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Cite this: DOI: 10.1039/c4nr04227a
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Cytotoxicity of nucleus-targeting fluorescent gold nanoclusters† Jing-Ya Zhao,a Ran Cui,a Zhi-Ling Zhang,a Mingxi Zhang,b Zhi-Xiong Xiec and Dai-Wen Pang*a Gold nanoclusters (AuNCs) with ultra small sizes and unique fluorescence properties have shown promising potential for imaging the nuclei of living cells. However, little is known regarding the potential cytotoxicity of AuNCs after they enter the cell nucleus. The aim of this study is to investigate whether and how nucleus-targeting AuNCs affect the normal functioning of cells. Highly stable, water-soluble and bright fluorescent Au25NCs (the core of each nanocluster is composed of 25 gold atoms) were synthesized. Specific targeting of Au25NCs to the cell nucleus was achieved by conjugating the TAT peptide to the Au25NCs. Cell viability, cell morphology, cell apoptosis/necrosis, reactive oxygen species (ROS) level and mitochondrial membrane potential examinations were performed on different cell lines exposed to the nucleus-targeting Au25NCs. We found that the nucleus-targeting Au25NCs caused cell apoptosis in a dose-dependent manner. A possible mechanism for the cytotoxicity of the nucleus-targeting Au25NCs
Received 25th July 2014, Accepted 28th August 2014
was proposed as follows: the nucleus-targeting Au25NCs induce the production of ROS, resulting in the
DOI: 10.1039/c4nr04227a
oxidative degradation of mitochondrial components, in turn leading to apoptosis via a mitochondrial damage pathway. This work facilitates a better understanding of the toxicity of AuNCs, especially
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nucleus-targeting AuNCs.
1.
Introduction
Labeling the organelles with fluorophores in living cells is essential for studying complex intracellular events. Among all the organelles, the cell nucleus, which contains hereditary information, is considered to be the control center of the cell. Fluorescent labeling of the nuclei in living cells allows us to visualize specific biological processes (e.g. mitosis),1,2 locate the cell nucleus in real-time imaging3 and determine the cell cycle and morphology of the cell nucleus.4 Thus, it has played an important role in the fields of biological research and medical diagnosis. In the past few decades, organic dyes and fluorescent proteins (FPs) have been widely used for imaging the cell nucleus.5 However, organic dyes lack photostability, which limits their use in the long-time imaging of the cell nucleus.
a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, P. R. China. E-mail: [email protected]; Fax: +86 27 68754067; Tel: +86 27 68756759 b State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, P. R. China c College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4nr04227a
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FPs are also sensitive to photobleaching and it is often difficult to construct and express FP fusion proteins, which requires tedious workloads operated by biology experts. With the development of nanotechnology, a series of fluorescent nanomaterials, such as semiconductor quantum dots, carbon dots and nanodiamonds, have broadened our tools for visualizing biology.6–8 However, these nanomaterials could hardly reach the cell nucleus because of their relatively large hydrodynamic sizes, which restrain their free movement across the nuclear pores.9,10 Recently, gold nanoclusters (AuNCs), composed of a few to a hundred gold atoms, have attracted considerable attention.11,12 AuNCs bridge the gap between organogold complexes (2 nm) and show numerous attractive features, such as strong photoluminescence, intrinsic magnetism, chiral optical properties and catalytic activities.11 Among all the kinds of AuNCs, Au25NCs (the core of each NC is composed of 25 gold atoms), with a size of ∼1.2 nm, have been extensively studied.13 Their ultra small size facilitates their entrance into the cell nucleus through nuclear pores.14 Furthermore, highly fluorescent Au25NCs can be prepared under mild conditions and can be functionalized for applications as biological labels.15,16 With these advantages, Au25NCs have been recognized as promising materials for imaging the nuclei of living cells. Nonetheless, their toxicity is still a concern because multiple studies have
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reported the damaging effects of AuNCs.17–19 For example, Pan et al. have found that AuNCs showed size-dependent cytotoxicity.18 Cell necrosis and cell apoptosis were observed when cells were exposed to AuNCs with a diameter of 1.4 nm and 1.2 nm, respectively. Recently, Zhang et al. studied in vivo renal clearance and toxicity of Au25NCs.19 They found that BSA-protected Au25NCs were hardly metabolized and were accumulated in the liver and spleen. In addition, these clusters caused kidney damage, infection and inflammation in mice. In contrast, GSH-protected Au25NCs were metabolizable and caused a repairable toxicity response. However, up to now, the biocompatibility of nucleus-targeting Au25NCs, which is significant for their future biomedical applications, has not yet been fully studied. Herein, strongly fluorescent Au25NCs were synthesized and modified with TAT peptide, a widely used nuclear localization peptide, for imaging the cell nucleus. The toxic effects of nucleus-targeting Au25NCs were systematically studied on a cellular and subcellular level. In addition, a possible mechanism for the cytotoxicity induced by nucleus-targeting Au25NCs was proposed.
2. Materials and methods 2.1.
Materials
Wheat germ agglutinin (WGA, homogeneous by SDS-PAGE) was purchased from Vector. HAuCl4·4H2O (analytical grade) and NaOH (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. Streptavidin (SA), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and reduced glutathione (GSH) were purchased from Amresco. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Sigma-Aldrich. The biotinylated HIV-1 TAT protein-derived peptide (biotin-TAT, TAT peptide sequence: YGRKKRRQRRR, purity: 95%) was chemically synthesized by HD Biosciences Co. Ltd. Fluorescein isothiocyanate-labeled Annexin V (AnnexinV-FITC), the reactive oxygen species (ROS) assay kit and the mitochondrial membrane potential assay kit were purchased from the Beyotime Institute of Biotechnology. Dulbecco’s modified Eagle’s medium (DMEM) and single channel apoptosis and necrosis assay kit were purchased from Invitrogen. Human lung epithelial carcinoma (A549) cells, African green monkey kidney epithelial (Vero) cells and Madin-Darby canine kidney (MDCK) cells were obtained from the China Center for Type Culture Collection. 2.2.
Synthesis of Au25NCs
Preparation of the Au(I) precursor. 67.2 mg of GSH was added to 3 mL of a 1% (w/v) aqueous solution of HAuCl4 under vigorous stirring at room temperature. The pH value of the solution was adjusted to 2.0–3.0 using 10 mol L−1 NaOH and a cloudy precipitate, known as the Au(I)SG complex was obtained. The resulting precipitate was collected by centrifugation and washed with water.
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Preparation of the AuNCs. In a typical synthesis, the Au(I) precursor was dissolved in a 0.01 mol L−1 solution of NaOH. Then 4 mg of WGA in 500 μL of water was mixed with 500 μL of the Au(I) precursor solution (3.4 mmol L−1) under vigorous stirring at 37 °C. The pH value of the solution was adjusted to 12.2 with NaOH (10 mol L−1) immediately. The reaction was allowed to last for 4 h at 37 °C with stirring. The as-prepared AuNCs were purified using a centrifugal filter device (Millipore). The molar concentration of the AuNCs was calculated by dividing the total molar concentration of gold atoms (determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES)) by the number of gold atoms per NC (determined by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry). 2.3.
Characterization
High resolution transmission electron microscopy (HR-TEM) images were performed on a JEOL JEM-2100 transmission electron microscope. The sizes of the Au25NCs were analyzed by Nano Measurer software. The absorption spectra and fluorescence spectra of the Au25NCs were collected on a Shimadzu UV-2550 UV-vis spectrometer and a Horiba Jobin Yvon Inc Fluorolog-3 spectrophotometer, respectively. The matrixassisted laser desorption ionization-time of flight (MALDITOF) mass spectra were collected with a Shimadzu AXIMATOF2 mass spectrometry equipped with a 337 nm nitrogen laser with a 3 ns pulse width. All mass spectra were performed in positive ion mode with an accelerating voltage of 20 kV. The DLS data were obtained on a Malvern Nano-ZS ZEN3600 zetasizer. 2.4.
Cell culture
The Vero, A549 and MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2-humidified environment. 2.5. Preparation of TAT–Au25NCs and live cell labeling with TAT–Au25NCs 12 nmol of Au25NCs were allowed to incubate with 0.5 mg of EDC-activated SA in 500 μL of PBS ( pH 7.2, 0.1 mol L−1) under gentle shaking for 2 h at 37 °C. The SA-Au25NCs conjugates were purified by an Amicon centrifugal filter unit. The TAT– Au25NCs conjugates were prepared by mixing SA-Au25NCs with biotin-TAT at a molar ratio of 1 : 2 at room temperature. For live cell labeling, cells were cultured in a 3.5 cm glassbottomed Petri dish for 24 h and then incubated with 2.4 μM TAT–Au25NCs for 1 h at 37 °C. Unless otherwise stated, the above method was used for all experiments. Fluorescence images were acquired using an Olympus BX51 microscope and a PerkinElmer UltraView VOX laser confocal microscope. 2.6.
Colocalization assay
For the colocalization assay, Hoechst 33342 (5 μg mL−1) was chosen as a contrast agent for cell nucleus staining. Fluorescence images were collected using a laser confocal fluorescence microscope. The Au25NCs and Hochest 33342 were
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excited by a 488 nm laser and 405 nm laser, respectively. The fluorescence signals were separated using a 640 ± 20 nm filter and a 485 ± 20 nm filter. Line profile analysis of the selected cells was performed using Image Pro Plus software.
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2.7.
Apoptosis assay
For cell morphology analysis, Vero cells were incubated with 2.4 μmol L−1 TAT–Au25NCs for 1 h at 37 °C. Then, the cells were supplied with fresh medium and observed under the fluorescence microscope immediately. For the phosphatidylserine externalization assay, TAT– Au25NCs labeled Vero cells were incubated with Annexin V-FITC for 15 min at 4 °C and then observed under the fluorescence microscope immediately. For flow cytometry analysis, the A549, Vero and MDCK cells were plated in 12 well plates at a density of 1 × 105 cells mL−1 one day before use and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 2 h at 37 °C. The cells were harvested and incubated with AnnexinV-Alexa Fluor 488 and SYTOX Green for 15 min at 4 °C. After incubation, the stained cells were analyzed by flow cytometry (Beckman) as soon as possible. Cells treated with 1 mM H2O2 and 0.1 μM actinomycin D were used as a necrosis positive control and an apoptosis positive control, respectively. 2.9.
L−1 biotin-TAT were used as a control. The JC-1 dye was excited using a 488 nm laser and the fluorescence signals of the monomers and aggregates were separated using a 525 ± 20 nm filter and a 585 ± 15 nm filter, respectively.
Cell viability assay
The A549, Vero and MDCK cells were cultured overnight in 96 well plates at a density of 5 × 104 cells per well and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 24 h at 37 °C. After treatment, the cells were incubated with 0.5 mg mL−1 MTT for 4 h at 37 °C. The suspensions in each well were removed carefully and the purple formazan product was dissolved using DMSO with gentle shaking for 10 min in the dark. The absorbance at 570 nm of each well was recorded using the microplate reader (Thermo Scientific MULTISKAN GO). 2.8.
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3. Results and discussion 3.1.
Synthesis and characterization of fluorescent Au25NCs
Highly fluorescent and water-soluble Au25NCs were synthesized by protein-mediated reactions. Previous studies indicated that a appropriate stabilizer, mild reductant and gold precursor in the appropriate valence state play key roles in the synthesis of AuNCs.16,20,21 In this work, WGA was chosen as the reducing and capping agent because the rich tyrosine residues (4.1%) of WGA can reduce a Au(III) or Au(I) complex at pH > 10 and the rich cysteine residues (18.4%) of WGA can subsequently bind to the reduced gold atoms via Au–S bonds to form stabilized nanoclusters.16 In the preparation of AuNCs, the Au(I) complex was incubated with WGA at pH ∼ 12.2 under vigorous stirring at 37 °C. HR-TEM images suggested that uniform and monodisperse AuNCs with an average diameter of 1.2 ± 0.2 nm (statistical analysis of at least 200 AuNCs) were obtained (Fig. 1A and 1C). The AuNCs exhibited bright red photoluminescence (Fig. 1B) with an emission peak at 640 nm (Fig. 1D) and a quantum yield of 8.2% (using Rhodamine 6G as a reference). These nanoclusters were stable during storage at 4 °C and no obvious change in fluorescence was observed (Fig. S1 in ESI†) at high salt concentrations (4 mol L−1 NaCl) as well as in an acidic solution ( pH 4.5). When compared with reported methods,16 the reaction time was greatly shorten by
ROS assay
The A549, Vero and MDCK cells were plated in 12 well plates at a density of 1 × 105 cells mL−1 one day before use and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 2 h at 37 °C. The cells were harvested and incubated with 500 μL of 10 μM 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 min at 37 °C. After incubation, the cells were analyzed by flow cytometry immediately. Cells exposed to Rosup at a concentration of 50 mg L−1 for 20 min at 37 °C were used as a positive control. 2.10. Mitochondrial membrane potential assay Vero cells were exposed to 2.4 μmol L−1 TAT–Au25NCs for 2 h at 37 °C. The cells were incubated with 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) dye for 20 min at 37 °C. After incubation, the cells were imaged on a laser confocal fluorescence microscope immediately. Vero cells with no treatment and treated with 4.8 μmol
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Fig. 1 (A) HR-TEM image of the Au25NCs. (B) Bright-field and fluorescence photographs of the Au25NCs. (C) The size distribution histogram of the Au25NCs. (D) UV-vis (black line) and PL (red line) spectra of the Au25NCs.
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adopting a Au(I) complex as the gold precursor (Fig. S2 in ESI†). The photoluminescence was observed 7 min after the reaction. Moreover, the reaction was completed in 4 h, which was confirmed by the intensity of fluorescence. MALDI-TOF mass spectrometry was used to determine the molecular weight of the as-prepared AuNCs. As shown in Fig. S3,† no signal greater than 34.6 kDa, the molecular weight of WGA, was detected in the as-prepared sample. The structure of the protein was easily destroyed in strong alkaline solutions and WGA might dissociate into two identical subunits with a molecular weight of 17.3 kDa during the reaction.22,23 Thus, the peak at m/z 17.3 kDa in the as-prepared sample might correspond to free subunit of WGA. Therefore, we believe that the peaks appearing at m/z 22.3 kDa, 11.15 kDa and 5.5 kDa corresponded to [one subunit of WGA + Au25]+, [one subunit of WGA + Au25]2+ and [one subunit of WGA + Au25]4+, respectively. Accordingly, we assign the as-prepared products to (one subunit of WGA)-stabilized Au25NCs with molecular weight of ∼22.3 kDa. 3.2. Uptake of TAT–Au25NCs and their localization in living cells To transport Au25NCs into the cell nucleus, the TAT peptide, which has been widely used for delivering protein and small nanoparticles into cell nucleus,24,25 was conjugated to the Au25NCs via a biotin-streptavidin link. Firstly, Au25NCs were modified with SA through a EDC-mediated coupling. The hydrodynamic sizes of the Au25NCs increased from 2.7 nm to 6.0 nm after conjugation with SA (Fig. S4 in ESI†). Then, the TAT–Au25NCs complex was obtained through the high affinity biotin-streptavidin system by incubating the biotin-TAT with SA-Au25NCs. Fluorescence imaging was carried out to demonstrate the specific targeting of TAT–Au25NCs to the nuclei of living cells. In this study, A549, Vero and MDCK cells were chosen as our cell models. The intrinsic fluorescence of Au25NCs was utilized to visualize their intracellular distributions. All the cell lines treated with 2.4 μM TAT–Au25NCs showed bright red fluorescence, whereas the control groups treated with Au25NCs, SA-Au25NCs or a mixture of biotin-TAT and Au25NCs showed no obvious signal (Fig. S5 in ESI†). Moreover, we found that the fluorescence signals of Au25NCs were mainly in the central region of the cells, which may be the cell nuclei. To delineate their intracellular distribution, a laser confocal scanning microscope was used to image the TAT–Au25NCs and nuclei simultaneously. TAT–Au25NCs can be seen mostly in the nuclei after incubation with the cells for 1 h (Fig. 2A), as demonstrated by the blue fluorescence from Hoechst 33342 lighting up the nuclei. Also, line profile analysis of the observations was performed, which can quantify the intensity of the fluorescence signals from the TAT–Au25NCs and Hoechst 33342 and indicates the distribution of the two signals. As shown in Fig. 2B, the distribution of the two signals on the white line in the inset was consistent. The nice match in both fluorescence peaks from the two signals confirmed that the TAT–Au25NCs and Hoechst
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Fig. 2 (A) Laser confocal scanning microscopic images of Vero cells (a–c), MDCK cells (d–f ) and A549 cells (g–i) labeled with the TAT– Au25NCs (red) and Hoechst 33342 (blue). Cells were incubated with 2.4 μM TAT–Au25NCs for 1 h at 37 °C. TAT–Au25NCs: 488 nm/640 ± 20 nm (excitation laser/emission filter). Hoechst 33342: 405 nm/485 ± 20 nm (excitation laser/emission filter). Merge: merge of the bright field, TAT–Au25NCs and Hoechst 33342. (B) Line profiles indicating the distribution of the TAT–Au25NCs (red line) and Hoechst 33342 (blue line) on the white line shown in the inset. Line profile analysis of the Vero cells (a), MDCK cells (b) and A549 cells (c) was performed using Image Pro Plus software.
33342 were colocalized. The above results indicated that the TAT–Au25NCs specifically targeted the cell nucleus. The excellent stability of Au25NCs permits them to be used for long-time imaging of the cell nucleus (Fig. S6 in ESI†). However, their toxicity is still a concern since their small size also brings high surface energy, which would greatly enhance the interactions of the Au25NCs with intracellular components and may cause acute cytotoxicity. 3.3.
Cytotoxicity of TAT–Au25NCs
Subsequently, we monitored the viability of those cells using an MTT assay to figure out whether the nucleus-targeting Au25NCs would interfere with the normal function of the cells. MTT, a yellow dye, is reduced to purple formazan by succinate dehydrogenase in the mitochondria of viable cells and the absorbance of the product can be quantified. Thereby, it is a colorimetric assay essentially dependant on the conversion of substrate to product and the amount of color formation is proportional to the number of viable cells.
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Fig. 3 MTT assay on Vero, MDCK and A549 cells treated with different concentrations of the TAT–Au25NCs (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 24 h at 37 °C.
As shown in Fig. 3 and S7,† there was a significant reduction in cell viability with an increasing concentration of TAT– Au25NCs (Fig. 3), while no detectable effect on the viability of cells cultured with Au25NCs, biotin-TAT and TAT-SA was observed (Fig. S7 in ESI†). The viability of the cells was less than 70% after being exposed to 2.4 μM TAT–Au25NCs for 24 h, which indicated the toxicity of TAT–Au25NCs. To understand how nucleus-targeting Au25NCs interfere with the normal function of cells, microscopic observations of the TAT–Au25NCs labeled cells were carried out since the most noticeable effect following exposure of cells to toxic nanomaterials was a change of cell morphology. Vero cells were incubated with 2.4 μM TAT–Au25NCs for 1 h to allow efficient nuclear targeting. Then real-time microscopic imaging was used to observe the morphology of the TAT–Au25NCs labeled Vero cells. Representative images at 0 min and 420 min postlabeling are shown in Fig. 4A. At 0 min post-labeling, Vero cells were found to experience shrinkage and membrane ruffling (white circles). With increasing time, the situation got worse. Cell shrinkage, accompanied with a large amount of cell membrane curly, ruffling and blebbing, happened at 420 min post-labeling (white circles), whereas there was no morphological change in untreated, biotin-TAT treated and TAT-SA treated Vero cells (Fig. S8 in ESI†). The above observations are consistent with the morphological characteristics of cells undergoing apoptosis.26 To confirm that, we detected the existence of phosphatidylserine (PS) on the cell surface. It has been demonstrated that PS, normally in cytoplasmic face of plasma membrane, will be translocated to the cell surface as cell apoptosis occurs, which can characterize the apoptotic cells.27 Due to high affinity of Annexin V with PS, we can track the PS further by incubating the cells with AnnexinV-FITC. As shown in Fig. 4B, membranes of TAT–Au25NCs labeled Vero cells exhibited green fluorescence, demonstrating that PS was exposed on their external plasma membranes after treatment with the TAT–Au25NCs. This was also consistent with the
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shrinkage of the corresponding cells observed under the bright field. To study this phenomenon on a larger scale, we used AnnexinV-Alexa Fluor 488 and SYTOX Green apoptosis assay kit to analyze cells treated with TAT–Au25NCs by flow cytometry. AnnexinV-Alexa Fluor 488 is similar to AnnexinV-FITC, which can bind to externalized PS and shows green fluorescence. SYTOX Green is a type of green-fluorescent nucleic acid stain that can only penetrate cells with damaged membranes.28 Cell membranes of apoptotic cells externalize but remain integrated, whereas those of necrotic cells externalize and are damaged. Therefore, live cells show little green fluorescence, apoptotic cells show green fluorescence and necrotic cells show brighter green fluorescence after treatment with these probes, which can be distinguished by flow cytometry measurements (Fig. S9 in ESI†). Quantitative flow cytometry results showed that apoptosis induced by the TAT–Au25NCs was dose-dependent (Fig. 5) and no significant necrosis was observed. Nearly 20% of the cells underwent apoptosis upon treatment with 2.4 μM TAT–Au25NCs for 2 h. These results clearly showed that nucleus-targeting Au25NCs could cause apoptosis. 3.4.
Mechanism of cytotoxicity of TAT–Au25NCs
Apoptosis is usually stimulated by two pathways, the extrinsic pathway and the intrinsic pathway.29,30 Ligation of death receptors, such as CD95 receptor, is the key way to activate the extrinsic pathway while mitochondrial damage, resulting from oxidative stress, is primarily responsible for the intrinsic pathway. It has been suggested that cell apoptosis induced by nanoparticles is mainly attributed to the generation of ROS.31 Thus, we measured the ROS generated in the TAT–Au25NCs labeled cells by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay kit. After diffusion into cells, nonfluorescent DCFH-DA is deacetylated by cellular esterases into nonfluorescent 2′,7′-dichlorodihydrofluorescein (DCFH), which is then rapidly oxidized by ROS into highly green fluorescent 2′,7′dichlorofluorescein (DCF).32 The fluorescence intensity of DCF was measured by flow cytometry, and is shown in Fig. 6. Compared with untreated cells, the TAT–Au25NCs treated cells showed brighter fluorescence, demonstrating that ROS were induced following the exposure of cells to TAT–Au25NCs and the amount of ROS increased dramatically with increasing concentrations of TAT–Au25NCs. The increase of ROS in cells exposed to 2.4 μM TAT–Au25NCs was comparable to that in cells treated with a strong oxidant. The large surface area-to-volume ratios of Au25NCs and the high activity of their surface gold atoms might be responsible for the generation of ROS.33 Usually, ROS is associated with the oxidative degradation of mitochondrial protein and lipid, leading to mitochondrial damage.30 A significant feature of mitochondrial damage is the loss of mitochondrial membrane potential, which can be measured by JC-1 dye. JC-1 dye exhibits potential-dependent accumulation in mitochondria. In the intact mitochondria, the mitochondrial membrane presents relatively high potential
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Fig. 4 (A) Microscopic images of the TAT–Au25NCs labeled Vero cells. Fluorescence images of Vero cells at 0 min (a) and 420 min (c) post-labeling and the corresponding bright field images of Vero cells at 0 min (b) and 420 min (d) post-labeling. Vero cells were exposed to 2.4 μM TAT–Au25NCs for 1 h at 37 °C. (B) The bright field image (a) and the corresponding fluorescence image (b) of Vero cells labeled with the TAT–Au25NCs (red) and stained with AnnexinV-FITC, the magnification of a single Vero cell (c).
and JC-1 forms red fluorescence aggregates. However, in the damaged mitochondria, the mitochondrial membrane potential is relatively low and JC-1 exists as green fluorescence monomers. As shown in Fig. 7, in comparison with the control, cells treated with 2.4 μM TAT–Au25NCs showed a substantial reduction in the red/green fluorescence intensity ratio, demonstrating mitochondrial damage. The damage of mitochondria will accompany with the release of apoptogenic factors, which consequently activate the cell apoptotic process.34
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Taking into account the above results, we proposed a mechanism for the cytotoxicity induced by the TAT–Au25NCs as shown in Fig. 8. Firstly, the TAT–Au25NCs entered the cell nucleus (Fig. 2) and induced the production of ROS (Fig. 6). The formation of ROS in turn caused oxidative damage to the mitochondria (Fig. 7). Then, the apoptotic process was activated via a mitochondrial damage pathway and the characteristics of apoptotic cells appeared, including PS externalization (Fig. 4B), cell shrinkage (Fig. 4A and 4B), cell membrane ruffling and blebbing (Fig. 4A).
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Fig. 5 Apoptosis assay on A549 (A), Vero (B) and MDCK (C) cells exposed to TAT–Au25NCs at concentrations from 0 to 2.4 μmol L−1 for 2 h at 37 °C. Cells treated with 0.1 μM actinomycin D and 1 mM H2O2 were used as the apoptosis positive control and the necrosis positive control, respectively.
4.
Conclusions
In summary, we have systematically studied and investigated the cytotoxicity of nucleus-targeting Au25NCs. Several lines of evidence strongly suggest that nucleus-targeting Au25NCs cause cell apoptosis. Firstly, nucleus-targeting Au25NCs cause obvious changes in cell morphology after they have entered
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Fig. 6 Flow cytometry plots of the DCF fluorescence intensity in A549 (A), Vero (B) and MDCK (C) cells treated with the TAT–Au25NCs at concentrations ranging from 0 to 2.4 μmol L−1 for 2 h at 37 °C. Cells exposed to Rosup at a concentration of 50 mg L−1 were used as a positive control.
the nuclei of cells, including cell shrinkage, cell membrane curly, ruffling and blebbing. Secondly, nucleus-targeting Au25NCs induce the externalization of PS. Thirdly, nucleus-targeting Au25NCs cause a significant increase in the percentage
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National Natural Science Foundation of China (21105075), and the 111 Project (111-2-10).
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Notes and references
Fig. 7 Laser confocal scanning microscopic image (e) and the corresponding bright field image (f ) of Vero cells treated with 2.4 μM TAT– Au25NCs for 2 h at 37 °C and then stained with JC-1 dye. Vero cells with no treatment (a and b) and treated with biotin-TAT (c and d) were used as a control.
Fig. 8 A schematic of the proposed mechanism for the cytotoxicity of TAT–Au25NCs. TAT–Au25NCs enter the cell nucleus and induce the formation of ROS, which damage mitochondria and then cause apoptosis via a mitochondrial damage pathway. Finally, the characteristics of apoptotic cells, such as PS externalization, cell shrinkage, cell membrane ruffling and blebbing, appear.
of apoptotic cells. We have also demonstrated that the nucleus-targeting Au25NCs enter the cell nucleus and induce the production of ROS, resulting in mitochondrial damage and thereby leading to cell apoptosis. This work provides a better understanding of the toxicity of AuNCs, which is important for their future biomedical applications. It might also show the potential of AuNCs for anticancer therapy.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, no. 2013CB933904), the
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1 T. Kanda, K. F. Sullivan and G. M. Wahl, Curr. Biol., 1998, 8, 377–385. 2 K. Yamauchi, M. Yang, P. Jiang, N. Yamamoto, M. X. Xu, Y. Amoh, K. Tsuji, M. Bouvet, H. Tsuchiya, K. Tomita, A. R. Moossa and R. M. Hoffman, Cancer Res., 2005, 65, 4246–4252. 3 R. D. Phair and T. Misteli, Nature, 2000, 404, 604–609. 4 F. Belloc, P. Dumain, M. R. Boisseau, C. Jalloustre, J. Reiffers, P. Bernard and F. Lacombe, Cytometry, 1994, 17, 59–65. 5 R. M. Martin, H. Leonhardt and M. C. Cardoso, Cytometry, Part A, 2005, 67A, 45–52. 6 M. Baker, Nat. Methods, 2010, 7, 957–962. 7 S. L. Liu, Z. L. Zhang, E. Z. Sun, J. Peng, M. Xie, Z. Q. Tian, Y. Lin and D. W. Pang, Biomaterials, 2011, 32, 7616–7624. 8 S. L. Liu, Z. Q. Tian, Z. L. Zhang, Q. M. Wu, H. S. Zhao, B. Ren and D. W. Pang, Biomaterials, 2012, 33, 7828– 7833. 9 L. Cao, X. Wang, M. J. Meziani, F. S. Lu, H. F. Wang, P. J. G. Luo, Y. Lin, B. A. Harruff, L. M. Veca, D. Murray, S. Y. Xie and Y. P. Sun, J. Am. Chem. Soc., 2007, 129, 11318– 11319. 10 K. K. Liu, C. C. Wang, C. L. Cheng and J. I. Chao, Biomaterials, 2009, 30, 4249–4259. 11 R. C. Jin, Nanoscale, 2010, 2, 343–362. 12 L. Shang, S. J. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401–418. 13 J. F. Parker, C. A. Fields-Zinna and R. W. Murray, Acc. Chem. Res., 2010, 43, 1289–1296. 14 Y. L. Wang, J. J. Chen and J. Irudayaraj, ACS Nano, 2011, 5, 9718–9725. 15 Y. L. Wang, Y. Y. Cui, Y. L. Zhao, R. Liu, Z. P. Sun, W. Li and X. Y. Gao, Chem. Commun., 2012, 48, 871–873. 16 J. P. Xie, Y. G. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888–889. 17 M. Tsoli, H. Kuhn, W. Brandau, H. Esche and G. Schmid, Small, 2005, 1, 841–844. 18 Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau and W. Jahnen-Dechent, Small, 2007, 3, 1941–1949. 19 X. D. Zhang, D. Wu, X. Shen, P. X. Liu, F. Y. Fan and S. J. Fan, Biomaterials, 2012, 33, 4628–4638. 20 M. X. Zhang, R. Cui, Z. Q. Tian, Z. L. Zhang and D. W. Pang, Adv. Funct. Mater., 2010, 20, 3673–3677. 21 R. Cui, M. X. Zhang, Z. Q. Tian, Z. L. Zhang and D. W. Pang, Nanoscale, 2010, 2, 2120–2125. 22 K. Chaudhari, P. L. Xavier and T. Pradeep, ACS Nano, 2011, 5, 8816–8827. 23 H. Kawasaki, K. Hamaguchi, I. Osaka and R. Arakawa, Adv. Funct. Mater., 2011, 21, 3508–3515.
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24 E. Vivès, P. Brodin and B. Lebleu, J. Biol. Chem., 1997, 272, 16010–16017. 25 J. M. de la Fuente and C. C. Berry, Bioconjugate Chem., 2005, 16, 1176–1180. 26 G. Häcker, Cell Tissue Res., 2000, 301, 5–17. 27 V. A. Fadok, D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton and P. M. Henson, J. Immunol., 1992, 148, 2207–2216. 28 L. J. Jones and V. L. Singer, Anal. Biochem., 2001, 293, 8–15. 29 S. Fulda and K. M. Debatin, Oncogene, 2006, 25, 4798–4811.
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PAPER
Cite this: DOI: 10.1039/c4nr04227a
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Cytotoxicity of nucleus-targeting fluorescent gold nanoclusters† Jing-Ya Zhao,a Ran Cui,a Zhi-Ling Zhang,a Mingxi Zhang,b Zhi-Xiong Xiec and Dai-Wen Pang*a Gold nanoclusters (AuNCs) with ultra small sizes and unique fluorescence properties have shown promising potential for imaging the nuclei of living cells. However, little is known regarding the potential cytotoxicity of AuNCs after they enter the cell nucleus. The aim of this study is to investigate whether and how nucleus-targeting AuNCs affect the normal functioning of cells. Highly stable, water-soluble and bright fluorescent Au25NCs (the core of each nanocluster is composed of 25 gold atoms) were synthesized. Specific targeting of Au25NCs to the cell nucleus was achieved by conjugating the TAT peptide to the Au25NCs. Cell viability, cell morphology, cell apoptosis/necrosis, reactive oxygen species (ROS) level and mitochondrial membrane potential examinations were performed on different cell lines exposed to the nucleus-targeting Au25NCs. We found that the nucleus-targeting Au25NCs caused cell apoptosis in a dose-dependent manner. A possible mechanism for the cytotoxicity of the nucleus-targeting Au25NCs
Received 25th July 2014, Accepted 28th August 2014
was proposed as follows: the nucleus-targeting Au25NCs induce the production of ROS, resulting in the
DOI: 10.1039/c4nr04227a
oxidative degradation of mitochondrial components, in turn leading to apoptosis via a mitochondrial damage pathway. This work facilitates a better understanding of the toxicity of AuNCs, especially
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nucleus-targeting AuNCs.
1.
Introduction
Labeling the organelles with fluorophores in living cells is essential for studying complex intracellular events. Among all the organelles, the cell nucleus, which contains hereditary information, is considered to be the control center of the cell. Fluorescent labeling of the nuclei in living cells allows us to visualize specific biological processes (e.g. mitosis),1,2 locate the cell nucleus in real-time imaging3 and determine the cell cycle and morphology of the cell nucleus.4 Thus, it has played an important role in the fields of biological research and medical diagnosis. In the past few decades, organic dyes and fluorescent proteins (FPs) have been widely used for imaging the cell nucleus.5 However, organic dyes lack photostability, which limits their use in the long-time imaging of the cell nucleus.
a Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, P. R. China. E-mail: [email protected]; Fax: +86 27 68754067; Tel: +86 27 68756759 b State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, P. R. China c College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4nr04227a
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FPs are also sensitive to photobleaching and it is often difficult to construct and express FP fusion proteins, which requires tedious workloads operated by biology experts. With the development of nanotechnology, a series of fluorescent nanomaterials, such as semiconductor quantum dots, carbon dots and nanodiamonds, have broadened our tools for visualizing biology.6–8 However, these nanomaterials could hardly reach the cell nucleus because of their relatively large hydrodynamic sizes, which restrain their free movement across the nuclear pores.9,10 Recently, gold nanoclusters (AuNCs), composed of a few to a hundred gold atoms, have attracted considerable attention.11,12 AuNCs bridge the gap between organogold complexes (2 nm) and show numerous attractive features, such as strong photoluminescence, intrinsic magnetism, chiral optical properties and catalytic activities.11 Among all the kinds of AuNCs, Au25NCs (the core of each NC is composed of 25 gold atoms), with a size of ∼1.2 nm, have been extensively studied.13 Their ultra small size facilitates their entrance into the cell nucleus through nuclear pores.14 Furthermore, highly fluorescent Au25NCs can be prepared under mild conditions and can be functionalized for applications as biological labels.15,16 With these advantages, Au25NCs have been recognized as promising materials for imaging the nuclei of living cells. Nonetheless, their toxicity is still a concern because multiple studies have
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reported the damaging effects of AuNCs.17–19 For example, Pan et al. have found that AuNCs showed size-dependent cytotoxicity.18 Cell necrosis and cell apoptosis were observed when cells were exposed to AuNCs with a diameter of 1.4 nm and 1.2 nm, respectively. Recently, Zhang et al. studied in vivo renal clearance and toxicity of Au25NCs.19 They found that BSA-protected Au25NCs were hardly metabolized and were accumulated in the liver and spleen. In addition, these clusters caused kidney damage, infection and inflammation in mice. In contrast, GSH-protected Au25NCs were metabolizable and caused a repairable toxicity response. However, up to now, the biocompatibility of nucleus-targeting Au25NCs, which is significant for their future biomedical applications, has not yet been fully studied. Herein, strongly fluorescent Au25NCs were synthesized and modified with TAT peptide, a widely used nuclear localization peptide, for imaging the cell nucleus. The toxic effects of nucleus-targeting Au25NCs were systematically studied on a cellular and subcellular level. In addition, a possible mechanism for the cytotoxicity induced by nucleus-targeting Au25NCs was proposed.
2. Materials and methods 2.1.
Materials
Wheat germ agglutinin (WGA, homogeneous by SDS-PAGE) was purchased from Vector. HAuCl4·4H2O (analytical grade) and NaOH (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. Streptavidin (SA), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and reduced glutathione (GSH) were purchased from Amresco. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Sigma-Aldrich. The biotinylated HIV-1 TAT protein-derived peptide (biotin-TAT, TAT peptide sequence: YGRKKRRQRRR, purity: 95%) was chemically synthesized by HD Biosciences Co. Ltd. Fluorescein isothiocyanate-labeled Annexin V (AnnexinV-FITC), the reactive oxygen species (ROS) assay kit and the mitochondrial membrane potential assay kit were purchased from the Beyotime Institute of Biotechnology. Dulbecco’s modified Eagle’s medium (DMEM) and single channel apoptosis and necrosis assay kit were purchased from Invitrogen. Human lung epithelial carcinoma (A549) cells, African green monkey kidney epithelial (Vero) cells and Madin-Darby canine kidney (MDCK) cells were obtained from the China Center for Type Culture Collection. 2.2.
Synthesis of Au25NCs
Preparation of the Au(I) precursor. 67.2 mg of GSH was added to 3 mL of a 1% (w/v) aqueous solution of HAuCl4 under vigorous stirring at room temperature. The pH value of the solution was adjusted to 2.0–3.0 using 10 mol L−1 NaOH and a cloudy precipitate, known as the Au(I)SG complex was obtained. The resulting precipitate was collected by centrifugation and washed with water.
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Preparation of the AuNCs. In a typical synthesis, the Au(I) precursor was dissolved in a 0.01 mol L−1 solution of NaOH. Then 4 mg of WGA in 500 μL of water was mixed with 500 μL of the Au(I) precursor solution (3.4 mmol L−1) under vigorous stirring at 37 °C. The pH value of the solution was adjusted to 12.2 with NaOH (10 mol L−1) immediately. The reaction was allowed to last for 4 h at 37 °C with stirring. The as-prepared AuNCs were purified using a centrifugal filter device (Millipore). The molar concentration of the AuNCs was calculated by dividing the total molar concentration of gold atoms (determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES)) by the number of gold atoms per NC (determined by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry). 2.3.
Characterization
High resolution transmission electron microscopy (HR-TEM) images were performed on a JEOL JEM-2100 transmission electron microscope. The sizes of the Au25NCs were analyzed by Nano Measurer software. The absorption spectra and fluorescence spectra of the Au25NCs were collected on a Shimadzu UV-2550 UV-vis spectrometer and a Horiba Jobin Yvon Inc Fluorolog-3 spectrophotometer, respectively. The matrixassisted laser desorption ionization-time of flight (MALDITOF) mass spectra were collected with a Shimadzu AXIMATOF2 mass spectrometry equipped with a 337 nm nitrogen laser with a 3 ns pulse width. All mass spectra were performed in positive ion mode with an accelerating voltage of 20 kV. The DLS data were obtained on a Malvern Nano-ZS ZEN3600 zetasizer. 2.4.
Cell culture
The Vero, A549 and MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2-humidified environment. 2.5. Preparation of TAT–Au25NCs and live cell labeling with TAT–Au25NCs 12 nmol of Au25NCs were allowed to incubate with 0.5 mg of EDC-activated SA in 500 μL of PBS ( pH 7.2, 0.1 mol L−1) under gentle shaking for 2 h at 37 °C. The SA-Au25NCs conjugates were purified by an Amicon centrifugal filter unit. The TAT– Au25NCs conjugates were prepared by mixing SA-Au25NCs with biotin-TAT at a molar ratio of 1 : 2 at room temperature. For live cell labeling, cells were cultured in a 3.5 cm glassbottomed Petri dish for 24 h and then incubated with 2.4 μM TAT–Au25NCs for 1 h at 37 °C. Unless otherwise stated, the above method was used for all experiments. Fluorescence images were acquired using an Olympus BX51 microscope and a PerkinElmer UltraView VOX laser confocal microscope. 2.6.
Colocalization assay
For the colocalization assay, Hoechst 33342 (5 μg mL−1) was chosen as a contrast agent for cell nucleus staining. Fluorescence images were collected using a laser confocal fluorescence microscope. The Au25NCs and Hochest 33342 were
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excited by a 488 nm laser and 405 nm laser, respectively. The fluorescence signals were separated using a 640 ± 20 nm filter and a 485 ± 20 nm filter. Line profile analysis of the selected cells was performed using Image Pro Plus software.
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2.7.
Apoptosis assay
For cell morphology analysis, Vero cells were incubated with 2.4 μmol L−1 TAT–Au25NCs for 1 h at 37 °C. Then, the cells were supplied with fresh medium and observed under the fluorescence microscope immediately. For the phosphatidylserine externalization assay, TAT– Au25NCs labeled Vero cells were incubated with Annexin V-FITC for 15 min at 4 °C and then observed under the fluorescence microscope immediately. For flow cytometry analysis, the A549, Vero and MDCK cells were plated in 12 well plates at a density of 1 × 105 cells mL−1 one day before use and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 2 h at 37 °C. The cells were harvested and incubated with AnnexinV-Alexa Fluor 488 and SYTOX Green for 15 min at 4 °C. After incubation, the stained cells were analyzed by flow cytometry (Beckman) as soon as possible. Cells treated with 1 mM H2O2 and 0.1 μM actinomycin D were used as a necrosis positive control and an apoptosis positive control, respectively. 2.9.
L−1 biotin-TAT were used as a control. The JC-1 dye was excited using a 488 nm laser and the fluorescence signals of the monomers and aggregates were separated using a 525 ± 20 nm filter and a 585 ± 15 nm filter, respectively.
Cell viability assay
The A549, Vero and MDCK cells were cultured overnight in 96 well plates at a density of 5 × 104 cells per well and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 24 h at 37 °C. After treatment, the cells were incubated with 0.5 mg mL−1 MTT for 4 h at 37 °C. The suspensions in each well were removed carefully and the purple formazan product was dissolved using DMSO with gentle shaking for 10 min in the dark. The absorbance at 570 nm of each well was recorded using the microplate reader (Thermo Scientific MULTISKAN GO). 2.8.
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3. Results and discussion 3.1.
Synthesis and characterization of fluorescent Au25NCs
Highly fluorescent and water-soluble Au25NCs were synthesized by protein-mediated reactions. Previous studies indicated that a appropriate stabilizer, mild reductant and gold precursor in the appropriate valence state play key roles in the synthesis of AuNCs.16,20,21 In this work, WGA was chosen as the reducing and capping agent because the rich tyrosine residues (4.1%) of WGA can reduce a Au(III) or Au(I) complex at pH > 10 and the rich cysteine residues (18.4%) of WGA can subsequently bind to the reduced gold atoms via Au–S bonds to form stabilized nanoclusters.16 In the preparation of AuNCs, the Au(I) complex was incubated with WGA at pH ∼ 12.2 under vigorous stirring at 37 °C. HR-TEM images suggested that uniform and monodisperse AuNCs with an average diameter of 1.2 ± 0.2 nm (statistical analysis of at least 200 AuNCs) were obtained (Fig. 1A and 1C). The AuNCs exhibited bright red photoluminescence (Fig. 1B) with an emission peak at 640 nm (Fig. 1D) and a quantum yield of 8.2% (using Rhodamine 6G as a reference). These nanoclusters were stable during storage at 4 °C and no obvious change in fluorescence was observed (Fig. S1 in ESI†) at high salt concentrations (4 mol L−1 NaCl) as well as in an acidic solution ( pH 4.5). When compared with reported methods,16 the reaction time was greatly shorten by
ROS assay
The A549, Vero and MDCK cells were plated in 12 well plates at a density of 1 × 105 cells mL−1 one day before use and then exposed to different concentrations of the TAT–Au25NCs conjugates (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 2 h at 37 °C. The cells were harvested and incubated with 500 μL of 10 μM 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 min at 37 °C. After incubation, the cells were analyzed by flow cytometry immediately. Cells exposed to Rosup at a concentration of 50 mg L−1 for 20 min at 37 °C were used as a positive control. 2.10. Mitochondrial membrane potential assay Vero cells were exposed to 2.4 μmol L−1 TAT–Au25NCs for 2 h at 37 °C. The cells were incubated with 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) dye for 20 min at 37 °C. After incubation, the cells were imaged on a laser confocal fluorescence microscope immediately. Vero cells with no treatment and treated with 4.8 μmol
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Fig. 1 (A) HR-TEM image of the Au25NCs. (B) Bright-field and fluorescence photographs of the Au25NCs. (C) The size distribution histogram of the Au25NCs. (D) UV-vis (black line) and PL (red line) spectra of the Au25NCs.
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adopting a Au(I) complex as the gold precursor (Fig. S2 in ESI†). The photoluminescence was observed 7 min after the reaction. Moreover, the reaction was completed in 4 h, which was confirmed by the intensity of fluorescence. MALDI-TOF mass spectrometry was used to determine the molecular weight of the as-prepared AuNCs. As shown in Fig. S3,† no signal greater than 34.6 kDa, the molecular weight of WGA, was detected in the as-prepared sample. The structure of the protein was easily destroyed in strong alkaline solutions and WGA might dissociate into two identical subunits with a molecular weight of 17.3 kDa during the reaction.22,23 Thus, the peak at m/z 17.3 kDa in the as-prepared sample might correspond to free subunit of WGA. Therefore, we believe that the peaks appearing at m/z 22.3 kDa, 11.15 kDa and 5.5 kDa corresponded to [one subunit of WGA + Au25]+, [one subunit of WGA + Au25]2+ and [one subunit of WGA + Au25]4+, respectively. Accordingly, we assign the as-prepared products to (one subunit of WGA)-stabilized Au25NCs with molecular weight of ∼22.3 kDa. 3.2. Uptake of TAT–Au25NCs and their localization in living cells To transport Au25NCs into the cell nucleus, the TAT peptide, which has been widely used for delivering protein and small nanoparticles into cell nucleus,24,25 was conjugated to the Au25NCs via a biotin-streptavidin link. Firstly, Au25NCs were modified with SA through a EDC-mediated coupling. The hydrodynamic sizes of the Au25NCs increased from 2.7 nm to 6.0 nm after conjugation with SA (Fig. S4 in ESI†). Then, the TAT–Au25NCs complex was obtained through the high affinity biotin-streptavidin system by incubating the biotin-TAT with SA-Au25NCs. Fluorescence imaging was carried out to demonstrate the specific targeting of TAT–Au25NCs to the nuclei of living cells. In this study, A549, Vero and MDCK cells were chosen as our cell models. The intrinsic fluorescence of Au25NCs was utilized to visualize their intracellular distributions. All the cell lines treated with 2.4 μM TAT–Au25NCs showed bright red fluorescence, whereas the control groups treated with Au25NCs, SA-Au25NCs or a mixture of biotin-TAT and Au25NCs showed no obvious signal (Fig. S5 in ESI†). Moreover, we found that the fluorescence signals of Au25NCs were mainly in the central region of the cells, which may be the cell nuclei. To delineate their intracellular distribution, a laser confocal scanning microscope was used to image the TAT–Au25NCs and nuclei simultaneously. TAT–Au25NCs can be seen mostly in the nuclei after incubation with the cells for 1 h (Fig. 2A), as demonstrated by the blue fluorescence from Hoechst 33342 lighting up the nuclei. Also, line profile analysis of the observations was performed, which can quantify the intensity of the fluorescence signals from the TAT–Au25NCs and Hoechst 33342 and indicates the distribution of the two signals. As shown in Fig. 2B, the distribution of the two signals on the white line in the inset was consistent. The nice match in both fluorescence peaks from the two signals confirmed that the TAT–Au25NCs and Hoechst
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Fig. 2 (A) Laser confocal scanning microscopic images of Vero cells (a–c), MDCK cells (d–f ) and A549 cells (g–i) labeled with the TAT– Au25NCs (red) and Hoechst 33342 (blue). Cells were incubated with 2.4 μM TAT–Au25NCs for 1 h at 37 °C. TAT–Au25NCs: 488 nm/640 ± 20 nm (excitation laser/emission filter). Hoechst 33342: 405 nm/485 ± 20 nm (excitation laser/emission filter). Merge: merge of the bright field, TAT–Au25NCs and Hoechst 33342. (B) Line profiles indicating the distribution of the TAT–Au25NCs (red line) and Hoechst 33342 (blue line) on the white line shown in the inset. Line profile analysis of the Vero cells (a), MDCK cells (b) and A549 cells (c) was performed using Image Pro Plus software.
33342 were colocalized. The above results indicated that the TAT–Au25NCs specifically targeted the cell nucleus. The excellent stability of Au25NCs permits them to be used for long-time imaging of the cell nucleus (Fig. S6 in ESI†). However, their toxicity is still a concern since their small size also brings high surface energy, which would greatly enhance the interactions of the Au25NCs with intracellular components and may cause acute cytotoxicity. 3.3.
Cytotoxicity of TAT–Au25NCs
Subsequently, we monitored the viability of those cells using an MTT assay to figure out whether the nucleus-targeting Au25NCs would interfere with the normal function of the cells. MTT, a yellow dye, is reduced to purple formazan by succinate dehydrogenase in the mitochondria of viable cells and the absorbance of the product can be quantified. Thereby, it is a colorimetric assay essentially dependant on the conversion of substrate to product and the amount of color formation is proportional to the number of viable cells.
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Fig. 3 MTT assay on Vero, MDCK and A549 cells treated with different concentrations of the TAT–Au25NCs (0, 0.15, 0.3, 0.6, 1.2 and 2.4 μM) for 24 h at 37 °C.
As shown in Fig. 3 and S7,† there was a significant reduction in cell viability with an increasing concentration of TAT– Au25NCs (Fig. 3), while no detectable effect on the viability of cells cultured with Au25NCs, biotin-TAT and TAT-SA was observed (Fig. S7 in ESI†). The viability of the cells was less than 70% after being exposed to 2.4 μM TAT–Au25NCs for 24 h, which indicated the toxicity of TAT–Au25NCs. To understand how nucleus-targeting Au25NCs interfere with the normal function of cells, microscopic observations of the TAT–Au25NCs labeled cells were carried out since the most noticeable effect following exposure of cells to toxic nanomaterials was a change of cell morphology. Vero cells were incubated with 2.4 μM TAT–Au25NCs for 1 h to allow efficient nuclear targeting. Then real-time microscopic imaging was used to observe the morphology of the TAT–Au25NCs labeled Vero cells. Representative images at 0 min and 420 min postlabeling are shown in Fig. 4A. At 0 min post-labeling, Vero cells were found to experience shrinkage and membrane ruffling (white circles). With increasing time, the situation got worse. Cell shrinkage, accompanied with a large amount of cell membrane curly, ruffling and blebbing, happened at 420 min post-labeling (white circles), whereas there was no morphological change in untreated, biotin-TAT treated and TAT-SA treated Vero cells (Fig. S8 in ESI†). The above observations are consistent with the morphological characteristics of cells undergoing apoptosis.26 To confirm that, we detected the existence of phosphatidylserine (PS) on the cell surface. It has been demonstrated that PS, normally in cytoplasmic face of plasma membrane, will be translocated to the cell surface as cell apoptosis occurs, which can characterize the apoptotic cells.27 Due to high affinity of Annexin V with PS, we can track the PS further by incubating the cells with AnnexinV-FITC. As shown in Fig. 4B, membranes of TAT–Au25NCs labeled Vero cells exhibited green fluorescence, demonstrating that PS was exposed on their external plasma membranes after treatment with the TAT–Au25NCs. This was also consistent with the
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shrinkage of the corresponding cells observed under the bright field. To study this phenomenon on a larger scale, we used AnnexinV-Alexa Fluor 488 and SYTOX Green apoptosis assay kit to analyze cells treated with TAT–Au25NCs by flow cytometry. AnnexinV-Alexa Fluor 488 is similar to AnnexinV-FITC, which can bind to externalized PS and shows green fluorescence. SYTOX Green is a type of green-fluorescent nucleic acid stain that can only penetrate cells with damaged membranes.28 Cell membranes of apoptotic cells externalize but remain integrated, whereas those of necrotic cells externalize and are damaged. Therefore, live cells show little green fluorescence, apoptotic cells show green fluorescence and necrotic cells show brighter green fluorescence after treatment with these probes, which can be distinguished by flow cytometry measurements (Fig. S9 in ESI†). Quantitative flow cytometry results showed that apoptosis induced by the TAT–Au25NCs was dose-dependent (Fig. 5) and no significant necrosis was observed. Nearly 20% of the cells underwent apoptosis upon treatment with 2.4 μM TAT–Au25NCs for 2 h. These results clearly showed that nucleus-targeting Au25NCs could cause apoptosis. 3.4.
Mechanism of cytotoxicity of TAT–Au25NCs
Apoptosis is usually stimulated by two pathways, the extrinsic pathway and the intrinsic pathway.29,30 Ligation of death receptors, such as CD95 receptor, is the key way to activate the extrinsic pathway while mitochondrial damage, resulting from oxidative stress, is primarily responsible for the intrinsic pathway. It has been suggested that cell apoptosis induced by nanoparticles is mainly attributed to the generation of ROS.31 Thus, we measured the ROS generated in the TAT–Au25NCs labeled cells by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay kit. After diffusion into cells, nonfluorescent DCFH-DA is deacetylated by cellular esterases into nonfluorescent 2′,7′-dichlorodihydrofluorescein (DCFH), which is then rapidly oxidized by ROS into highly green fluorescent 2′,7′dichlorofluorescein (DCF).32 The fluorescence intensity of DCF was measured by flow cytometry, and is shown in Fig. 6. Compared with untreated cells, the TAT–Au25NCs treated cells showed brighter fluorescence, demonstrating that ROS were induced following the exposure of cells to TAT–Au25NCs and the amount of ROS increased dramatically with increasing concentrations of TAT–Au25NCs. The increase of ROS in cells exposed to 2.4 μM TAT–Au25NCs was comparable to that in cells treated with a strong oxidant. The large surface area-to-volume ratios of Au25NCs and the high activity of their surface gold atoms might be responsible for the generation of ROS.33 Usually, ROS is associated with the oxidative degradation of mitochondrial protein and lipid, leading to mitochondrial damage.30 A significant feature of mitochondrial damage is the loss of mitochondrial membrane potential, which can be measured by JC-1 dye. JC-1 dye exhibits potential-dependent accumulation in mitochondria. In the intact mitochondria, the mitochondrial membrane presents relatively high potential
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Fig. 4 (A) Microscopic images of the TAT–Au25NCs labeled Vero cells. Fluorescence images of Vero cells at 0 min (a) and 420 min (c) post-labeling and the corresponding bright field images of Vero cells at 0 min (b) and 420 min (d) post-labeling. Vero cells were exposed to 2.4 μM TAT–Au25NCs for 1 h at 37 °C. (B) The bright field image (a) and the corresponding fluorescence image (b) of Vero cells labeled with the TAT–Au25NCs (red) and stained with AnnexinV-FITC, the magnification of a single Vero cell (c).
and JC-1 forms red fluorescence aggregates. However, in the damaged mitochondria, the mitochondrial membrane potential is relatively low and JC-1 exists as green fluorescence monomers. As shown in Fig. 7, in comparison with the control, cells treated with 2.4 μM TAT–Au25NCs showed a substantial reduction in the red/green fluorescence intensity ratio, demonstrating mitochondrial damage. The damage of mitochondria will accompany with the release of apoptogenic factors, which consequently activate the cell apoptotic process.34
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Taking into account the above results, we proposed a mechanism for the cytotoxicity induced by the TAT–Au25NCs as shown in Fig. 8. Firstly, the TAT–Au25NCs entered the cell nucleus (Fig. 2) and induced the production of ROS (Fig. 6). The formation of ROS in turn caused oxidative damage to the mitochondria (Fig. 7). Then, the apoptotic process was activated via a mitochondrial damage pathway and the characteristics of apoptotic cells appeared, including PS externalization (Fig. 4B), cell shrinkage (Fig. 4A and 4B), cell membrane ruffling and blebbing (Fig. 4A).
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Fig. 5 Apoptosis assay on A549 (A), Vero (B) and MDCK (C) cells exposed to TAT–Au25NCs at concentrations from 0 to 2.4 μmol L−1 for 2 h at 37 °C. Cells treated with 0.1 μM actinomycin D and 1 mM H2O2 were used as the apoptosis positive control and the necrosis positive control, respectively.
4.
Conclusions
In summary, we have systematically studied and investigated the cytotoxicity of nucleus-targeting Au25NCs. Several lines of evidence strongly suggest that nucleus-targeting Au25NCs cause cell apoptosis. Firstly, nucleus-targeting Au25NCs cause obvious changes in cell morphology after they have entered
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Fig. 6 Flow cytometry plots of the DCF fluorescence intensity in A549 (A), Vero (B) and MDCK (C) cells treated with the TAT–Au25NCs at concentrations ranging from 0 to 2.4 μmol L−1 for 2 h at 37 °C. Cells exposed to Rosup at a concentration of 50 mg L−1 were used as a positive control.
the nuclei of cells, including cell shrinkage, cell membrane curly, ruffling and blebbing. Secondly, nucleus-targeting Au25NCs induce the externalization of PS. Thirdly, nucleus-targeting Au25NCs cause a significant increase in the percentage
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National Natural Science Foundation of China (21105075), and the 111 Project (111-2-10).
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Notes and references
Fig. 7 Laser confocal scanning microscopic image (e) and the corresponding bright field image (f ) of Vero cells treated with 2.4 μM TAT– Au25NCs for 2 h at 37 °C and then stained with JC-1 dye. Vero cells with no treatment (a and b) and treated with biotin-TAT (c and d) were used as a control.
Fig. 8 A schematic of the proposed mechanism for the cytotoxicity of TAT–Au25NCs. TAT–Au25NCs enter the cell nucleus and induce the formation of ROS, which damage mitochondria and then cause apoptosis via a mitochondrial damage pathway. Finally, the characteristics of apoptotic cells, such as PS externalization, cell shrinkage, cell membrane ruffling and blebbing, appear.
of apoptotic cells. We have also demonstrated that the nucleus-targeting Au25NCs enter the cell nucleus and induce the production of ROS, resulting in mitochondrial damage and thereby leading to cell apoptosis. This work provides a better understanding of the toxicity of AuNCs, which is important for their future biomedical applications. It might also show the potential of AuNCs for anticancer therapy.
Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, no. 2013CB933904), the
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