Antimicrobial Gold Nanoclusters Kaiyuan Zheng, Magdiel I. Setyawati,* David Tai Leong,* and Jianping Xie* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 S Supporting Information *
ABSTRACT: Bulk gold (Au) is known to be chemically inactive. However, when the size of Au nanoparticles (Au NPs) decreases to close to 1 nm or sub-nanometer dimensions, these ultrasmall Au nanoclusters (Au NCs) begin to possess interesting physical and chemical properties and likewise spawn different applications when working with bulk Au or even Au NPs. In this study, we found that it is possible to confer antimicrobial activity to Au NPs through precise control of their size down to NC dimension (typically less than 2 nm). Au NCs could kill both Gram-positive and Gram-negative bacteria. This wide-spectrum antimicrobial activity is attributed to the ultrasmall size of Au NCs, which would allow them to better interact with bacteria. The interaction between ultrasmall Au NCs and bacteria could induce a metabolic imbalance in bacterial cells after the internalization of Au NCs, leading to an increase of intracellular reactive oxygen species production that kills bacteria consequently. KEYWORDS: gold nanoclusters, silver nanoclusters, antimicrobial agents, metal nanoparticles, reactive oxygen species
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In nanomedicine applications, several recent studies have shown that further decreasing the size of silver (Ag) NPs to the NC range could produce enhanced antimicrobial agents with wide-spectrum applicability that allows us to remain ahead of the antimicrobial resistance.25,26 Nevertheless, the enhanced antimicrobial effectiveness was not accompanied by desirable biocompatibility in the mammalian cell model, raising safety concerns for its clinical application.27 This is expected as the antimicrobial activity of Ag NCs is mainly derived from the generation of Ag+ ions, which are potentially toxic to both the targeted bacteria cells and the host human cells alike. Clearly, there is a need for an alternative wide-spectrum antimicrobial agent with higher biocompatibility (over Ag NCs) to ensure the clinical translation. Unlike silver, gold, the “noblest” among the metals, is inert, highly stable, and would not easily dissociate into ions.28 These noble qualities contribute to the widely accepted notion of Au NPs as being highly biocompatible in mammalian system, both in vitro and in vivo.29 This biocompatibility in mammalian cells is also observed when the size is further reduced to the NC range. For example, utilizing the in vitro colorectal cell model, we observed no cytotoxic and genotoxic effect on the cells exposed with Au NCs.30 Interestingly, Au NCs were found to instead boost cell metabolism and overall cell proliferation. In animal studies, ultrasmall Au NCs showed improved tumor uptake and high renal clearance.31,32 It is also this inertness that
anotechnology advancement has brought much excitement to the material science field.1,2 The excitement is not unwarranted as material scientists are able to precisely control the physicochemical properties down to atomic precision to yield advanced materials with extraordinary properties, such as nanoparticles (NPs) with quantum size effects.1,2 Among the tunable physicochemical properties, the size-dependent properties of NPs receive the most attention. The exploration of control size has resulted in particular applications of NPs, such as the surface-enhanced Raman scattering (SERS)-based analysis, green catalysts, superior magnetic resonance imaging agent, etc.3−6 Controlling NPs’ size also allows the material scientists to modulate their biochemical and biophysical properties, including cell uptake, biodistribution, pharmacokinetics, cytotoxicity, targeting efficiency, etc.7−13 It is also possible to push the NPs’ effectiveness further by reducing the size of metal NPs to a value comparable to the Fermi wavelength of electrons (∼1 nm), producing the ultrasmall metal nanoclusters (NCs).14−16 Ultrasmall metal NCs (with core sizes less than 2 nm) hold discrete electronic states and characteristic geometric structures, which offer them several intriguing molecular-like properties, such as well-defined molecular structure, quantized charging, HOMO−LUMO transition, molecular magnetism, molecular chirality, and strong luminescence.17−23 An interesting example is that gold (Au) is highly catalytic in the form of Au NCs. It was reported that Au55 NCs (consisting of 55 Au atoms) showed efficient catalytic activity for selective oxidation of styrene by dioxygen, whereas even slightly larger Au NPs (>2 nm) were completely inactive.24 © 2017 American Chemical Society
Received: March 24, 2017 Accepted: June 8, 2017 Published: June 8, 2017 6904
DOI: 10.1021/acsnano.7b02035 ACS Nano 2017, 11, 6904−6910
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Figure 1. Characterizations of the as-prepared Au NPs and NCs protected by MHA. (a) UV−vis absorption spectrum of Au NPs; insets are a representative TEM image (left) and photograph (right). (b) UV−vis absorption spectrum of Au NCs (the distinctive absorption peaks for Au25 NCs are marked); insets are a representative TEM image (left) and photograph (right). (c) UV−vis absorption spectrum of Au(I)−MHA complexes; inset shows its photograph in solution. The top panel shows the simplified structure of each sample. Color scheme: yellow, gold; green, sulfur of MHA ligand. The crystal structure of Au NCs was adapted from ref 39.
many in the field including us assumed that Au would not be a viable antimicrobial material. Instead, Au NP-based antimicrobial strategies evolved around grafting known antimicrobial compounds, such as ampicillin, antimicrobial peptides, and cationic or zwitterionic ligands, on the surface of large Au NPs, presumably using Au NPs as passive drug carriers.33−37 Now, since an enhanced antimicrobial activity was observed when the size of the Ag NPs was reduced to the NC range, we hypothesized that through size control, Au NPs, when reduced down to Au NCs, might begin to exhibit some special biological properties, such as antimicrobial activity, contrary to the popular assumption of inertness. Here, we demonstrated that ultrasmall Au NCs possess surprisingly high wide-spectrum antimicrobial activity, which is however absent in their larger Au NP counterparts. In addition, we elucidated the possible mechanism that drives the antimicrobial activity of ultrasmall Au NCs.
(NCs contain 25 Au atoms).22 Moreover, the Au NCs do not show any peak at ∼520 nm, which indicates that the asprepared Au NCs were homogeneously formed without any Au NP byproduct. As shown in the inset photo of Figure 1b, the Au NC solution is reddish-brown. This solution color is also indentical to that for thiolated Au25 NCs. In addition, from the inset TEM image of Figure 1b, the size of Au NCs was found to be less than 2 nm. As a result, the surface to volume ratio of Au NCs to Au NPs is approximately 3-fold. The molecular formula of the as-prepared Au NCs was determined to be Au25MHA18. This was evident from the electrospray ionization mass spectrometry (ESI-MS) analysis depicted in Figure S2, in which three sets of peaks at m/z 1262, 1514, and 1892 were observed. These peaks correspond to [Au25(MHA)18 − 5H]6−, [Au25(MHA)18 − 4H]5−, and [Au25(MHA)18 − 3H]4−, respectively. The detailed assignments are presented in the middle and bottom panels of Figure S2. For the structure of Au25MHA18, it has a centered icosahedral Au13 as the core. This core is protected by 12 Au(I) and 18 thiolate ligands, which are divided into six pairs of [SR−Au(I)−SR−Au(I)−SR] motifs (SR represents thiolate ligand).39 Compared to the Au NPs, our data suggest that the Au NCs possess better-defined structure and size. Consequently, the Au NCs could be expected to behave in a more molecular-like manner when compared to the Au NPs. In good agreement with the above data, the hydrodynamic diameter of Au NCs was ∼4 nm, whereas that of the Au NPs was ∼10 nm (Figure S3). The ζ-potential values of Au NCs and NPs were determined to be −30.6 ± 2.5 and −29.5 ± 1.6 eV, respectively. The similar ζ-potential value was expected as both Au NPs and NCs are protected by the same thiolate ligand. In addition, we also prepared Au(I)−MHA complexes that will be utilized as a reference group later in the study. The Au(I)−MHA complexes were produced by mixing the Au(III) ions and MHA in the absence of reducing agent. As shown in Figure 1c, the complexes do not show any absorption peak, and the color of the solution remains colorless, further confirming the absence of Au NCs or NPs in solution.
RESULTS AND DISCUSSION As size is the decisive factor in our hypothesis, it is pivotal for the Au NPs used in this study to have different sizes while keeping their respective surface groups the same. As such, we utilized 6-mercaptohexanoic acid (MHA) as protecting ligand in the synthesis of both Au NPs and NCs. As shown in Figure 1a, the UV−vis absorption spectrum of Au NPs shows one absorption peak at ∼520 nm, which is the characteristic surface plasmon resonance peak of small Au NPs (typically 2 nm). Au NCs could kill both the Grampositive and Gram-negative bacteria, showing their potential as a wide-spectrum bactericide. This wide-spectrum antimicrobial activity is attributed to the Au NCs’ ultrasmall size, allowing them to possess high surface to volume ratio. Consequently, when the Au NCs were internalized, this active surface could induce a metabolic imbalance in the cells, which leads to an increase of intracellular ROS production, conferring bacteria killing effect as the end result.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b02035. Experimental section of bacterial-related tests, such as cell death analysis, ROS measurement, half-maximal inhibitory concentration (IC50) test, microarray gene expression profiling, and cytotoxicity test; TEM images, ESI mass spectra, representative fluorescence images, ROS test results, cytotoxicity results (PDF)
EXPERIMENTAL SECTION Materials. Ultrapure water (18.2 MΩ) was used throughout the study. All glassware and magnetic stir bars were washed with aqua regia, rinsed with abundant ethanol and ultrapure water, and dried in an oven before use. All chemicals were commercially available and used as received: gold(III) chloride trihydrate (HAuCl4·3H2O), 6mercaptohexanoic acid (MHA), sodium borohydride (NaBH4), agar, and 2′,7′-dichlorofluorescein diacetate (DCFH-DA), lysozyme, lysostaphin, paraformaldehyde (PFA), poly(acrylic acid) (PAA), and Triton X-100 were purchased from Sigma-Aldrich; sodium hydroxide (NaOH) was from Merck; ethanol was from Fisher; Luria−Bertani (LB) was from Becton Dickinson; Hoechst 33342, SYTOX green nucleic acid stain, and ProLong Gold antifade reagent with DAPI were purchased from Life Technologies. Staphylococcus aureus (ATCC 25923) was a kind gift from Prof. Tan Kai Soo (National University of Singapore, NUS). Pseudomonas aeruginosa (NRRL-B 3509) and Bacillus subtilis (NRRL-NRS 762) were kind gifts from Prof Ting Yen Peng (NUS). Escherichia coli (ATCC 700926) was obtained from the American Type Culture Collection (ATCC, USA). Staphylococcus epidermidis (NBRC 12993) was obtained from the National Institute of Technology and Evaluation Biological Resource Center (NBRC, Japan). Instruments. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine the concentration of Au atoms in solution, which was measured with the iCAP 6000 series, Thermo Scientific. UV−vis absorption spectra of the samples were recorded by a Shimadzu UV-1800 photospectrometer. The size of Au NCs was determined by electrospray ionization (ESI) mass spectrometry on a Bruker microTOF-Q system. TEM images were taken on a JEOL JEM 2010 microscope operating at 200 kV. Hydrodynamic diameter and ζ-potential were measured on Malvern DLS (dynamic light scattering). Optical density at 600 nm (OD600) of bacterial cells and fluorescence intensity of dyes were measured on a Biotek H4FM microplate reader. The bacteria fluorescent images were taken with an epifluorescence microscope Leica DMI6000. Synthesis of Au NCs, Au NPs, and Au(I) Complex. MHAProtected Au NCs. The synthesis of Au NCs followed a reported method.22 In a typical synthesis, freshly prepared aqueous solutions of HAuCl4 (20 mM, 0.25 mL) and MHA (10 mM, 1 mL) were mixed in water (3.35 mL), leading to the formation of white Au(I)−MHA complexes. After that, an aqueous NaOH solution (1 M, 0.3 mL) was added to dissolve the mixture. A freshly prepared NaBH4 solution (112 mM) was obtained by dissolving 43 mg of NaBH4 in 2 mL of NaOH solution (1 M), followed by the addition of 8 mL of ultrapure water. After that, 0.1 mL of NaBH4 solution was added into Au(I)− MHA complexes, and the Au NCs were collected after 3 h. After synthesis, a stirred ultrafiltration cell (model 8010, Millipore Corporation, USA) with a semipermeable membrane of 3000 Da molecular weight cutoff was used to purify Au NCs in solution. MHA-Protected Au NPs. Freshly prepared aqueous solutions of HAuCl4 (20 mM, 0.25 mL) and MHA (10 mM, 0.1 mL) were mixed in water (4.65 mL), leading to the formation of white Au(I)−MHA
AUTHOR INFORMATION Corresponding Authors
*E-mail:
[email protected] (M.I.S.). *E-mail:
[email protected] (D.T.L.). *E-mail:
[email protected] (J.X.). ORCID
David Tai Leong: 0000-0001-8539-9062 Jianping Xie: 0000-0002-3254-5799 Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS This work was financially supported by Ministry of Education, Singapore, under Grant R-279-000-481-112. K.Z. acknowledges the National University of Singapore for her research scholarship. REFERENCES (1) Daniel, M.-C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications Toward Biology, Catalysis, and Nanotechnology. Chem. Rev. 2004, 104, 293−346. (2) Dreaden, E. C.; Alkilany, A. M.; Huang, X.; Murphy, C. J.; ElSayed, M. A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41, 2740−2779. (3) Kneipp, J.; Kneipp, H.; Kneipp, K. SERS−A Single-Molecule and Nanoscale Tool for Bioanalytics. Chem. Soc. Rev. 2008, 37, 1052− 1060. (4) Zhou, X.; Xu, W.; Liu, G.; Panda, D.; Chen, P. Size-Dependent Catalytic Activity and Dynamics of Gold Nanoparticles at the SingleMolecule Level. J. Am. Chem. Soc. 2010, 132, 138−146. (5) Alloyeau, D.; Ricolleau, C.; Mottet, C.; Oikawa, T.; Langlois, C.; Le Bouar, Y.; Braidy, N.; Loiseau, A. Size and Shape Effects on the Order-Disorder Phase Transition in CoPt Nanoparticles. Nat. Mater. 2009, 8, 940−946. (6) Li, B.; Setyawati, M. I.; Zou, H.; Dong, J.; Luo, H.; Li, N.; Leong, D. T. Emerging Zero-Dimensional Transition Metal Dichalcogenides Nanostructures for Sensors, Biomedicine, and Clean Energy. Small 2017, DOI: 10.1002/smll.201700527. (7) Albanese, A.; Tang, P. S.; Chan, W. C. The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems. Annu. Rev. Biomed. Eng. 2012, 14, 1−16. 6909
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