Protein-Guided Formation of Silver Nanoclusters and Their Assembly

Letter pubs.acs.org/JPCL

Protein-Guided Formation of Silver Nanoclusters and Their Assembly with Graphene Oxide as an Improved Bioimaging Agent with Reduced Toxicity Niloy Kundu,† Devdeep Mukherjee,‡ Tapas Kumar Maiti,‡ and Nilmoni Sarkar*,† †

Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, West Bengal, India Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India

‡

S Supporting Information *

ABSTRACT: As an emerging category of fluorescent metal nanoclusters (NCs), protein-based NCs are considered as one of the promising candidates for the biomedical applications because of their luminescent properties and inherent biocompatibilities. Protein-capped silver NCs impregnated onto graphene oxide (GO) sheets can be internalized into the K562 cell, a human erythroleukemic cell line, and the Ag NCs/GO assembly can act as a synergistic drug carrier for Imatinib, a first-generation tyrosine kinase inhibitor. Further, Ag NCs adsorbed on GO have a great potential to be used as X-ray computer tomography (CT) imaging contrasting agents, and CT images show significant contrast enhancement of bone tissues in mice models. Overall, this assembly can exhibit great potential in the field of biomedical application and therapeutic studies.

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makes them ideal for biological assays and cell imaging compared to usual fluorescent labels. On the other hand, two-dimensional atomically thin graphene oxide (GO) has attracted considerable interest in several applications such as supercapacitors, nonlinear optics, sensors, drug delivery, and so forth13,14 due to its long-range π conjugation and strong thermal and electrical conductive properties. The presence of different functional groups such as carboxylic acid and hydroxyl or epoxy groups creates an opportunity to covalently modify the drug molecules that can be used for targeted drug delivery in a cell. Thus, it becomes more of a concern about the delivery of small molecules like nucleotides, drugs, and peptides to specific tissues. Earlier studies show that graphene, including GO and reduced graphene oxide (RGO), is biocompatible and a perfect platform for the adsorption of different metal and metal oxide nanoparticles, fluorescent molecules, drug molecules, and so forth.15,16 Moreover, the infrared thermal properties of GO make them attractive cancer therapeutic agent.17 In this regard, fluorescent NC/GO nanocomposites can offer a new and effective multimodal therapeutic material to detect and target different cells, and it can also be an interesting alternative for cancer therapeutic techniques. MTT assays show the excellent viability of these NCs, which broadens the opportunity for biological imaging. The adsorption of NCs on a GO surface and the internalization into K562 cells are supervised by

n the last few decades, metal nanoclusters (NCs) have attracted interest in different fields of research and applications that include catalysis, chemical sensing, biomedicine, optoelectronic, and so forth.1,2 The size-dependent optical properties, high water solubility, biocompatibility, and low toxicity of metal NCs provide a new direction for designing promising fluorescent probes for different biomedical applications.3,4 The size of the metal NCs is close to 1 nm, and it is comparable to the Fermi wavelength of the conduction electrons, which leads to different molecule-like phenomena such as luminescence and charging properties. Unlike semiconducting quantum dots (QDs), which are larger in dimensions and involve different toxic metal species, noble metal NCs are highly appreciated in biomedical research because of their low toxicity.5 However, there are some drawbacks associated with the metal NCs in terms of low quantum yield (compared to QDs), stability, and so forth. Synthetic protocols for the preparation of metal NCs using different templates such as DNA, polymers, dendrimers, and proteins have been reported in the literature.6−9 Thiolprotected gold (Au) NCs emit fluorescence in the blue to IR region, although the quantum yields of these NCs are relatively low (0.001−0.1%).10 Ying et al. first reported a simple, one-pot, green synthetic route of metal NCs using protein as a template at physiological temperature (37 °C).11 Mukherjee et al. reported the formation of blue-emitting silver NCs (Ag-HSA (human serum albumin) NCs) with high quantum yield (>11%).12 The near-infrared emission properties of these Ag NCs avoid interference from many biological moieties, which © 2017 American Chemical Society

Received: March 11, 2017 Accepted: May 3, 2017 Published: May 3, 2017 2291

DOI: 10.1021/acs.jpclett.7b00600 J. Phys. Chem. Lett. 2017, 8, 2291−2297

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Figure 1. (a) HR-TEM image of synthesized Ag-HSA NCs. The dark spots or the bigger aggregates seen in the image may be the aggregate of clusters. Bigger particles are formed due to the addition of excess NaBH4, and the sharpness of the image is reduced due to the presence of protein corona. (b) Size distribution profile of Ag-BSA and Ag-HSA NCs obtained from DLS. (c,d) Steady-state excitation and emission spectra of Ag-HSA NCs (c) and Ag-BSA NCs (d). (e) Fitted FCS traces of Ag-BSA and Ag-HSA NCs in water (λexc = 488 nm). (f,g) Fitted FCS traces of Ag-BSA NCs (f) and Ag-HSA NCs (g) in the presence of different concentrations of GO.

fluorescence correlation spectroscopy (FCS) measurement. Fluorescence lifetime imaging microscopy (FLIM) study further suggests that the Ag NCs/GO assembly can act as a synergistic drug carrier for Imatinib mesylate, a first-generation tyrosine kinase inhibitor. Besides, over the past decade, the immense success of X-ray microcomputed tomography (μCT) technology in medical application has provided an opportunity to augment image quality so as to retrieve more information from a given clinical subject. In line with this, development of appropriate contrast agents has also gained much interest. While aiming at optimal contrast enhancement, the pharmacokinetics and toxicity assessment of the agent are also of major concern. Here, we propose that Ag NCs/GO assembly can significantly enhance the contrast of bone tissues performed on mice models. Protein-mediated highly fluorescent Ag NCs are prepared following the literature procedure.12 For the synthesis, we used two globular proteins, human serum albumin (HSA) and bovine serum albumin (BSA), as a template. The detailed description of the synthesis of the NCs is given in the Supporting Information. The formation of the NCs was indicated by the change in color of the solution from colorless to reddish brown for both NCs, and the absorbance profile of both NCs shows that the color of the Ag-HSA NCs is more intense compared to that of Ag-BSA NCs (Figure S1a, Supporting Information). The synthesized Ag-BSA/HSA NCs exhibit outstanding photophysical properties close to the visible region. Formation of the NCs is confirmed by MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) and subsequent high-resolution transmission electron microscopy (HR-TEM) measurements. The synthesized NCs are spherical in nature, as evidenced by the HR-TEM image (Figure 1a), and

the EDAX (energy-dispersive analysis of X-rays) spectrum obtained from the HR-TEM images confirms the presence of a Ag atom in the spherical particles (Figure S2a, Supporting Information). Further, the zeta potential of the Ag-HSA NCs is measured to determine the surface potential of the NCs. The zeta potential value of the Ag-HSA NCs is negative (−40 mV). It is believed that at pH > 10, the tyrosine residue of the HSA reduces the Ag salt. Thus, the phenol functional group of tyrosine becomes more negatively charged after the reaction, and it creates a more negative surface charge compared to native protein. In addition, the hydrodynamic radii of the NCs are determined from the dynamic light scattering (DLS) measurements (Figure 1b). For Ag-HSA and Ag-BSA NCs, they are 2.6 and 2 nm, respectively. MALDI-TOF measurement of Ag-HSA NCs shows the base peak at m/z = 67915 Da, and the corresponding double charged peak is centered at 33900 Da (Figure S3, Supporting Information). The difference of the base peak between Ag-HSA NCs and HSA protein alone (66500 Da) reveals that 14 Ag atoms make up the core of the NC in the case of Ag-HSA NCs. Similarly, for Ag-BSA NCs, eight Ag atoms form the cluster. Anand et al.12 hypothesized the mechanistic details of the formation of these NCs. When silver nitrate is added to the protein solution, the Ag+ is entrapped in the various scaffolds inherently present in the protein and stabilized by electrostatic interaction with the different ionic groups present in the protein. Further, NaOH is added to enable the reduction property of tyrosine, and it reduces Ag+ to Ag. Addition of NaBH4 into the reaction mixture overshadows the reduction property of tyrosine and increases the reaction rate. Besides, it also controls the growth of the NCs. The bulky HSA/BSA protein stabilizes the Ag+ ion, and it experiences a constraint environment that increases its fluorescence (Scheme 2292

DOI: 10.1021/acs.jpclett.7b00600 J. Phys. Chem. Lett. 2017, 8, 2291−2297

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The Journal of Physical Chemistry Letters Scheme 1. Chemical Structure of Synthesized GO and the Drug Imatinib Mesylate and the Schematic Diagram of the Synthesized Protein-Stabilized Ag Nanoclusters

Figure 2. (a) FLIM images of K562 cells treated with Ag-BSA NCs (i) and Ag-HSA NCs (ii). The left panel of the image is the intensity image, and the right panel represents the lifetime image. Scale bars in images (i) and (ii) represent 2 and 1.6 μm, respectively. (b) Lifetime distribution of AgBSA and Ag-HSA NCs obtained from the FLIM images. (c) FACS (fluorescence-activated cell sorting) histogram of K562 cells treated with NCs. The x axis in FACS shows the fluorescence intensity of each K562 cell, and the y axis shows the cell count and mean fluorescence intensity of both NCs are plotted in figure (iii). (d) FCS traces of Ag NCs incubated in K562 cells.

also agree that the size of the Ag-HSA NCs is higher than that of Ag-BSA NCs. The adsorption of different biomolecules including protein, DNA, drug, and fluorophores on GO surface is well reported in the literature. Silver-based NPs or NCs stabilized with nucleic acid decorated on GO are used for sensing applications and for the detection of various infectious pathogens genes.19 FCS can be used as a tool to understand the adsorption of these synthesized NCs on the GO surface. The flake nature of the GO is established from HR-TEM measurement (Figure S2b, Supporting Information). FESEM (field emission scanning electron microscopy) and AFM (atomic force microscopy) images of GO further signify that the lateral dimension of GO varies from region to region and it is less than 10 μm (Figure

1). The emission spectra for Ag-BSA (λexc = 440 nm) and AgHSA ((λexc = 480 nm) are centered at 510 and 620 nm, respectively (Figure 1c,d). Due to the formation of different sized NCs (as evidenced from the HR-TEM image), the fluorescence property of Ag-HSA may show a broad spectrum,12 and the emission spectrum of Ag-HSA NCs shows a second contribution at ∼530 nm when they are excited at 440 nm (Figure S1b, Supporting Information). The excitation spectra of both NCs are also shown in Figure 1c,d. The calculated quantum yields for Ag-HSA and Ag-BSA NCs are 0.13 and 0.10, respectively, and the values are quite higher than other literature-reported NCs.10 Now, the Jellium model predicts that as the size of the NCs decreases it shows a blue shift in the emission maxima.18MALDI-TOF measurements 2293

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Figure 3. (i,ii) FLIM images of K562 cells incubated with Ag-BSA (i) and Ag-HSA NCs (ii). (b,c) The cell is treated with GO (50 μg/mL) and Imatinib. Relative fluorescence intensity variations along cross sections in (a−c) are shown in (d). (iii) K562 cell proliferation study after 48 h of exposure to Imatinib (2 μM), GO, and Ag NCs of different concentrations. (a) Green, orange, and red columns represent the variation of GO concentration as 20, 50, and 100 μg/mL, respectively. (b,c) Concentrations of Ag-HSA (b) and Ag-BSA (c) NCs are varied as 1, 2, and 4 mg/mL. (d) Yellow, orange, and red columns represent cells treated with Imatinib alone; Imatinib, Ag-HSA NCs (2 mg/mL), and GO (100 μg/mL); and Imatinib, Ag-BSA NCs (2 mg/mL), and GO (100 μg/mL), respectively.

Now, to determine the number of bound fluorophores on the GO surface, FCS autocorrelation curves are fitted in another way. The diffusion times (τD) are fixed at their highest value, and only the contributions to the diffusion times (αi) are allowed to vary.20The fraction of bound NCs (f) obtained from fitting of the FCS traces is plotted against the concentration of GO (Figure S4, Supporting Information). Thus, the binding constants obtained for Ag-BSA and Ag-HSA NCs are (1.72 ± 0.05) and (2.74 ± 0.17) mL/mg, respectively, and the corresponding free energies of binding for Ag-BSA and AgHSA NCs are −1.34 and −2.50 kJ/mol, respectively. Ag NCs with high quantum yield are beneficial to cellular staining and imaging.3 For this reason, the NCs are treated to K562 cells to test if they could be used as a specific fluorescent probe for cell imaging. K562 cells are the first established human immortalized myelogenous leukemia line obtained in blast crisis. The intracellular localization of these NCs in K562 cells is confirmed by the FLIM images. Figure 2a shows the time domain images of the K562 cells treated with Ag NCs. The intensity image is calculated from the photons in all time channels of the pixels and in the FLIM images, the lifetime obtained for each pixel is enclosed by color. FLIM images show that both Ag NCs are localized in the cytoplasm as well as the nucleus of the cell. Due to multiple localizations of these NCs, the lifetime distribution obtained from the FLIM images is quite broad in nature, and a bimodal distribution is observed, which clearly signifies multiple locations of the NCs in the cells (Figure 2b). Bhattacharyya et al. recently determined the

S2c,d, Supporting Information). The normalized FCS traces of Ag-BSA/HSA NCs in water are shown in Figure 1e. In bulk water, the FCS traces are well fitted with a 3D diffusion model. For Ag-BSA and Ag-HSA NCs in water, the diffusion coefficients are obtained as 169.8 and 152.7 μm2 s−1, respectively. From the diffusion coefficient, following the Stokes−Einstein equation, the hydrodynamic radii of the NCs can also be predicted. For the Ag-BSA and Ag-HSA NCs, the hydrodynamic radii are calculated to be 1.29 and 1.43 nm, respectively, in good agreement with the values obtained from DLS measurements. The FCS traces of these NCs in the presence of difference concentrations of GO are shown in Figure 1f,g, and FCS traces are fitted with a two-component diffusion model.20 The fast component is assigned to the NCs that are freely diffusing in water, and the slower component contributes to the NCs that are adsorbed on the GO (Table S1, Supporting Information). With increasing concentration of GO, more probe molecules are adsorbed on the GO surface and the diffusion of the molecules become restricted. Thus, the contribution to the slower component (α2) of the diffusion coefficient is gradually increased with increasing concentration of GO. However, for two different NCs, a different extent of decrease in the diffusion coefficient is observed, and much slower diffusion is observed for Ag-HSA NCs compared to that of Ag-BSA NCs. Besides, the adsorption of NCs on the GO surface is also evidenced by HR-TEM and FLIM images (Figure S2e−g, Supporting Information) 2294

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is due to the adsorption of NCs on the GO surface. The lifetime distribution and the area of each deconvoluted spectrum of NCs in the absence or presence of GO in K562 cells remain almost similar, as evidenced in Figure S6, Supporting Information. Thus, the unaltered lifetime in the presence of GO signifies that the surrounding environment of Ag NCs in the presence and absence of GO remained the same. A very short component in the lifetime distribution (∼100 ps) is observed in case of Ag-HSA NCs (Figure S6b, Supporting Information), which can be attributed to the Ag-HSA NCs that are adsorbed on GO in solution. Thus, we can conclude that the localization of the NCs does not change into the K562 cells in the presence of GO. The viability of K562 cells following treatment with NCs, GO, and a first-generation tyrosine kinase inhibitor, Imatinib mesylate, is evaluated by MTT assay. Imatinib reduces the growth of cancer cells by blocking the BCR-ABL enzyme that is generated through reciprocal translocation of chromosomes 9 and 22. In most reports, Ag nanoparticles have been shown to possess toxicity with IC50 ≈ 2−5 μg/mL after 48 h of treatment.24,25 Ag NPs exert antiproliferative activity by increasing intracellular reactive oxygen species and modulating mitochondrial membrane potentials.26 In contrast, in the present study, after 48 h of incubation with increasing concentration of BSA- and HSA-Ag NCs up to 4 mg/mL, above ∼80% viability of K562 cells is observed (Figure 3iii). It was argued that several factors may be responsible for reducing the toxic effects of Ag NPs, namely, size, shape, degree of uptake, and the presence of ligand molecules.27 Importantly, the presence of protein moieties, hitherto termed protein coronas, has been shown to be a critical toxicity modulator of these NCs.28 In fact, such remarkable cell viability even after 48 h of incubation with NCs confirms the stability and strong association of the protein coronas with NCs. Consistent with earlier report by Miclaus et al., it appears that Ag NCs are either partially or completely sulfidated with strongly associated HSA or BSA coronas.29 GO possesses a higher capacity of adsorption mainly due to its high surface area to volume ratio. Therefore, we hypothesize that immobilizing Imatinib and NCs on the surface of GO would be a better way for drug delivery. Results show concentration-dependent inhibition of cell viability (Figure 3iii,a) following 48 h of treatment with GO, whereas 2 μM Imatinib caused significant inhibition in cell proliferation. However, as compared to Imatinib alone, 8−12% further reduction in cell viability is observed after coadministering K562 cells with NCs and Imatinib immobilized on GO (Figure 3iii,d). Such reduction may be attributed to the synergistic effects of GO and Imatinib.17 Further, FLIM study also supports the conclusion (Figure S7, Supporting Information). The lifetime of Ag NCs associated with K562 cells and their cellular distribution do not change after treatment with Imatinib alone or an Imatinib/GO assembly. However, partial reduction in the intensity of images corresponds to more adsorption of the drug to the GO surface. Although GO in combination with NCs and Imatinib did not exert a drastic reduction in the viability of CML cells compared to Imatinib alone, in the future it would be interesting to study the potential of these formulations in addressing the challenges related to the primary resistance against Imatinib in CML patients, which mainly arises due to an impaired drug influx/ efflux ratio through the cell membrane.29

polarity in different regions of a cell, and they concluded that the polarity around the nucleus region is much higher than that in the cytoplasm.21 Thus, on the basis of their experimental evidence, the higher and lower lifetime of Ag NCs contributes to the fluorophores located at the cytoplasm and nucleus in K562 cells, respectively, and it is also supported by the FLIM images. From the lifetime distribution, it is found that the NCs are mainly localized in the cytoplasm. Now, the area of each deconvoluted spectra is calculated from the lifetime distribution, and the fraction of the area of each deconvoluted spectra with respect to the overall distribution is determined. Therefore, it can be concluded that more Ag-HSA NCs are localized in the nucleus compared to Ag-BSA NCs. Internalization of Ag NCs is also confirmed by taking the FLIM images at different Z values (Z being vertical).3 A typical section image of the cells demonstrated that both NCs are present in the cell nuclear fraction (Figure S5i,ii, Supporting Information). Further, by comparing the fluorescence image with nuclear Hoechst 3552 dye, we confirm that the intracellular Ag NCs are present in both the nucleus and the cytoplasm of the K562 cell (Figure S5iii, Supporting Information). In this aspect, Li et al.3 proposed that nucleolin, a carrier protein that transfers molecules between the cell surface and nucleus, is highly expressed in malignant cells and nucleolin-mediated endocytosis might be the possible reason for the internalization of Ag NCs in K562 cells. Next, the cellular uptake efficiency of the NCs was quantified through a flow cytometer (Figure 2c), and it appears that after 2 h of incubation of K562 cells Ag-HSA NCs show ∼42% higher accumulation compared to Ag-BSA NCs (Figure 2c,iii). Further, internalization of these NCs into K562 cells is confirmed by the FCS measurements (Figure 2d). FCS traces in K562 cells are fitted by a single-component diffusion model, and the diffusion coefficient values are tabulated in Table S2, Supporting Information. However, the simple diffusion model that is used to describe the normal Fickian diffusion cannot fit the FCS traces. The FCS traces are fitted by the following equation ⎛ 1⎜ 1 G (τ ) = ⎜ N⎜1 + τ τD ⎝

⎞⎛ ⎟⎜ 1 α ⎟⎜ 1 ⎟⎜ 1 + 2 ω ⎠⎝

( )

⎞0.5 ⎟ α⎟ ⎟ ⎠

( ) τ τD

(1)

In the above equation, α describes the extent of deviation from the normal diffusion. The abundance of different biomacromolecules in the cytoplasm and nucleus of a cell causes a state of molecular crowding, and translational motion in a crowding environment cannot be described by the normal (Fickian) diffusion.22 In this case, we have observed anomalous subdiffusion (α < 1) of the two NCs in K562 cells. Recently, Bhattacharyya et al. found that a cancer cell provides a more crowded and rigid environment compared to a normal cell.18 Moreover, the cytoplasm of a cancer cell is more viscous compared to bulk water.23 Thus, the much slower diffusion coefficient in the K562 cell is due to the more viscous and rigid environment than that of bulk water. Noncovalent functionalization of Ag NCs with GO were studied earlier by FCS measurements and can be utilized in several applications ranging from biosensing to cell imaging.16 The internalization of Ag NCs adsorbed on GO in the K562 cells is shown by the FLIM images, as described in Figure 3i,ii. The fluorescence intensity of the NCs is significantly quenched when the K562 cells are incubated with GO (Figure 3), which 2295

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Figure 4. (a) Preprocessed CT image of Swiss albino mice treated with a Ag NCs and GO assembly. (b) Postprocessed image of mice treated with a Ag-HSA NC/GO assembly with top, right, front, and 3D views. (The postprocessed image of control sample is shown in Figure S8).

and NCs on the surface of GO would be a better way for drug delivery. Finally, X-ray CT data show that this GO/Ag NCs assembly can significantly enhance the postrendering volume of the bones in mice models; thus, it can be used as a contrast agent for CT imaging. Overall, protein-stabilized Ag NCs impregnated onto GO sheets undoubtedly broaden the applications of functionalized Ag NCs in biological areas.

Ag or Au NPs have good X-ray attenuation properties, which enable their use as in vivo imaging agents through X-ray computed tomography (CT).30 Compared to other nanoparticle-based contrast agents, lower concentrations of BSA or HSA NC/GO were used to avoid viscosity problems.31 Earlier, Zou et al. reported 37.1 mM Ag nanoparticle as the lowest concentration for visualization through μCT in rats.32 However, Ag nanoparticles at this concentration might exert significant toxicity to the surrounding tissues. Because we used a 10 mM concentration of NCs in the present study (considering the initial concentration of AgNO3), consistent with the previous observation, we could not locate NCs in the mice. However, the post-BSA or HSA NC/GO injection in vivo CT scan showed significant contrast enhancement of the bone tissues plausibly due to the accumulation of NC/GO composites (Figure 4a). In addition, considerable postrendering volume enhancement in the vertebral column and other bone tissues was observed following injection with BSA or HSA NC/GO. While control mice showed volumes in the bone tissues in the range of 600−700 mm3, NC/GO-treated mice showed the same in the range of 900−1000 mm3 (Figures 4b and S8). Therefore, this NC/GO-based platform offers great promise to be used as a contrast agent in X-ray CT imaging applications. In conclusion, Ag NCs are synthesized using HSA and BSA as templates. HSA and BSA both are globular proteins. These NCs readily interact with GO while keeping their morphology and fluorescent properties. The adsorption of the NCs on a GO surface is evidenced by the FCS measurements. FCS study suggests that more Ag-HSA NCs are adsorbed on the GO surface compared to that of Ag-BSA NCs. The high quantum yield of these NCs allows clear imaging of the morphology of the cells. However, as an imaging agent, Ag-HSA NCs provide better results compared to Ag-BSA NCs due to their high quantum yield. Further, the internalization of the NCs into the K562 cells is shown by the FLIM and flow cytometry measurements. MTT assays show the excellent viability of these NCs, and FLIM study further suggests that the Ag NCs/ GO assembly can act as a synergistic drug carrier for Imatinib, and as NCs readily adsorbed on GO, immobilizing Imatinib

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b00600. Experimental section, table of diffusion coefficients of NCs in the presence of GO and K562 cells, UV−vis spectra of NCs, EDAX analysis of Ag NCs, TEM, FESEM, and FLIM images of GO, MALDI-TOF spectra, determination of fraction-bound Ag NCs on GO, Z stack FLIM images of K562 cells and fluorescence images of K562 cells stained with Hoechst 3552 dye, lifetime distribution of NCs in K562 cells, and postprocessed CT images (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 91-3222-255303. ORCID

Nilmoni Sarkar: 0000-0002-8714-0000 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.S. gratefully acknowledges SERB (Grant No: IR/S1/LU001/2013 dated 3/24/2015), the Department of Science and Technology (DST) and Council of Scientific Industrial Research (CSIR), and the Government of India for providing generous research grants. N.K is thankful to IIT Kharagpur, and D.M acknowledges CSIR for their research fellowships. We are 2296

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(20) Kundu, N.; Roy, A.; Banik, D.; Kuchlyan, J.; Sarkar, N. Graphene Oxide and Pluronic Copolymer Aggregates−Possible Route to Modulate the Adsorption of Fluorophores and Imaging of Live Cells. J. Phys. Chem. C 2015, 119, 25023−25035. (21) Ghosh, S.; Chattoraj, S.; Mondal, T.; Bhattacharyya, K. Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy. Langmuir 2013, 29, 7975− 7982. (22) Weiss, M.; Elsner, M.; Kartberg, F.; Nilsson, T. Anomalous Subdiffusion Is a Measure for Cytoplasmic Crowding in Living Cells. Biophys. J. 2004, 87, 3518−3524. (23) Mastro, A. M.; Babich, M. A.; Taylor, W. D.; Keith, A. D. Diffusion of a Small Molecule in the Cytoplasm of Mammalian Cells. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 3414−3418. (24) Dziedzic, A.; Kubina, R.; Bułdak, R. J.; Skonieczna, M.; Cholewa, K. Silver Nanoparticles Exhibit the Dose-Dependent Anti-Proliferative Effect against Human Squamous Carcinoma Cells Attenuated in the Presence of Berberine. Molecules 2016, 21, 365. (25) de Luna, L. A. V.; de Moraes, A. C. M.; Consonni, S. R.; Pereira, C. D.; Cadore, S.; Giorgio, S.; Alves, O. L. Comparative in Vitro Toxicity of a Graphene Oxide-Silver Nanocomposite and the Pristine Counterparts Toward Macrophages. J. Nanobiotechnol. 2016, 14, 1− 17. (26) Ma, W.; Jing, L.; Valladares, A.; Mehta, S. L.; Wang, Z.; Li, P. A.; Bang, J. J. Silver Nanoparticle Exposure Induced Mitochondrial Stress, Caspase-3 Activation and Cell Death: Amelioration by Sodium Selenite. Int. J. Biol. Sci. 2015, 11, 860−867. (27) Gorka, D. E.; Osterberg, J. S.; Gwin, C. A.; Colman, B. P.; Meyer, J. N.; Bernhardt, E. S.; Gunsch, C. K.; DiGulio, R. T.; Liu, J. Reducing Environmental Toxicity of Silver Nanoparticles through Shape Control. Environ. Sci. Technol. 2015, 49, 10093−10098. (28) Miclăuş, T.; Beer, C.; Chevallier, J.; Scavenius, C.; Bochenkov, V. E.; Enghild, J. J.; Sutherland, D. S. Dynamic Protein Coronas Revealed as a Modulator of Silver Nanoparticle Sulphidation in Vitro. Nat. Commun. 2016, 7, 11770. (29) Thomas, J.; Wang, L.; Clark, R. E.; Pirmohamed, M. Active Transport of Imatinib Into and Out of Cells: Implications for Drug Resistance. Blood 2004, 104, 3739−3745. (30) Guo, R.; Wang, H.; Peng, C.; Shen, M.; Pan, M.; Cao, X.; Zhang, G.; Shi, X. X-ray Attenuation Property of DendrimerEntrapped Gold Nanoparticles. J. Phys. Chem. C 2010, 114, 50−56. (31) Rabin, O.; Manuel Perez, J.; Grimm, J.; Wojtkiewicz, G.; Weissleder, R. An X-ray Computed Tomography Imaging Agent Based on Long-Circulating Bismuth Sulphide Nanoparticles. Nat. Mater. 2006, 5, 118−122. (32) Zou, J.; Hannula, M.; Misra, S.; Feng, H.; Labrador, R. H.; Aula, A. S.; Hyttinen, J.; Pyykkö, I. Micro CT Visualization of Silver Nanoparticles in the Middle and Inner Ear of Rat and Transportation Pathway after Transtympanic Injection. J. Nanobiotechnol. 2015, 13, 5.

thankful to Mr. Subhodeep Jana for the analysis of the CT images.

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REFERENCES

(1) Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine. Acc. Chem. Res. 2008, 41, 1578−1586. (2) Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Gold Nanoparticles in Chemical and Biological Sensing. Chem. Rev. 2012, 112, 2739−2779. (3) Li, J.; Zhong, X.; Cheng, F.; Zhang, J.-R.; Jiang, L.-P.; Zhu, J. J. One-Pot Synthesis of Aptamer-Functionalized Silver Nanoclusters for Cell-Type-Specific Imaging. Anal. Chem. 2012, 84, 4140−4146. (4) Zheng, J.; Zhang, C. W.; Dickson, R. M. Highly Fluorescent, Water-Soluble, Size-Tunable Gold Quantum Dots. Phys. Rev. Lett. 2004, 93, 077402. (5) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Lett. 2004, 4, 11−18. (6) Guo, W.; Yuan, J.; Dong, Q.; Wang, E. Highly SequenceDependent Formation of Fluorescent Silver Nanoclusters in Hybridized DNA Duplexes for Single Nucleotide Mutation Identification. J. Am. Chem. Soc. 2010, 132, 932−934. (7) Xu, H.; Suslick, K. S. Sonochemical Synthesis of Highly Fluorescent Ag Nanoclusters. ACS Nano 2010, 4, 3209−3214. (8) Le Guével, X.; Hötzer, B.; Jung, G.; Hollemeyer, K.; Trouillet, V.; Schneider, M. Formation of Fluorescent Metal (Au, Ag) Nanoclusters Capped in Bovine Serum Albumin Followed by Fluorescence and Spectroscopy. J. Phys. Chem. C 2011, 115, 10955−10963. (9) Mohanty, J. S.; Xavier, P. L.; Chaudhari, K.; Bootharaju, M. S.; Goswami, N.; Pal, S. K.; Pradeep, T. Luminescent, Bimetallic Au Ag Alloy Quantum Clusters in Protein Templates. Nanoscale 2012, 4, 4255−4462. (10) Negishi, Y.; Chaki, N. K.; Shichibu, Y.; Whetten, R. L.; Tsukuda, T. Origin of Magic Stability of Thiolated Gold Clusters: A Case Study on Au25(SC6H13)18. J. Am. Chem. Soc. 2007, 129, 11322−11323. (11) Xie, J.; Zheng, Y.; Ying, J. Y. Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters. J. Am. Chem. Soc. 2009, 131, 888−889. (12) Anand, U.; Ghosh, S.; Mukherjee, S. Toggling Between Blueand Red-Emitting Fluorescent Silver Nanoclusters. J. Phys. Chem. Lett. 2012, 3, 3605−3609. (13) Chen, D.; Feng, H.; Li, J. Graphene Oxide: Preparation, Functionalization, and Electrochemical Applications. Chem. Rev. 2012, 112, 6027−6053. (14) Georgakilas, V.; Tiwari, J. N.; Kemp, K. C.; Perman, J. A.; Bourlinos, A. B.; Kim, K. S.; Zboril, R. Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials, Biosensing, Catalytic, and Biomedical Applications. Chem. Rev. 2016, 116, 5464− 5519. (15) Yoo, E.; Honma, I.; et al. Enhanced Electrocatalytic Activity of Pt Subnanoclusters on Graphene Nanosheet Surface. Nano Lett. 2009, 9, 2255−2259. (16) Kim, T. H.; Shah, S.; Yang, L.; Yin, P. T.; Hossain, M. K.; Conley, B.; Choi, J. W.; Lee, K. B. Controlling Differentiation of Adipose- Derived Stem Cells Using Combinatorial Graphene HybridPattern Arrays. ACS Nano 2015, 9, 3780−3790. (17) Wang, C.; Li, J.; Amatore, C.; Chen, Y.; Jiang, H.; Wang, X. M. Gold Nanoclusters and Graphene Nanocomposites for Drug Delivery and Imaging of Cancer Cells. Angew. Chem., Int. Ed. 2011, 50, 11644− 11648. (18) Chattoraj, S.; Bhattacharyya, K. Fluorescent Gold Nanocluster Inside a Live Breast Cell: Etching and Higher Uptake in Cancer Cell. J. Phys. Chem. C 2014, 118, 22339−22346. (19) Liu, X.; Wang, F.; Aizen, R.; Yehezkeli, O.; Willner, I. Graphene Oxide/Nucleic-Acid-Stabilized Silver Nanoclusters: Functional Hybrid Materials for Optical Aptamer Sensing and Multiplexed Analysis of Pathogenic DNAs. J. Am. Chem. Soc. 2013, 135, 11832−11839. 2297

DOI: 10.1021/acs.jpclett.7b00600 J. Phys. Chem. Lett. 2017, 8, 2291−2297