Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real

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Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells Xiaojuan Wang, Yanan Wang, Hua He, Xiqi Ma, Qi Chen, Shuai Zhang, Baosheng Ge, Shengjie Wang, Werner M. Nau, and Fang Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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ACS Applied Materials & Interfaces

Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells

Xiaojuan Wanga,b,*, Yanan Wangb, Hua Hea,b, Xiqi Mab, Qi Chenb, Shuai Zhangc, Baosheng Gea,b, Shengjie Wanga,b, Werner M. Nauc and Fang Huanga,b,* a

State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East

China), Qingdao 266555, China b

Centre for Bioengineering and Biotechnology, China University of Petroleum (East

China), Qingdao 266555, China c

Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus

Ring 1, 28759 Bremen, Germany *Corresponding Author Tel: 86-532-86983455, Fax: 86-532-86983455, Email：[email protected] (X. Wang) Tel: 86-532-86981560, Fax: 86-532-86981560, Email ： [email protected] (F. Huang)

1

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Abstract Nucleoli are important subnuclear structures inside cells. We report novel fluorescent gold nanoclusters (K‒AuNCs) that are able to stain the nucleoli selectively and make it possible to explore the nucleolar morphology with fluorescence imaging technique. This novel probe is prepared through an easy synthesis method by employing a tripeptide (Lys-Cys-Lys) as the surface ligand. The properties, including deep-red fluorescence emission (680 nm), large Stocks shift, broad excitation band, low cytotoxicity and good photostability, endow this probe with potential for bioanalytical applications. Because of their small size and their positively charged surface, K‒AuNCs are able to accumulate efficiently at the nucleolar regions and provide precise morphological information. K‒AuNCs are also used to monitor the nucleolar dynamics along the reverse transformation process of malignant cells, induced by the agonist of protein A, 8-Chloro-cyclic adenosine monophosphate. This gives a novel approach for investigating the working mechanism of anti-tumor drugs. Keywords: fluorescence, bioimaging, gold nanoclusters, nucleolus, reverse transformation

2


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1. Introduction Fluorescence microscopy is one of the most rapidly expanding analytical techniques

employed

in

modern

medical

and

biological

research.1

This

straightforward tool provides not only a high degree of sensitivity, but also the possibility to specifically target structural components and dynamic processes in fixed as well as living cells and tissues. A great variety of fluorescent probes have been constructed for staining biological macromolecules, monitoring local environmental variables, or illuminating specific structural regions. Nucleoli are important subnuclear structures inside cells. Although they are widely known to serve as the site where ribosomal RNAs (rRNAs) are synthesized and processed, their biological functions have not yet been fully elucidated.2-5 Previous research has demonstrated that the nucleolar morphology changes dynamically in physiological and pathological situations, guiding the surgical pathologist in cancer diagnosis.6-8 The features of nucleoli, such as shape, size and numbers, could be important indications in the evolution of malignant lesions. Despite the significant role played by nucleoli, there is so far only one commercially available nucleolar probe, known as “SYTO RNA-Select”, which emits green fluorescence. Over the past decade, novel nucleoli staining fluorophores have been developed. In particular, metal coordination complexes containing europium, iridium, lanthanide and ruthenium have been reported for nucleoli staining.9-16 Alternatively, low molecular weight organic probes have been designed to detect RNA in nucleoli and in the cytoplasm.17-20 Shen et al. also prepared ligand modified quantum dots for nucleolar labelling.21 All the presently known fluorophores have the common feature, however, that they either require multi-step syntheses or suffer from photobleaching or potential toxicity. There if therefore a pressing need for 3



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fluorophores that remedy these shortcomings. In our previous work, luminescent nanomaterials based on graphene quantum dots (nGQDs) have been used to specifically stain nucleoli.22 Although nGQDs provide good biocompatibility, their fluorescence emission is limited to the short-wavelength range (λem = 447 nm) and overlaps with the autofluorescence of biological samples. In this paper, we report novel biocompatible nucleolar fluorescent probes with emission at longer wavelength range (> 650 nm) and excellent photostability. Gold nanoclusters (AuNCs) have attracted much attention for fluorescence bioimaging analysis because of their ultra-small size, good biocompatibility and extraordinary stability.23-29 Our novel probe is prepared through an easy one-pot synthesis method by employing a short tripeptide Lys-Cys-Lys (KCK, Scheme 1) as the stabilizing ligand. The reaction mechanism of similar procedure has been systematically investigated by Xie’s group.30 As reported, the AuNCs products had a core-shell structure as Au(0)@Au(I)-thiolate. We demonstrate that – compared with conventionally reported glutathione-ligated gold nanoclusters (G‒AuNCs), that mainly localize in the cytoplasm after cellular uptake – KCK-modified clusters (K‒AuNCs) are able to accumulate selectively and luminesce efficiently precisely in the nucleolar regions. Moreover, we have characterized K‒AuNCs in detail and used them as fluorescent probes to monitor the nucleolar dynamics during reverse transformation of malignant cells induced by the agonist of protein A, 8-Chloro-cyclic adenosine monophosphate (8-Chloro-cyclic AMP). 2. Materials and methods 2.1. Materials Hydrogen tetrachloroaurate (HAuCl4•4H2O) was purchased from Shanghai 4


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Jiuyue Chemical Company. The KCK tripeptide was synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. The quality was checked with MALDI-TOF mass spectroscopy before use (Figure S1, MALDI-TOF mass spectrometer, Microflex LRF,

Bruker).

L-Glutathione

in

its

reduced

form

(GSH),

tris(2-carboxyethyl)phosphine (TCEP), and 8-cl-cAMP were obtained from Sigma−Aldrich. High-glucose Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640 medium and fetal bovine serum (FBS) were from Life Technologies. SYTO RNA-Select was purchased from Thermo-Fisher Scientific Inc. All chemicals were used as received without further purification. All solutions were prepared with ultra-pure water (18.2MΩ) purified on a Millipore system (Millipore, USA). 2.2. Syntheses of K‒AuNCs and G‒AuNCs and their characterization To obtain K‒AuNCs, freshly prepared AuHCl4 solution (20 mM, 500 µL) was mixed with KCK tripeptide (20 mM, 500 µL) and TCEP (20 mM, 500 µL) while stirring at 70 °C for 15 min. Subsequently, NaOH (1.5 M, 50 µL) and NaBH4 (0.1 M, 8 µL) were dropped into the mixture and reacted at 70 °C for 10 h.30 The resulting mixture was purified using Amicon Ultra Centrifugal Filter Units (UFC201024PL, 10kDa cutoff, Millipore Corp) to remove excess reagents and unbound peptides. The pure K‒AuNCs solution can be stored at 4 °C for at least 3 months with negligible changes in optical properties. G‒AuNCs were prepared as previously described, with minor modifications.29-31 Freshly prepared AuHCl4 solution (20 mM, 500 µL) was mixed with GSH (20 mM, 1000 µL) and kept at 70 °C for 12 h. The resulting mixture was purified as described for K‒AuNCs. Ultraviolet-visible (UV-Vis) spectra were recorded on a Shimadzu UV-2450 spectrometer. Steady-state fluorescence spectra were collected on a FluoroMax-4 5



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fluorometer. FT-IR spectra were obtained using a Nicolet 6700 FT-IR spectrometer. X-ray photoelectron spectroscopy (XPS) measurements were carried out with Thermo 250 ESCALAB. All optical measurements were performed at room temperature under ambient conditions. The quantum yield (QY) of K‒AuNC was measured using a Rhodamine 6G (QY = 95%) ethanol solution as the reference standard. Fluorescence lifetimes were determined by time-correlated single-photon counting (FLS920, Edinburgh Instruments Ltd.) with a 373 nm diode laser (PicoQuant, LDH-P-C 375, FWHM ca. 50 ps) for excitation. The obtained fluorescence decay traces were fitted with a tri-exponential function as following to get the value of pre-exponential factors (α1, ɑ2, ɑ3), the characteristic lifetimes (τ1, τ2, τ3) and the background constant A.32-34 y = A + α − + α − + α −

(1)

The intensity-averaged lifetime was calculated as 〈 〉 = + +

(2)

where = ⁄∑ ∙ 100%, and i = 1, 2, 3. High-resolution transmission electron microscopic (HRTEM) images were measured on a JEM-2100 electron microscope at 200 kV. The zeta potential of the synthesized AuNCs was determined on a NanoZS Zetasizer (Malvern, UK). 2.3. Cell culture and drug treatment The fibrosarcoma cell line HT1080 was purchased from the Cell Bank of the Shanghai Institute for Biological Science, Chinese Academy of Sciences (Shanghai, China). Cells were incubated in a RPMI1640 medium supplemented with 10% fetal bovine serum in a humidified incubator at 37 °C in which the CO2 level was kept constant at 5%. Reverse transformation of HT1080 followed the literature.7 Cells (4 × 104) were 6


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seeded onto coverslips and precultured for 24 h. The culture medium was subsequently discarded, the cells were washed three times with PBS, and maintained in serum-containing nutrient medium supplemented with 8-chloro-cyclic AMP (60 mM) for 1-3 days as indicated for the individual experiments. 2.4. AuNCs staining and confocal imaging For normal HT1080 staining, cells (1 × 105) were seeded onto 12-mm sterile coverslips in a 24-well plate and cultured for 24 h, washed three times with PBS, and then incubated with culture medium containing AuNCs (400 µg/mL) at 37 ºC for 2 h. After washing three times with cold PBS, the cells were fixed for 20 min in 400 µL of 4% paraformaldehyde and rinsed three times with PBS. For reversed transformation monitoring, the 8-chloro-cyclic AMP treated cells on coverslips were washed three times with PBS and stained with K‒AuNCs as described above. After staining, the side of the coverslip with the fixed cells was overlaid with a glass slide with 5 µL of 50% glycerol/PBS (v/v). Fluorescence images were taken with a confocal laser scanning microscope (CLSM, Nikon Al, Nikon, Japan). K– AuNCs were excited with a 488nm laser and emission was collected between 662-737 nm. G–AuNCs were excited with a 405nm laser and emission was collected between 552-617 nm. 2.5. Photostability testing HT1080 cells were incubated in culture medium containing K‒AuNCs (400 µg/mL) or SYTO RNA-Select (5 µM) and imaged by confocal microscopy. The stained cells were irradiated with the appropriate laser (488 nm for K‒AuNC and 405 nm for SYTO RNA-Select), and the emission signal was collected between 662-737 nm and 500-530 nm, respectively. The cell images were scanned once every 20 s for 6 7



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min. The fluorescence microscopic images were processed with Nikon’s NIS-Elements AR software on the CLSM instrument. The initial intensity referred to the first scan. 2.6. Cytotoxicity assay HT1080 cells of exponential phase were seeded on 96-well plates at 2 × 104 cells/well and incubated with 1640 medium supplemented with 10% fetal bovine serum in a humidified incubator for 24 h at 37 °C in a humidified 5% CO2 atmosphere. On the day of experiments, the cells were washed with PBS buffer and incubated with fresh medium-containing K–AuNCs at 37 °C for 12 or 24 h. In the control wells, only neat medium was added. The cells were washed twice with PBS, and 100 µL of MTT solution (5 mg/mL) was subsequently added to each well. The plates were incubated at 37 °C for 4 h. The precipitated formazan was dissolved in 150 µL of dimethyl sulfoxide. The absorbance of each sample at 490 nm was measured by using a microplate autoreader (Molecular Devices, M2e). The cell viability ratio was calculated according to A490 nm/A0,490 nm (control) after background subtraction. 3. Results and discussion 3.1. Synthesis and characterization of K–AuNCs Peptides have already been successfully employed as stabilizing agents in AuNCs synthesis.30,

35, 36

Compared with protein-ligated AuNCs, short-peptide-stabilized

AuNCs remain below a critical size that facilitates permeability in cells and tissues as well as renal excretion.26,

36, 37

Previous work has also shown that the positively

charged surface is crucial for the accumulation of the nanoparticles at the nucleolar regions.22, 38 We therefore applied a tripeptide KCK as the stabilizing ligand in this project (Scheme 1), which offers not only a side-chain thiol residue, but also two 8


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positively charged amino groups. The nucleolar targeting gold nanoclusters were obtained through a one-pot synthetic strategy, which simplifies their preparation and ensures a sufficiently small size of the product to enable subcellular migration. AuNCs stabilized with cysteine-containing short peptides have been systematically investigated by Xie’s group and a Au(0)@Au(I)-thiolate core-shell structure was suggested.30 In order to demonstrate the effect of surface charge, a conventional negatively charged tripeptide, glutathione, was employed to synthesize a counterpart type of gold clusters, G‒AuNCs. The one-pot synthesis was carried out under optimized mild conditions (Figure S2). Deep-red-emitting K–AuNCs were obtained (QY = 12.4 %), which can be kept at 4 °C in the dark for 3 months without detectable changes in their dispersity and fluorescence properties (Figure S3). The morphology and size of K‒AuNCs and G‒ AuNCs were characterized with high-resolution transmission electron microscopy (HRTEM, Figure 1). The results showed good dispersity and homogeneous size distribution. The average diameters of the K‒AuNC and G‒AuNC gold cores were found to be 1.6 and 2.6 nm, respectively, by HRTEM, close to the expected values.26, 29, 36

Most importantly, these values fall below the dominating channel of nuclear pore

complexes (~5.2 nm in diameter), which is crucial for nanomaterials to migrate through the nuclear envelope and reach the nucleolar regions.22 K‒AuNCs were further evaluated using spectroscopy. The FT-IR spectra showed that the peak at ~2530 cm–1 of the KCK peptide, which corresponds to S-H stretching vibration mode, disappeared in the spectrum of K‒AuNCs, indicating that all KCK peptides were ligated on the AuNCs (Figure S4). HAADF-STEM image and the chemical element mapping results also proved the colocalization of S, N, O with Au, (Figure S5). X-ray photoelectron spectroscopy (XPS) measurements were carried out 9



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to analyze the composition and valence states of Au in K‒AuNCs (Figure S6). The XPS survey spectrum exhibits four distinct peaks which were assigned to Au4f, C1s, N1s, and O1s, respectively. A peak of S2p also suggests the coexistence of gold and peptides. The high-resolution Au4f spectrum shows two diffraction peaks corresponding to 4f7/2 and 4f5/2, respectively, which could be resolved into four peaks with binding energies at about 83.80 eV (Au(0) 4f7/2), 84.20 eV (Au(I) 4f7/2), 87.45 eV (Au(0) 4f5/2), and 88.05 eV (Au(0) 4f5/2).39, 40 This result suggests that both Au(0) and Au(I) exist in K‒AuNCs, consistent with literature results.41-43 UV-Vis spectra confirmed the absence of a surface plasmon resonance peak at 520 nm for both, K‒AuNCs and G‒AuNCs, as expected for ultra-small nanoclusters (Figure 2A). The absorption spectra of K‒AuNCs showed an onset at 580 nm, very different from that at 420 nm for G‒AuNCs. Additionally, when excited at 400 nm, G‒AuNCs showed a fluorescence emission band centered at 590 nm, whereas K‒ AuNCs emitted deep-red fluorescence peaking at 680 nm (Figure 2B). The obvious spectroscopic differences between K‒AuNCs and G‒AuNCs demonstrate that the surface ligand has a pronounced effect on the photophysical properties of AuNCs. AuNCs are known to display long-lived fluorescence with multi-exponential fluorescence decay behavior, which is attributed to multiple metal−ligand energy transfer processes.32, 44 Although the steady state optical spectra of K‒AuNCs clearly differ from those of G‒AuNCs, the intensity-averaged fluorescence lifetime of K‒ AuNCs was determined to be 2.48 ± 0.05 µs (Figure 2C) similar to G‒AuNCs (2.40 ± 0.05µs, Figure S7). It should be noted that K‒AuNCs display broad excitation spectra, ranging from 330 to 550 nm, which are accessible to many excitation light sources used for imaging. The effects of temperature, ion concentration and pH on the luminescence of K‒ 10


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AuNCs were also investigated (Figure S8). It was found that the fluorescence intensity showed less than 30% variation when the condition of the surrounding medium changed in much broader ranges than the physiological condition. The broad excitation window, long-wavelength emission, long fluorescence lifetime and good stability endow K‒AuNCs with excellent properties as fluorescent probes for biological analysis. The electrostatic properties of the gold nanoclusters were characterized by agarose-electrophoresis and zeta potential measurements (Figures 2D and 2E). As expected, after loading in the middle line of the same gel, K‒AuNCs migrated towards the cathode, whereas G‒AuNCs migrated towards the anode under the same electric field, demonstrating the opposite charge on these two types of nanocluster. This was further verified through surface potential measurements, which gave values of 2.6 ± 0.2 mV and –28.8 ± 0.7 mV for K‒AuNCs and G‒AuNCs, respectively. It was noted that when the electrophoresis was complete, K‒AuNCs showed several overlaid bands, indicating that the product might be a mixture of AuNCs with slightly different structural formulae, consistent with the literature.30 3.2. Nucleoli staining with K‒AuNCs To investigate the intracellular targeting specificity of K‒AuNCs, fluorescence imaging was conducted with one normal cell line HEK293E and three different tumor cell lines, HT1080, A549 and HepG-2. After incubation with K‒AuNCs (400 µg/mL) for 2 h at 37 °C, the cells were imaged with a confocal microscope. As shown in Figure 3, sharp-contrast bright luminescent spots were evident within the nuclear regions of the K‒AuNC-loaded HT1080 cells. In contrast, cells incubated with G‒ AuNCs only showed fluorescence in the cytoplasm, leaving a completely dark nuclear area in each cell. The different localizations of these two types of AuNCs were further 11



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verified by the quantitative luminescence intensity profiles. Similar intracellular localization of K‒AuNCs was achieved in HEK293E, A549 and HepG-2 cells (Figure S9), where the fluorescence of K‒AuNCs was found not only in the cytoplasm but also as bright spots inside of the nuclear region. In order to confirm that the bright spots observed in the nucleus were related to the K‒AuNCs localizing at the nucleoli, HT1080 cells cultured under the same conditions were stained with K‒AuNCs as well as with the commercial nucleolus stain SYTO RNA-Select, for comparison (Figure 4). It can be seen that the bright spots in both images showed similar localization and morphology, strongly suggesting that K‒AuNCs are successfully localized in the nucleolar regions. There are two possible working mechanisms for the reported nucelolar stains: the relatively high concentration of probes in the nucleolar regions or the enhanced luminescence efficiency of probes upon binding with nucleic acids. The electrophoresis experiments showed that after mixing K‒AuNCs with the same amount of DNA or RNA, the mobility of the fluorescent complexes changed, demonstrating that K‒AuNCs were able to interact with nucleic acids (Figure S10). Compared with DNA, a higher ratio of RNA can interact with K‒AuNCs to form negatively charged complexes, indicating that the K‒AuNCs bind to RNA molecules with a higher affinity than DNA. However, spectroscopic experiments showed that after mixing with DNA or RNA in buffer solution, the fluorescence intensity of K‒ AuNCs did not show any enhancement (Figure S11); this control experiment ruled out the RNA-binding induced fluorescence enhancement and confirmed that K‒AuNCs do actually accumulate in the nucleoli regions. This conjecture was also supported by the phase-dark protuberance in the bright-field image (indicated by red arrows in Figure 4C), which showed a consistent localization with the luminescence spots in the 12


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corresponding fluorescence image. An endocytic inhibitor, cytocchalasin D, and two metabolic inhibitors, 2-deoxy-D-glucose and oligomycin, were used to treat the cells and check the uptake mechanism of K‒AuNCs. As summarized in Figure 5, after the cells were pretreated with each kind of inhibitor, which could individually block the energy dependent cellular uptake pathway, the nucleolar fluorescence did not show any obvious variation. The cellular uptake of K‒AuNCs is therefore probably energy independent, which must be related to passive diffusion or simple diffusion. Our experiments demonstrate the efficient uptake of K‒AuNCs into cells and their accumulation in the nucleolar regions. In contrast, although conventional G‒ AuNCs also show efficient uptake, they mainly localize in the cytoplasm. Since the two types of gold nanoclusters have similar sizes that fall below the channel size of nuclear pore complexes, it is therefore reasonable to suggest that the varying subcellular distribution is closely related to the different surface charges of the two types of AuNCs. The nucleolar targeting property has also been reported for other positively charged nanoparticles, such as graphene quantum dots and chitosan nanoparticles.22,

38

Additionally, the electrophoresis experiments showed that K‒

AuNCs were able to interact efficiently with RNA. We therefore suggest the selective accumulation of K‒AuNCs at the nucleolar regions is related to the surface potential dependent interactions between K‒AuNCs and RNA molecules. To establish that the physiological state of the cells was not adversely affected in the course of the staining processes and, in particular, to exclude any significant cytotoxicity of K‒AuNCs, MTT assays were carried out. As shown in Figure S12, HT1080 cells maintained over 90% viability after treatment with K‒AuNCs even at higher concentrations (500 µg/mL) and at much longer incubation times (12 h) than 13



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used in the staining process (400 µg/mL for 2 h), indicating that K‒AuNCs are sufficiently biocompatible to conduct cellular staining experiments and to utilize them in functional assays (see below). Taking advantage of the broad excitation and emission spectra of K‒AuNCs, the nucleolar morphology stained with K‒AuNCs could be detected in several channels when excited with different lasers (Figure S13). This facilitates multi-channel imaging to illuminate simultaneously different subcellular organelles. Additionally, when the cells stained with K‒AuNCs and SYTO RNA-Select were continuously scanned by confocal microscopy, the fluorescence of SYTO RNA-Select decayed much more rapidly than that of K‒AuNCs, indicating a pronouncedly better photostability of K‒ AuNCs (Figure 6). This result is of particular practical importance, because SYTO RNA-Select serves as the only stain presently commercially available and therefore as a performance reference point. 3.3. Monitoring the reverse transformation of malignant cells We have demonstrated the potential of K‒AuNCs as nucleoli-targeting imaging probes. An important application perspective in nucleolar research is the study of the mechanism of drug action and the evaluation of the associated therapeutic progress. 8-cl-cAMP is an analogue of the protein kinase regulatory subunit, which is able to inhibit the growth of a wide range of cancer cells in vitro and in vivo.45, 46 Previous work has reported that 8-cl-cAMP treatment can cause the reverse transformation of fibrosarcoma cells HT1080. During this particular process the nucleolar morphology changes dramatically, which has only been able to followed up to now by silver staining and phase-contrast microscopy.7, 46 Taking advantage of the much more sensitive fluorescence microscopy technique and the novel nucleolar probe K‒AuNCs, we are now able to monitor the nucleolar 14


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dynamics in the reverse transformation process more accurately and with the distinct advantage of real-time monitoring. As shown in Figure 7, the nucleoli of cells at various physiological and pathological situations were all successfully stained with our K‒AuNCs and showed the characteristic red fluorescence. Since K‒AuNCs accumulate in the nucleolar regions, the morphology of the nucleoli can also be observed in the phase-contrast images due to the much more condensed structure of gold clusters (Figure S14). It is immediately evident from the comparison of the images in Figures 7 and S13 that the fluorescence images resolve the nucleoli better. Based on statistical analyses, it was found that most untreated HT1080 cells (87.5%) had more than 3 nucleoli in each cell with an average number of 4.6; moreover, most nucleoli were irregularly shaped and without distinctive features (Figure 7). After exposure to 8-cl-cAMP and in the course of reverse transformation, the nucleoli became more homogeneous in shape and more rounded. In addition, the average number of nucleoli per cell decreased with time from 4.6 (0 h) to 3.9 (24 h), 2.4 (48 h), and 1.7 (72 h). The observed coalescence of the nucleoli is important evidence for the reorganization of chromatin domains for new gene expression, which provides valuable reference data for pathological research.47, 48 The functional assay further demonstrates that K‒AuNCs can be employed as fluorescent probes for analyzing biological and biochemical processes involving the nucleoli, which are of current interest but are largely unknown. 4. Conclusions A novel nucleolar probe, the ultra-small fluorescent gold nanoclusters K‒AuNCs has been developed. These are prepared through an easy synthesis by employing a positively charged tripeptide as the surface ligand. This probe exhibits a unique combination of desirable properties, including deep-red fluorescence emission, broad 15



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excitation band, low cytotoxicity and good photostability. They can be used to stain the nucleoli for visualizing them by fluorescence and bright-field microscopy. Moreover, K‒AuNCs can be successfully applied in functional assays for monitoring the nucleolar dynamics during the reverse transformation process of malignant cells, which has direct implications for studying the action of drugs mechanistically. Compared with small organic dyes, nanometer-sized K‒AuNCs have a large surface area, which creates the possibility of combining tethered therapeutic moieties together with the nucleoli-targeting ability to realize – and to monitor simultaneously – the delivery of specific drugs. Acknowledgements This study was supported by the National Natural Science Foundation of China (21373271 and 21273287), the National Key Basic Research Program of China (2012CB518000),

the

Natural

Science

Foundation

of

Shandong

Province

(ZR2014BM028), the Key Technologies R&D Program of Shandong Province (2015GSF118013), and the CSC (doctoral fellowship for SZ). Supporting Information MALDI-TOF Mass Spectrum of KCK peptide, fluorescence emission spectra of K‒AuNCs synthesized under different conditions, fluorescence emission spectra of as-prepared K‒AuNCs suspension and after keeping in fridge for 1 month, the HAADF-STEM image and the chemical element mapping result, FT-IR spectra of KCK peptide and K‒AuNCs, the survey spectrum and the high-resolution Au4f spectrum, photoluminescence decay profile of G‒AuNCs, the effects of temperature, ion concentration and pH value on the luminescence of K‒AuNCs, the nucleoli staining tests of three other cell lines, the electrophoresis analysis and fluorescence 16


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emission spectra of K‒AuNCs after mixing with DNA and RNA, MTT assays illustrating the HT1080 cell viability after incubated with K‒AuNCs, fluorescence microscopic images of K‒AuNCs stained HT1080 cells scanned under different working conditions, phase contrast microscopic images of HT1080 cells treated with 8-cl-cAMP (Figures S1-S13) can be found at http://

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Scheme 1. Synthesis of nucleoli-targeting K‒AuNCs by using a tripeptide, KCK, for stabilization.

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Figure 1. HRTEM image and size distribution of K‒AuNCs (A, B) in comparison with G‒AuNCs (C, D). Scale bar is 20 nm.

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Figure 2. Characterization of K‒AuNCs and G‒AuNCs. (A) UV-Vis absorption spectra. The inset shows the photographs of K‒AuNCs (left) and G‒AuNCs (right) aqueous suspensions at daylight. (B) Fluorescence excitation (λem = 680 nm for K‒AuNCs and λem = 590 nm for G‒AuNCs) and emission spectra (λex = 480nm for K‒AuNCs and λex = 400 nm for G‒AuNCs). The inset shows the images of K‒AuNCs (left) and G‒AuNCs (right) aqueous suspensions with 365 nm UV light illumination. (C) Photoluminescence decay profile of K‒AuNCs. λex /λem = 373 nm / 680 nm. (D) Gel-electrophoresis analysis and (E) Zeta potential measurements of two types of AuNCs.

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Figure 3. HT1080 cells stained with K‒AuNCs and G‒AuNCs, respectively. (A, D) Fluorescence images. (B, E) Bight-field images. (C, F) Relative fluorescence intensity profiles along the lines shown in (A) and (D), respectively.

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Figure 4. HT1080 cells stained with K‒AuNCs and with SYTO RNA-Select, respectively. (A, B) Fluorescence images. (C, D) Bight-field images.

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Figure 5. Effects of metabolic and endocytic inhibitors on cellular uptake of K‒AuNCs in HT1080 cells. (A) The cells were incubated in normal situation without any treatment. (B) The cells were stained with K‒AuNCs. (C-E) The cells were pretreated with 2-deoxy-D-glucose (50 mM), oligomycin (5 mM) and Cytochalasin D (5 mM) for 1 h at 37°C, respectively, and then stained with K‒AuNCs.

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Figure 6. (A) Comparison of relative dye photostability in K‒AuNCs and SYTO RNA-Select-stained nucleoli as monitored from confocal fluorescence images taken after different irradiation times. (B) Time courses of the fluorescence intensity decays for the cell images in panel A.

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Figure 7. Nucleolar phenotype of HT1080 fibrosarcoma cells and their reverse-transformed counterparts after treatment with 8-cl-cAMP for 24, 48 and 72 h. (A) Fluorescence images of cells stained with K‒AuNCs. Scale bar is 25 µm. (B) Nucleoli number analysis based on the fluorescence images in panel A.

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Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real

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