. A probability level of 95% (p < 0.05) was considered to be significantly different.
Conclusions In summary, uniform GDQDs with green fluorescence, deriving from graphdiyne oxide, were fabricated and its biocompatibility was evaluated both in vivo and in vitro. The prepared GDQDs possess excitation- and pH-dependent fluorescence emission, as well as strong resistance to photobleaching. They can conduct efficient cell imaging without disturbing cell viabilities or inducing the dysfunction of mitochondrial in vitro. The good biocompatibility of the GDQDs was further confirmed by hemolytic test, histopathological examination and serum biochemical analysis in vivo. Compared with reported carbon dots or graphene quantum dots, the unique chemical structure (sp-hybridized carbon atoms) and superior electrical properties (such as natural band-gap, intrinsic holes and electrons mobility) of graphdiyne grant more attractive potential to the derived GDQDs in catalysis.43 Therefore, the GDQDs are expected to conduct much richer functional bioimaging by combining optical and enzymatic properties for diagnosis and treatment of diseases. Current study is the first step for more diversified exploration of GDQDs and encourages further optimization.
Conflict of Interest 15
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There are no conflicts of interest to declare.
Acknowledgements This work was supported by grants from National Natural Science Foundation of China (91543127, 31571021, 51573191, 21790053), National Key R&D Program of China (2018YFA0208900, 2016YFA0200104), Innovation Group of the National Natural Science Foundation of China (11621505), National Science Fund for Excellent Young Scholars (31622026), and the Key Laboratory of Biomedical Effects of Nanomaterials and Nanosafety, CAS.
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Figure 1. (a) Schematic illustration of the preparation of GDQDs. (b) XPS spectra of GDYO and GDQDs. High-resolution C1s spectra of (c) GDYO and (d) GDQDs.
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Figure 2. Morphological characterization of GDQDs. (a) TEM image of GDQDs. (b) Size distribution of GDQDs. Inset, the photographs of GDQDs dissolved in water, cell culture medium (DMEM, 10% FBS) and PBS for one month. (c) High resolution TEM image of GDQDs. (d) AFM image of GDQDs deposited on a silicon wafer., (e) The height profiles along the lines in (d).
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Figure 3. The optical properties of the GDQDs. (a) UV-visable absorption spectra of GDYO (black) and GDQDs (red). Inset, photographs of GDQDs dispersed in water (left) and under 365 nm UV irradiation (right). (b) The fluorescence excitation and emission spectra of GDQDs. (c) The fluorescence spectra of GDQDs at different excitation wavelengths. (d) pHdependent fluorescence emission of GDQDs. Inset shows intensity of GDQDs with respect to pH values. (e) Time-resolved photoluminescence (TRPL) decay profile of GDQDs (pH = 5) recorded at room temperature (Ex: 371 nm; Em: 495 nm.). (f) Stability of GDQDs under UV (365 nm) irradiation in water for 5 h. Inset, photographs of GDQDs before and after 300 min irradiation.
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Figure 4. Cellular uptake and cytotoxicity of GDQDs. (a) Confocal laser scanning microscopic images of HUVEC cells incubated with GDQDs for different times, and (b) the corresponding quantitative analysis of fluorescence intensity. (c) 3D reconstruction of cell imaging. (d) Confocal fluorescence microscopy images of HUVEC cells treated with GDQDs for 12 h and stained with Lyso Tracker red. (e) The viabilities of HUVEC cells treated with GDQDs for 12 or 24 h by CCK-8 assay. (f) Mitochondrial membrane potential of HUVEC cells after treatment with GDQDs for 12 h and 24 h. HUVEC cells treated with H2O2 were used as positive control.
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Figure 5. Safety evaluation of GDQDs in vivo. (a) Hemolysis of the RBCs incubated with different concentrations of GDQDs at 37 ℃ in PBS. Inset, photographs of RBCs incubated with GDQDs. RBCs incubated in water is used as a positive control (hemolysis percentage = 100%). (b) Serum biochemistry analysis of the mice treated with the GDQDs. AST, aspartate transaminase; ALT, alanine transaminase; UA, uric acid; BUN, blood urea nitrogen; T-BIL, total bilirubin; CREA, creatinine. (c) Body weight profiles of mice treated with the GDQDs. (d) H&E staining of indicated tissue sections from the mice 14 days after treatment of the GDQDs.
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Graphical Abstract
Graphdiyne quantum dots (GDQDs) are fabricated from graphdiyne oxide with uniform size and good crystallization. The GDQDs exhibit excitation- and pH-dependent fluorescence emission. A good biocompatibility, which is confirmed both in vitro and in vivo, escorts the efficient cell imaging of the GDQDs.
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